Biomonitoring SOP

Dr. Kathleen L. Caldwell
Phone: 770-488-7990
Fax:
770-488-4097
Email: [email protected]
James L. Pirkle, M.D., Ph.D.
Director, Division of Laboratory Sciences

Important Information for Users
The Centers for Disease Control and Prevention (CDC) periodically refines these
laboratory methods. It is the responsibility of the user to contact the person listed on the
title page of each write-up before using the analytical method to find out whether any
changes have been made and what revisions, if any, have been incorporated.

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Table of Contents
Cross reference to DLS CLIA and Policy and Procedures . . . . . . . . . .. . . . . . . . . 4
Index of tables and figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1) Clinical Relevance & Summary of Test Principle
a. Clinical Relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
b. Test Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2) Limitations of Method; Interfering Substances and Conditions
a. Interferences Addressed by This Method
i. Argon Chloride (40Ar35Cl) on Arsenic (75As) . . . . . . . . . . . . . . . . . . . . . . . .

ii. Tin (114Sn) and Molybdenum Oxide (98Mo16O) on Cadmium (114Cd) . .

iii. Matrix Enhancement of Arsenic Signal . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
b. Limitations of Method (Interferences Remaining in Method)
i. Calcium Chloride (40Ca35Cl) on Arsenic (75As) . . . . . . . . . . . . . . . . . . . . . .

3) Procedures for Collecting, Storing, and Handling Specimens; Criteria for
Specimen Rejection
a. Procedures for Collecting, Storing, and Handling Specimens . . . . . . . . . . . . . 9
b. Criteria for Specimen Rejection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
c. Transfer or Referral of Specimens; Procedures for Specimen Accountability
and Tracking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 10
4) Safety Precautions
a. General Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
b. Radiation Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
c. Waste Disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
5) Instrument & Material Sources
a. Sources for ICP-MS Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
b. Sources for ICP-MS Parts & Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
c. Sources for ICP-MS Maintenance Equipment & Supplies . . . . . . . . . . . . . . . . 19
d. Sources for General Laboratory Equipment & Consumables . . . . . . . . . . . . . . 19
e. Sources for Chemicals, Gases, & Regulators . . . . . . . . . . . . . . . . . . . . . . . . . . 21
6) Preparation of Reagents and Materials
a. Internal Standard Intermediate Mixture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

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b. Diluent and Carrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
c. Base Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
d. ICP-DRC-MS Rinse Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
e. Standards and Calibrators
i. Multi-element Intermediate Stock Calibration Standard . . . . . . . . . . . . . . . 28
ii. Multi-element Intermediate Working Calibration Standards. . . . . . . . . . . . . 29
iii. Working Multi-element Calibrators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

iv. Multi-element Intermediate Stock Calibration Verification Standard . . . . .

v. Multi-element Intermediate Working Calibration Verification Standards . .

vi. Internal Quality Control Materials (“Bench” QC) . . . . . . . . . . . . . . . . . . . . .

7) Analytical Instrumentation & Parameters
a. Instrumentation & Equipment Setup
i. ICP-DRC-MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Modifications made to ICP-DRC-MS . . . . . . . . . . . . . . . . . . . . . . . . . .

2. Configuration of tubing for liquid handling . . . . . . . . . . . . . . . . . . . . . .

3. Cones used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4. Gases & Regulators setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5. Chiller / Heat Exchanger. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ii. Computer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

iii. Autosampler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

b. Parameters for Instrument and Method (see Table 1) . . . . . . . . . . . . . . . . . . . 37
8) Method Procedures
a. Quality Control
i. Types of Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ii. Calibration Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

b. Daily Analysis of Samples
i. Preparation of the Analytical Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . .

ii. Preparation of Samples for Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

iii. Specimen Storage and Handling During Testing . . . . . . . . . . . . . . . . . . . .

iv. Starting the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v. Monitoring the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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vi. Records of Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii. Transfer of Results to the Laboratory Database . . . . . . . . . . . . . . . . . . . . .

viii. Analyst Evaluation of Run Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix. Submitting Final Work for Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

x. Overnight operation (or Any Use of Auto Stop) . . . . . . . . . . . . . . . . . . . . . . . 49
c. Equipment Maintenance
i. ICP-MS Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
ii. Data Backup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
9) Interpretation of the Results
a. Reportable Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
b. Reference Ranges (Normal Values) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
c. Action Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
10) Method Calculations
a. Method Limit of Detection (LOD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
b. Method Limit of Quantitation (LOQ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
c. QC Limits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

11) Alternate Methods for Performing Test and Storing Specimens If Test System
Fails . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Appendix A (Ruggedness Test Results) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Appendix B (Tables) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

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IRAT-DLS Method Code: 3018 and 3018A

1.
2.
3.
4.

7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.

19.

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Cross reference to DLS CLIA and Policy and Procedures policy
Summary of Test Principle and Clinical Relevance
1.a, 1.b
Safety Precautions
4
Computerization; Data System Management
7.a.ii, 8.b.vi, 8.b.vii, 8.b.vix, 8.c,ii
Specimen Collection, Storage, and Handling Procedures; Criteria for Specimen
Rejection
3
Procedures for Microscopic Examinations; Criteria for Rejection of Inadequately
Prepared Slides
- As no microscope used in this process there are no procedures for
microscopic examinations; and as no slides are prepared for this analysis
there is no criteria for rejection of inadequately prepared slides
Preparation of Reagents, Calibrators (Standards), Controls, and All Other
Materials; Equipment and Instrumentation
5, 6, 7, 8
Calibration and Calibration Verification Procedures
8.a.ii
Procedure Operating Instructions; Calculations; Interpretation of Results
8, 9
Reportable Range of Results
9.a
Quality Control (QC) Procedures
8.a.i, 8.b.viii, 10.c
Remedial Action If Calibration or QC Systems Fail to Meet Acceptable Criteria
8.b.viii
Limitations of Method; Interfering Substances and Conditions
2
Reference Ranges (Normal Values)
9.b
Critical Call Results ("Panic Values")
9.c
Specimen Storage and Handling During Testing
8.b.iii
Alternate Methods for Performing Test or Storing Specimens If Test System Fails
11
Test Result Reporting System; Protocol for Reporting Critical Calls (If Applicable)
8.b.vi, 8.b.vii, 8.b.ix, 9.c.
Transfer or Referral of Specimens; Procedures for Specimen Accountability and
Tracking
3.c
References

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List of Tables
Table 1. Instrument and Method Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Table 2. Suggested maximum analyte concentrations for base urine . . . . . . . . . . . 73
Table 3. Concentrations of Analytes in the Multi-Element Intermediate Stock
Standard from High Purity Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Table 4. Preparation of Multi-element Intermediate Working Standards . . . . . . . . . 75
Table 5. Acceptable ways to perform two consecutive analytical runs, bracketing
with bench quality control samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Table 6. A typical SAMPLE/BATCH window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Table 7. Preparation of Multi-element Intermediate Working Standards . . . . . . . . . 78
Table 8. Range of Reporting and Calibration Verification Requirements . . . . . . . . . 79
Table 9. Boundary Concentrations for Urine Concentrations (µ/L) . . . . . . . . . . . .

Table 10. Reference Ranges for Urine Concentrations (from the Third National
Report on Exposure to Environmental Chemicals). All results in µg/L . . . 81
Table 11. References to Total Urine Arsenic Concentrations . . . . . . . . . . . . . . . . . . . 82

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List of Figures
Figure 1.

Configuration of tubing and devices for liquid handling
a. ESI SC4 Autosampler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Figure 2.

Figure 3.

Figure 4.

ELAN ICP-DRC-MS Method Screen Shots (12 element panel)
a. Timing Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

b. Processing Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c. Equations Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

d. Calibration Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

e. Sampling Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

f. Report Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ELAN ICP-DRC-MS Method Screen Shots (12 element panel)
a. Timing Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

b. Processing Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c. Equations Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

d. Calibration Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

e. Sampling Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

f. Report Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Appendix B (cont)
a. ESI SC4 Autosampler Screen Shots used (Main page) . . . . . . . . .

b. ESI SC4 Autosampler Screen Shots used (“Configure” page) . . . .

c. ESI SC4 Autosampler Screen Shots used (“Communication” page)

d. ESI SC4 Autosampler Screen Shots (“FAST” page) *As only* . . . .

e. ESI SC4 Autosampler Screen Shots (5x12 Rack Setup window) . .

100

f. ESI SC4 Autosampler Screen Shots (50mLTube Rack Setup) . . . .

100

g. ESI SC4 Autosampler Screen Shots (Rinse Station Rack Setup) . .

101

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1) Clinical Relevance & Summary of Test Principle
a. Clinical Relevance:
This method is used to achieve rapid and accurate quantification of fifteen
elements of toxicological and nutritional interest including Antimony (Sb), Arsenic
(As), Barium (Ba), Beryllium (Be), Cadmium (Cd), Cesium (Cs), Cobalt (Co),
Lead (Pb), Manganese (Mn), Molybdenum (Mo), Platinum (Pt), Strontium (Sr),
Thallium (TI), Tin (Sn), Tungsten (W), and Uranium (U). The method may be
used to screen urine when people are suspected to be acutely exposed to these
elements or to evaluate chronic environmental or other non-occupational
exposure. [1-4].
b. Test Principle:
Inductively coupled plasma dynamic reaction cell mass spectrometry (ICP-DRCMS) is a multi-element analytical technique capable of trace level elemental
analysis [1-4]. This ICP-DRC-MS method is used to measure either arsenic, a
15 element panel (Antimony, Barium, Beryllium, Cadmium, Cesium, Cobalt,
Lead, Manganese, Molybdenum, Platinum, Strontium, Thallium, Tin, Tungsten,
and Uranium), or any subgroup of the 15 element panel.
Liquid samples are introduced into the ICP through a nebulizer and spray
chamber carried by a flowing argon stream. By coupling radio-frequency power
into flowing argon, plasma is created in which the predominant species are
positive argon ions and electrons and has a temperature of 6000-8000 K. The
sample passes through a region of the plasma and the thermal energy atomizes
the sample and then ionizes the atoms. The ions, along with the argon, enter the
mass spectrometer through an interface that separates the ICP (at atmospheric
pressure, ~760 torr) from the mass spectrometer (operating at a pressure of 10-5
torr). The ions pass through a focusing region, the dynamic reaction cell, the
quadrupole mass filter, and finally are counted in rapid sequence at the detector
allowing individual isotopes of an element to be determined. The dynamic
reaction cell operates in one of two modes. In ‘standard’ mode the cell is not
pressurized and ions pass through the cell to the quadrupole mass filter
unaffected. In ‘DRC’ mode the cell is pressurized with a gas which will collide or
react with the incoming ions to either eliminate an interfering ion or change the
ion of interest to a new mass which is free from interference. In this method the
instrument is operated in DRC mode when analyzing for cadmium, manganese
and arsenic, but in standard mode when analyzing for all of the other analytes.
For arsenic, the reaction cell is pressurized with a mixture of hydrogen (10%) and
argon (90%) which causes the breakup of the 40Ar35Cl+ ion which would
otherwise interfere with detection of 75As at m/z 75. When analyzing for
cadmium, the reaction cell is pressurized with oxygen. The 98Mo16O+ ions which
would normally interfere with detection of 114Cd at m/z 114 react with the oxygen
in the cell creating 98Mo16O 2 + and 98Mo16O 3 + at masses which no longer
represent an interference to 114Cd analysis. The DRC is also pressurized with

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oxygen gas when analyzing for 55Mn. The 39K16O+ ions which would normally
interfere with the detection of 55Mn at m/z 55 react with the oxygen in the cell and
no longer represent interference to 55Mn analysis. Electrical signals resulting
from the detection of ions are processed into digital information that is used to
indicate first the intensity of the ions and then the concentration of the element.
This method was originally based on the method by Mulligan et al. [5]. The DRC
portions of the method are based on work published by Tanner et al. [2, 3]. Urine
samples are diluted 1+ 9 with 2% (v/v) concentrated nitric acid (and 1.5% ethanol
in the case of arsenic). The diluent for the 15 element panel contains iridium (Ir),
rhodium (Rh) for multi-internal standardization. The diluent for arsenic contains
gallium (Ga) for internal standardization. Nitric acid is used for the purpose of
solubilizing and stabilizing metals in solution. Internal standards are a constant
concentration in all blanks, calibrators and samples. Monitoring the instrument
signal ratio of a metal to its internal standard allows correction for instrument
noise and drift, and sample-to-sample matrix differences. Ethanol is used in the
case of arsenic for the purpose of providing a constant amount of signal
enhancement (carbon effect) across all blanks, calibrators, and samples.
2) Limitations of Method; Interfering Substances and Conditions
a. Interferences Addressed by This Method
i. Breakup of Argon Chloride (40Ar35Cl) Interference on Arsenic (75As) Using
DRC: The dynamic reaction cell of the ELAN ICP-DRC-MS is used in this
method to break apart the argon chloride (40Ar35Cl) interference on arsenic at
m/z 75 [6] which is common to urine analysis by ICP-MS (see Section 1.b for
an explanation of this process).
ii. Correction & Elimination of Interferences (114Sn, 98Mo16O) on Cadmium (114Cd).
1. Mathematical Correction for Tin (114Sn) Interference:
The correction equation (-0.026826*Sn118) is used in the “Equations” tab
of the method to correct the counts observed as m/z 114 to exclude counts
due to 114Sn.
2. Elimination of Molybdenum Oxide (98Mo16O) Interference Using DRC:
The dynamic reaction cell of the ELAN ICP-DRC-MS is used in this
method to eliminate interference from molybdenum oxide (98Mo16O) onto
cadmium at m/z 114 [7]. See Section 1.b for an explanation of this
process.
iii. Elimination of interference (39K16O) on manganese 55Mn using DRC:
The dynamic reaction cell of the ELAN ICP-DRC-MS is used in this method to
reduce the potassium oxide (39K16O) interference on manganese at m/z 55.
See section 1.b for an explanation of this process.
iv. Matrix Enhancement of Arsenic Signal:

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Matrix induced signal enhancement in ICP-MS analysis from carbon on arsenic
has been previously reported in the literature [8, 9]. When arsenic is being
determined by this method, ethanol (1.5% v/v) is added in the diluent and rinse
solutions to “normalize” the arsenic signal enhancement in all blanks,
calibrators, and samples.
b. Limitations of Method (Interferences Remaining in Method)
i. Calcium Chloride (40Ca35Cl) Interference on Arsenic (75As):
It has been determined that a small interference remains at m/z 75 when the
urine matrix contains both high chloride and high calcium levels [6]. Even at
extreme calcium and chloride levels, this interference is has not been found to
be significant (approximately 0.4 µg/L).
ii. Gallium Oxide (71Ga17O) Interference on Strontium (88Sr):
Arsenic only analysis requires Ga to be used as the internal standard and is
added to the diluent at a concentration of 10 ug/L (see section 6.c for the
preparation of diluent). Gallium should not be added to the diluent (for the
purpose of being used as an internal standard for As) when strontium is
measured (15 element panel) due to the formation of 71Ga17O+, which occurs
at the same m/z as 88Sr+. A 5 ug/L solution of Ga in 2% HNO 3 resulted in a
background equivalent concentration (BEC) of 50 ng/L for 88Sr. Based on
these results, the expected increase in 88Sr concentration in a diluted urine
sample is 1 ug/L.
3) Procedures for Collecting, Storing, and Handling Specimens; Criteria for
Specimen Rejection; Specimen Accountability and Tracking
a. Procedures for Collecting, Storing, and Handling Specimens: Specimen handling
conditions, special requirements, and procedures for collection and transport are
discussed in the division (DLS) Policies and Procedures Manual [10]. Copies are
available in branch, laboratory, and special activities specimen-handling offices.
An electronic copy is available at:
http://intranet.nceh.cdc.gov/dls/pdf/policiesprocedures/Policy_and_Procedures_
Manual.DLS.2002mod.pdf. In general,
i. No fasting or special diets are required before collection of urine.
ii. Use sterile, lot screened collectors for specimen acquisition.
iii. Urine specimens should be transported frozen (packed in dry ice during
shipment is preferred when possible).
iv. Once received, store long term at ≤ -20°C until time for analysis. Short-term
storage at 2-4°C is acceptable. Refreeze at ≤ -20°C portions of the sample

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that remain after analytical aliquots are withdrawn. Thawing and refreezing
samples has not been found to compromise sample results.
v. Acceptable containers for analytical aliquots include lot screened
polypropylene (PP) cryovials or tubes (i.e. 5 mL cryogenic vial or 15mL
centrifuge tube).
b. Criteria for Specimen Rejection: Specimen characteristics that may compromise
test results are indicated above. Reasons for rejection of a sample for analysis
include
i. Low volume: Optimal amount of urine is 1.8+ mL. The volume of urine used
for one analysis is 0.5 mL.
ii. Contamination: Improper collection procedures or collection devices can
contaminate the urine by contact with dust, dirt, etc.
In all cases, request a second urine specimen.
c. Transfer or Referral of Specimens; Procedures for Specimen Accountability and
Tracking: Location, status, and final disposition of the specimens will be tracked
at least by paper document in the “Study Folder” (created before analysts receive
the samples). Apart from this specimen tracking form, this folder will also contain
the paper print outs of results from analysis of the specimens. Maintain records
for a minimum of 3 years. Use only numerical identifiers for samples within the
laboratory (e.g., case ID numbers) in order to safeguard confidentiality. Only the
medical supervisor (MS) or project coordinator (PC) i.e. non CDC personnel
should have access to the personal identifiers.
4) Safety Precautions
a. General Safety
i. Observe all safety regulations as detailed in the Division (DLS) Safety Manual.
Additional information can be found in your lab’s chemical hygiene plan.
ii. Observe Universal Precautions when working with urine.
iii. Wear appropriate gloves, lab coat, and safety glasses while handling all
solutions. Consult the laboratory chemical hygiene plan.
iv. Exercise special care when handling and dispensing concentrated nitric acid.
Add acid to water. Nitric acid is a caustic chemical that is capable of causing
severe eye and skin damage. If nitric acid comes in contact with any part
of the body, quickly wash the affected area with copious quantities of
water for at least 15 minutes.

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v. Use secondary containment for containers of biological or corrosive liquids.
vi. The use of the foot pedal on the Micromedic Digiflex™ is recommended
because it reduces analyst contact with work surfaces that have been in
contact urine and also keeps the analyst’s hands free to hold the specimen
cups and autosampler tubes and to wipe off the tip of Micromedic Digiflex™.
vii. Training will be given before operating the ICP-DRC-MS, as there are many
possible hazards including ultraviolet radiation, high voltages, radio-frequency
radiation, and high temperatures. This information is also detailed in the
PerkinElmer ELAN® ICP-DRC-MS System Safety Manual.
viii. Use flash arrestors on oxygen and argon / hydrogen gas cylinders and properly
secure gas cylinders with safety harnesses.
ix. Wipe down all work surfaces at the end of the day with bleach-rite spray or
freshly prepared 10% (v/v) sodium-hypochlorite solution.
b. Radiation Safety: All personnel performing this method must successfully meet
requirements of a CDC-OHS radiation worker (RW) due to the use of natural
uranium in this method and observe all necessary radiation safety considerations
indicated in the CDC Radiation Safety Manual [11].
c. Waste Disposal: Operators of this method should take the CDC-OHS Hazardous
Chemical Waste Management Course (initial and yearly refreshers).
i. Waste to be Placed Into Biohazard Autoclave Bags & Pans:
1. All biological samples and diluted specimens (after analysis run).
2. All disposable plastic and paper which contact urine (autosampler tubes,
gloves, etc.).
3. Used non-glass/quartz ICP-MS consumables (i.e. probes, tubing, cones,
ion lenses).
ii. Waste to be Placed Into Sharps Containers: Pipette Tips, broken glass or
quartz instrument consumables (broken spray chambers, torches, nebulizers,
etc. . .). Large broken glass which will not fit in the sharps container should be
placed in a separate autoclave pan from other waste and labeled as “broken
glass” (see the “Autoclaving” section of the CDC safety policies and practices
manual located in the laboratory).
iii. Liquid Waste

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1. Waste discarded down sink: Only liquid waste from the ICP-DRC-MS
instrument can be discarded at the sink. Flush the sink with copious
amounts of water.
2. Waste to be Picked up by the Radiation Safety Office: Contact the
laboratory radiation inventory person and the CDC Radiation Safety Office
for disposal of any single element uranium standard, intermediate stock
standard, or intermediate working standard solutions.
3. Waste to be Picked up by Hazardous Waste Program: Submit request for
hazardous waste removal of all other liquid waste.

5) Instrument & Material Sources
a. Sources for ICP-MS Instrumentation
i. ICP-MS:
Inductively Coupled Plasma Dynamic Reaction Cell Mass
Spectrometer (ELAN® 6100 DRCPlus or ELAN® DRC II) (PerkinElmer Norwalk,
CT, www.perkinelmer.com).
1. DXi-FAST upgrade: Standard peristaltic pump replaced by DXi_FAST
micro-peristaltic pump / FAST actuator and valve combination unit. For
ELAN DRCII, part # DXI-54-P4-F6.
Refrigerated chiller
ii. Recirculating chiller / heat exchanger for ICP-MS:
®
Plus
(PolyScience 6105PE for ELAN 6100 DRC
instruments) or heat exchanger
(PolyScience 3370 for ELAN® DRC II instruments) (PerkinElmer Norwalk, CT,
www.perkinelmer.com).
iii. Autosampler: ESI SC-4 autosampler (Elemental Scientific Inc., Omaha, NE) or
equivalent.
iv. FAST Sample Introduction System (Elemental Scientific Inc., Omaha, NE).
1. FAST controller
2. FAST actuator: CTFE high-flow valve head like part number SC-0599-1210
(part number includes lines and probes).
b. Sources for ICP-MS Parts & Consumables
NOTE: The minimum number of spares recommended before reordering (if
owning one instrument) are listed as “# Spares =” in the descriptions below.
i. Adapter, PEEK: Securely connects 1.6mm O.D. PFA tubing to 0.03” I.D.
peristaltic tubing. Composed of three PEEK parts.

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1. Female nut for 1.6mm O.D. (1/16”) tubing. Like part P-420 (Upchurch
Scientific, Oak Harbor, WA, www.upchurch.com).
2. PEEK ferrule. Like part P-260x (10pk SuperFlangeless ferrule, Upchurch
Scientific, Oak Harbor, WA, www.upchurch.com).
3. Conical Adapter Body. Like part P-692 (Upchurch Scientific, Oak Harbor,
WA, www.upchurch.com).
ii. Bottles (for rinse solution): Four liter screw-cap polypropylene container with 2
luer connections (like catalog# SC-0305-1, Elemental Scientific Inc., Omaha,
NE., www.elementalscientific.com).
iii. Carboy and cap assembly for waste collection: 10-15 L, polypropylene
widemouth carboy (100 mm neck size) with handles and no spigot (Like part
#7BE-25126, Lab Safety Supply, Janesville, WI, www.lss.com) with cap
assembly
like
part
#
N0690271
(PerkinElmer,
Norwalk,
CT,
www.perkinelmer.com).
iv. Coolant, for Polyscience chiller or heat exchanger: Only PerkinElmer part #
WE01-6558 (PerkinElmer Norwalk, CT, www.perkinelmer.com) is approved for
use by PerkinElmer. # Spares = 6.
v. Cone, sampler (nickel/platinum): PerkinElmer part # WE021140/WE027802
(PerkinElmer Norwalk, CT, www.perkinelmer.com). Part # SC2011-Ni (Testing
has also found Spectron, Ventura, CA, www.spectronus.com cones to be
comparable). # Spares = 4.
vi. Cone, skimmer (nickel/platinum): PerkinElmer part # WE021137/WE027803
(PerkinElmer Norwalk, CT, www.perkinelmer.com). Part # SC2012-Ni (Testing
has also found Spectron, Ventura, CA, www.spectronus.com cones to be
comparable) # Spares = 4.
vii. Connector (for tubing): Use to connect 1/8” I.D. PVC tubing to 0.125” I.D
peristaltic pump tubing. Use part # 3140715 (PerkinElmer Norwalk, CT,
www.perkinelmer.com) or equivalent. # Spares = 4.
viii. Detector, electron multiplier: Like part # N8125001 (PerkinElmer Norwalk, CT,
www.perkinelmer.com). Available direct from manufacturer (part # 14210,
SGE Incorporated, Austin, Texas, http://www.etpsci.com) or various
distributors. # Spares = 1.
ix. FAST accessories
1. Valve: CTFE High-flow valve head for SC-FAST (uses ¼-28 fittings). Like
part # SC-0599-1010 (Elemental Scientific Inc., Omaha, NE.,
www.elementalscientific.com).
2. Stator: CTFE Stator for 6 port SC-FAST high flow valve (¼-28 fittings).
Like part # SC-0599-1010-01 (Elemental Scientific Inc., Omaha, NE.,
www.elementalscientific.com).
3. Rotor: Composite rotor for 6 port SC-FAST high flow valve (¼-28 fittings).
Like part # SC-0599-1010-05 (Elemental Scientific Inc., Omaha, NE.,
www.elementalscientific.com).

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4. Sample Loop:
a. Multielement analysis: 3 mL Teflon, white connector-nuts for high flow
valve head. Like part # SC-0315-30 (Elemental Scientific Inc., Omaha,
NE., www.elementalscientific.com).
b. Arsenic only analysis: Default volume is 1.5mL Teflon sample loop
with white nut connectors for high flow valve head of FAST sample
introduction system. This volume loop can be created by cutting 25%
off the length of a 2mL Teflon sample loop was cut to the 1.5mL length.
Like part # SC-0315-20 (Elemental Scientific Inc., Omaha, NE.,
www.elementalscientific.com). Volumes larger than 1.5mL can be
used, but will require longer loop fill (ESI software) and sample flush
(ELAN software) times, and proportionally larger volumes used in
sample preparation (Table 7 in the Appendix).
5. Probe, Autosampler: Teflon, carbon fiber support, 0.8mm i.d., blue marker,
1/4-28 fittings. Like part number SC-5037-3751 (Elemental Scientific Inc.,
Omaha, NE., www.elementalscientific.com). # Spares = 2.
6. Probe, Carrier Solution: Teflon, carbon fiber support, 0.5mm i.d., orange
marker, 1/4-28 fittings. Like part number SC-5037-3501 (Elemental
Scientific Inc., Omaha, NE., www.elementalscientific.com). # Spares = 2.
7. Tubing, FAST vacuum: Vacuum line for SC-FAST high flow valve,
connects to port #6, black nut for connection to valve head, natural brown
color nut on other end for connection to SC autosampler vacuum port. Like
part
#
SC-0321
(Elemental
Scientific
Inc.,
Omaha,
NE.,
www.elementalscientific.com).
8. Tubing, connects nebulizer to valve: See “Nebulizer, PolyPro-ST micro
flow”
x. Hose, for connection to chiller: Push on hose. I.D. = ½”, O.D. = ¾”. Use part
# PB-8 (per inch, Georgia Valve and Fitting, Atlanta, GA, www.swagelok.com)
or equivalent. Do not normally need spare hose (unless moving instrument
into a new location).
xi. Hose, for exhaust of ELAN: Available as part of ELAN installation kit from
Perkin Elmer (PerkinElmer Norwalk, CT, www.perkinelmer.com). Available
direct from manufacturer as part # S-LP-10 air connector (Thermaflex,
Abbeville, SC, www.thermaflex.net). Equivalent part may be substituted. #
Spares = 10 feet of 4” diameter and 10 feet of 6” diameter hose.
xii. Injector, quartz with ball joint: I.D. = 2.0 mm. PerkinElmer part # WE023948
(PerkinElmer Norwalk, CT, www.perkinelmer.com). Available direct from
manufacturer as part # 400-30 (Precision Glass Blowing, Centennial, CO,
www.precisionglassblowing.com) or from various distributors. # Spares = 2.
xiii. Injector support (for pass-through injector: PerkinElmer part # WE023951
(PerkinElmer Norwalk, CT, www.perkinelmer.com). Available direct from
manufacturer as part # 400-37 (Precision Glass Blowing, Centennial, CO,
www.precisionglassblowing.com) or from various distributors. # Spares = 2.

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xiv. Ion Lens:
PerkinElmer part # WE018034 (PerkinElmer Norwalk, CT,
www.perkinelmer.com). # Spares = 3.
xv. Nebulizer: PolyPro-ST micro flow polypropylene nebulizer with external 1/4-28
threaded connector for liquid delivery, low pressure version or equivalent. Like
part # ES-4040-7010 (Elemental Scientific Inc., Omaha, NE.,
www.elementalscientific.com). # Spares = 1. Different nebulizers may be
used, however, the nebulizer gas flow rate, sample flush time, read delay time,
loop fill time, loop size, urine sample dilution preparation volume, and sampleto-sample carry-over must be evaluated and optimized.
1. Gas connection:
a. Teflon tubing: 4mm o.d., 2.4mm i.d. Teflon tubing (like part # ES2502,
Elemental
Scientific
Inc.,
Omaha,
NE.,
www.elementalscientific.com). # Spares = 1.
b. Adapter kit: Plastic adapters to connect Teflon tubing (2.4mm i.d) to
¼” male Swagelok (compression) port on ICP-DRC-MS. Parts can
be obtained as components in a “gas fittings kit for microflow
nebulizer”, kit part # ES-2501-1000, Elemental Scientific Inc.,
Omaha, NE., www.elementalscientific.com). # Spares = 1.
2. Liquid connection: Connects nebulizer to port #3 of high flow FAST valve
head with green, 1/4- 28 fitting. Like part # SC-0317-0250 (Elemental
Scientific Inc., Omaha, NE., www.elementalscientific.com). # Spares = 2.
xvi. Nut: (for flanged connections of 1.59mm (1/16”) o.d. PFA tubing) Flanged, for
1/16” o.d. tubing, 1/4-28 threads. Use part # P-406x (pkg. of 10, Upchurch
Scientific, Oak Harbor, WA, www.upchurch.com) or equivalent. Use a Tefloncoated Viton o-ring with this nut instead of the stainless steel washer that
comes with part # P-406x). # Spares = 10.
xvii. Nut and Ferrule set, 1/8” Swagelok: Such as part # SS-200-NFSET (stainless
steel) or part # B-200-NFSET (brass) (Georgia Valve and Fitting, Atlanta, GA,
www.swagelok.com) or equivalent. For part numbers listed here a quantity of 1
means 1 nut, 1 front ferrule, and 1 back ferrule. Spares = 20.
xviii. Nut and Ferrule set, 1/4” Swagelok: Such as part # SS-400-NFSET (stainless
steel) or part # B-400-NFSET (brass) (Georgia Valve and Fitting, Atlanta, GA,
www.swagelok.com) or equivalent. For part numbers listed here a quantity of 1
means 1 nut, 1 front ferrule, and 1 back ferrule. Spares = 20.
xix. Oil, Welch Directorr Gold: For roughing pumps. Available direct from
manufacturer as part # 8995G-15 (1 gallon, Welch Rietschle Thomas, Skokie,
IL, www.welchvacuum.com) or from various distributors. Equivalent oil may be
substituted. # Spares = 4.
xx. O-ring: (for sampler cone) PerkinElmer part # N8120511 (pkg. of 5,
PerkinElmer, Shelton, CT, www.perkinelmer.com) or equivalent. # Spares = 20
o-rings.

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xxi. O-ring: (for skimmer cone) PerkinElmer part # N8120512 (pkg. of 5,
PerkinElmer, Shelton, CT, www.perkinelmer.com) or equivalent. # Spares = 20
o-rings.
xxii. O-ring: (for flanged connections of 1.59mm (1/16”) o.d. PFA tubing) Tefloncoated Viton o-ring, i.d. = 1/16", thickness = 1/16”, o.d. = 3/16”. Such as part #
V75-003 (O-rings West, Seattle, WA, www.oringswest.com) or equivalent. #
Spares = 20.
xxiii. O-ring: (for injector support).
1. Internal o-rings: ID = ¼”, OD = 3/8”, thickness = 1/16”. Need 2 o-rings per
injector support setup. PerkinElmer part # N8122008 (PerkinElmer,
Shelton, CT, www.perkinelmer.com) or equivalent (such as part # V75-010,
O-rings West, Seattle, WA, www.oringswest.com). # Spares = 20.
2. External o-rings: ID = 3/8”, OD = 1/2”, thickness = 1/16”. Need 2 o-rings
for each injector support setup.
PerkinElmer part # N8122009
(PerkinElmer, Shelton, CT, www.perkinelmer.com) or equivalent (such as
part # V75-012, O-rings West, Seattle, WA, www.oringswest.com). #
Spares = 20.
xxiv. O-ring: (for inside spray chamber at nebulizer port) Such as part # 120-56
(Precision Glass Blowing, Centennial, CO, www.precisionglassblowing.com).
Additional o-rings can sometimes be obtained free of charge or at reduced
price when acquired while purchasing spray chambers. # Spares = 20.
xxv. O-ring: (for inside of torch mount): Part # WE017284 (PerkinElmer, Shelton,
CT, www.perkinelmer.com). Do not substitute. The PerkinElmer o-ring is
specially metal impregnated to minimize RF leakage though the torch mount.
# Spares = 2.
xxvi. Photon Stop: PerkinElmer part # WE018278 (PerkinElmer, Shelton, CT,
www.perkinelmer.com). # Spares = 1.
xxvii. Plugs, Quick Change for Roughing Pump Oil: These plugs will only work on
the Varian roughing pumps which come standard on ELAN DRC II ICPMS
instruments. These plugs will not fit the Leybold pumps which come standard
on the ELAN DRC Plus instruments. Part # W1011013 (PerkinElmer, Shelton,
CT, www.perkinelmer.com). No spares typically needed.
xxviii. Probes
1. for ESI autosampler: Teflon, carbon fiber support, 0.8mm i.d., blue
marker, 1/4-28 fittings. Like part number SC-5037-3751 (Elemental
Scientific Inc., Omaha, NE., www.elementalscientific.com). # Spares = 2.
2. for carrier solution of FAST sample introduction system: Teflon, carbon
fiber support, 0.5mm i.d., orange marker, 1/4-28 fittings. Like part number
SC-5037-3501
(Elemental
Scientific
Inc.,
Omaha,
NE.,
www.elementalscientific.com). # Spares = 2.
xxix. RF coil.
PerkinElmer part # WE02-1816 (PerkinElmer, Shelton, CT,
www.perkinelmer.com) or equivalent. # Spares = 2.

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xxx. Screw, for Torch Mount: PerkinElmer part # WE011870. (PerkinElmer,
Shelton, CT, www.perkinelmer.com) or equivalent. # Spares = 3.
xxxi. Spray chamber, quartz concentric:
PerkinElmer part # WE025221
(PerkinElmer, Shelton, CT, www.perkinelmer.com) or equivalent. Available
direct from manufacturer as part # 400-20 (Precision Glass Blowing,
Centennial, CO, www.precisionglassblowing.com) or from various distributors.
# Spares = 2.
xxxii. Torch, quartz: PerkinElmer part # N812-2006 (PerkinElmer, Shelton, CT,
www.perkinelmer.com) or equivalent. Available direct from manufacturer as
part
#
400-10
(Precision
Glass
Blowing,
Centennial,
CO,
www.precisionglassblowing.com) or various distibutors. Damaged torches can
often be repaired for substantially lower cost than purchasing a new one by
companies such as Wilmad LabGlass (Buena, NJ, www.wilmad-labglass.com)
or Precision Glass Blowing (Centennial, CO, www.precisionglassblowing.com).
# New Spares = 2.
xxxiii. Tubing and adapter, for SC autosampler rinse station drain: Tygon tubing and
adapter to attach to back of SC autosampler for draining rinse station waste
(like part # SC-0303-002, Elemental Scientific Inc., Omaha, NE.,
www.elementalscientific.com).
xxxiv. Tubing and adapters, for SC autosampler rinse station filling: Teflon tubing
and adapters (to attach to back of SC autosampler for filling rinse stations and
to attach to rinse containers). Like part # SC-0302-0500, Elemental Scientific
Inc., Omaha, NE., www.elementalscientific.com).
xxxv. Tubing and nut, for FAST carrier solution: 0.5mm i.d. Teflon tubing (orange
marker) with red ¼-28 male nut. Connects to high flow FAST valve head, port
#2. Like part # SC-0316-0500 (Elemental Scientific Inc., Omaha, NE.,
www.elementalscientific.com).
xxxvi. Tubing, FAST vacuum: Vacuum line for SC-FAST high flow valve, connects to
port #6, black nut for connection to valve head, natural brown color nut on
other end for connection to SC autosampler vacuum port. Like part # SC-0321
(Elemental Scientific Inc., Omaha, NE., www.elementalscientific.com).
xxxvii. Tubing, main argon delivery to instrument: I.D. = 1/8”, O.D. = ¼”. Such as part
# C-06500-02 (pkg. of 100ft, polypropylene, Fisher Scientific International,
Hampton, NH, www.fishersci.com) or equivalent. # Spares = 50ft.
xxxviii. Tubing, PFA: I.D. = 0.5mm, O.D. = 1.59mm (1/16”). Used to transfer liquid
between rinse solution jug and peristaltic pump tubing
The Perfluoroalkoxy (PFA) copolymer is a form of Teflon®. Such as part #
1548 (20ft length, Upchurch Scientific, Oak Harbor, WA, www.upchurch.com)
or equivalent. # Spares = 20ft.
xxxix. Tubing, peristaltic, 0.045” i.d. (rinse station feed): Standard PVC, 2-stop (red /
red) peristaltic pump tubing, i.d. = 0.045”. PerkinElmer part # N0680375,
(PerkinElmer, Shelton, CT, www.perkinelmer.com) or equivalent. # Spares = 6
packs of 12 tubes.

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xl. Tubing, peristaltic, 0.03” i.d. (carrier solution for ESI autosampler): use either
1. Standard PVC, 2-stop (black / black) peristaltic pump tubing, i.d. = 0.03”.
PerkinElmer
part
#
09908587
(PerkinElmer,
Shelton,
CT,
www.perkinelmer.com) or equivalent. # Spares = 6 packs of 12 tubes.
2. Standard PVC, 3-stop (black/ black/black) peristaltic pump tubing, i.d. 0.76
mm.
Spectron part # SC0056 (Spectron, Ventura, CA,
www.spectronus.com) or equivalent. #Spares = 6 packs of 12 tubes. Use
this type of tubing with ESI DXi micro-peristaltic pump.
xli. Tubing, peristaltic, 0.125” i.d. (spray chamber drain): use either
1. Standard PVC, 2-stop (black / white) peristaltic pump tubing, i.d. = 0.125”
or equivalent. PerkinElmer part # N812-2012 (PerkinElmer, Shelton, CT,
www.perkinelmer.com) or equivalent. # Spares = 6 packs of 12 tubes.
2. Standard Santoprene, 3-stop (grey/ grey/ grey) peristaltic pump tubing, i.d.
1.30 mm.
Spectron part # SC0311 (Spectron, Ventura, CA,
www.spectronus.com) or equivalent. #Spares = 6 packs of 12 tubes. Use
this type of tubing with ESI DXi micro-peristaltic pump.
xlii. Tubing, PVC, i.d. = 1/8”, o.d. = 3/16”. May be used to transfer liquid
1. between spray chamber waste port and peristaltic pump
2. between peristaltic pump and liquid waste jug
Like part # 14-169-7A (pkg. of 50ft, Fisher Scientific International, Hampton,
NH, www.fishersci.com) or equivalent. # Spares = 20ft.
xliii. Tubing, Stainless Steel, o.d. = 1/8”, wall thickness = 0.028”: Used to connect
DRC gas cylinders to ELAN DRC gas ports. Also used to replace plastic
tubing in the DRC gas path within the ELAN. Like part # SS-T2-S-028-20 (20ft,
Georgia Valve and Fitting, Atlanta, GA, www.swagelok.com) or equivalent.
Spares = 20ft.
xliv. Tubing, Teflon, corrugated, ¼” o.d.: Connects to the auxiliary and plasma gas
side-arms of the torch. Part # WE015903 (PerkinElmer, Shelton, CT,
www.perkinelmer.com). # Spares = 2.
xlv. Tubing, vinyl (argon delivery to nebulizer): Vinyl Tubing, 1/8" ID x 1/4" OD.
Like part # EW-06405-02 (Cole Parmer, Vernon Hills, Illinois,
www.coleparmer.com) or equivalent. Equivalent tubing material may be
substituted. # Spares = 10ft.
xlvi. Union Elbow, PTFE ¼” Swagelok: Connects argon tubing to torch auxiliary
gas sidearm on bayonet mount ELAN ICPMS instruments. Like part # T-400-9
(Georgia Valve and Fitting, Atlanta, GA, www.swagelok.com) or equivalent.
Spares = 2.
xlvii. Union Tee, PTFE, ¼” Swagelok: Connects argon tubing to torch plasma gas
sidearm and holds igniter inside torch sidearm on bayonet mount ELAN ICPMS
instruments. Like part # T-400-3 (Georgia Valve and Fitting, Atlanta, GA,
www.swagelok.com) or equivalent. Spares = 2.

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c. Sources for ICP-MS Maintenance Equipment & Supplies
i. Anemometer: Like digital wind-vane anemometer (Model 840032, SPER
Scientific LTD., Scottsdale, AZ, www.sperscientific.com) or equivalent. Use to
verify adequate exhaust ventilation for ICP-MS (check with hoses fully
disconnected).
ii. Pan, for changing roughing pump oil: Like part # 53216 (United States Plastics
Corporation, Lima, OH, www.usplastic.com) or equivalent. # On hand = 1.
iii. Container, to hold acid baths for glassware: Polypropylene or polyethylene
containers with lids (must be large enough for torch, injector, or spray chamber
submersion). May be purchased from laboratory or home kitchen supply
companies. # On hand = 4.
iv. Cotton swabs: Any vendor. For cleaning of cones and glassware.
v. Cutter (for 1/8” o.d. metal tubing): Terry tool with 3 replacement wheels. Like
part # TT-1008 (Chrom Tech, Inc., Saint Paul, MN, www.chromtech.com) or
equivalent.
vi. Getter Regeneration Kit: Part # WE023257 (PerkinElmer, Shelton, CT,
www.perkinelmer.com). Use this as needed (at least annually) to clean the
getter in the pathway of channel A DRC gas.
vii. Magnifying glass: Any 10x + pocket loupe for inspection of cones and other
ICP-MS parts. Plastic body is preferred for non-corrosion characteristics. Like
part # 5BC-42813 (Lab Safety Supply, Janesville, WI, www.labsafety.com).
viii. Screw Driver, for Ion Lens Removal: Screw driver with long, flexible shaft, and
2mm ball-Allen end for removal of ion lens screws (if lens is not in quickrelease mount), part # W1010620. Extra 2mm bits, part # W1010598
(PerkinElmer, Shelton, CT, www.perkinelmer.com).
ix. Toothbrush: Any vendor. For cleaning ion lens and glassware.
x. Ultrasonic bath: Like ULTRAsonik™ Benchtop
Bloomfield, CT, www.neytech.com) or equivalent.

Cleaners

(NEYTECH,

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Pittsburgh, PA, www.fishersci.com) have both been used. Acid rinse before
use. Equivalent containers may be substituted.
iv. Cups for urine collection: Like polypropylene 4.5 oz cup, catalog # 354013
(Becton Dickinson Labware, Franklin Lakes, NJ, www.bd.com) or equivalent.
Each lot of cups used must be lot screened (tested to be free of trace metal
contamination). Clear plastics tend to have lowest trace metal contamination.
v. Gloves: Powder-free, low particulate nitrile (like Best CleaN-DEX™ 100%
nitrile gloves, any vendor). Equivalent nitrile or latex gloves may be
substituted.
vi. Paper towels: For general lab use, any low-lint paper wipes such as
KIMWIPES®EX-L Delicate Task Wipers or KAYDRY®EX-L Delicate Task
Wipers (Kimberly-Clark Professional, Atlanta, GA, www.kcprofessional.com).
For sensitive applications in cleanrooms, a wipe designed for cleanroom use
may be desired such as the Econowipe or Wetwipe (Liberty, East Berlin, CT,
www.liberty-ind.com).
vii. Pipette (for preparation of urine dilutions to be analyzed): Micromedic DigiflexCX Automatic™ pipette equipped with 10.0-mL dispensing syringe, 2 mL
sampling syringe, 0.75-mm tip, and foot pedal (Titertek, Huntsville, AL,
http://www.titertek.com/).
viii. Pipettes (for preparation of intermediate stock working standards & other
reagents): Like Brinkmann Research Pro Electronic pipettes (Brinkmann
Instruments, Inc., Westbury, NY, http://www.brinkmann.com/home/). 5-100 µL
(catalog #4860 000.070), 20-300 µL (catalog #4860 000.089), 50-1000 µL
(catalog #4860 000.097), 100-5000 µL (catalog #4860 000.100). Note: pipette
catalog numbers are without individual chargers. Can purchase individual
chargers (pipette catalog numbers will differ) or a charging stand that will hold
four pipettes (catalog #4860 000.860). When purchasing pipette tips (epTips),
purchase one or more boxes, then “reloads” for those boxes after that: 5-100
µL (box catalog # 22 49 133-4, reload catalog # 22 49 153-9), 20-300 µL (box
catalog # 22 49 134-2, reload catalog # 22 49 154-7), 50-1000 µL (box catalog
# 22 49 135-1, reload catalog # 22 49 155-5), 100-5000 µL (box catalog # 22
49 138-5, reload catalog # 22 49 198-9, bulk bag catalog # 22 49 208-0).
Equivalent pipettes and tips can be substituted.
ix. Tubes for sample analysis (for autosampler): Like polypropylene 15-mL
conical tubes, BD Falcon model #352097 (Becton Dickinson Labware, Franklin
Lakes, NJ, www.bd.com). Equivalent tubes may be substituted which are
shown by lot screening to be free of trace metal contamination. Clear plastics
tend to have lowest trace metal contamination. Blue colored caps have also
been used successfully for this method.
x. Tubes for storage of intermediate working stock standards: Like polypropylene
50-mL conical tubes, BD Falcon model #352098 (Becton Dickinson Labware,
Franklin Lakes, NJ, www.bd.com). For use in storage of intermediate working
stock standards. Equivalent tubes may be substituted which are shown by lot
screening to be free of trace metal contamination. Clear plastics tend to have

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lowest trace metal contamination. Blue colored caps have also been used
successfully for this method.
xi. Vortexer: Like MV-1 Mini Vortexer (VWR, West Chester, PA, www.vwr.com).
Used for vortexing urine specimens before removing an aliquot for analysis.
Equivalent item can be substituted.
xii. Water purification system: Like NANOpure DIamond Ultrapure Water System
(Barnstead International, Dubuque, Iowa, www.barnstead.com). For ultra-pure
water used in reagent and dilution preparations. An equivalent water
purification unit capable of producing >18 Mega-ohm·cm water may be
substituted.
e. Sources of Chemicals, Gases, and Regulators
i. Acid, Hydrochloric acid: Veritas™ double-distilled grade, 30-35% (GFS
Chemicals Inc. Columbus, OH, www.gfschemicals.com). This is referred to as
“concentrated” hydrochloric acid in this method write-up.
For use in
preparation of intermediate working stock standards.
An equivalent
hydrochloric acid product may be substituted, but it must meet or exceed the
purity specifications of this product for trace metals content.
ii. Acid, Nitric acid: Veritas™ double-distilled grade, 68-70% (GFS Chemicals Inc.
Columbus, OH, www.gfschemicals.com). For use in diluent, rinse solution,
intermediate working stock standards, and QC pool preparations. This is
referred to as “concentrated” nitric acid in this method write-up. An equivalent
nitric acid product may be substituted, but it must meet or exceed the purity
specifications of this product for trace metals content.
iii. Ethanol (EtOH): USP dehydrated 200 proof (Pharmco Products, Inc.) or
equivalent.
iv. Argon Gas (for plasma & nebulizer) and Regulator: High purity argon
(>99.999% purity, Specialty Gases Southeast, Atlanta, GA, www.sgsgas.com)
for torch and nebulizer. Minimum tank source is a dewar of liquid argon (180250L). Bulk tank (1500+L is preferred).
1. Regulator for argon (at dewar): Stainless steel, single stage, specially
cleaned regulator with 3000 psig max inlet, 0-100 outlet pressure range,
CGA 580 cylinder connector, and needle valve shutoff on delivery side
terminating in a ¼” Swagelok connector.
Part number
KPRAFPF415A2AG10 (Georgia Valve and Fitting, Atlanta, GA,
www.swagelok.com). An equivalent regulator from an alternate vendor may
be substituted. # Spares = 1.
2. Regulator for argon (between bulk tank and PerkinElmer filter regulator):
Single Stage 316SS Regulator, with 0-300 psi Inlet Gauge, 0-200 psi
Outlet Gauge, Outlet Spring Range, 0-250 psi, ¼” Swagelok Inlet
Connection, ¼ turn Shut off Valve on Outlet with ¼” Swagelok Connection
and Teflon Seals. Part number KPR1GRF412A20000-AR1 (Georgia Valve

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and Fitting, Atlanta, GA, www.swagelok.com). An equivalent regulator from
an alternate vendor may be substituted. # Spares = 1.
3. Regulator for argon (PerkinElmer filter regulator on back of ELAN): Argon
regulator filter kit. Catalog number N812-0508 (PerkinElmer, Shelton, CT,
www.perkinelmer.com).
v. Argon / hydrogen: Argon (90%) / hydrogen (10%) for DRC channel A. Initial
purity of argon = 99.9997+% (“Research grade 5.7”). Initial purity of hydrogen
= 99.9999+% (“Research Grade 6.0”). Mixture is typically purchased in cylinder
size 35 (6”x24”) (Airgas South, Atlanta, GA, www.airgas.com).
1. Regulator for argon / hydrogen: Stainless steel, two stage, specially
cleaned regulator with 3000 psig max inlet, 0-25 outlet pressure range,
CGA 350 cylinder connector, and needle valve shutoff on delivery side
terminating in a ¼” Swagelok connector.
Like part number
KCYADPF412A2AD10 (Georgia Valve and Fitting, Atlanta, GA,
www.swagelok.com). An equivalent regulator from an alternate vendor may
be substituted. # Spares = 1.
2. Flash Arrestor (Stainless steel): Like part # 6104 (Matheson Tri Gas,
Montgomeryville, PA, www.mathesontrigas.com) or equivalent.
vi. Disinfectant, for work surfaces: Bleach-rite spray (any distributor). On-site
dilutions of bleach (1part bleach + 9 parts water) may be substituted, but must
be re-made daily.
vii. Oxygen: Oxygen (“Research Grade Research Grade 5.0”, 99.9999% purity)
for DRC channel B. Typically purchased in cylinder size 300 (9.5” x 54”)
(Airgas South, Atlanta, GA, www.airgas.com).
1. Regulator for oxygen: High purity brass body with monel trim, two stage
regulator. Stainless steel is not used for this application due to safety
concerns of working with oxygen at high pressure [12]. For one regulator,
order the following parts, and ask that they be tested and assembled
(Engineered Specialty Products, Kennesaw, GA, www.espgauges.com).
a. Tescom part # 44-3410S24-555
Regulator body: Brass bar stock, two stage, Monel trim, TFE seats,
Elgiloy diaphragms, Cv=0.05, 3000 psig max inlet, 1-25 psig outlet
range, 1/4 FNPT inlet / outlet / gauge ports, O 2 cleaned to ASTMG93
and CGA4.1.
b. Tescom part # 60500-3000N
Inlet pressure gauge: 2" diameter, 0-3000 psig range , O 2 cleaned, ¼”
MNPT bottom, brass.
c. Tescom part # 60500-0015N
Delivery pressure gauge: 2" diameter, 0-15 psig range , O 2 cleaned, ¼”
MNPT bottom, brass.
d. Tescom part # 63842-540-B

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NPT to CGA Adaptor: ¼” NPT to CGA 540 adapter, brass.
e. Swagelok part # B-200-1-4:
Adapter: Brass male connector, ¼” MNPT to 1/8” Swagelok (Georgia
Valve and Fitting, Atlanta, GA, www.swagelok.com).
An equivalent regulator from an alternate vendor may be substituted.
# Spares = 1.
Like part # 6103 (Matheson Tri Gas,
2. Flash Arrestor (brass):
Montgomeryville, PA, www.mathesontrigas.com) or equivalent.
viii. Standard, Gallium: Like 1,000 mg/L, item # PLGA2-2Y. (SPEX Industries,
Inc., Edison, NJ, www.spexcsp.com). Used as an internal standard in diluent.
Any vendor whose standards are traceable to the National Institute for
Standards and Technology may be substituted. The standard must have low
trace metal contamination. Gallium is only used in the diluent for the
measurement of arsenic (As).
ix. Standard, Iridium: Like 1,000 mg/L iridium, item # PLIR3-2Y (SPEX Industries,
Inc., Edison, NJ, www.spexcsp.com). Used as an internal standard in diluent.
Any vendor whose standards are traceable to the National Institute for
Standards and Technology may be substituted. The standard must have low
trace metal contamination.
x. Standard, Multi-element intermediate stock standard: Item number SM-2107003 (High Purity Standards, Charleston, SC, http://www.hps.net/). This is a
custom mix solution (see Table 3 in Appendix B for concentrations). This
solution is diluted to prepare the intermediate stock working standards, which
are in turn diluted to prepare the working calibrators. This solution can be
prepared in-house from NIST traceable single element stock solutions if
necessary.
xi. Standard, Rhodium: Like 1,000 mg/L, item # PLRH3-2Y. (SPEX Industries,
Inc., Edison, NJ, www.spexcsp.com). Used as an internal standard in diluent.
Any vendor whose standards are traceable to the National Institute for
Standards and Technology may be substituted. The standard must have low
trace metal contamination.
xii. Standard, single element stock standards for preparation of urine quality
control pools: National Institute of Standards and Technology (NIST) Standard
Reference Materials (SRMs) 3103a (As), 3105a (Be), 3113 (Co), 3134 (Mo),
3108 (Cd), 3102a (Sb), 3111a (Cs), 3104a (Ba), 3163 (W), 3128 (Pb), 3140
(Pt), 3158 (TI), and 3164 (U) (National Institute of Standards and Technology
(NIST), Office of Standard Reference Materials, Gaithersburg, MD,
www.nist.gov). Other sources of standards can be used if they are NIST
traceable.
xiii. Triton X-100™ surfactant: Like “Baker Analyzed” TritonX-100™ (J.T. Baker
Chemical Co., www.jtbaker.com). Another source may be substituted, but it
must be free of trace-metal contamination.

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6) Preparation of Reagent and Materials.
a. Internal Standard Intermediate Mixture
i. For Urine 15 Element (Ir and Rh): Preparation of single intermediate solution
containing all internal standards will simplify the addition of the internal
standards into the final diluent solution. This solution can be purchased rather
than prepared. To prepare 200 mL of the Intermediate internal standard
solution:
1. Partially fill a 200 mL acid-washed volumetric flask (PP, PMP, or Teflon™)
with >18 Mega-ohm·cm water (approximately 100-150 mL).
2. Carefully add 4 mL of double-distilled, concentrated nitric acid. Mix into
solution.
3. Add 0.8 mL of 10,000 ug/mL Rh standard. If initial Rh standard
concentration is different, adjust volume proportionally.
4. Add 0.8 mL of 10,000 ug/mL Ir standard. If initial Ir standard concentration
is different, adjust volume proportionally.
5. Fill to mark (200mL) and mix thoroughly.
6. Label should include “Internal Standard Intermediate Mixture. 40 ug/mL
Rh, Ir, and. 2% (v/v) HNO3”, “Store at room temperature”, preparation
date, expiration date 1 year from preparation date, and preparer’s initials.
ii. For Arsenic (Ga): Dilution of a Ga standard to be used as an internal standard
in the final diluents solution. This solution can be purchased rather than
prepared. To prepare 200 mL of the Intermediate internal standard solution:
1. Partially fill a 200 mL acid-washed volumetric flask (PP, PMP, or Teflon™)
with >18 Mega-ohm·cm water (approximately 100-150 mL).
2. Carefully add 4 mL of double-distilled, concentrated nitric acid. Mix into
solution.
3. Add 0.8 mL of 10,000 ug/mL Ga standard. If initial Ga standard
concentration is different, adjust volume proportionally.
4. Fill to mark (200mL) and mix thoroughly.
5. Label should include “Internal Standard Intermediate Mixture. 40 ug/mL
Ga, 2% (v/v) HNO3”, “Store at room temperature”, preparation date,
expiration date 1 year from preparation date, and preparer’s initials.
b. Diluent and Carrier
i. Purpose: All samples (blanks, calibrators, QC, or patient samples) are
combined with the diluent during the sample preparation step before analysis.
This is where the internal standards are added which during the analysis will
compensate for instrumental variations on the analyte signal. If using the
FAST sample introduction system, the diluent is also used as the carrier
solution.

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ii. Preparation:
1. Diluent Preparation for 15 element method (not including arsenic)
– NO ETHANOL
a. Contents: An aqueous solution of 10 microgram/L Rh, Ir, and in 2%
(v/v) double-distilled nitric acid.
b. Preparation (4L) & storage: This solution does not have to be made up
in a volumetric flask. The important thing about the concentration of
the internal standards is that they be consistent within all samples in
one run. To prepare different volumes of diluent, add proportionally
larger or smaller volumes of the solution constituents.
i.

Acid-rinse a 4 L container (material may be polypropylene (PP),
polymethylpentene (PMP), or Teflon™).

ii.

Partially fill the 4 L container with >18 megaohm·cm water.

iii.

Carefully add 80 mL double-distilled, concentrated nitric acid and
mix.

iv.

Add 1 mL of the 40 ug/mL Rh, Ir, internal standard solution. If
other concentrations are used, the volume added should be
adjusted proportionally.

Make up to volume (4 L) with >18 megaohm·cm water.

vi.

Store at room temperature and prepare as needed.

vii.

Label should include “10 µg/L Rh, and Ir,”, “2% (v/v) HNO 3 ”, “Store
at room temperature”, preparation date, expiration date (1 year
from prep), and preparer’s initials.

2. Diluent Preparation for urine arsenic method – CONTAINS ETHANOL
a. Contents: An aqueous solution of 10 microgram/L Ga in 2% (v/v)
double-distilled nitric acid and 1.5% (v/v) ethanol.
b. Preparation (4L) & storage: This solution does not have to be made up
in a volumetric flask. The important thing about the concentration of
the internal standards is that they be consistent within all samples in
one run. To prepare different volumes of diluent, add proportionally
larger or smaller volumes of the solution constituents.
i.

Acid-rinse a 4 L container (material may be polypropylene (PP),
polymethylpentene (PMP), or Teflon™).

ii.

Partially fill the 4 L container with >18 megaohm·cm water.

iii.

Carefully add 80 mL double-distilled, concentrated nitric acid and
mix.

iv.

Carefully add 60 mL dehydrated 200 proof ethanol and mix.

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Add 1 mL of the 40 ug/mL Ga internal standard solution. If other
concentrations are used, the volume added should be adjusted
proportionally.

vi.

Make up to volume (4 L) with >18 megaohm·cm water.

vii.

Store at room temperature and prepare as needed.

viii.

Label should include “10 µg/L Ga”, “2% (v/v) HNO 3 ”, “1.5% (v/v)
Ethanol”, “Store at room temperature”, preparation date, expiration
date (1 year from prep), and preparer’s initials.

c. Base Urine
i. Purpose: This urine pool material will be mixed with the intermediate working
calibrators just prior to analysis to matrix-match the calibration curve to the
urine matrix of the unknown samples.
ii. Contents: A mixture of multiple urine sources collected from anonymous
donors are used to approximate an average urine matrix.
iii. Preparation & Storage:
1. Collect urine anonymously by placing screened containers and collection
cups in the restrooms with a sign stating the reason the specimens are
being collected, the name of the investigator to contact for additional
information, and requesting that people provide a urine specimen
(complete details can be found in CDC protocol #3994, ProTrack #
DLSITN0313).
2. Once the urine is collected from donors, it should be analyzed to ensure
that concentrations of the analytes in this method are relatively low, so as
to not interfere with the proper measurement of calibrators (see Table 2 in
Appendix B for suggested maximum base urine concentrations).
3. Once screened, mix the urine collections together in a larger container (i.e.
acid washed polypropylene (PP), polymethylpentene (PMP), or Teflon™)
and stir for 30+ minutes on a large stir plate (acid wash large Teflon™ stir
bar before use).
4. For short term storage, store at 2-4°C. For long-term storage, dispense
into smaller-volume tubes (i.e., 50-mL acid-washed or lot screened
polypropylene tubes) and store at ≤ -20°C.
5. Labels on 50mL tubes should include “Base Urine for Multi-element
Method”, “Store Long Term at
≤ 20° C”, “Store Short Term at 2 -4° C”,
preparation date, expiration date 3 years from prep date, and preparer’s
initials.
d. ICP-DRC-MS Rinse Solution
i. Purpose: Pump this solution into the sample introduction system between
samples to prevent carry-over of the analytes of interest from one sample
measurement to the next.
ii. Preparation:

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1. Intermediate Triton X-100 Solution: To avoid the process of dissolving
pure Triton X-100 on a daily basis, prepare an intermediate 2% Triton X100™ / 5% (v/v) double-distilled, nitric-acid solution for daily use.
a. To prepare 2L of Intermediate Triton X-100 Solution:
i.

Partially fill a 2 L acid-washed bottle (PP, PMP, or Teflon™) with
>18 Mega-ohm·cm water (approximately 1-1.5 L).
Use of
volumetric flask is not required.

ii.

Add 20 mL of Triton X-100™ and stir until completely dissolved.
Use a Teflon™ stir bar and stir plate if necessary (acid wash stir
bar before use).

iii.

Carefully add 100 mL of double-distilled, concentrated nitric acid.

iv.

Fill to 2 L and stir thoroughly.

Label should include “2% Triton X-100™ / 5% (v/v) HNO3”, “Store
at room temperature”, preparation date, expiration date 1 year from
preparation date, and preparer’s initials.

2. Rinse Solution Preparation for 15 element method (not including arsenic)
– NO ETHANOL
a. Contents: A 0.002% Triton X-100™, 5% (v/v) double-distilled nitric
acid solution.
b. Preparation & Storage: To Prepare 4 L of the Final Rinse Solution,
i.

Partially fill a 4 L acid-washed bottle (PP, PMP, or Teflon™ ) with
>18 Mega-ohm·cm water (approximately 2-3 L). Use of volumetric
flask is not required.

ii.

Add 4 mL of the 2% Triton X-100™ / 5% (v/v) double-distilled,
nitric-acid intermediate stock solution and mix well.

iii.

Carefully add 200 mL of double distilled concentrated nitric acid
and mix well.

iv.

Fill to 4 L using >18 Megaohm·cm water.

Store at room temperature and prepare as needed. To prepare
volumes other than specified here, add proportionally larger or
smaller volumes of the solution constituents.

vi.

Label should include “0.002% Triton X-100™ / 5% (v/v) HNO3”,
“Store at room temperature”, preparation date, expiration date one
year from preparation date, and preparer’s initials.

3. Rinse Solution Preparation for arsenic method – INCLUDES ETHANOL
a. Contents: A 0.002% Triton X-100™, 5% (v/v) double-distilled nitric
acid solution and 1.5% (v/v) ethanol.
b. Preparation & Storage: To Prepare 4 L of the Final Rinse Solution,

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Partially fill a 4 L acid-washed bottle (PP, PMP, or Teflon™ ) with
>18 Mega-ohm·cm water (approximately 2-3 L). Use of volumetric
flask is not required.

ii.

Add 4 mL of the 2% Triton X-100™ / 5% (v/v) double-distilled,
nitric-acid intermediate stock solution and mix well.

iii.

Carefully add 200 mL of double distilled concentrated nitric acid
and mix well.

iv.

Carefully add 60 mL dehydrated 200 proof ethanol and mix well.

Fill to 4 L using >18 Megaohm·cm water.

vi.

Store at room temperature and prepare as needed. To prepare
volumes other than specified here, add proportionally larger or
smaller volumes of the solution constituents.

vii.

Label should include “0.002% Triton X-100™ / 5% (v/v) HNO3,
1.5% (v/v) ethanol”, “Store at room temperature”, preparation date,
expiration date one year from preparation date, and preparer’s
initials.

e. Standards and Calibrators
i. Multi-element Intermediate Stock Calibration Standard
1. Purpose: This master solution will be diluted to prepare five intermediate
working calibrators.
2. Contents: An aqueous solution containing all 16 elements of interest (15
element panel analytes, arsenic, and elements for future R&D (see
certificate of analysis), but does not include the internal standards).
Concentrations are listed in Table 3 of the Appendix. Matrix is 2% (v/v)
HNO3 and 1% (v/v) HCl with traces of HF in >18 Mega-ohm·cm water.
3. Preparation (Purchase) & Storage:
a. Purchasing from vendors: Either purchased as a NIST-traceable
custom mixture, or prepared in-house.
Current vendor & preparation process: Currently purchased
from High Purity Standards (Charleston, SC, part number
SM-2107-003).
b. In-house Preparation: Standard may be made in the lab from NISTtraceable single element standards.
c. Storage: Store at room temperature. Label with additional information
such as “store at room temperature”, date received, date opened, and
initials of person to first open.

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ii. Multi-element Intermediate Working Calibration Standards
1. Purpose: Use each day of analysis to prepare the final five working
calibrators that will be placed on the autosampler.
2. Content: Five aqueous dilutions of the multi-element intermediate stock
calibration standard solution in 2% (v/v) double-distilled nitric acid and 1%
(v/v) hydrochloric acid. Final concentrations are listed in Table 4 of the
Appendix.
3. Preparation & Storage: Different volumes may be prepared by adding
proportionally larger or smaller volumes of solution constituents.
a. Cleaning flasks: Acid-rinse three 100-mL, one 200-mL, one 500-mL
PP, and one 2 L PP (or PMP) volumetric flasks. Check their
cleanliness by comparing the counts observed on the ICP-DRC-MS for
1% (v/v) HNO 3 before and after contact with the flasks. Mark each
flask according to intended use. Dedicate to purpose.
b. HNO 3 & HCl Diluent Preparation: In the cleaned 2L flask, add 1-1.5L
>18 Megaohm· cm water, 40 mL high purity concentrated HNO 3 , and
20 mL high purity concentrated HCl. Fill to the mark and mix
thoroughly. Use this diluent to fill the remaining flasks during
preparation of the intermediate working calibration standards.
c. Dilutions & Storage:
i.

Partially fill the 100 mL, 200 mL, and 500 mL flasks with the HNO 3
& HCl diluent (50-75% full).

ii.

Using the volumes listed (Table 4 of the Appendix) pipette the
appropriate volume of the multi-element intermediate stock
calibration standard solution into each of the five volumetric flasks.
Dilute each to the volumetric mark with the HNO 3 & HCl diluent
using a pipette for the final drops. Mix each solution thoroughly.
Final concentrations are listed in Table 4 of the Appendix.

iii.

Once mixed, transfer to acid-cleaned, labeled, 50-mL containers
(PP, PMP, or Teflon™) for storage. Labels should include
information such as “Multi-element Urine Working Calibrators”, “2%
(v/v) HNO3, 1% (v/v) HCl”, date of preparation, expiration date (1
year from date of preparation), “store at room temperature”, initials
of preparer, and concentrations for each element.

iii. Working Multi-element Calibrators
1. Purpose: The working multi-element calibrators will be analyzed in each
run to provide a signal-to-concentration response curve for each analyte in
the method. The concentration of an analyte in a patient urine sample
dilution is determined by comparing the observed signal from the dilution of
the patient urine sample to the response curve from the working multielement calibrators.

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2. Content: Dilutions (1:100) of the corresponding five intermediate working
calibration standards. The dilutions are described in Table 7 of the
Appendix.
3. Preparation & Use: Made immediately prior to analysis when the
intermediate working calibration standards are mixed with base urine
(Section 7.b) and diluent (Section 7.a) using a Digiflex automatic pipetter.
See Table 7 of the Appendix and section 8.b.2 for details of sample
preparation.
iv. Multi-element Intermediate Stock Calibration Verification Standard
1. Purpose: This is the master solution from which all working calibration
verification standards will be prepared. It will be diluted to prepare
intermediate working calibration verification standards which are in turn
diluted and used to verify the accuracy of instrument response to analyte
concentrations greater than the calibration range. This stock solution
contains all elements needed for both the arsenic and the 12 element
panel.
2. Contents: The concentrations of the elements in the intermediate stock
calibration standards are listed in Table 3 of the Appendix. For long shelf
life, these four aqueous solutions have different matrices which are
optimized to the elements in each (this was recommended for the
calibration verification stock standard solutions because the elemental
concentrations were very high compared to the concentrations in the
calibration stock standard solution.
a. Solution A: HNO 3 (10%), HF (0.5%)
b. Solution B: HCl (10%), trace HNO 3
c. Solution C: HCl (1%)
d. Solution D: HCl (2%)
3. Preparation (Purchase) & Storage:
a. Purchasing from vendors:
The intermediate stock calibration
verification standard solutions may be purchased as custom mixtures
from any vendor which prepares multi-element solutions that are
traceable to the National Institute for Standards and Technology
(NIST) for their accuracy.
Current vendor & preparation process: Currently it is
purchased from High Purity Standards (Charleston, SC, part
number SM-2107-012, solutions A, B, C, and D).
b. In-house Preparation: If outside laboratories were not available to
prepare the intermediate stock calibration standard solution, it is also
possible to make it in the laboratory from single element standards
which are NIST traceable.
c. Storage: Due to the uranium content, and in keeping with the
guidance of the CDC radiation safety manual [11], the intermediate

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stock standards must be kept in a lockbox. Store the solutions at room
temperature. Label these bottles from HPS with additional information
such as “store at room temperature”, date received, date opened, and
initials of person to first open.
v. Multi-element Intermediate Working Calibration Verification Standards
1. Purpose: Verification of accuracy of instrument response to analyte
concentrations greater than the calibration range
2. Content:
The intermediate working calibration verification standard
solutions used in this method are aqueous dilutions of the multi-element
intermediate stock calibration verification standard solution in 2% (v/v)
double-distilled nitric acid and 1% (v/v) hydrochloric acid containing all 13
elements of interest (does not include the internal standards). The
concentrations of the 13 elements in the intermediate stock calibration
verification standard are listed in Table 8 in Appendix B.
a. Preparation & Storage: Prepare the Intermediate Calibration
Verification Standards for analysis just as a Intermediate Working
calibrators are prepared, but using volumes and concentrations from
Table 8 in Appendix B.
vi. Internal Quality Control Materials (“Bench” QC)
1. Purpose: Internal (or “bench”) quality control (QC) materials are used to
evaluate the accuracy and precision of the analysis process, and to
determine if the analytical system is “in control” (is producing results that
are acceptably accurate and precise). They are included in the beginning
and at the end of each analytical run.
2. Content: The internal (or “bench”) quality control (QC) materials used in
this method are pooled human urine, acidified to 1-2% (v/v) HNO 3 , and
may have been spiked to reach a desired concentration. The analyte
concentrations in the “low QC” are in the low-normal concentration range.
The analyte concentrations in the “high QC” are in the high-normal
concentration range.
3. Preparation & Storage: Quality control materials can be either prepared by
and purchased from an external laboratory or prepared within the CDC
laboratories. Quality control must always be traceable to the National
Institute for Standards and Technology (NIST). The CDC laboratory
currently prepares its own bench QC materials using the following
procedures:
a. Collection of urine: Collect urine anonymously by placing screened
containers and / or collection cups in the restrooms with a sign stating
the reason the specimens are being collected, the name of the
investigator to contact for additional information, and requesting that
people provide a urine specimen (complete details can be found in
CDC protocol #3994, ProTrack # DLSITN0313). Volume of urine to

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collect is dependent on the desired pool size. This write-up will
assume a 10-L pool size for both the low and high bench QC.
b. Screening Urine: Screen collected samples for metal content before
mixing together to make 2 separate base urine pools (for preparing the
low and high bench QC materials).
Samples can be screened
individually or after combining several together (reduces number of
analyses).
i.

Keep urine refrigerated whenever possible to minimize microbial
growth.

ii.

Because this is only a quick screen of the metal content, the
number of replicates in the urine method can be reduced to one in
order to reduce analysis time.

iii.

Analyte concentrations in the the final urine pool to be spiked for
the low bench QC pool should be in the low-normal population
range. Analyte concentrations in the final urine pool to be spiked
for the high bench QC pool should be less than some pre-selected
target concentration values in the high normal population range.
See the Second National Report on Human Exposure to
Environmental Chemicals for estimations of the normal population
ranges for metals (http://www.cdc.gov/exposurereport/).

c. Combining Collected Urine: Be attentive not to combine only diluted
matrix urine samples into the low pool and only concentrated matrix
urine samples into the high pool. The goal is for combining samples is
to approach an ‘average’ matrix for each pool.
i.

Graduate four acid-washed 10-L carboys (PP or PMP) in 0.5 L
increments (two will be used for decanting into).

ii.

Combine collected urine samples into two separate acid-washed
10-L carboys (PP or PMP), according to their concentrations, for
the low bench and high bench QC pools.

iii.

Mix each urine pool using large acid washed, Teflon™ coated stir
bars and large stir plates. Keep urine refrigerated whenever
possible.

iv.

Acidify each urine pool to 1% (v/v) HNO3 by adding the
appropriate volume of double distilled HNO3. Stir for 30+ min on
large stir plates.

d. Settling out of solids:
i.

Refrigerate the urine (no stirring) for 1-3 days to allow for settling
out of solids.

ii.

For each urine pool, decant the urine into another of the acidwashed 10-L carboys to remove the urine from the solids settled
out on the bottom of the carboy.

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Repeat steps (i) and (ii) until minimal solids are left at the bottom of
the carboy after sitting overnight.

e. Spiking of urine
i.

Analyze a sample of each urine pool. Record these results for
future recovery calculations.

ii.

Use these results to determine target analyte concentrations
possible for the pools

iii.

Calculate the volume of single element standards needed to spike
each pool to the desired concentrations.

iv.

While stirring the pools on large stir plates, spike each pool with
calculated volumes of single element standards (all spiking
standards used must be traceable to NIST).

Continue to stir pools for 30+ minutes after spiking, then reanalyze.

vi.

Repeat steps 4 and 5 until all analytes reach target concentrations
keeping track of the total volume of spiking solution added to each
urine pool.

f. Dispensing and Storage of urine
i.

Container Types: Dispense urine into lot screened containers (i.e.
– 5 or 15 mL polypropylene tubes). If possible, prepare tubes of
QC which have only enough volume for one typical run + 1 repeat
analysis. This allows for one vial of QC to be used per day of
analysis, reducing chances of contamination of QC materials due
to multi-day use.

ii.

Labels: Place labels on vials after dispensing and capping if the
vials are originally bagged separately from the caps. This
minimizes the chance for contamination during the process.
Include at least the name of QC pool (text and bar code), date of
preparation, and a vial number on the labels.

iii.

Dispensing: Dispensing can be accomplished most easily using a
Digiflex automatic pipetter in continuous cycling dispense mode.
This process should be done in a clean environment (i.e. a class
100 cleanroom area or hood).
1. Allow urine pool to reach room temperature before dispensing
(to prevent temperature gradients possibly causing
concentration gradients across the large number of vials being
dispensed and to prevent condensation problems during
labeling of vials). This may require leaving the carboy of urine
at room temperature overnight before dispensing.
2. Replace the tubing attached to the dispensing syringe (left
when looking at front of Digiflex) with a length of clean
Teflon™ tubing long enough to reach into the bottom of the
10L carboy while it is sitting on the stir plate.

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3. Check cleanliness of Digiflex before use by analyzing 1-2%
(v/v) HNO3 which has been flushed through the Digiflex with a
portion of the same solution which has not been through the
Digiflex.
4. Approximately one hour before dispensing begins,
a. With the large stir plate close to the left side of the Digiflex,
begin stirring the urine pool to be dispensed.
b. Also during this time, flush the Digiflex with urine from the
pool to be dispensed. Place the ends of the tubing
attached to both the sample and dispensing syringes into
the carboy of urine so that urine won’t be used up during
this process. Be sure to secure both ends of tubing in the
carboy with Parafilm so they will not come out during the
flushing process.
5. After dispensing the urine into the vials, cap the vials and label
them. Placing labels on vials after capping minimizes the
chance for contamination during the process.
iv.

Homogeneity Testing: After dispensing, check homogeneity of
analyte concentrations in pool aliquots by analysis of every Nth
sample dispensed (where N ~ 20 - 50 depending on the pool size).
Sample more heavily from the beginning and the ending portions
of the tubes dispensed (these are the regions where most
homogeneity problems occur).
Keep samples pulled for
homogeneity analysis in the sequence that they were dispensed
for the purpose of looking for trends in concentrations. Once
dispensed and homogeneity has been shown to be good
throughout the tubes of a pool, store tubes at≤ -20°C and pull
tubes out as needed for analysis.

Storage: Urine pools should be stored long term at ≤ -20°C. Short
term storage (several days) at refrigerator temperature (~ 2-4°C).

7) Analytical Instrumentation & Parameters
(see Section 6 for details on hardware used, including sources)
a. Instrumentation & Equipment Setup:
i. ICP-DRC-MS: Inductively Coupled Plasma Dynamic Reaction Cell Mass
Spectrometer ELAN® 6100 DRCPlus or ELAN® DRC II.
1. Modifications made to ICP-DRC-MS
a. Plastic tubing for between mass flow controllers and dynamic reaction
cell have been replaced with stainless steel. Stainless steel tubing is

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preferred between the reaction gas cylinder / regulator and the back of
the ICP-DRC-MS instrument.
b. A second mass flow controller has been added (channel B) for use
with oxygen.
c. Standard built-in peristaltic pump replaced by DXi-FAST microperistaltic pump / FAST actuator unit.
2. Configuration of tubing for liquid handling:
a. Sample introduction system:
Valve connections must match this
i. SC-FAST valve setup:
description for urine total arsenic analysis. See Appendix B,
Figure 1a.
1. Port 1: 1.5mL sample loop (white nut). See “Loop, for FAST
valve” in section 5.b. for details.
2. Port 2: 0.5 mm ID probe (red nut) for carrier solution. See
“Probes” in section 5.b. for details.
3. Port 3: nebulizer line (green nut) for transfer of liquid to
nebulizer. See “Nebulizers” in section 5.b. for details.
4. Port 4: sample loop (white nut). See “Loop, for FAST valve” in
section 5.b. for details.
5. Port 5: 0.8 mm ID probe (blue nut) for diluted samples. See
“Probes, for ESI autosampler” in section 5.b. for details.
6. Port 6: vacuum line (black nut). See “Tubing, FAST vacuum”
in section 5.b. for details.
ii.

SC autosampler setup for non-FAST applications:
See Appendix B, Figure 1b.

b. Tubing connection between autosampler rinse station and rinse
solution reservoir: Tubing of different inner diameters can be
obtained from Elemental Scientific, their distributors, or custom built
in the lab to optimize the rinse station fill rate between samples.
Rinse station should not go empty at any point.
c. Tubing for autosampler rinse station waste removal: Use minimum
drain tubing to make this connection. If this tube is too long, the
rinse station will not drain properly.
d. Rinse solution jug: Leave one of the caps on the top of the rinse jug
loose to allow air venting into the jug as liquid is removed.
Otherwise the jug will collapse on itself as the liquid is removed and
a vacuum is created inside. Use secondary containment tray and
label appropriately (see solution preparation instructions).

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e. Waste solution jug: Use secondary containment tray and label
appropriately (see solution preparation instructions).
f. Configuration of tubing and probe for carrier solution: Use a
‘peristaltic to Teflon tubing adapter’ (see consumables descriptions
in section 5.b) for connections.
3. Cones used
Nickel and platinum cones have been used.
4. Gases & Regulators setup:
a. Argon: Argon stored as liquid in a dewar (180-250L) or bulk tank.
Gaseous argon used for plasma and nebulizer.
i.

ii.

iii.

Regulator for argon source (if a dewar): Keep the inlet pressure
(headspace pressure of liquid argon dewar) above 100 psi. Set
delivery pressure to 60-100psi to allow for pressure drop across
tubing that stretches to the instrument. See Section 6.f. for part
numbers and details.
Step down regulator (if source of argon is a bulk tank): Place this
single stage regulator in the lab so that incoming argon pressure
can be monitored and adjusted. Set delivery pressure to 60-100
psig. See Section 6.f. for part numbers and details.
Regulator at ICP-DRC-MS: Single stage “argon regulator filter
kit” supplied with the ICP-DRC-MS. Set the delivery pressure
depending on the specifications for that model of ELAN ICP-DRCMS instrument (see the PE Hardware Manual). This will be 52±1
psi for instruments having a 0-60psi gauge and 60+1 for
instruments having a 0-100psi gauge. See Section 6.f. for
regulator part numbers.

b. Argon (90%) / hydrogen (10%) mixture for DRC channel A. NOTE:
Only for arsenic analysis.
i.

Regulator for Ar / H 2 gas mixture: Set delivery pressure to 5-7
psig. See Section 6.f. for part numbers and details.

ii.

Flash arrestor: Stainless steel flash arrestor is used on outlet side
of regulator. See Section 6.f. for part numbers and details.

c. Oxygen (99.999+%) gas for DRC channel B.
cadmium and manganese analysis
i.

NOTE:

Only for

Regulator for O 2 gas: Set the delivery pressure = 5-7 psig. See
Section 6.f. for part numbers and details.

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Flash arrestor: Brass flash arrestor is used on outlet side of
regulator. See Section 6.f. for part numbers and details.

5. Chiller / Heat Exchanger: Refrigerated chiller (for ELAN® 6100 DRCPlus
instruments) or heat exchanger (for ELAN® DRC II instruments). For
refrigerated chiller, set temperature control to 18°C.
ii. Computer: Dell Optiplex GX150, GX270, or GX280 have all been used.
Processors used have included Pentium III (1 GHz) through Pentium IV (2.8
GHz). Recommend 512Mb - 1Gb RAM. External hard disk drive for nightly
backups of data connects via USB port. Software used includes Windows XP
Professional, service pack 2 and ELAN v3.3.
iii. Autosampler: ESI SC4 autosampler with (arsenic) or without (multi-element)
FAST sample introduction. Rack calibration, tubing ID for rinse supply,
additional rinse time, probe movement speeds, and probe depth is optimized
per autosampler (see Table 1 in Appendix B for default settings).
b. Parameters for Instrument and Method: See Table 1 in Appendix B for a
complete listing of the instrument and method parameters. Also, see Figures 2a2g and 3a-3g in Appendix B for images of the ELAN method screens (15 element
panel and arsenic respectively).
8) Method Procedures
a. Quality Control: Quality control procedures implemented in this method are
defined by the Division Procedures and Practices Guidelines and include two
types of QC systems which are both subjected to the complete analytical
process.
The data from these materials are then used to estimate
methodological imprecision and to assess the magnitude of any time-associated
trends. The concentrations of these materials should cover the expected
concentration range of the analytes for the method. Before QC materials can be
used to judge patient analytical runs, acceptable QC concentration limits must be
calculated from the concentration results observed in at least 20 characterization
runs. During the 20 characterization runs, previously characterized QCs or pools
with target values assigned by outside laboratories should be included to
evaluate the analysis. The process of limits calculation is performed using the
laboratory database and the SAS division QC characterization program.
i. Types of Quality Control:
1. “Bench QC”: The bench QC pools used in this method comprise two levels
of concentration spanning the “low-normal” and “high-normal” ranges of the
analyte of interest. The intent of bench QC is for the analyst to evaluate
the performance of the analytical system on the day of analysis. The
analyst inserts both the “low” and the “high” bench QC specimens two
times in each analytical run (a set of consecutive assays performed without
interruption) so that judgments may be made on the day of analysis. The

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first analysis of the two bench QC pools is done after the calibration
standards are analyzed but before any patient samples are analyzed (so
that judgments on the calibration curves may be made before analysis of
patient samples). The second analysis of the two bench QC pools is done
at the end of the run (approximately 20 patient samples total). If more
patient samples are analyzed on the same calibration curve after the
second run of the bench QC, both the low-normal and high-normal bench
QC must be reanalyzed before and after the additional samples. For
example, the schemes shown in Table 5 in Appendix B are both
acceptable ways to analyze multiple consecutive “runs”.
2. “Blind QC”: When possible, “blind” QC samples are QC materials placed in
vials, labeled, and processed so that they are indistinguishable from the
subject samples handled by the analyst. Ideally, the supervisor decodes
and reviews the results of the blind specimens without the analyst knowing
of their presence in the runs. When it is not possible to have blind QC
materials processed so that they are indistinguishable by the analyst from
the patient samples, it is acceptable for the analyst to randomly insert into
the run a QC material which only the QC reviewer knows the acceptable
concentration limits for. At least one low-normal concentration and one
high-normal concentration QC material should be kept in the laboratory for
this purpose.
3. External Reference Materials: Materials produced by laboratories outside
of the CDC which have assigned target concentrations can be helpful in
verifying method performance.
Some examples include Standard
Reference Materials (SRM) from the National Institute of Standards and
Technology (NIST) (i.e. SRM 2670a low & 2607a high) and samples from
previous challenges of proficiency testing programs (i.e. Centre de
Toxicologie du Quebec (CTQ)). However, only the results for the bench
and blind QC materials are used to determine if the run results can be
used.
ii. Calibration Verification:
a. Bi-annual tests as defined in the DLS Policy and Procedures manual: CLIA
requires the verification of accuracy of instrument response to analyte
concentration be completed at least every 6 months. NIST traceable
calibrators are analyzed in each run to define this response up to the
concentration of the highest calibrator in the run. To verify accuracy of
instrument response at concentrations higher than the highest calibrator in
each run, analyze a NIST traceable standard with very high concentrations
(see Table 8 in Appendix B for concentrations) at least every 6 months.
Prepare the Calibration Verification Standard for analysis just as a working
calibrator is prepared. Use the “Urine Blank” as the blank when it is analyzed.
If the observed concentrations for the Calibration Verification Standard are not
within 10% of the target value (see Table 8 in Appendix B) the lab supervisor
should be notified and the issue should be investigated. Do not substitute

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external reference materials (i.e. biological samples from a PT program) for
the Calibration Verification Standard when performing this. Solutions needed
for the Calibration Verification checks can be purchased from standards
vendors (i.e.SPEX, High Purity Standards, etc . . .) or prepared in-house from
NIST traceable single element standards.
Always verify that normal
background levels have been re-achieved through adequate rinse time
following analysis of elevated standards for calibration verification.
b. As-needed confirmations (per supervisor discretion): When a sample result is
greater than the highest calibrator in the run, the supervisor may request that
the result be confirmed in an analysis run which includes a standard or
external reference material with equivalent (within 10%) or greater
concentration than the sample. In order to avoid needless contamination of
the instrument with high concentrations of analytes, the analyst should use the
lowest appropriate calibration verification solution concentrations to meet the
need.
For infrequent verification needs, the calibration verification stock solutions
can be used to prepare verification standards to appropriate concentrations.
This will, however, introduce elevated concentrations of all elements in the
method to the sample introduction system. Frequent measurement of these
very high concentrations can result in high background levels in the instrument
which are difficult to rinse out and which may limit the ability to measure low
concentrations.
For frequent verification needs (i.e. when certain studies have many elevated
results on particular elements) or when a concentration higher than those
shown in Table 8 in Appendix B needs to be verified, use NIST-traceable
single element stock standards to prepare single element verification
standards. This will limit the exposure of the instrument to elevated
concentrations
of
only
the
elements
needing
verification.
An external reference material (i.e. historical proficiency testing sample) can
be substituted in place of the Calibration Verification Standard sample in these
situations IF
i.

The target value has been assigned by an external source (i.e.
NIST, or the proficiency testing program).

ii.

The concentration of the external reference material is within 10%
or is higher than the concentration of the material you need it to
confirm.

iii.

There is confidence that there is no contamination of previously
used external reference material.

iv.

A note to file is made that this was done.

If the observed concentrations are not within 10% of the target
value the lab supervisor should be notified and the issue should be
investigated.

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Always verify that normal background levels have been re-achieved through
adequate rinse time following analysis of calibration verification standard 3 or
higher (including samples with those concentrations).
b. Daily Analysis of Samples
i. Preparation of the Analytical Equipment
For further details on any part of this description, see the ITN Daily Startup
SOP for ELAN ICPMS instruments.
1. Power on the computer, printer, and autosampler, and log into the
operating system.
2. Peristaltic pump: Set up the peristaltic pump tubing with proper tension for
the sample rinse station.
3. Software: Starting the ESI software before starting the ELAN software may
improve stability of software.
4. Daily Pre-Ignition Maintenance Checks: Perform daily maintenance checks
as described in the IRAT Daily Startup SOP for ELAN instruments (i.e., Ar
supply pressure, interface components cleanliness and positioning,
interface pump oil condition, vacuum pressure, etc.). Make appropriate
notes in the Daily Maintenance Checklist and Instrument Log Book.
5. Start the Plasma: In the INSTRUMENT window of the software (or on the
front of the ELAN), press the “Start” button to ignite the plasma.
6. Aspirate rinse (multi-element method) or carrier (arsenic method) solution:
Send Probe to Rinse Station (multi-element method) or manually place
carrier probe into carrier solution (arsenic method).
7. Start the peristaltic pump: Start the peristaltic pump by pressing the
appropriate arrow in the DEVICES window (make sure that the rotational
direction is correct for the way the tubing is set up in the peristaltic pump).
Set the pump speed to a slow speed in the DEVICES window during warmup.
8. Warm-up time: Allow approximately 30 to 45 minutes warm-up time for the
ICP-DRC-MS after igniting the plasma. This warm-up time is for the RF
generator. There will be another “Stability time” for the DRC later in this
procedure.
9. Optimizations and Daily Performance Check: After this warm-up time,
perform a daily performance check and any optimizations necessary (as
described in the ITN Daily Startup SOP for ELANs). Include Be (m/z 9) in
the daily performance check. Fill in the Daily Maintenance Checklist
according to the optimization procedures performed.
a. Magnesium (24Mg) may have high RSDs due to the use of Triton-X100
in the rinse solution. Avoid this problem by either temporarily using
non-Triton-containing rinse solution during the daily check, or repeating
the daily check multiple times in succession with no rinse time
between.

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10. Software setup for Analysis:
a. Workspace (files & folders): Click on “Open Workspace” from the
“File” menu. Select the workspace file “CDC_urine multi-element.wrk”
(or one customized for user preferences). Select “Review Files” from
the “File” menu. Verify & set up the correct files and data directories
for your analysis (See Table 1 in Appendix B).
b. Samples / Batch Window: Update the window to reflect the current
sample set. The only fields which need to be filled in include the
autosampler location, sample identification (id), measurement action,
method, sample flush time, sample flush speed, read delay time, read
delay & analysis speed, wash time, wash speed. Use a bar code
scanner to input data whenever possible. See Table 1 in Appendix B
for times and speeds. Save the Sample window file and re-use it on
other days by simply replacing the sample IDs for the patient samples.
1. DRC Stability Time: Best analyte-to-internal standard ratio
stability is obtained after 1-1.5 hrs of analysis of urine samples
using the DRC method. Analyze enough “dummy” urine
sample dilutions prior to any DRC analysis run to fill 1-1.5
hours of analysis time (not necessary if analyzing only a
subgroup of the method containing no DRC analytes). If
analyzing the full set of method analytes, 10 samples will be
sufficient. See Table 5 in Appendix B for example of setup in
the Samples / Batch window.
2. Urine vs. Aqueous Method Files:
a. The difference: There are two method files for this one
method (see Table 1 in Appendix B). It is necessary to use
both to accomplish each run because the current
PerkinElmer software will not allow for more than one blank
per method file. The ONLY DIFFERENCE between these
two files is on the Sampling tab where one lists the
autosampler positions of the urine blank and urine
calibrators (the “urblk” method file) and the other lists the
autosampler position of the aqueous blank (the “aqblk”
method file).
b. Use: The ONLY TIME when it matters which of these files
is used is when the measurement action includes “Run
blank” or “Run standards”. When the measurement action
is only ‘run sample’, it does not matter whether the “urblk”
or “aqblk” method file is used. Analysts typically follow the

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pattern below, however, for the sake of consistency and as
a reminder of which blank must be used for which type of
sample. See Table 6 in Appendix B.
i. The “urblk” method file: Use to analyze the initial urine
blank (blank for the calibration curve), the urine
calibrators, and the urine blank checks (urblkchk1 &
urblkchk2) at the very beginning of the run. The urine
blank method defines the autosampler location of the
urine blank and the urine calibration standards.
ii. The “aqblk” method file must be used to analyze all QC
materials and patient samples. The aqueous blank
method defines the aqueous blank in autosampler
location.
3. Notation of Dilutions: To designate an extra dilution of a
sample, edit the sample ID to reflect the level of dilution being
performed (i.e., A 1:2 dilution of sample 1 would be reflected in
the sample ID “sample 1 (2x dilution)”. This sample ID will be
edited during the data-import process to the database so that
it is recognized as the appropriate sample. Do not use the
ELAN® software to automatically correct for sample dilutions.
Extra dilution is performed on urine samples whose
concentration is greater than the concentrations listed in Table
8 in Appendix B (linearity of the method has been documented
up to these concentrations).
ii. Preparation of Samples for Analysis (See Table 7 in Appendix B)
1.

Thaw the frozen urine specimens; allow them to reach ambient
temperature.

DRC stability “dummy urine matrix”. Prepare 50+mL of standard 2 or
standard 3 to be analyzed for 1-1.5 hr before the beginning of the run.
This can be prepared using 50mL polypropylene tubes or a wide-mouth
bottle (which can be put on the autosampler in place of one of the tube
trays).

Set up a series of 15-mL polypropylene tubes corresponding to the
number of blanks, standards, QCs, and patient samples to be analyzed.

Prepare the following solutions in the 15-mL falcon tubes using the
Micromedic Digiflex™ (see Table 3 in Appendix B for a summary).
a. Aqueous Blank: Prepare two aqueous blanks consisting of 1,000 µL of
>18 Mega-ohm·cm water and 9,000 µL of diluent. One will be the
actual aqueous blank and the other will be a backup (“Aqueous Blank
Check”) in case the original aqueous blank gets contaminated.
b. Urine Blank: Prepare two urine blank dilutions consisting of 900 µL of
base urine (same material used to prepare the urine calibration
standards), 100 µL of >18 Mega-ohm·cm water, and 9,000 µL of

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diluent. One of these urine blanks will be the blank for the calibration
standards; the other will be analyzed twice after standard 5 as
UrBlkChk1 and UrBlkChk2, respectively. Results from the UrBlkChks
will be used to determine the method limit of detection.
c. Calibrators: Prepare the working calibration standards as 100 µL of
the appropriate aqueous intermediate working calibration standard,
900 µL of base urine, and 9,000 µL of diluent.
d. Patient & QC Samples: Before taking an aliquot for analysis, mix the
sample so that no particulates remain on the bottom of the tube.
Prepare urine sample dilutions as 4,500 µL of diluent and 500 µL of the
urine sample.
e. Cap all of the blanks, standards, and samples and mix them well.
Uncap them and place them in the autosampler of the ELAN® ICPMS
in the order that was entered in the Samples / Batch window of the
ELAN software.
iii. Specimen Storage and Handling During Testing: Specimens may be left at
room temperature during analysis in case confirmation analyses must be
made. Take stringent precautions to avoid external contamination by the
metals to be determined. Specimens may be stored short term at refrigerated
temperatures, but should be stored long term (>4 weeks) at ≤ -20 °C.
iv. Starting the Analysis: To begin analysis, highlight (click and drag with the
mouse) the table rows of the samples that should be included in the run, and
then click on “Analyze Batch.”
v. Monitoring the Analysis: Initiate work in a timely manner so that the run may
be monitored. Make every effort to complete analysis within the work day so
that the entire run can be monitored. If it is not possible to complete the
analysis by the end of the work day, the run may be left to complete itself
unattended as long as appropriate planning is made for either overnight
operation or Auto Stop (see below).
Monitor the analysis for the following:
1. DRC stability (analyte / internal standard ratio stability)
After the analysis of the DRC stability “dummy” samples, these results can
be reviewed to determine if sufficient stability of the analyte-to-internal
standard ratio was reached before beginning analysis. Importing data into
an MS Excel template file is useful to visualizing magnitude of drift.
2. Proper operation of the instrument.
3. Contaminated blanks.
4. Linear calibration curves.
a. Typical correlation coefficients will be 0.999 to 1.000.
b. The ELAN software generates a “simple linear” calibration curve (using
a least squares calculation) for each of the 13 elements in this method.

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The curves are generated using the results from analysis of the urine
blank and the 5 external urine calibrators whose concentrations are
defined in the Calibration tab of the Method file. Specifically, the
software plots the “net intensity” (y-axis) versus the analyte
concentration (x-axis). The “net intensity” is the blank subtracted ratio
of the measured intensity for the analyte to the measured intensity of
the associated internal standard and is calculated as follows:

net int ensity =

Analyte Meas Intensity sample
Internal Std Meas. Intensity sample

−

Analyte Meas Intensity Blank
Internal Std Meas Intensity Blank

`
5. Bench QC results within the acceptable limits.
If an analyte result for the beginning QC material(s) falls outside of the 99%
limits, then the following steps are recommended:
a. If a particular calibration standard is obviously in error, remake a new
dilution at the Digiflex of that working calibrator, reanalyze it, and
reprocess the sample analyses using this new result as part of the
calibration curve.
b. Prepare a fresh dilution of the failing QC material and reanalyze it.
c. Prepare fresh dilutions at the Digiflex of all of the calibration standards
(working urine multi-element standards) and reanalyze the entire
calibration curve using the freshly prepared standards.
If these three steps do not result in correction of the out-of-control values
for QC materials, consult the supervisor for other appropriate corrective
actions. Do not report analytical results for runs that are not in statistical
control.
6. Good precision among replicates.
7. Consistent measured intensities of the internal standards.
Some sample-to-sample variations are to be expected. However the
intensities should be within a few percent of one another, and should
fluctuate around an average value (not drift continuously in one direction).
8. Elevated patient results. After any sample having a concentration greater
than the third calibration verification standard (see CV3 in Table 8 of
Appendix B), verify the instrument background levels have returned to
normal before proceeding in analysis to the next sample, adding additional
rinsing time if necessary. If this cannot be done in real-time, the sample
analyzed immediately after the elevated sample should be repeated for
confirmation (within 10% of the original result) prior to reporting. Report
the first analytically verified result.

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vi. Records of Results: Run results will be documented daily in both electronic
and paper form.
1. Electronic Records:
a. Transfer of Results to the Laboratory Information System / Database:
Transfer data electronically between computers or software to reduce
errors. When keyboard entry must be used, proofread transcribed
data after entry.
b. Long-Term Storage of ELAN software files: Files used and produced
by the ELAN software in analyzing samples will be backed up long
term on compact disk and kept a minimum of three years.
2. Paper Records: The paper copy of the results from the run should be put
into the study folder(s) and should include
a. A summary of the calibration curve statistics.
b. A printout of analysis of each measurement made during the run.
c. Optional, but helpful, is a printout of the DRC stability check
measurements in graphical form.
d. On the front sheet of the printed records, write the following
i.

Analyst initials

ii.

Instrument ID

iii.

Date of Analysis

iv.

Run # for the day on this instrument

Study ID and Group Number

vi.

Database batch ID (Not known until the run is imported into the
database)

Every analytical run
vii. Transfer of Results to the Laboratory Database:
performed for the analysis of patient samples should be entered into the
laboratory results database unless the run is not useable for obvious reasons
(i.e. the run is stopped for some reason before ending QC is analyzed, no
internal standard spiked into the diluent, etc. . . ).
1. Data Export Process (from ELAN® software to .TXT file): If the data file
was not created during the initial analysis, reprocess the data of interest
either with “original conditions” option, or by loading the files and folders
used during the analysis. In the ELAN® ICP-DRC-MS software, select
“Review Files” from the “File” menu. From this window, you must open the
files and directories that were used when collecting the data of the run that
you wish to export. (If the analysis has just ended, all of these files and
directories will still be open.) NOTE: A second copy of the ELAN®
software can be run as an Edit/Reprocess copy without affecting an
ongoing analysis by the first copy of the software running in Windows.
After you open the relevant files, go to the “Report” page in the METHOD

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window. Deselect the box that prints a paper copy of data and select the
box that sends data to a file. Select the “Report Options Template” named
“CDC_Database Output.rop” and type in a report filename using a format
such as “2005-0714a_group55.txt” to designate data from analysis of
group 55 from July 14, 2005, run #1. Under “Report Format”, choose the
“Use Separator” option, and under the “File Write” section choose
“Append.” Finally, reprocess the data of interest. (See PerkinElmer
ELAN® ICPMS Software Manual.) Make sure you apply the aqueous
blank to all sample and quality control material analyses.
2. Data Import Process (from .TXT file to Microsoft Access™ database):
a. Move the .TXT file to the appropriate subdirectory on the network drive
where exported data are stored. Directories for data storage are
named according to instrument \ year \ month\, such as
I:\Instruments\ELANDRC2A\2005\07\.
b. Using the ITN Database Frontends, import the instrument file into the
database. On the GoTo window, click on “Add Sample Results to
Database”, then “Import Instrument Data File”.
c. Enter the appropriate information to identify the instrument, assay,
analysis date & time, run number, analyst, calibrator lot number and
prep date used (use the “IS Lot Number” field) and study. If other than
default values for Method LOD, High Calibrator, Rep Delta Limit, and
units were used in the run, document what was used by clicking on the
“View/Set Batch Parameters” button, changing the appropriate values,
and then clicking “Back”.
d. Press the “Import” button, then browse to the correct network folder to
select the file which contains the results from the run. Select the file
and click “OK”.
e. In the “Import Instrument Results” table, pressing the “Find X’s” button
will show only those samples whose sample ID is not recognized as a
valid QC pool ID or sample ID for this study. (Sample IDs are set up
when the study is logged into the database.) Corrections to sample
IDs and dilution factors can be made in this table (e.g., correction of
transcription errors and adjustment for level of dilution). If samples
were diluted for analysis, both the sample ID and the dilution factor
need to be edited in this table before the values are transferred to the
database (the Replace command under the Edit window is helpful in
this case). When corrections to sample IDs are made, press the
“Check IDs” button to re-evaluate the sample IDs. Any sample or
analyte row marked “Not Recognized” will not be transferred to the
database when the “Transfer” button is pressed. Once transferred
into the database, the data should be evaluated for QC pass / fail, then
set with the with the appropriate settings for QC accept / reject, final
value status, and comment(s). See the database programmers for
more detail on working in the database.

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viii. Analyst Evaluation of Run Results:
1. Bench Quality Control: After completing a run, and importing the results
into the database, export the QC results to the SAS program where the run
will be judged to be in or out of control. The QC limits are based on the
average and standard deviation of the beginning and ending analyses of
each of the bench QC pools, so it will not be possible to know if the run is
officially accepted or rejected until it is completed.
a. Quality Control Rules: The SAS program applies the division QC rules
to the data as follows:
i.

If both QC run means (low & high bench QC) are within 2Sm limits
and individual results are within 2Si limits, then accept the run.

ii.

If 1 of the 2 QC run means is outside a 2Sm limit - reject run if:
1. Extreme Outlier – Run mean is beyond the characterization
mean +/- 4Sm
2. 1 3S Rule - Run mean is outside a 3Sm limit
3. 2 2S Rule - Both run means are outside the same 2Sm limit
4. 10 X-bar Rule – Current and previous 9 run means are on
same side of the characterization mean

iii.

If one of the 4 QC individual results is outside a 2Si limit - reject
run if:
1. R 4S Rule – Within-run ranges for all pools in the same run
exceed 4Sw (i.e., 95% range limit)

Note: Since runs have multiple results per pool for 2 pools, the R 4S
rule is applied within runs only.
Abbreviations:
Si = Standard deviation of individual results (the limits are not shown
on the chart unless run results are actually single
measurements).
Sm = Standard deviation of the run means (the limits are shown on the
chart).
Sw = Within-run standard deviation (the limits are not shown on the
chart).
b. Implications of QC Failures: If the division SAS program declares the
run out of control” for any analyte, use the following to determine the
implications on usability of the data from the run.
i.

For 1 or 2 analytes: ONLY the analytes which were “out of control”
are invalid for reporting from the run. Set all run results for those 1
or 2 analytes as “QC Rejected” in the database.

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A
ii.

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For 3 or more analytes: All results, regardless of analyte, are
invalid for reporting from the run.
Set all run results for all
analytes as “QC Rejected” in the database. Note in the batch
comment field why all results were marked QC rejected.

2. Patient Results:
a. Elevated Results:
i.

Boundaries Requiring Confirmatory Measurement:
1. Results Greater than the First Upper Boundary (1UB):
Concentrations observed greater than the “first upper
boundary” (defined in the laboratory database as the “1UB”)
should be confirmed by repeat analysis of a new sample
preparation. The concentration assigned to the 1UB for an
element is determined by study protocol but default
concentrations are in Table 9 in Appendix B. Report the
original result, as long as the confirmation is within 10% of the
original.
Continue repeat analysis until a concentration can
be confirmed.
2. Results Greater Than Highest Calibrator: When a sample
result is greater than the highest calibrator in the run, the
supervisor may request that the result be confirmed in an
analysis run which includes a standard or external reference
material with equivalent (within 10%) or greater concentration
than the sample.
3. Results Greater Than Calibration Verification Tested: Perform
an extra dilution on any urine sample whose concentration is
greater than those listed in Table 8 in Appendix B (the linearity
of the method has been documented up to these
concentrations). See Table 7 in Appendix B for description of
sample
preparation
with
extra
dilution.
4. Uranium Isotope Ratio Measurement for Elevated Uranium
Concentrations: A uranium 235/238 isotope ratio analysis is
performed for all urine uranium samples where the urine total
uranium concentration is greater than the 2UB boundary (see
Table 9 in Appendix B).

ii.

Inadequate Precision in Confirmation of a Measurement: If a
sample is reanalyzed to obtain a confirmation of an initially
elevated result, the confirmation should be within 10% of the
original result.

iii.

Analyst Reporting of Elevated Results: Concentrations observed
greater than the “second upper boundary” (defined in the
laboratory database as the “2UB”) should be reported to the QC
reviewer as an “elevated result”. The concentration assigned to

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IRAT-DLS Method Code: 3018 and 3018A

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the 2UB for an element is determined by study protocol but default
concentrations are in Table 9 in Appendix B. The analyst should
report any patient results confirmed to be greater than the second
upper boundary to the QC reviewer as an “elevated result”. There
is no routine notification for elevated levels for the metals
determined in this method. The protocol for supervisors reporting
elevated results to medical personnel is defined according to the
study protocol.
b. Inadequate Precision Within One Measurement: If the range of the
three replicate readings (maximum replicate concentration value minimum replicate concentration value) for a single sample analysis is
greater than the criteria listed in Table 9 in Appendix B (“>Lim Rep
Delta” in the database) and the range of the three replicate readings is
greater than 10% of the observed concentration, do not use the
measurement for reporting. Repeat the analysis of the sample.
ix. Submitting Final Work for Review: Once results have been imported,
reviewed, and set as final in the database by the analyst,
1. Submit an email to the QC reviewer informing them of the readiness of the
data for final review. The email should include
a. Instrument ID, run Date, run number, study ID, group ID.
b. Any bench QC failures (include reasons if known).
c. Any patient sample result greater than the 2UB boundaries (see Table
9 in Appendix B).
d. Anything out of the ordinary about this analytical work which could
have a bearing on the availability (i.e. insufficient sample to analyze),
accuracy, or precision of the results.
2. Include all items called for by the study folder cover sheet in the study
folder (i.e. printouts from the ICP-MS, bench QC evaluation) together in the
study folder before submitting the folder for review when analysis is
complete.
x. Overnight Operation or Using Auto Stop: Make every effort to complete
analysis within the work day so that the entire run can be monitored. If it is not
possible to complete the analysis by the end of the work day, the run may be
left to complete itself unattended as long as appropriate planning is made for
either overnight operation or Auto Stop.
1. 24 hrs / day operation in DRC mode:
a. To reduce startup time in the mornings, the analyst is encouraged to
operate the ELAN in DRC mode 24hrs/day during the work week. This
eliminates the need for daily 45 minute RF generator warm-up, and
possibly the need for DRC stability time (if the DRC gas is not off for
extended periods of time before analysis). To maintain the instrument
in DRC mode when not analyzing patient samples, setup multiple
sample rows in the Samples / Batch window with autosampler position

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IRAT-DLS Method Code: 3018 and 3018A

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n zero (rinse station of autosampler) and wash time of 1800s (30
minutes).
Repeat this sample row enough times to keep the
instrument in analysis mode overnight (1 sample with 15 minute wash
will take ~ 25 minutes).
2. AutoStop: If 24 hrs / day ELAN operation is not desired, the instrument
can shut the plasma off unattended after analysis. Setup this as follows:
a. On the “Auto Start / Stop” tab of the Instrument window, enable the
Auto Stop feature.
b. Press the “Change” button within the Auto Stop box and set the
Delayed shutdown time to 5 minutes. This will rinse the sample
introduction system of urine matrix before turning off the plasma.
c. It will be necessary to replace the sample peristaltic pump tubing the
next day since it will have been clamped shut overnight.
c. Equipment Maintenance: Analysts are expected to follow a 4-day analysis / 1day maintenance schedule in the laboratory.
i. ICPMS Maintenance: On the maintenance day, perform all maintenance per
the Inorganic Toxicology and Nutrition Branch ELAN ICP-MS Weekly
Maintenance SOP. All equipment maintenance should be documented in the
instrument logbook.
ii. Data Backup: Data on the ELAN computer will be backed up via two backup
routines.
1. Daily Backups to External Hard Drive: Automatic backups of the “elandata”
directory and all subdirectories should be programmed to occur each night
onto an external hard disk.
2. Weekly Backup to CD: Backup all files in the active “elandata” directory
and all subdirectories onto one recordable compact disc during the weekly
maintenance SOP. When the active “elandata” directory on the ICP-DRCMS computer hard drive becomes too large to fit onto a single recordable
compact disk, the oldest data can be removed from the computer to make
it easier to backup the entire directory weekly. This can usually be done
annually.
a. Backup the oldest data on the hard drive to two duplicate compact
disks and verify that the files on the CD are readable
b. Label them with the name of the instrument, the date range of the data,
the current date, your name, and “Copy 1 of 2” or “Copy 2 of 2”
c. After verifying that the CDs are readable, the oldest, backed up data
can be deleted from the ICP-MS computer hard drive.
d. It is best to not store duplicate copies in the same location.
9) Interpretation of the Results
a. Reportable Range: Urine multi-element values are reportable in the range
between the method LOD (see Appendix, Table 8 in Appendix B) and the highest

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concentration verified accurate by bi-annual calibration verification tests (see
Appendix, Table 8 in Appendix B). For example, if a urine cadmium value is less
than the method LOD of 0.042, report it as < 0.042 µg/L). Above the highest
concentration verified, extra dilutions are made of the urine sample to bring the
concentration within the verified range.
b. Reference Ranges (Normal Values): In this method the 95% reference ranges
(see Appendix, Table 10 in Appendix B) for these elements in urine fall within the
range of the calibrators.
c. Action Levels: Due to the uncertainty of the health implications of elevated
concentrations of many of the elements determined with this method, there is no
routine notification for elevated levels of every analyte determined with this
method. The present NRC standard for workplace removal is 15 µg/L of U in
urine [13]. Other action levels for reporting to supervising physicians are
determined on a study-by-study basis.
10) Method Calculations
a. Method Limit of Detection (LODs): The method detection limits for elements in
blood specimens are defined as 3 times s 0, where s 0 is the estimate of the
standard deviation at zero analyte concentration. S 0 is taken as the y-intercept
of a linear or 2nd order polynomial regression of standard deviation versus
concentration (4 concentration levels of the analytes in blood each measured 60
times across at least a 2-month timeframe). Method LODs are re-evaluated
periodically.
b. Method Limit of Quantitation (LOQ): The Division of Laboratory Sciences does
not currently utilize limits of quantitation in regards to reporting limits [10].
c. QC Limits: Quality control limits are calculated based on concentration results
obtained in at least 20 separate runs. It is preferable to perform separate
analyses on separate days and using multiple calibrator lot numbers,
instruments, and analysts to best mimic real-life variability. The statistical
calculations are performed using the SAS program developed for the Division of
Laboratory Sciences (DLS_QC_compute_char_stats.sas).
11) Alternate Methods for Performing Test and Storing Specimens If Test System
Fails:
If the analytical system fails, the analysis may be setup on other ELAN DRC
instruments in the laboratory. If no other instrument is available, store the
specimens at ~4°C until the analytical system can be restored to functionality. If
interruption longer than 4 weeks in anticipated, then store urine specimens at
≤ -20°C.

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IRAT-DLS Method Code: 3018 and 3018A

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Appendix A. Ruggedness Testing Results
Parameter Test #1 (15 Element Panel): Evaluate the impact on analysis results if the
RF Power is increased to 1600W (instrument maximum) or decreased to 1150W (by
20%) for the analytical run.
Test Details:
1. Three different RF power settings were tested in separately prepared, consecutive
runs on the instrument without turning off the plasma. At least 15 minutes
stabilization time was allowed between each run after the RF power was changed.
“Junk urine” samples (20) were analyzed between the beginning and ending QC of
each run. All other method parameters were kept per method.
2. Run #1 (method default, 1450W).
3. Run #2 (Decreased RF power by 20% to 1150W).
4. Run #3 (Increased RF power to instrument maximum, 1600W).

HU-04311_UMP3_e

LU-04310_UMP3_e

Parameter Test 1 Results (Table 1 of 3 for 15 Element Panel). All concentrations in
ug/L. Test performed 3/24/2010 by Denise Tevis using ELAN DRC-2N.
Sample
RF Power Tested
Ba
Be
Cd
Co
ID
Characterized
0.76
0.69
0.32
0.42
Mean
(±2SD Range)
(0.65 - 0.86) (0.59 - 0.79) (0.28 - 0.36) (0.38 - 0.47)
1150W
0.67
0.62
0.28
0.39
(reduced)
1450W
0.70
0.66
0.31
0.32¥
(per method)
1600W
0.75
0.75
0.33
0.42
(increased)
Characterized
5.01
5.28
1.62
1.88
Mean
(±2SD Range)
(4.54 - 5.24) (4.48 - 6.07) (1.47 - 1.78) (1.66 - 2.09)
1150W
4.93
5.75
1.6
1.96
(reduced)
1450W
5.55
5.28
1.75
1.67
(per method)
1600W
4.85
5.82
1.58
1.90
(increased)
¥Data are not statistically different according to the expected precision of the method (QC 3SD =
0.072)

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IRAT-DLS Method Code: 3018 and 3018A

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Appendix A. Ruggedness Testing Results. (continued)

HU-04311_UMP_e

LU-04310_UMP_e

HU-04311_UMP_e

LU-04310_UMP_e

Parameter Test 1 Results (Table 2 of 3 for 15 Element Panel). All concentrations in
ug/L. Test performed 3/23/2011 by Denise Tevis using ELAN DRC-2N.
Sample
RF Power
Cs
Mo
Pb
Pt
ID
Tested
Characterized
2.38
19.3
0.42
0.10
Mean
(±2SD Range) (2.25 - 2.51) (18.6 - 20.0) (0.37 - 0.48)
(0.07 - 0.13)
1150W
2.13¥
17.2*
0.42
0.09
(reduced)
1450W
2.32
18.9
0.41
0.09
(per method)
1600W
2.42
18.8
0.43
0.10
(increased)
Characterized
9.82
136
2.95
0.85
Mean
(±2SD Range) (9.03 - 10.6) (131 - 142) (2.82 - 3.08)
(0.71 - 1.00)
1150W
9.62
136
3.03
0.93
(reduced)
1450W
10.56
133
2.89
1.02#
(per method)
1600W
9.55
135
3.05
1.08#
(increased)
Sample
RF Power
Sb
Tl
W
U
ID
Tested
Characterized
0.19
0.18
0.22
0.014
Mean
(±2SD Range) (0.17 - 0.21) (0.17 - 0.19) (0.19 - 0.24) (0.011 - 0.016)
1150W
0.21
0.16
0.19
0.017α
(reduced)
1450W
0.16
0.19
0.22
0.014
(per method)
1600W
0.20
0.18
0.22
0.017 α
(increased)
Characterized
0.66
0.58
0.94
0.128
Mean
(±2SD Range) (0.60 - 0.71) (0.55 - 0.61) (0.90 - 0.99) (0.115 - 0.141)
1150W
0.69
0.59
0.90
0.153 α
(reduced)
1450W
0.61
0.57
0.93
0.126
(per method)
1600W
0.66
0.60
0.91
0.150 α
(increased)
¥Data are not statistically different; expected precision of the method (QC 3SD = 0.2)
*Data are not statistically different; expected precision of the method (QC 3SD = 1.08)
#Data are not statistically different; expected precision of the method (QC 3SD = 0.22)
α
Data are not statistically different; expected precision of the method (QC 3SD = 0.02)

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IRAT-DLS Method Code: 3018 and 3018A

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Appendix A. Ruggedness Testing Results. (continued)
Parameter Test 1 Results (Table 3 of 3 for 15 Element Panel). All concentrations in
ug/L. Test performed 3/23/2011 by Denise Tevis using ELAN DRC-2N.

Seronorm Trace
Elements Urine§

NYDOH UE09-06‡

NYDOH UE09-05‡

Sample ID

RF Power Tested

Characterized Mean
(±2SD Range)

1.37
(1.55 -1.19)

2.2
(2.0-2.8)

1.22

2.9

1450W (per method)

0.98

2.8

1600W (increased)

1.30

3.0

Characterized Mean
(±2SD Range)

31.1
(26.3 -35.9)

61
(55.0 - 67.0)

29.4

67.4

1450W (per method)

23.8

68.5

1600W (increased)

30.0

66.7

Characterized Mean

12.3

54.6

110

(±2SD Range)

(10.9 - 13.7)

(51.9 - 57.3)

(104 -116)

10.4

62.3

113

1450W (per method)

8.46

62.3

111

1600W (increased)

10.5

61.4

111

1150W

(reduced)

§Purchased from Sero AS, Billingstad, Norway.
‡ Purchased from Wadsworth Center, New York State Department of Health

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Appendix A. Ruggedness Testing Results. (continued)
Parameter Test #1 (Arsenic): Evaluate the impact on analysis results if the set RF
Power is increased to 1600W (instrument maximum) or decreased to 1150W (by 20%)
for the analytical run.
Test Details:
1. Three different RF power settings were tested in separately prepared, consecutive
runs on the instrument without turning off the plasma. At least 15 minutes
stabilization time was allowed between each run after the RF power was changed.
“Junk urine” samples (40) were analyzed between the beginning and ending QC of
each run. All other method parameters were kept per method.
2. Run #1 (method default, 1450W).
3. Run #2 (Decreased RF power by 20% to 1150W).
4. Run #3 (Increased RF power to instrument maximum, 1600W).
Parameter Test 1 Results (Arsenic). All concentrations in ug/L. Test
performed 3/26/10 by Graylin Mitchell using ELAN DRC-2G.
QC Pool ID

LU-04310_UMP_e

RF Power Tested

Characterized Mean
Characterized 2SD Range
Characterized 3SD Range

3.74
3.21 – 4.27
2.95 – 4.53

1150W

(Reduced)

3.72

1450W

(Per Method)

4.10

1600W

(Increased)

3.66

Characterized Mean
Characterized 2SD Range
Characterized 2SD Range
HU-04311_UMP_e

55.8
53.3 – 58.3
52.1 – 59.6

1150W

(Reduced)

55.6

1450W

(Per Method)

58.8

1600W

(Increased)

52.2

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Appendix A. Ruggedness Testing Results. (continued)
Parameter Test #2 (cadmium and manganese): Evaluate the impact on analysis results
if the cell gas flow rate is increased or decreased by 20% for the analytical run.
Test Details:
1. Three different cell gas flow rates were tested in separately prepared, consecutive
runs on the instrument without turning off the plasma. Samples were prepared with
diluent containing 400 ppm K and 60 ppb Mo in addition to the internal standards. At
least 15 minutes stabilization time was allowed between each run after the cell gas
flow rate was changed. “Junk urine” samples (20) were analyzed between the
beginning and ending QC of each run
2. Run #1 (method default = 2.3 mL/min)
3. Run #2 (decreased cell gas flow rate by 20% to 1.8 mL/min).
4. Run #3 (increased cell gas flow rate by 20% to 2.75 mL/min).

Seronorm Trace
Elements
HU-04311_UMP3_e LU-04310_UMP3_e
Urine§

Parameter Test 2 Results (Table 1 of 2 for 15 element). All concentrations in ug/L. Test
performed 4/4/11 by Denise Tevis using ELAN DRC-2N.
Cell gas Flow Rate
Sample ID
Cd
Mn
Tested
Characterized Mean
0.32
(±2SD Range)
(0.28 - 0.36)
1.8 mL/min (reduced)

0.29

2.3 mL/min (per method)

0.29

2.75 mL/min (increased)

0.28

Characterized Mean
(±2SD Range)

1.62
(1.47 - 1.78)

1.8 mL/min (reduced)

1.65

2.3 mL/min (per method)

1.62

2.75 mL/min (increased)

1.57

Characterized Mean
(±2SD Range)
1.8 mL/min (reduced)

12.3
(8.70 - 15.9)
11.0

2.3 mL/min (per method)

10.1

2.75 mL/min (increased)
§Purchased from Sero AS, Billingstad, Norway.

10.7

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Appendix A. Ruggedness Testing Results. (continued)

NYDOH UE 10-10‡

NYDOH UE 10-04‡

Parameter Test 2 Results (Table 2 of 2 for 15 element). All concentrations in ug/L.
Test performed 4/4/11 by Denise Tevis using ELAN DRC-2N.
Cell gas Flow Rate
Sample ID
Cd
Mn
Tested
Characterized Mean
24.5
(±2SD Range)
(19.7 - 29.3)
1.8 mL/min (reduced)

27.0

2.3 mL/min (per
method)
2.75 mL/min
(increased)
Characterized Mean
(±2SD Range)

2.1
(1.1 - 3.1)

1.8 mL/min (reduced)

1.82

24.4
26.2

2.3 mL/min (per
1.60
method)
2.75 mL/min
1.78
(increased)
‡ Purchased from Wadsworth Center, New York State Department of Health

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Appendix A. Ruggedness Testing Results. (continued)
Parameter Test #2 (Arsenic): Evaluate the impact on analysis results if the cell gas flow
rate is increased or decreased by 20% for the analytical run.
Test Details:
1. Three different cell gas flow rates were tested in separately prepared, consecutive
runs on the instrument without turning off the plasma. At least 15 minutes
stabilization time was allowed between each run after the cell gas flow rate was
changed. “Junk urine” samples (40) were analyzed between the beginning and
ending QC of each run (diluent was prepared to a 1% HCl matrix so ArCl+
interference removal would be challenged).
2. Run #1 (method default = 0.95 mL/min).
3. Run #2 (decreased cell gas flow rate by 20% to 0.76 mL/min).
4. Run #3 (increased cell gas flow rate by 20% to 1.14 mL/min).
Parameter Test 2 Results (Arsenic).
Test performed 5/13/10 by Graylin Mitchell using ELAN DRC2-G.
QC
Pool ID

LU-04310_UMP_e

Cell Gas Flow Rate Tested

As (ug/L)

Characterized Mean
Characterized 2SD Range
Characterized 3SD Range

3.74
3.21 – 4.27
2.95 – 4.53

0.76 mL/min

(Reduced)

0.95 mL/min (Method Default)
1.14 mL/min

(Increased)

Characterized Mean
Characterized 2SD Range
Characterized 2SD Range
HU-04311_UIMP_e

0.76 mL/min

(Reduced)

0.95 mL/min (Method Default)
1.14 mL/min

(Increased)

4.35
4.02
4.03
55.8
53.3 – 58.3
52.1 – 59.6
57.6
54.2
59.2

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Appendix A. Ruggedness Testing Results. (continued)
Parameter Test #3 (DRC elements: cadmium and manganese): Evaluate the impact on
analysis results if the RPq is increased or decreased by 20% for the analytical run.
Test Details:
1. Three RPq settings were tested for cadmium and manganese in separately
prepared, consecutive runs on the instrument without turning off the plasma.
Samples were prepared with diluent containing 400 ppm K and 60 ppb Mo in
addition to the internal standards. At least 15 minutes stabilization time was allowed
between each run after DRC RPq was changed. “Junk urine” samples (20) were
analyzed between the beginning and ending QC of each run.
2. Run #1 (instrument default DRC RPq: 0.45).
3. Run #2 (~20% decrease; DRC RPq: 0.35).
4. Run #3 (~20% increase; DRC RPq: 0.55 ).

LU04310_UMP3_e

0.32

(±2SD Range)
0.35 (reduced)
0.45 (typical)
0.55 (increased)

(0.28 - 0.36)
0.32
0.31
0.29

Characterized Mean

1.62

(±2SD Range)
0.35 (reduced)
0.45 (typical)
0.55 (increased)
Characterized Mean
(±2SD Range)
0.35 (reduced)
0.45 (typical)
0.55 (increased)
Characterized Mean
(±2SD Range)
0.35 (reduced)
0.45 (typical)
0.55 (increased)
Characterized Mean
(±2SD Range)
0.35 (reduced)
0.45 (typical)
0.55 (increased)

(1.47 - 1.78)
1.58
1.48
1.46

NYDOH UE NYDOH UE
09-05‡
09-06‡

Seronorm
Trace
Elements
Urine§

Characterized Mean

HU04311_UMP3_e

Parameter Test 4 Results. All concentrations in ug/L. Test performed 3/30/11 by
Denise Tevis using ELAN DRC-2N.
Sample ID
RPQ Tested
Cd
Mn

§Purchased from Sero AS, Billingstad, Norway.
‡ Purchased from Wadsworth Center, New York State Department of Health

12.3
(8.70 - 15.9)
10.7
11.8
11.0
31.1
(26.3 - 35.9)
29.3
32.1
30.9
1.8
(1.0 - 2.6)
1.28
1.41
1.29

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Appendix A. Ruggedness Testing Results. (continued)
Parameter Test #3 (Arsenic): Evaluate the impact on analysis results if the RPq is
increased or decreased by 20% for the analytical run.
Test Details:
1. Three different RPQ settings were tested for Cadmium in separately prepared,
consecutive runs on the instrument without turning off the plasma. At least 15
minutes stabilization time was allowed between each run after DRC RPQ was
changed. “Junk urine” samples (40) were analyzed between the beginning and
ending QC of each run. The diluent included 1% HCl.
2. Run #1 (previous method default DRC RPQ: 0.75).
3. Run #2 (decreased DRC RPQ 20%: 0.62).
4. Run #3 (increased DRC RPQ 20%: 0.88 ).
5. Additional test Run #4 (increased DRC RPQ: 0. 25).
6. Additional test Run #5 (increased DRC RPQ: 0. 45).
Parameter Test 3 Results (Arsenic).
Test performed 3/18/10 by Gulchekhra Shakirova.
QC
RPQ Tested
Pool ID
Characterized Mean
Characterized 2SD Range
Characterized 3SD Range
LU-04310_UMP_e

HU-04310_UMP_e

As (ug/L)
3.74
3.21 – 4.27
2.95 – 4.53

DRC RPQ: 0.38

3.92

DRC RPQ: 0.48

3.93

DRC RPQ: 0.60

3.71

DRC RPQ: 0.72

3.70

Characterized Mean
Characterized 2SD Range
Characterized 2SD Range

55.8
53.3 – 58.3
52.1 – 59.6

DRC RPQ: 0.38

54.9

DRC RPQ: 0.48

54.9

DRC RPQ: 0.60

55.2

DRC RPQ: 0.72

54.2

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Appendix A. Ruggedness Testing Results. (continued)
Parameter Test #4 (cadmium and manganese): Evaluate the impact on analysis results
if the axial field voltage (AFV) is increased or decreased by 20% for the analytical run.
Test Details:
1. Three different DRC AFV were tested in separately prepared, consecutive runs on
the instrument without turning off the plasma. Samples were prepared with diluent
containing 400 ppm K and 60 ppb Mo in addition to the internal standards. At least
15 minutes stabilization time was allowed between each run after the axial field
voltage was changed. “Junk urine” samples (20) were analyzed between the
beginning and ending QC of each run. The diluent including 60 ug/L Molybdenum.
2. Run #1 (instrument default DRC AFV = 375)
3. Run #2 (decreased DRC AFV by 20% to 300).
4. Run #3 (increased DRC AFV by 17% to 450).

NYDOH UE NYDOH UE
10-06‡
09-06‡

Seronorm
Trace
Elements
Urine§

HULU04311_UMP3_e 04310_UMP3_e

Parameter Test 4 Results. All concentrations in ug/L. Test performed 4/5/11 by
Denise Tevis using ELAN DRC-2N.
Sample ID
AFV Tested
Cd
Mn
Characterized Mean

0.32

(±2SD Range)

(0.28 - 0.36)

300 (reduced)
375 (typical)
450 (increased)

0.32
0.34
0.31

Characterized Mean

1.62

(±2SD Range)

(1.47 - 1.78)

300 (reduced)
375 (typical)
450 (increased)
Characterized Mean
(±2SD Range)
300 (reduced)
375 (typical)
450 (increased)
Characterized Mean
(±2SD Range)
300 (reduced)
375 (typical)
450 (increased)
Characterized Mean
(±2SD Range)
300 (reduced)
375 (typical)
450 (increased)

1.62
1.64
1.61

§Purchased from Sero AS, Billingstad, Norway.
‡ Purchased from Wadsworth Center, New York State Department of Health

Appendix A. Ruggedness Testing Results. (continued)

12.3
(8.70 - 15.90)
10.8
10.7
10.8
31.1
(26.3 - 35.9)
32.0
32.0
31.9
1.4
(0.2 - 2.6)
0.81
0.98
1.02

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Parameter Test #4 (Arsenic): Evaluate the impact on analysis results if the axial field
voltage (AFV) is increased or decreased for the analytical run.
Test Details:
1. Four different DRC AFV were tested in separately prepared, consecutive runs on the
instrument without turning off the plasma. At least 15 minutes stabilization time was
allowed between each run after the axial field voltage was changed. “Junk urine”
samples (40) were analyzed between the beginning and ending QC of each run.
2. Run #1 (method default DRC AFV = 250)
3. Run #2 (increased DRC AFV to 300).
4. Run #3 (decreased DRC AFV to 200).

Parameter Test 4 Results (Arsenic).
Test performed 6/2/10 by Graylin Mitchell using ELAN DRC2-G.
QC
Axial Field Voltage Tested
As (ug/L)
Pool ID
Characterized Mean
3.74
Characterized 2SD Range
3.21 – 4.27
Characterized 3SD Range
2.95 – 4.53
LU-04310_UMP_e

200

(Reduced)

4.51

250

(Typical)

4.37

300

(Increased)

4.08

Characterized Mean
Characterized 2SD Range

55.8
53.3 – 58.3

200

(Reduced)

54.2

250

(Typical)

55.9

300

(Increased)

54.0

HU-04311_UMP_e

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Appendix A. Ruggedness Testing Results. (continued)
Parameter Test #5 (15 Element Panel): Method descriptions and SOP assume
preparation and analysis on same day. Evaluate the impact on analysis results if the
analytical run is prepared to analyze but circumstances do not allow for analysis to
occur until 24 or 48 hours later.
Test Details:
1. Three separate run sets (A, B, and C) were prepared at one sitting from the same
starting materials. Set ‘A’ was analyzed immediately per the assumption of the
method. Set’s ‘B’ and ‘C’ were stored at room temperature for 24 and 48 hours,
respectively before analysis. “Junk urine samples (20) were analyzed between the
beginning and ending QC of each run, making each a normal length run. All other
method parameters were kept per method.
2. On day two, a fresh run set (“D”) was prepared and analyzed immediately for
comparison to results from set “B” (Run 2 of the day. Results not shown).
3. On day three, another fresh run set (“E”) was prepared and analyzed immediately for
comparison to results from set “C” (Run 2 of the day. Results not shown).

HULU04311_UMP 04310_UMP
3_e
3_e

Parameter Test 5 Results (Table 1 of 2 for 15 Element Panel). All concentrations in ug/L.
Test begun 3/23/11 by Denise Tevis using ELAN DRC-2N.
Time from
Sample
Preparation to
Ba
Be
Cd
Co
ID
Analysis
Characterized Mean
0.76
0.69
0.32
0.42
(±2SD Range)
(0.65 - 0.86) (0.59 - 0.79) (0.28 - 0.36) (0.38 - 0.47)
freshly prepared
0.70
0.66
0.32
0.32
24 hours
0.70
0.69
0.31
0.42
48 hours
0.86
0.67
0.31
0.43
Characterized Mean
5.01
5.28
1.62
1.88
(±2SD Range)
(4.54 - 5.24) (4.48 - 6.07) (1.47 - 1.78) (1.66 - 2.09)
freshly prepared
5.55
3.48
1.75
1.67
24 hours
4.66
5.76
1.57
1.94
48 hours
5.12
5.49
1.67
1.84
¥Data are not statistically different according to the expected precision of the method (QC 3SD = 0.072)

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Appendix A. Ruggedness Test #5 Results (continued)

HU-04311_UMP_e LU-04310_UMP_e

Parameter Test 5 Results (Table 2 of 3 for 15 Element Panel). All concentrations in ug/L.
Test begun 3/23/11 by Denise Tevis using ELAN DRC-2N.
Time from
Sample
Preparation to
Cs
Mo
Pb
Pt
ID
Analysis

HU-04311_UMP_e LU-04310_UMP_e

Sample
ID

Characterized Mean

2.38

19.3

0.42

0.10

(±2SD Range)

(2.25 - 2.51)

(18.6 - 20.0)

(0.37 - 0.48)

(0.07 - 0.13)

freshly prepared
24 hours
48 hours

2.32
2.35
2.30

18.9
19.2
19.0

0.41
0.44
0.46

0.09
0.11
0.10

Characterized Mean

9.82

136

2.95

0.85

(±2SD Range)

(9.03 - 10.6)

(131 - 142)

(2.82 - 3.08)

(0.71 - 1.00)

freshly prepared
24 hours
48 hours
Time from
Preparation to
Analysis

10.6
9.36
10.0

133
134
132

2.89
3.08
3.04

1.02
1.03
1.12

Characterized Mean

0.19

0.18

0.22

0.014

(±2SD Range)

(0.17 - 0.21)

(0.17 - 0.19)

(0.19 - 0.24)

(0.011 - 0.016)

freshly prepared
24 hours
48 hours

0.16
0.19
0.19

0.19
0.18
0.19

0.22
0.21
0.22

0.014
0.013
0.014

Characterized Mean

0.61

0.58

0.94

0.128

(±2SD Range)

(0.60 - 0.71)

(0.55 - 0.61)

(0.90 - 0.99)

(0.115 - 0.141)

freshly prepared
24 hours
48 hours

0.61
0.65
0.69

0.57
0.60
0.59

0.93
0.90
0.90

0.126
0.128
0.126

* Data are not statistically different according to the expected precision of the method (QC 3SD = 0.02)
¥Data are not statistically different according to the expected precision of the method (QC 3SD = 0.2)
*Data are not statistically different according to the expected precision of the method (QC 3SD = 1.08)
#Data are not statistically different according to the expected precision of the method (QC 3SD = 0.22)

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Appendix A. Ruggedness Testing Results. (continued)

Seronorm Trace NYDOH
Elements Urine§ UE09-06‡

NYDOH
UE09-05‡

Parameter Test 5 Results (Table 3 of 3 for 15 Element Panel). All concentrations in
ug/L. Test begun 3/23/11 by Denise Tevis using ELAN DRC-2N.
Sample
Time from Preparation
ID
to Analysis
Mn
Sn
Sr
Characterized Mean
1.37
2.2
(±2SD Range)
(1.55 -1.19)
(2.0-2.8)
freshly prepared
0.98
2.8
24 hours
1.26
2.6
48 hours
1.47
2.6
Characterized Mean
31.1
61
(±2SD Range)
(26.3 -35.9)
(55.0 - 67.0)
freshly prepared
23.8
68.5
24 hours
30.6
62.8
48 hours
31.9
61.4
Characterized Mean

12.3

54.6

110

(±2SD Range)
freshly prepared
24 hours
48 hours

(10.9 - 13.7)
8.47
10.9
10.4

(51.9 - 57.3)
62.3
57.5
58.3

(104 -116)
111
112
114

§Purchased from Sero AS, Billingstad, Norway.
‡ Purchased from Wadsworth Center, New York State Department of Health

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Appendix A. Ruggedness Test #5 Results (continued)
Parameter Test #5 (Arsenic): Method descriptions and SOP assume preparation and
analysis on same day. Evaluate the impact on analysis results if the analytical run is
prepared to analyze but circumstances do not allow for analysis to occur until 24 or 48
hours later.
Test Details:
1. Three separate run sets (A, B, and C) were prepared at one sitting from the same
starting materials. Set ‘A’ was analyzed immediately per the assumption of the
method. Set’s ‘B’ and ‘C’ were stored at room temperature for 24 and 48 hours,
respectively before analysis. “Junk urine samples (20) were analyzed between the
beginning and ending QC of each run, making each a normal length run. All other
method parameters were kept per method.
2. On day two, a fresh run set (“D”) was prepared and analyzed immediately for
comparison to results from set “B” (Run 2 of the day. Results not shown).
3. On day three, another fresh run set (“E”) was prepared and analyzed immediately for
comparison to results from set “C” (Run 2 of the day. Results not shown).

Parameter Test 4 Results (Arsenic).
Test performed 5/26-28/10 by Graylin Mitchell using ELAN DRC2-G.
QC
Axial Field Voltage Tested
As (ug/L)
Pool ID
Characterized Mean
3.74
Characterized 2SD Range
3.21 – 4.27
Characterized 3SD Range
2.95 – 4.53
LU-04310_UMP_e

Fresh Preparation

3.40

After 24 Hours

3.37

After 48 Hours

3.41

Characterized Mean
Characterized 2SD Range

55.8
53.3 – 58.3

Fresh Preparation

54.7

After 24 Hours

53.8

After 48 Hours

53.5

HU-04311_UMP_e

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Appendix B
Table 1. Instrument and Method Parameters. Parameters are the same for arsenic
and the 15 element panel unless otherwise noted.
Instrument: PerkinElmer ELAN DRCPlus or DRC II ICP-MS
Autosampler: ESI SC4 Autosampler with FAST sample introduction system
Optimization Window Parameters
RF power 1.45 KW
Plasma Gas Flow (Ar) 15 L/min
Auxiliary Gas Flow (Ar) 1.2 L/min
Nebulizer Gas Flow (Ar) ~0.90 – 1.0 L/min (optimized as needed for
sensitivity)
Ion Lens Voltage(s) AutoLens (optimized as needed for sensitivity)
QRO, CRO, CPV, Discriminator Optimized per instrument by service engineer, or
Threshold advanced user.
Parameters of x-y alignment, nebulizer gas flow, AutoLens voltages, mass calibration,
and detector voltages are optimized regularly. Optimization file name = default.dac.
Configurations Window Parameters
Cell Gas Changes Pause Times Pressurize Delay (From Standard to DRC) = 30
Exhaust Delay (From DRC to Standard mode) = 30
Flow Delay (Gas changes while in DRC mode) = 30
Channel Delay (channel change in DRC mode) = 30
File Names & Directories
Method file names
Dataset Create a new dataset subfolder each day. Name as
“2011-0718” for all work done on July 18, 2011
Sample File Create for each day’s work
Report file name For sample results printouts
cdc_quant comprehensive_multielement.rop

Tuning
Optimization
Calibration
Polyatomic
Report Options Template
(transferring results to the
database)

For calibration curve information
CDC_Quant Comprehensive (calib curve info).rop
Default.tun
Default.dac
N/A
elan.ply
CDC_Database Output.rop
Report Format Options: select only “Use Separator”
File Write Option: Append
Report File name: include date, instrument, and
group being analyzed in file name (i.e. 20050311b_DRC2A_HM-0364.txt)

Method Parameters
Method Parameters: Timing Page (see Figures 2a and 3a in the Appendix)
Sweeps/reading 70
Readings/replicate 1
Replicates 3
Enable QC Checking Off

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Table 1. Instrument and Method Parameters. Parameters are the same for arsenic
and the 15 element panel unless otherwise noted.
Isotopes Monitored For Arsenic
and Internal Standard (use 71Ga as an internal standard)
Associations 71Ga (70.9249), 75As (74.9216)
(Exact Mass)
For 15 Element Panel
Group 1 (use 103Rh as an internal standard)
9
Be (9.0122), 59Co (58.9332), 88Sr (87.9056) ,98Mo
(97.9055), 103Rh (102.905), 118Sn (117.902), 121Sb
(120.904), 133Cs (132.905), 138Ba (137.905)
Group 2 (use 193Ir as an internal standard)
184
W (183.951), 193Ir (192.963), 195Pt (194.965),
(204.975), 208Pb (207.977), 238U (238.05)

Dwell Times

Scan Mode
DRC channel A Gas
Flow Rate

205

Group 3 (use 103Rh as an internal standard)
103
Rh (102.905), 114Cd (113.904), 55Mn (54.9381),
30 ms for 59Co, 88Sr,98Mo, 118Sn,103Rh in Standard
mode, 121Sb, 133Cs, 138Ba, 184W, 193Ir, 205Tl, and 208Pb
50 ms for 71Ga , 75As and 55Mn in DRC mode
100 ms for 9Be, 195Pt, 238U, 103Rh in DRC mode, and
114
Cd
Peak Hopping for all isotopes (1 MCA channel)
For Arsenic
10% hydrogen / 90% argon
(5-7 psig delivery pressure)
Typically 0.7 to 1.3 mL/min *
*(optimized per instrument, and periodically verified)

For 15 Element Panel
Not used.
DRC channel B Gas For Arsenic
Flow Rate Not used.
For 15 Element Panel
Oxygen (5-7 psig delivery pressure)
Typically 2.0 – 2.5 mL/min *
* (optimized instrument, and periodically verified)
RPa 0 for all isotopes

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Table 1. Instrument and Method Parameters. Parameters are the same for arsenic
and the 15 element panel unless otherwise noted.
RPq For Arsenic
DRC Mode (As group): Typically* 0.65 - 0.75 for
71
Ga (70.9249), 75As (74.9216). Use the same RPQ
for each.
For 15 Element Panel
Standard Mode: 0.25 for all standard mode isotopes
DRC Mode (Cd and Mn group): default 0.45 *,
typical range 0.35 - 0.55 for 114Cd (113.904) and
55
Mn (54.9381), and 103Rh (102.905) in DRC mode.
Use the same RPQ for each.
(* Optimize per instrument, and periodically verified)
Method Parameters: Processing Page (see Figures 2b and 3b in the Appendix)
Detector mode Pulse
Process Spectral Peak N/A
AutoLens On
Isotope Ratio Mode Off
Enable Short Settling Time Off
Blank subtraction After internal standard
Measurement units Cps
Process Signal Profile N/A
Method Parameters: Equations Page (see Figures 2c and 3c in the Appendix)
Equations On 208Pb, use “+ Pb 206 + Pb 207”
On 238U, use “+ U 235”
On 114Cd, use “- 0.027250 * Sn 118”
Method Parameters: Calibration Page (see Figures 2d and 3d in the Appendix)
Calibration Type External Std.
Curve type Simple Linear
Sample units “µg/L” or “ppb”
Calibration Standard Be: 0.1, 0.3, 1, 3, 10
Concentrations (µg/L) Co: 0.075, 0.225, 0.75, 2.25, 7.5
Sr: 3, 9, 30, 90, 300
Mo: 3, 9, 30, 90, 300
Sn: 0.3, 0.9, 3, 9, 30
Sb: 0.08, 0.24, 0.8, 2.4, 8
Cs: 0.2, 0.6, 2, 6, 20
Ba: 0.2, 0.6, 2, 6, 20
W: 0.06, 0.18, 0.6, 1.8, 6
Pt: 0.025, 0.075, 0.25, 0.75, 2.5
Tl: 0.04, 0.12, 0.4, 1.2, 4
Pb: 0.1, 0.3, 1, 3, 10
U: 0.005, 0.015, 0.05, 0.15, 0.5
Cd: 0.08, 0.24, 0.8, 2.4, 8
Mn: 0.1, 0.3, 1, 3, 10
As: 2, 6, 20, 60, 200
Method Parameters: Sampling Page (see Figures 2e and 3e in the Appendix)

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Table 1. Instrument and Method Parameters. Parameters are the same for arsenic
and the 15 element panel unless otherwise noted.
“Peristaltic Pump Under On
Computer Control”
Autosampler If using ESI autosampler
Tray Autosampler Type: AS-93plus
Port Tray Name: esi.try
Sampling Device
(either use shortcut in
C:\Elandata\Autosampler\AS-93 or
link to file in C:\program files\esi\esi sc\esi.try)
Port: GPIB1
Sampling Device: None
If using other autosampler
Refer to autosampler user guide.
Sample Flush FAST Defaults For Arsenic
6s at 6 rpm (standard ELAN peristaltic pump)
6s at 3rpm (ESI micro-peristaltic pump)
FAST Defaults For 15 Element Panel
6s at 6 rpm (standard ELAN peristaltic pump)
6s at 3rpm (ESI micro-peristaltic pump)
Can be optimized as needed to adequately fill the
FAST loop. As a matter of lab practice, set this time
to equal the loop fill time in the ESI FAST program.
As long as the combined time of sample flush + read
delay is equal to the time required for signal to reach
stability, analytical measurement will be good.
Read Delay Default For Arsenic
24s at 6 rpm (standard ELAN peristaltic pump)
24s at 3rpm (ESI micro-peristaltic pump)
Default For 15 Element Panel
30s at 6 rpm (standard ELAN peristaltic pump)
30s at 3rpm (ESI micro-peristaltic pump)
Can be optimized as needed to reach signal stability
before beginning analysis. As a matter of lab
practice, set this time equal to the total time required
for the signal to reach stability minus the loop fill
time. As long as the combined time of sample flush
+ read delay is equal to the time required for signal
to reach stability, analytical measurement will be
good.

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Table 1. Instrument and Method Parameters. Parameters are the same for arsenic
and the 15 element panel unless otherwise noted.
Wash Default For Arsenic
20s at 6 rpm (standard ELAN peristaltic pump)
20s at 3rpm (ESI micro-peristaltic pump)
Can be optimized to allow for changes in FAST loop
rinsing (must be greater than total time of steps in
FAST program after the initial “on rinse” command).
Default For 15 Element Panel
50s at 6 rpm (standard ELAN peristaltic pump)
50s at 3 rpm (default for entire panel)
Autosampler Locations of Blanks For Arsenic
and Standards For calibration curve (points to urine blank)
CDC_UMP3_DLS3018A_Urine Arsenic_urblk.mth
Urine Blank and Calibration Stds 1 – 5 in
autosampler positions 102 – 107 by default, but can
be customized.
For QC & patient sample analysis (points to aqueous
blank)
CDC_UMP3_DLS3018A_Urine Arsenic_aqblk.mth
Aqueous Blank in autosampler position 119, but can
be customized.
For 15 Element Panel
For calibration curve (points to urine blank)
CDC_UMP3_DLS3018_15 elem_urblk.mth
Urine Blank and Calibration Stds 1 – 5 in
autosampler positions 101 – 106, but can be
customized.
For QC & patient sample analysis (points to aqueous
blank)
CDC_UMP3_DLS3018_15 elem_aqblk.mth
Aqueous Blank in autosampler position 119, but can
be customized.
FAST Parameters: See Figures 4a through 4g in Appendix B for details
Configuration File default.sc
(saved at C:\Program Files\ESI\ESI-SC\)
FAST program For Arsenic
CDC_UMP3_DLS3018A_Urine Arsenic_SCFAST.txt
For 15 Element Panel
Urine 15 element_mthUMP3_DLS3018_SCFAST.txt

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Table 1. Instrument and Method Parameters. Parameters are the same for arsenic
and the 15 element panel unless otherwise noted.

Potential Emergency Response Modifications:
Cadmium: Analyze cadmium in standard mode with rhodium as
the internal standard. Set dwell time to 50ms, DRC
gas flow to 0, and RPq to 0.25.
Arsenic: • Use pure argon in place of 10% hydrogen 90%
argon for the DRC gas. A tee can be setup on
the main argon delivery line for the ICP-MS to
provide this argon for the DRC. No modifications
of the DRC gas flow rate necessary.
•

Analyze arsenic along with the 15-element
method to create a 16-element panel. Diluent
and reagents should not include ethanol. Use
revised QC limits for arsenic (will have “_CT” at
the end of the QC pool name).
Non-FAST sample introduction If the FAST sample introduction system is not
system: available on any instruments, the method can still be
implemented, but these changes will need to be
made in the ELAN (and ESI software if present).
•
•
•
•

Sample Flush: Default is ~90s at 10 rpm. Set so
that solution reaches nebulizer.
Read Delay: Default is 20s at 10rpm. Set for
best reproducibility of replicate measured
intensities.
Wash: Default is 120s at 24rpm. Set to prevent
significant carry-over from one sample to the
next.
If using ESI autosampler without FAST, disable
FAST in the ESI software before running
analysis.

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A

Page

Appendix B (continued)
Table 2. Suggested maximum analyte concentrations for base urine.
Analyte
Concentration (µg/L)
Be
0.5
Co
0.25
Mo
30
Sb
0.2
Cs
3
Ba
2
W
0.2
Pt
0.25
Tl
0.2
Pb
0.75
U
0.03
Cd
0.25
Mn
0.1
Sr
80
Sn
3
As
5

73 of 103

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Table 3. Concentrations of Analytes in the Multi-Element Stock Standard from
High Purity Standards.
Stock
Stock
Calibration
Calibration Verification
Analyte
Standard Conc. (mg/L)
Standard Conc. (mg/L)
High Purity Standards
High Purity Standards
Item # SM-2107-029
Item # SM-2107-035
Be
Co
Mo
Sb
Cs
Ba
W
Pt
TI
Pb
U
As
Cd
Sr
Sn
Mn
A
C

200 A
300 A
1800 B
200 A
1000 C
300 B
200 A
700D
50 A
500 A
40 B
6000 A
200A
8000 D
600 A
200 A

10
7.5
300
8
20
20
6
2.5
4
10
0.5
200
8
300
30
10

Solution A: HNO 3 (10%), HF (0.5%)
Solution C: HCl (1%)

B
D

Solution B: HCl (10%), trace HNO 3
Solution D: HCl (5%)

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Appendix B (continued)
Table 4. Preparation of Multi-element Intermediate Working Standards
Standard #
1
2
3
4
5
Volume of
500
200
100
100
100
Flask (mL)
Volume
Spike of
0.050
0.060
0.100
0.300
1.00
Int. Stock
Std. (mL)
Concentrations (ug/L) ‡
‡
Be*
1 (0.1)
3 (0.3) ‡
10 (1.0) ‡
30 (3.0) ‡
100 (10.0) ‡
Co*
0.75 (0.075) ‡ 2.25 (0.225) ‡ 7.5 (0.75) ‡ 22.5 (2.25) ‡
75 (7.5) ‡
‡
‡
‡
‡
Mo*
30 (3.0)
90 (9.0)
300 (30)
900 (90)
3000 (300) ‡
‡
‡
‡
‡
Sb*
0.8 (0.08)
2.4 (0.24)
8 (0.8)
24 (2.4)
80 (8.0) ‡
Cs*
2 (0.2) ‡
6 (0.6) ‡
20 (2.0) ‡
60 (6.0) ‡
200 (20) ‡
‡
‡
‡
‡
Ba*
2 (0.2)
6 (0.6)
20 (2.0)
60 (6.0)
200 (20) ‡
†
‡
‡
‡
‡
W
0.6 (0.06)
1.8 (0.18)
6 (6.0)
18 (1.8)
60 (6.0) ‡
†
‡
‡
‡
‡
Pt
0.25 (0.025)
0.75 (0.075)
2.5 (0.25)
7.5 (0.75)
25 (2.5) ‡
TI†
0.4 (0.04) ‡
1.2 (0.12) ‡
4 (0.4) ‡
12 (1.2) ‡
40 (4.0) ‡
†
‡
‡
‡
‡
Pb
1 (0.1)
3 (0.3)
10 (1.0)
30 (3.0)
100 (10) ‡
†
‡
‡
‡
‡
U
0.05 (0.005)
0.15 (0.015)
0.5 (0.05)
1.5 (0.15)
5 (0.5) ‡
Cd*
0.8 (0.08) ‡
2.4 (0.24) ‡
8 (0.8)‡
24 (2.4) ‡
80 (8.0) ‡
‡
‡
‡
‡
Sr
30 (3.0)
90 (9.0)
300 (30)
900 (90)
3000 (300)‡
‡
‡
‡
‡
Sn
3 (0.3)
9 (0.9)
30 (3.0)
90 (9.0)
300 (30)‡
‡
‡
‡
‡
Mn*
1 (0.1)
3 (0.3)
10 (1.0)
30 (3.0)
100 (10) ‡
As¥
20 (2.0) ‡
60 (6.0) ‡
200 (20) ‡
600 (60) ‡
2000 (200) ‡
* Rh-103 used as internal standard
†
Ir-193 used as internal standard
¥
Ga-71 used as internal standard
‡
A further 1:10 dilution occurs when added to base urine. Enter concentrations in parentheses
into the ELAN software (method window, calibration page).

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Appendix B (continued)
Table 5. Acceptable ways to perform two consecutive analytical runs, bracketing
with bench quality control samples.
Setup 1
Setup 2 (typical)
Run #1
Run #1
Calibration Standards
Calibration Standards
Low Bench QC
Low Bench QC
High Bench QC
High Bench QC
patient samples
patient samples
Low Bench QC
Low Bench QC
High Bench QC
High Bench QC
Run #2
Low Bench QC
High Bench QC
patient samples
Low Bench QC
High Bench QC

Run #2
Calibration Standards
Low Bench QC
High Bench QC
patient samples
Low Bench QC
High Bench QC

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Appendix B (continued)
Table 6. A typical SAMPLE/BATCH window.
AS
Sample ID
Measurements Action
Location*
236
DRCstability1
Run sample
236
DRCstability2
Run sample
236
DRCstability3
Run sample
236
DRCstability4
Run sample
Continue DRC stability samples . . .
236
DRCstability9
Run sample
236
DRCstability10£ Run sample
101
UrBlkChk1
Run blank, standards, and
sample **
113
UrBlkChk2
Run sample
120
Aq Blk Check
Run blank and sample ¥
130
L Bench QC
Run sample
160
H Bench QC
Run sample
301
Sample 1
Run sample
302
Sample 2
Run sample
303
Sample 3
Run sample
129
L Bench QC
Run sample
159
H Bench QC
Run sample

Method
....15elem_urblk.mth
....15elem_urblk.mth
....15elem_urblk.mth
....15elem_urblk.mth
....15elem_urblk.mth
....15elem_urblk.mth
....15elem_urblk.mth
....15elem_urblk.mth
....15elem_aqblk.mth
....15elem_aqblk.mth
....15elem_aqblk.mth
....15elem_aqblk.mth
....15elem_aqblk.mth
....15elem_aqblk.mth
....15elem_aqblk.mth
....15elem_aqblk.mth

* The exact autosampler positions of QCs and patient samples do not have to be those shown
above, but the order in which these are run should be as shown above.
** When executing this row, the ELAN will first analyze the urine blank at AS position 114, then
standards 1-5 at autosampler positions 102-106, then the “UrBlkChk1” sample at A/S position
100. The sampling information about AS positions 102-106 are stored in the “urblk” method file
and can be customized.
¥ When executing this row, the ELAN will first analyze the aqueous blank at AS position 119,
then the “Aq Blk Check” at AS position 120. The sampling information about AS positions 119 is
stored in the “urblk” method file and can be customized.
£ A larger number of DRC stability samples will need to be analyzed to make this stability period
1-1.5 hrs when measuring only arsenic (~55 measurements = 1hour).

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Appendix B (continued)
Table 7. Preparation of samples, working standards, and QC materials for
analysis
Total volume of prepared sample may be changed, from what is presented here.
However, volumes for each component should be adjusted proportionally.
AQ Intermediate
Patient or
Water
Base
Diluent
Working
QC urine
Dilution ID
(µL)
Urine (µL)
(µL)
Standard (µL)
sample (µL)
AQ Blank
1000
9000 *
Urine Blank and
100
900
9000 *
UrBlkChk
Working
Calibration
900
100
9000 *
Standards
Patient Urine or
500
4500
Urine-Based QC
Patient Urine
500
500
9000 *
2x Dilution H
Patient Urine
900
100
9000*
10x Dilution H
* 9000 µL diluent is best dispensed from the Digiflex™ as 2 4500-µL portions (i.e.- When
preparing a Working Calibration Standard dilution, dispense 4500 µL diluent + 100 µL water in
one cycle of Digiflex™, then 4500 µL diluent + 900 µL base urine in the next cycle of the
Digiflex™ to prepare a 10 mL total volume dilution.)
H
Extra dilution is performed on urine samples whose concentration is greater than the
concentrations listed in Table 8 in the Appendix (linearity of the method has been documented
up to these concentrations). Any extra level of dilution can be prepared as long as the 9:10 ratio
of diluent to total dilution volume is maintained. Use of the lowest possible dilution level is
preferred because matrix differences may lead to different observed concentration results as the
sample dilution becomes greater (i.e. 2x dilution is preferred over 10x if 2x is sufficient to dilute
analyte into the documented linearity range).

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Appendix B (continued)
Table 8. Range of Reporting and Calibration Verification Concentrations
Standard #
CV-1
CV-2
CV-3
Volume of Flask
100
100
100
(mL)
Volume Spike of Int. Sol A
0.150
0.250
0.500
Stock Std. (mL)
Sol B
0.100
0.500
1.000
Sol C
0.025
0.125
0.250
Sol D
0.050
0.100
0.200
Analyte
Concentration (ug/L)
Be*
300 (30) ‡
500 (50) ‡
1,000 (100) ‡
Co*
450 (45) ‡
750 (75) ‡
1,500 (150) ‡
‡
‡
Mo*
1,800 (180)
9,000 (900)
18,000 (1,800) ‡
‡
‡
Sb*
300 (30)
500 (50)
1,000 (100) ‡
Cs*
250 (25) ‡
1,250 (125) ‡
2,500 (250) ‡
‡
‡
Ba*
300 (30)
1,500 (150)
3,000 (300) ‡
†
‡
‡
W
300 (30)
500 (50)
1,000 (100) ‡
Pt†
350 (35) ‡
700 (70) ‡
1,400 (140) ‡
†
‡
‡
Tl
75 (7.5)
125 (12.5)
250 (25) ‡
†
‡
‡
Pb
750 (75)
1,250 (125)
2,500 (250) ‡
†
‡
‡
U
40 (4)
200 (20)
400 (40) ‡
Cd*
300 (30) ‡
500 (50) ‡
1,000 (100) ‡
‡
‡
Mn
300 (30)
500 (50)
1,000 (100) ‡
‡
‡
Sr
4,000(400)
8,000 (800)
16,000 (1600) ‡
Sn
900 (90) ‡
1,500 (150) ‡
3000 (300) ‡
¥
‡
‡
As
9,000 (900)
15,000 (1,500)
30,000 (3,000) ‡
* Rh-103 used as internal standard
†
Ir-193 used as internal standard
¥
Ga-71 used as internal standard
‡
A further 1:10 dilution occurs when added to base urine.

* If observed results are not within 10% of target, investigate the problem
with the involvement of the lab supervisor.

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Appendix B (continued)
Table 9. Boundary Concentrations for Urine Concentrations (µ/L).
1st Upper
Boundary
(“1UB”) *
0.2
2.83
293.5
0.8
16.5
17.1
1.38
0.2
0.62
7.8
0.277
2.54
4
400
25
100

Analyte
Be
Co
Mo
Sb
Cs
Ba
W
Pt
TI
Pb
U
Cd
Mn
Sr
Sn
As
st

2nd Upper
Boundary
(“2UB”) **
0.4
5.66
587
1.6
33
34.2
2.76
0.4
1.24
15.6
0.554
5.08
8
800
50
200

Range
Maximum
(“Lim Rep Delta”) †
0.3
0.3
4.0
0.2
0.5
0.4
0.2
0.2
0.2
0.3
0.03
0.3
0.2
3
0.5
10

* Typically, the 1 upper boundary (1UB) is the 99 percentile of non-weighted, non-creatinine
corrected concentration results from the NHANES 1999-2000 subset groups. Concentrations
observed greater than the “first upper boundary” (defined in the laboratory database as the
“1UB”) should be confirmed by repeat analysis of a new sample preparation. The concentration
assigned to the 1UB for an element is determined by study protocol but default concentrations
are listed in this table. Report the original result, as long as the confirmation is within 10% of the
original. Continue repeat analysis until a concentration can be confirmed.
nd

At the discretion of the
** Typically the 2 upper boundary (2UB) is set to 2x the 1UB.
supervisor, the 1UB may vary per study according to the concerns of the study. Regardless of
the study, report patient results confirmed to be greater than the 2UB to the QC reviewer as an
“elevated result”.
† Range maximum is the range of the three replicate readings for a single sample analysis. This
value is also called the “Lim RepDelta” in the database which handles data for the Inorganic and
Radiation Analytical Toxicology Branch. If the range of replicate readings is greater than the
range maximum, and represents greater than a 10% relative standard deviation for the
measurement, do not use the measurement for reporting.

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Appendix B (continued)
Table 10. Reference Ranges for Urine Concentrations (from the Third National Report
on Exposure to Environmental Chemicals [14] ). All results in µg/L.
Survey
Geometric
Analyte
50th
75th
90th
95th
N
Years
Mean
Be
99-00
≤ 0.13 *
≤ 0.13
≤ 0.13
≤ 0.13
≤ 0.13
2465
01-02
≤ 0.13
≤ 0.13
≤ 0.13
≤ 0.13
≤ 0.13
2690
Co
99-00
0.375
0.400
0.630
0.940
1.32
2465
01-02
0.379
0.410
0.610
0.930
1.27
2690
Mo
99-00
45.9
50.7
84.9
134
178
2257
01-02
45.0
52.4
83.3
124
165
2690
Sb
99-00
0.132
0.130
0.210
0.330
0.420
2276
01-02
0.134
0.130
0.180
0.260
0.340
2690
Cs
99-00
4.35
4.80
7.10
9.60
11.4
2464
01-02
4.81
5.49
7.91
10.4
12.6
2690
Ba
99-00
1.50
1.50
3.00
5.40
6.80
2180
01-02
1.52
1.63
3.12
5.22
7.48
2690
W
99-00
0.093
0.090
0.180
0.320
0.500
2338
01-02
0.082
0.060
0.150
0.300
0.450
2652
Pt
99-00
≤ 0.04 **
≤ 0.04
≤ 0.04
≤ 0.04
≤ 0.04
2465
01-02
≤ 0.04
≤ 0.04
≤ 0.04
≤ 0.04
≤ 0.04
2690
TI
99-00
0.176
0.200
0.280
0.400
0.450
2413
01-02
0.165
0.180
0.270
0.360
0.440
2653
Pb
99-00
0.766
0.800
1.30
2.10
2.90
2465
01-02
0.677
0.600
1.20
2.00
2.60
2690
U
99-00
0.008
0.007
0.013
0.026
0.046
2464
01-02
0.009
0.008
0.014
0.029
0.046
2690
Cd
99-00
0.193
0.232
0.475
0.858
1.20
2257
01-02
0.210
0.229
0.458
0.839
1.20
2690
As †
See Table 11 in Appendix B for urine arsenic reference values.
* Results were lower than the method detection limit of 0.13 ug/L.
** Results were lower than the method detection limit of 0.04 ug/L.
†
Urine As was not included in the Third National Report on Exposure to
Environmental Chemicals.

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Appendix B (continued)
Table 11. References to Total Urine Arsenic Concentrations
Reference
Group Type Sampled
Concentration (µg/L)
Normal
Stokinger, 1981 [15]
<100
Fowler, 1977 [16]
15
1 – 80
Iffland, 1994 [17]
(Generally < 10)
Elevated
After seafood consumption
300

Iffland, 1994 [17]

Gerhardsson et al., 1996
[18]

200

Copper smelter workers

5 - 952
vs. 5 - 365

Wood treatment workers
vs. comparison group (11)

25.9 – 667

Seafood-preferring population

74.1
378.1

Low-Inhalation exposure
High-Inhalation exposure (12)

50 – 100

High intake of seafood or
increased exposure of inorganic
arsenic from food or air

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Appendix B (continued)
Table 12.
Analyte

Reference to urine Mn, Sn, or Sr concentrations
Reference

Normal (i.e. non-exposed)
Mn
Health Canada, 2010[19]
Mn
Heitland et al., 2006[20]
Mn
Heitland et al., 2006[20]
Mn
Paschal et al., 1998[21]
Mn
ASTDR[22]
Sr
Heitland et al., 2006[20]
Sr
Heitland et al., 2006[20]
Sr
Usuda et al., 2006
Sn
Heitland et al., 2006[20]
Sn
Heitland et al., 2006[20]
Sn
Paschal et al., 1998[21]
Elevated (exposed)

Concentration (ug/L)

Group Type
Sampled

0.15
0.1
0.087
1.19
1 to 8
154
166
143.9¥
1.2
8.6
6.29

General population
German children
German adults
NHANES III
General population
German children
German adults
Japanese adults
German children
German adults
NHANES III

Moreno et al., 2010[23]

5.2

Gil et al., 2011[24]

0.43 +/- 4,00

Wang et al., 2011[25]

3.15 +/- 3.45

Moreno et al., 2010[23]

49.2

Juliao et al., 2007[26]

0.45 +/- 0.93

Children living in a
mine tailings zone
in Mexico
Iron and steel
industry workers in
Spain
e-waste dismantling
workers in China
Children living in a
mine tailings zone
in Mexico
Workers in a
niobium mine in
Brazil

concentration is a geometric mean and measurements were made via ICP-AES

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Appendix B (continued): Figures
Figure 1a. Configuration of tubing and devices for liquid handling using FAST
sample introduction.
Below shows the correct connections to the 6-port FAST valve. The two diagrams show
the differences in liquid flow directions when the valve changes from “Load” to “Inject”
This change is internal to the valve. The shift of the valve cannot be seen, but it can be
heard, and felt (with hand on the valve). The light indicators on the actuator body also
indicate the valve position.

The connections to the valve are color-coded (see section 7.a.2).
Enable the FAST program in the ESI software before running the method, but
optimizations can be done in either FAST or non-FAST mode.

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A

Page

85 of 103

Optimize
Per Instrument
(DRC mode only)

Appendix B (continued).
Figure 2a. ELAN ICP-DC-MS Method Screen Shots (timing page, 15 element
panel).

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A

Page

86 of 103

Appendix B (continued).
Figure 2b. ELAN ICP-DC-MS Method Screen Shots (processing page, 15 element
panel).

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A

Page

87 of 103

Appendix B (continued).
Figure 2c. ELAN ICP-DC-MS Method Screen Shots (equation page, 15 element
panel).

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A

Page

88 of 103

Appendix B (continued).
Figure 2d. ELAN ICP-DC-MS Method Screen Shots (calibration page, 15 element
panel).

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A

Page

89 of 103

Appendix B (continued).
Figure 2e. ELAN ICP-DC-MS Method Screen Shots (sampling page, 15 element
panel).

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A

Page

90 of 103

Appendix B (continued).
Figure 2f. ELAN ICP-DC-MS Method Screen Shots (report page, 15 element
panel).

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A

Page

91 of 103

Appendix B (continued).
Figure 3a. ELAN ICP-DC-MS Method Screen Shots (timing page, arsenic).

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A

Page

92 of 103

Appendix B(continued).
Figure 3b. ELAN ICP-DC-MS Method Screen Shots (processing page, arsenic).

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A

Page

93 of 103

Appendix B (continued).
Figure 3c. ELAN ICP-DC-MS Method Screen Shots (equation page, arsenic).

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A

Page

94 of 103

Appendix B (continued).
Figure 3d. ELAN ICP-DC-MS Method Screen Shots (calibration page, arsenic).

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A

Page

95 of 103

Pump speed shown is for ESI microperistaltic pump.
ELAN standard pump speed is 6 rpm.

Appendix B (continued).
Figure 3e. ELAN ICP-DC-MS Method Screen Shots (sampling page, arsenic).

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A

Page

96 of 103

Appendix B (continued).
Figure 3f. ELAN ICP-DC-MS Method Screen Shots (report page, arsenic).

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A

Page

97 of 103

Appendix B (continued).
Figure 4a. ESI SC4 Autosampler Screen Shots used (Main page). Additional flush
times and “Max Rinse Time” are default, but can be optimized for best reduction of
elemental carry-over between samples. Tray types can be changed to allow for
different volumes of diluted sample digests. ‘FAST control’ must be enabled before
start of method, but does not need to be used in instrument optimization (pre-analysis)
steps. Rinse and additional flush times for eliminating carry-over from one sample to
the next while using the minimum amount of rinse solution.
A rinse time of -1 causes the rinse station to be skipped.
A rinse time of 0 causes the probe to only dip into the station, but spends no time there.
Additional flush times can be optimized to keep the rinse station full while not using too
much rinse solution. The inner diameter size of the tubing providing the rinse solution to
the rinse station determines how quickly the station will fill. Various sizes are available
for purchase or can be made in the laboratory.

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A

Page

98 of 103

Appendix B (continued).
Figure 4b. ESI SC4 Autosampler Screen Shots used (“Configure” page). “High
Speed” option is to only be used for ‘High Speed’ models of the SC4 (look for “HS” in
serial number). Speeds and accel / decel values can be optimized per analyst
preference and to minimize droplet splatter off of probe.

Figure 4c . ESI SC4 Autosampler Screen Shots used (“Communication” page).
Communication ports will differ depending on available ports on instrument control
computer.

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A

Page

99 of 103

Appendix B (continued).
Figure 4d. ESI SC4 Autosampler Screen Shots (“FAST” page) *used for Arsenic
only*. Timer A can be optimized to achieve proper filling of loop with diluted sample
digestate. Timers B, C, D, E, and F control rinsing the loop after analysis and can be
optimized for eliminating carry-over from one sample to the next while using the
minimum amount of rinse solution. File should be saved with the name “Urine
Arsenic_methITU001B_HPS2107-003_SCFAST.txt”. It can be found in the directory
C:\Program Files\ESI\ESI-SC\.
Manually clicking the “Load” button prior to starting analysis will ensure the position of
the actuator is always the same at the beginning of the analysis.
Manually clicking the “Vacuum On” button prior to starting the analysis will help initial
sample uptake to be consistent (the vacuum pump may be slow to start for the first
sample if this is not done, possibly resulting in loop filling inconsistencies).

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A

Page

100 of 103

Appendix B (continued).
Figure 4e. ESI SC4 Autosampler Screen Shots (5x12 Rack Setup window).
Settings are approximate. To be sure the loop is filled, the probe should go down close
to the bottom of the cup, but not touch. Optimize retraction speed for least droplet
splatter.

Figure 4f. ESI SC4 Autosampler Screen Shots (50mL Tube Rack Setup window).
Settings are approximate. To be sure the loop is filled, the probe should go down close
to the bottom of the cup, but not touch. Optimize retraction speed for least droplet
splatter.

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A

Page

101 of 103

Appendix B (continued).
Figure 4g. ESI SC4 Autosampler Screen Shots (Rinse Station Rack Setup
Window). Settings are approximate. Optimize down height for best probe cleaning,
and retraction speed for least droplet splatter.

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A

Page

102 of 103

References
1.

Thomas, R., Practical Guide to ICP-MS (Practical Spectroscopy). 2003, New
York, NY: Marcel Dekker 336.
2.
Tanner, S.D., Baranov, Vladimir I, Theory, Design, and Operation of a Dynamic
Reaction Cell for ICP-MS. Atomic Spectroscopy, 1999. 20(2): p. 45-52.
3.
Tanner, S.D., V.I. Baranov, and D.R. Bandura, Reaction cells and collision cells
for ICP-MS: a tutorial review. Spectrochimica Acta Part B-Atomic Spectroscopy,
2002. 57(9): p. 1361-1452.
4.
PerkinElmer SCIEX Instruments, ELAN DRC II Hardware Guide. 2001, Canada.
5.
Mulligan, K.J., T.M. Davidson, and J.A. Caruso, Feasibility Of The Direct Analysis
Of Urine By Inductively Coupled Argon Plasma Mass-Spectrometry For
Biological Monitoring Of Exposure To Metals. Journal Of Analytical Atomic
Spectrometry, 1990. 5(4): p. 301-306.
6.
Jarrett, J.M., Total Urine Arsenic Biomonitoring Using Inductively Coupled
Plasma Mass Spectrometry with a Dynamic Reaction Cell. 2005, Centers for
Disease Control and Prevention.
7.
Jarrett, J.M., Elimination of Molybdenum Oxide Interference In Urine Cadmium
Analysis Using Inductively Coupled Plasma Reaction Cell Mass Spectrometry.
2004, Centers for Disease Control and Prevention.
8.
Larsen, E.H. and S. Sturup, Carbon-enhanced Inductively Coupled Plasma Mass
Spectrometric Detection of Arsenic and Selenium and Its Application to Arsenic
Speciation. Journal Of Analytical Atomic Spectrometry, 1994. 9: p. 1101-1105.
9.
Amarasiriwardena, C.J., et al., Determination of the total arsenic concentration in
human urine by inductively coupled plasma mass spectrometry: a comparison of
the accuracy of three analytical methods. Analyst, 1998. 123(3): p. 441-445.
10.
Office of Health and Safety in the Division of Laboratory Sciences, Policies and
Procedures Manual. 2002, Division of Laboratory Sciences (DLS), National
Center for Environmental Health, Centers for Disease Control and Prevention,
Public Health Service, Department of Health and Human ServicesCenters for
Disease Control and Prevention, .
11.
Centers for Disease Control and Prevention (CDC) Radiation Safety Committee,
CDC/ATSDR Occupational Health and Safety Manual (Radiation Safety chapter).
Centers for Disease Control and Prevention, Public Health Service, Department
of Health and Human ServicesCenters for Disease Control and Prevention.
12.
Heitland, P. and H.D. Koster, Biomonitoring of 37 trace elements in blood
samples from inhabitants of northern Germany by ICP-MS. Journal of Trace
Elements in Medicine and Biology, 2006. 20(4): p. 253-262.
13.
U.S. Nuclear Regulatory Commission, Regulatory guide 8.22 (revision 1).
Bioassay at uranium mills. 1988: Atlanta, GA.
14.
Centers for Disease Control and Prevention, Third National Report on Human
Exposure to Environmental Chemicals, http://www.cdc.gov/exposurereport. 2005.
15.
Stokinger, H.E., The metals, in Patty’s industrial hygiene and toxicology
G. Clayton and F. Clayton, Editors. 1981, John Wiley and Sons: New York. p. 14932060.
16.
Fowler, B.A., in Toxicology of trace elements
R. Goyer and M. Mehlman, Editors. 1977, John Wiley and Sons: New York. p. p. 79.

Urine Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: 3018 and 3018A
17.

18.

19.
20.
21.
22.

23.

24.

25.

26.

Page

103 of 103

Iffland, R., Arsenic, in Handbook on metals in clinical and analytical chemistry, H.
Seiler, A. Sigel, and H. Sigel, Editors. 1994, Marcel Dekker, Inc.: New York. p.
238-250.
Gerhardsson, L. and S. Skerfving, Concepts on biological markers and
biomonitoring for metal toxicity, in Toxicology of metals, L. Chang, Editor. 1996,
CRC Press: Boca Raton, Florida. p. 98.
Report on Human Biomonitoring of Environmental Chemicals in Canada. 2010,
Health Canada: Ottawa.
Heitland, P. and H.D. Koster, Biomonitoring of 30 trace elements in urine of
children and adults by ICP-MS. Clinica Chimica Acta, 2006. 365(1-2): p. 310-318.
Paschal, D.C., et al., Trace metals in urine of United States residents: Reference
range concentrations. Environmental Research, 1998. 76(1): p. 53-59.
Agency for Toxic Substances and Disease Registry (ATSDR). 2000.
Toxicological profile for Manganese. Atlanta, G.U.S.D.o.H.a.H.S., Public Health
Service. , Toxicological Profile for Manganese, ATSDR, Editor. 2000. p. 15.
Moreno, M.E., et al., Biomonitoring of metal in children living in a mine tailings
zone in Southern Mexico: A pilot study. International Journal of Hygiene and
Environmental Health, 2010. 213(4): p. 252-258.
Gil, F., et al., Biomonitorization of cadmium, chromium, manganese, nickel and
lead in whole blood, urine, axillary hair and saliva in an occupationally exposed
population. Science of the Total Environment, 2011. 409(6): p. 1172-1180.
Wang, H.M., et al., Urinary heavy metal levels and relevant factors among people
exposed to e-waste dismantling. Environment International, 2011. 37(1): p. 8085.
Juliao, L., et al., Exposure of workers in a mineral processing industry in Brazil.
Radiation Protection Dosimetry, 2007. 125(1-4): p. 513-515.

Division of Laboratory Sciences
Laboratory Protocol
Analytes:

Cadmium, Lead, Manganese, Mercury, and Selenium

Matrix:

Whole Blood

Method:

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS

Method Code:

DLS 3016

Branch:

Inorganic Radionuclides and Analytical Toxicology

Prepared By:

Deanna M. Jones, PhD
author's name

Supervisor:

date

signature

date

Jeffery M. Jarrett, MS
supervisor's name

Branch Chief:

signature

Robert L Jones PhD
Branch Chief

signature and date

Adopted:
date

Updated:
date

Director's Signature Block:
Reviewed:
signature

date

signature

date

signature

date

signature

date

Modifications/Changes: see Procedure Change Log

Procedure Change Log
Procedure: Blood Metals Panel 2 by (BMP2) ICP-DRC-MS
DLS Method Code: 3016

Date

Changes Made

4/1/2011

1UB and 2UB for Mn changed from 15
to 25 ug/L and from 30 to 50 ug/L,
respectively.
Limit Rep Delta for Mn changed from
1.0 to 2.0.
Revised matrix of internal standard
intermediate to 1% v/v HNO3.
Changed BMN 1UB (25 ug/L to 20
ug/L) and 2UB (50ug/L to 35 ug/L).
Supporting references added.
Added comment to CV standard
tables regarding use of gravimetric
preparation.

Rev’d
By
(Initials)
JJ

4/1/2011
7/28/2011
8/9/2011
10/7/2011

Date
Rev’d

Laboratory Procedure Manual
Analytes:

Matrix:
Method:

Cadmium, Lead, Manganese,
Mercury, and Selenium
Whole Blood
Blood Metals Panel 2 (BMP2) ICP-DRC-MS

Method No: DLS 3016
Revised:
As performed by: Inorganic Radionuclides and Toxicology
Division of Laboratory Sciences
National Center for Environmental Health
Contact:

Jeffery M. Jarrett, MS
Phone: 770-488-7906
Fax:
770-488-4097
Email: [email protected]
Dr. Jim Pirkle, MD, PhD, Director
Division of Laboratory Sciences

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 1 of 88
Table of Contents

Cross reference to DLS CLIA and Policy and Procedures ........................................ 4
Index of tables ............................................................................................................... 5
List of Figures ............................................................................................................... 6
1) Clinical Relevance & Summary of Test Principle
a. Clinical Relevance ............................................................................................. .. 7
b. Test Principle ...................................................................................................... 10
2) Limitations of Method; Interfering Substances and Conditions
a. Interferences Addressed by This Method ........................................................... 12
i. Argon dimer (40Ar2+) on 80Se+
ii. Argon Nitride (40Ar15N+), Argon Hydroxide (38Ar16O1H+)
iii. Chlorine Oxide (37Cl18O+), Iron Hydride (54Fe1H+)
3) Procedures for Collecting, Storing, and Handling Specimens; Criteria for
Specimen Rejection
a. Procedures for Collecting, Storing, and Handling Specimens ........................... 13
b. Criteria for Specimen Rejection ......................................................................... 14
c. Transfer or Referral of Specimens; Procedures for Specimen Accountability
and Tracking ....................................................................................................... 14
4) Safety Precautions
a. General Safety.................................................................................................... 14
b. Waste Disposal................................................................................................... 15
5) Instrument & Material Sources
a. Sources for ICP-MS Instrumentation .................................................................. 16
b. Sources for ICP-MS Parts & Consumables ....................................................... 16
c. Sources for ICP-MS Maintenance Equipment & Supplies .................................. 21
d. Sources for General Laboratory Equipment & Consumables ............................. 22
e. Sources for Chemicals, Gases, & Regulators..................................................... 23
6) Preparation of Reagents and Materials
a. Internal Standard Intermediate Mixture .............................................................. 26
b. 20% Triton X-100 intermediate solution ............................................................ 26
c. Diluent ................................................................................................................ 27

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 2 of 88

d. DRC Stability Test Solution ................................................................................ 27
e. Base Blood ......................................................................................................... 28
f. ICP-DRC-MS Rinse Solution .............................................................................. 28
g. Single-element Stock Standards for preparation of intermediate stock calibration
standard ............................................................................................................. 29
h. 3 % (v/v) HCl ...................................................................................................... 29
i. Intermediate Stock Calibration Standard ............................................................ 29
j. Intermediate Working Calibration Standard ........................................................ 31
k. Working Calibration Standards ........................................................................... 31
l.

Single-Element Stock Standards For Preparation of Intermediate Stock
Calibration Verification Standard ........................................................................ 32

m. Intermediate Stock Calibration Verification Standard ......................................... 32
n. Intermediate Working Calibration Verification Standard ..................................... 33
o. Internal Quality Control Materials (“Bench” QC) ................................................. 33
7) Analytical Instrumentation & Parameters
a. Instrumentation & Equipment Setup
i. ICP-DRC-MS ................................................................................................... 36
ii. Sample introduction system setup................................................................... 37
iii. Cones .............................................................................................................. 38
iv. Gases & Regulators setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v. Chiller / Heat Exchanger. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vi. Computer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii. Autosampler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

b. Parameters for Instrument and Method (see Table 1) . . . . . . . . . . . . . . . . . . . 39
8) Method Procedures
a. Quality Control
i. Types of Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ii. Calibration Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

b. Daily Analysis of Samples
i. Preparation of the Analytical Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . .

ii. Preparation of Samples for Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 3 of 88

iii. Specimen Storage and Handling During Testing . . . . . . . . . . . . . . . . . . . .

iv. Starting the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v. Monitoring the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vi. Records of Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii. Transfer of Results to the Laboratory Database . . . . . . . . . . . . . . . . . . . . .

viii. Analyst Evaluation of Run Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix. Submitting Final Work for Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

x. Overnight operation (or Any Use of Autostop) . . . . . . . . . . . . . . . . . . . . . . . 50
c. Equipment Maintenance
i. ICP-MS Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
ii. Data Backup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
9) Interpretation of the Results
a. Reportable Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
b. Reference Ranges (Normal Values) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
c. Action Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
10) Method Calculations
a. Method Limit of Detection (LOD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
b. Method Limit of Quantitation (LOQ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
c. QC Limits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
11) Alternate Methods for Performing Test and Storing Specimens If Test System
Fails . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Appendix A (Critical Parameter Test Results)
Critical Parameter Test #1……………………………………………………………………53
Critical Parameter Test #2……………………………………………………..……………..54
Critical Parameter Test #3……………………………………………………………………55
Critical Parameter Test #4……………………………………………………………………57
Critical Parameter Test #5……………………………………………………………………59
Appendix B (Tables referenced in the method) ……………………………..………...60
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

1.
2.
3.
4.
5.

6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.

Page 4 of 88

Cross reference to DLS CLIA and Policy and Procedures policy
Summary of Test Principle and Clinical Relevance
1) a. b.
Safety Precautions
4) a.b.c.
Computerization; Data System Management
8) b.vi vii ix
Specimen Collection, Storage, and Handling Procedures; Criteria for Specimen
Rejection
3) a.b.
Procedures for Microscopic Examinations; Criteria for Rejection of Inadequately
Prepared Slides
- As no microscope is used in this process there are no procedures for
microscopic examinations and therefore no slide rejection criteria.
Preparation of Reagents, Calibrators (Standards), Controls, and All Other
Materials; Equipment and Instrumentation
5) a. i ii iii b. 6) a. b. c. d. e. 7) a. b. c. d. 8) c. i ii
Calibration and Calibration Verification Procedures
8) ii
Procedure Operating Instructions; Calculations; Interpretation of Results
8) b. i ii iv v x
Reportable Range of Results
9) a.
Quality Control (QC) Procedures
8) a. i
Remedial Action If Calibration or QC Systems Fail to Meet Acceptable Criteria
8) ii 1, ii 2, e.
Limitations of Method; Interfering Substances and Conditions
2) a. b
Reference Ranges (Normal Values)
9) b.
Critical Call Results ("Panic Values")
9) c.
Specimen Storage and Handling During Testing
8) b. iii
Alternate Methods for Performing Test or Storing Specimens If Test System Fails
11)
Test Result Reporting System; Protocol for Reporting Critical Calls (If Applicable)
9) c.
Transfer or Referral of Specimens; Procedures for Specimen Accountability and
Tracking
3) c.
References

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 5 of 88
List of Tables

Table 1. Instrument and Method Parameters . . . . . . . . . . . . . . . . . . . . . . . . … 60 - 62
Table 2. Suggested maximum analyte concentrations for base blood and Quality
control material. . . . . . . . . . . ………………………………………………….…62
Table 3. Preparation of Intermediate Stock Calibration Solution from NIST primary
standards. . . . . . . . . . . . . . . . . . . . . . . . . . . …………………………………..62
Table 4. Preparation of Intermediate Stock Calibration Solution from single element
stock calibrator solutions without Pb……………………………………………63
Table 5. Preparation of Intermediate Working Standards……………………………….63
Table 6. Preparation of samples, working standards, and QC materials for analysis..64
Table 7. Preparation and Final Concentrations of Intermediate Stock Calibration
Verification Standards. . . . . . . . ……………………………………………......65
Table 8. Preparation and Final Concentrations of Intermediate Working Calibrator
Verification Standards. . . . . . . . ………………………………………………...65
Table 9. Acceptable ways to perform two consecutive analytical runs, bracketing with
bench quality control samples…………………………………………………...67
Table 10. A typical SAMPLE/BATCH window. . . . . . . . . . . . ………………………….68
Table 11. Boundary Concentrations for Whole Blood Concentrations (g/L)…………..68
Table 12. Reference Ranges for Blood Concentrations………………………………….69

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 6 of 88
List of Figures

Figure 1.

Figure 2.

ELAN ICP-DRC-MS Method Screen Shots
a. Timing Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

b. Processing Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c. Equations Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

d. Calibration Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

e. Sampling Page (Aq blank) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

f. Sampling Page (BldBlank)………………………………………………

g. Report Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ESI SC4 Autosampler Screen Shots
a.

Main Page…………………………………………………………………. 78

Configure Page..………………………………………………………….. 79

Communication Page……………………………………………………. 80

FAST Page……………………………………………………………… 81

5x12 Rack Setup window…………………………………………………82

50 mL Tube Rack Setup window………………………………………….83

Rinse Station Rack Setup Window………………………………………84

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 7 of 88

1) Clinical Relevance & Summary of Test Principle
a. Clinical Relevance:
Metals ions affect human health in various ways. Some metals (i.e. lead,
cadmium, and mercury) show only deleterious effects on human health. Some
(i.e. selenium and manganese) play an essential role in the human biological
system if within certain concentration ranges, while negative health implications
are observed when concentrations in biological systems are in deficit or excess.
Determination of a person’s level of environmental exposure to chemicals
through direct measurement of the substances or their metabolites in human
specimens such as blood is called biomonitoring. Biomonitoring reduces the
uncertainty of determining levels of exposure over making these determinations
through calculations of estimated dose based on analysis of environmental
samples and assumptions about exposure pathways[1].
Biomonitoring
measurements are the most health-relevant assessments of exposure because
they indicate the amount of the chemical that actually gets into people from all
environmental sources (e.g., air, soil, water, dust, or food) combined, rather than
the amount that may get into them. The laboratory method described here is a
multi-element technique for monitoring the concentrations of cadmium (Cd), lead
(Pb), manganese (Mn), mercury (Hg), and selenium (Se) in whole human blood
for the purpose of biomonitoring.
There is no known biological role of mercury in the human body. The main
sources of mercury intake in humans are fish, dental amalgams, and
occupational exposures[2]. The main organs affected by mercury are the brain
and the kidneys. Exposure of childbearing-aged women is of particular concern
because of the potential adverse neurologic effects of Hg in fetuses. The health
effects of mercury are diverse and depend on the form of mercury encountered
and the severity and length of exposure. The general population may be
exposed to three forms of mercury:
elemental, inorganic, and organic
(predominantly methyl). However, this method tests only for the total amount of
mercury in the blood without regard to chemical form. In the general population,
total blood mercury is due mostly to the dietary intake of organic forms which are
formed through microbial action from inorganic mercury that has deposited in
aquatic environments and bioaccumulated through the food chain (especially into
large predatory fish)[3]. Exposure to inorganic or elemental mercury (e.g. dental
amalgams or occupational exposures) is particularly reflected in urine excretion
rather than blood. Psychic and emotional disturbances are the initial signs of
chronic intoxication by elemental mercury vapors or salts.
Parasthesia,
neuralgias, renal disease, digestive disturbances, and ocular lesions may
develop[4]. Massive exposure over a longer period of time results in violent
muscular spasms, hallucinations, delirium, and death[5].
Except for
methylmercury exposures, blood is considered useful if samples are taken within
a few days of exposure. This is because most forms of mercury in the blood
decrease by one-half every three days if exposure has been stopped. Thus,

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mercury levels in the blood provide more useful information after recent
exposures than after long-term exposures. Several months after an exposure,
mercury levels in the blood and urine are much lower. Table 12 in Appendix B
lists reference concentrations which have been reported in the literature.
There is no known biological role of lead in the human body. Lead, a naturally
occurring metal, has had many different commercial uses from which a person
can be exposed either in the occupational / manufacturing process or by the
manufactured products such as paint (paint chips, or dust and soil contaminated
from deteriorating paint), solder or pipes (only now in older homes), gasoline
(now outlawed for all but specialized applications), glazes on pottery, hobby uses
(e.g. stained glass), commercial products (e.g. batteries, lead-containing jewelry),
home remedy medicines containing lead compounds and non-Western
cosmetics. Soil may contain lead naturally, or from man-made uses of lead such
as paint (near older homes), gasoline (near roadways), mining, manufacturing,
and disposal. The main target for lead toxicity is the nervous system, both in
adults and children. The developing biological systems of children are most
sensitive to the effects of Pb, where effects are being recognized even at blood
lead levels <10 g/dL[6]. In its initial phase, acute lead poisoning is associated
with anorexia, dyspepsia, and constipation followed by diffuse paroxysmal
abdominal pain. Lead exposure may cause encephalopathy, particularly in
children[7]. The alkyl lead species are highly toxic to the central nervous
system[8]. The primary screening method for lead exposure is blood lead, which
primarily reflects recent exposures (excretory half-life in blood is approximately
30 days)[9]. Lead in blood is primarily (99%) in the red blood cells. Table 12 in
Appendix B lists reference concentrations which have been reported in the
literature.
There is no known biological role of cadmium in the human body. The
predominant commercial use of cadmium is in battery manufacturing. Other uses
include pigment production, coatings and plating, plastic stabilizers, and
nonferrous alloys. Since 2001, U.S. cadmium use has declined in response to
environmental concerns. In the United States, for nonsmokers the primary
source of cadmium exposure is from the food supply. People who regularly
consume shellfish and organ meats will have higher exposures. In general, leafy
vegetables such as lettuce and spinach, potatoes and grains, peanuts,
soybeans, and sunflower seeds contain high levels of cadmium due to
bioaccumulation from the soil. Tobacco leaves accumulate high levels of
cadmium from the soil, and smoking is the primary non-occupational source of
cadmium exposure for smokers. Generally, the critical organ for Cd is the
kidney. Kidney dysfunction is one of the most characteristic signs of exposure to
Cd. Workers in an environment with high exposure levels have developed
proteinuria, renal glucosuria, aminoaciduria, hypercalciuria, phosphaturia, and
polyuria. Chronic obstructive lung disease of varying degrees of severities is
frequently seen in Cd workers. Concentration of cadmium in blood of healthy
unexposed adults are in the range 0.1 – 4 g/L[10]. Newborn babies are

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practically free of Cd[11]. Exposure to high concentration of fumes appearing
from heated cadmium metal or compounds has led to acute poisoning and in
some cases to the death of workers[7]. Principal symptoms reported were
respiratory distress due to chemical pneumonitis and edema. It has been
estimated that 8 hrs exposure to 5 gm Cd/m3 will be lethal[7]. Ingestion of high
amounts of Cd may lead to a rapid onset with severe nausea, vomiting, and
abdominal pain. Cadmium levels in blood, urine, feces, liver, kidney, hair, and
other tissues have been used as biological indicators of exposure to cadmium.
Blood cadmium levels are principally indicative of recent exposure(s) to cadmium
rather than whole-body burdens[12-15]. Urine cadmium levels primarily reflect
total body burden of cadmium, although urine levels do respond somewhat to
recent exposure[16]. Table 12 in Appendix B lists reference concentrations
which have been reported in the literature.
Manganese (Mn) is a trace element essential to humans and is associated with
the formation of connective and bony tissue, growth and reproductive functions
and with carbohydrate and lipid metabolism [17]. Manganese is also a known
neurotoxin but little information exists about levels of manganese that cause
toxicity. Symptoms of manganese toxicity are similar to Parkinson’s Disease and
can also include disorientation, memory impairment, anxiety and compulsive
behavior [18]. There is much concern for the levels of manganese in humans
whom are occupationally exposed to it [19-25]. Recently, there are growing
concerns over exposure due to contamination of drinking water with manganese
[26-28] and as a result of methylcyclopentadienyl mangangese tricarbonyl (MMT)
used as an anti-knocking additive in gasoline[29-35]. Populations suffering from
iron deficiencies may be particularly susceptible to manganese toxicity because
iron deficiency may lead to an accumulation of manganese in the central nervous
system [32]. To fully understand the essentiality and toxicity of manganese,
further investigations are needed regarding the levels of manganese in biological
matrices. Group average levels in blood appear to be related to manganese body
burden, while average urinary excretion levels appear to be most indicative of
recent exposures[36]. On an individual basis the correlation between the level of
workplace exposure and the levels in blood or urine has always been found to be
a reliable predictor of exposure[20, 36-38]. Manganese in blood or urine may be
useful in detecting groups with above-average current exposure, but
measurements of manganese in these body fluids in individuals may only be
related to exposure dose after the exposure has ceased. In addition to individual
variability, another factor that limits the usefulness of measuring manganese in
blood, urine, or feces as a measure of excess manganese exposure is the
relatively rapid rate of manganese clearance from the body. Excess manganese
in blood is rapidly removed by the liver and excreted into the bile, with very little
excretion in urine[39, 40]. Thus, levels of manganese in blood or urine are not
expected to be the most sensitive indicators of exposure[41]. Table 12 in
Appendix B lists reference concentrations which have been reported in the
literature.

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Selenium is an essential element that is required to maintain good health but
both selenium deficiency and excessive levels of selenium are associated with
several disorders[42, 43]. Selenium is a naturally occurring mineral element that
is distributed widely in nature in most rocks and soils. Most processed selenium
is used in the electronics industry, but it is also used: as a nutritional supplement;
in the glass industry; as a component of pigments in plastics, paints, enamels,
inks, and rubber; in the preparation of pharmaceuticals; as a nutritional feed
additive for poultry and livestock; in pesticide formulations; in rubber production;
as an ingredient in antidandruff shampoos; and as a constituent of fungicides.
Radioactive selenium is used in diagnostic medicine. In the body, selenium is
incorporated into proteins to make selenoproteins, which are important
antioxidant enzymes. The antioxidant properties of selenoproteins help prevent
cellular damage from free radicals. Free radicals are natural by-products of
oxygen metabolism that may contribute to the development of chronic diseases
such as cancer and heart disease[43, 44]. Other selenoproteins help regulate
thyroid function and play a role in the immune system[45-48]. Human selenium
deficiency is rare in the U.S. but is seen in other countries where soil
concentration of selenium is low[49]. There is evidence that selenium deficiency
may contribute to development of a form of heart disease, hypothyroidism, and a
weakened immune system[50, 51]. There is also evidence that selenium
deficiency does not usually cause illness by itself. Rather, it can make the body
more susceptible to illnesses caused by other nutritional, biochemical or
infectious stresses[52]. Symptoms of very high exposure to selenium, a
condition called selenosis, include gastrointestinal upsets, hair loss, white blotchy
nails, garlic breath odor, fatigue, irritability, and mild nerve damage[42].
Selenium can be detected in the blood, feces, urine, hair, and nails of exposed
individuals, however, field studies have used primarily blood or urine levels to
indicate the degree of selenium exposure[53]. Table 12 in Appendix B lists
reference concentrations which have been reported in the literature.
The laboratory method presented here can be used to achieve rapid and
accurate quantification of five elements of toxicological and nutritional interest
including cadmium (Cd), lead (Pb), mercury, manganese (Mn) and selenium (Se)
in whole human blood. The method may be used to screen blood when people
are suspected to be acutely exposed to these elements or to evaluate chronic
environmental or other non-occupational exposure.
b. Test Principle:
This method directly measures the Cd, Mn, Hg, Pb, and Se content of whole
blood specimens using mass spectrometry after a simple dilution sample
preparation step.
During the sample dilution step, a small volume of whole blood is extracted from
a larger whole blood patient specimen after the entire specimen is mixed
(vortexed) to create a uniform distribution of cellular components. This mixing
step is important because some metals (e.g. Pb) are known to be associated

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mostly with the red blood cells in the specimen and a uniform distribution of this
cellular material must be produced before a small volume extracted from the
larger specimen will accurately reflect the average metal concentration of all
fractions of the larger specimen. Coagulation is the process in which blood forms
solid clots from its cellular components. If steps are not taken to prevent this
process from occurring, i.e. addition of anti-coagulant reagents such as EDTA in
the blood collection tube prior to blood collection, blood will immediately begin to
form clots once leaving the body and entering the tube. These clots prevent the
uniform distribution of cellular material in the blood specimen even after rigorous
mixing, making a representative sub-sample of the larger specimen unattainable.
It is important that prior to or during sample preparation the analyst identify any
sample having clots or micro-clots (small clots). Consequently, blood samples
containing clots should not be analyzed by this method due to the inhomogeneity
issues and expected results from the sample should be documented as not
reportable.
Dilution of the blood in the sample preparation step prior to analysis is a simple
dilution of 1 part sample + 1 part water + 48 parts diluent. The effects of the
chemicals in the diluent are to release metals bound to red blood cells making
them available for ionization, reduce ionization suppression by the biological
matrix, prevent clogging of the sample introduction system pathways by
undissolved biological solids, and allow introduction of internal standards to be
utilized in the analysis step. Tetramethylammonium hydroxide (TMAH, 0.25%
v/v) and Triton X-100 (0.05%) in the sample diluent solubilizes blood
components. Triton X-100 also helps prevent biological deposits on internal
surfaces of the instrument’s sample introduction system and reduce collection of
air bubbles in sample transport tubing. Ammonium pyrrolidine dithiocarbamate
(APDC) in the sample diluent (0.25%) aids in solubilizing metals released from
the biological matrix. Ethyl alcohol in the sample diluent (1%) aids solubility of
blood components and aids in aerosol generation by reduction of the surface
tension of the solution. The internal standards, rhodium, iridium and tellurium,
are at a constant concentration in all blanks, calibrators, QC, and samples.
Monitoring the instrument signal ratio of a metal to its internal standard allows
correction for instrument noise and drift, and sample-to-sample matrix
differences.
Liquid samples are introduced into the mass spectrometer through the inductively
coupled plasma (ICP) ionization source. The liquid diluted blood sample is
forced through a nebulizer which converts the bulk liquid into small droplets in an
argon aerosol. The smaller droplets from the aerosol are selectively passed
through the spray chamber by a flowing argon stream into the ICP. By coupling
radio-frequency power into flowing argon, plasma is created in which the
predominant species are positive argon ions and electrons and has a
temperature of 6000-8000 K. The small aerosol droplets pass through a region
of the plasma and the thermal energy vaporizes the liquid droplets, atomizes the
molecules of the sample and then ionizes the atoms. The ions, along with the

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argon, enter the mass spectrometer through an interface that separates the ICP
(at atmospheric pressure, ~760 torr) from the mass spectrometer (operating at a
pressure of 10-5 torr). The ions first pass through a focusing region, then the
dynamic reaction cell (DRC), the quadrupole mass filter, and finally are
selectively counted in rapid sequence at the detector allowing individual isotopes
of an element to be determined.
Generally, the DRC operates in one of two modes. In ‘vented’ (or ‘standard’)
mode the cell is not pressurized and ions pass through the cell to the quadrupole
mass filter unaffected. In ‘DRC’ mode, the cell is pressurized with a gas for the
purpose of causing collisions and/or reactions between the fill gas and the
incoming ions. In general, collisions or reactions with the incoming ions
selectively occur to either eliminate an interfering ion, change the ion of interest
to a new mass, which is free from interference, or collisions between ions in the
beam and the DRC gas can focus the ion beam to the middle of the cell and
increase the ion signal. In this method, the instrument is operated in DRC mode
when analyzing for manganese, mercury and selenium. For selenium, the DRC
is pressurized with methane gas (CH4, 99.999%) which reduces the signal from
40
Ar2+ while allowing the 80Se+ ions to pass relatively unaffected through the DRC
on toward the analytical quadrupole and detector. Manganese and mercury are
both measured when the DRC is pressurized with oxygen gas (O2, 99.999%).
They are analyzed at the same flow rate of oxygen to the DRC cell to avoid
lengthening analysis time due to pause delays that would be necessary if
different gas flows were used for the two analytes. The oxygen reduces the ion
signal from several interfering ions (37Cl18O+, 40Ar15N+, 38Ar16O1H+, 54Fe1H+) while
allowing the Mn+ ion stream to pass relatively unaffected through the DRC on
toward the analytical quadrupole and detector. In the case of mercury, collisional
focusing of the mercury ions occurs, increasing the observed mercury signal at
the detector by approximately a factor of two (2x).
Once ions pass through the DRC cell and electrically selected for passage
through the analytical quadrupole, electrical signals resulting from the ions
striking the discrete dynode detector are processed into digital information that is
used to indicate the intensity of the ions. The intensity of ions detected while
aspirating an unknown sample is correlated to an elemental concentration
through comparison of the analyte:internal standard signal ratio with that
obtained when aspirating calibration standards. This method was originally
based on the method by Lutz et al.[54] The DRC portions of the method are
based on work published by Tanner et al. [55, 56].
2) Limitations of Method; Interfering Substances and Conditions
a. Interferences Addressed by This Method
i. Reduction of argon dimer (40Ar2+) interference on selenium (80Se+) using ICPDRC-MS: 40Ar2+ is a polyatomic ion formed in the plasma as a result of a

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reaction between the plasma gas (Ar) and itself. The dynamic reaction cell of
the ELAN ICP-DRC-MS is used to reduce ion signals from polyatomic ions via
ion-molecule reaction chemistry [56, 57]. In the reaction cell, methane (CH4)
molecules react with 40Ar2+ ions through a charge transfer reaction. The
products of the reaction are 40Ar+ (ion at a different mass) and 40Ar (neutral).
The background ion signal at m/z 80 is reduced by six orders of magnitude
because of this reaction.
ii. Reduction of argon nitride (40Ar15N+), argon hydroxide (38Ar16O1H+)
interference on manganese (55Mn) using ICP-DRC-MS: 40Ar15N+ and
38
Ar16O1H+ are polyatomic ions formed in the plasma as a result of reactions
between the plasma gas (Ar) and atmospheric gases (N2, O2) or the solvent
(H2O). The dynamic reaction cell of the ELAN ICP-DRC-MS is used to reduce
ion signals from polyatomic ions via ion-molecule reaction chemistry[56, 57].
In the reaction cell, oxygen molecules react with 40Ar15N+ and 38Ar16O1H+ ions
through either charge transfer reactions or oxygen transfer reactions. The
products of the reactions are either neutral molecules and are not detected
(charge transfer), or a new ion with higher mass (oxygen transfer). In either
case, attenuation of the background ion signal at m/z 55 occurs.
iii. Reduction of 37Cl18O+, 39K16O+, 54Fe1H+ interferences on manganese (55Mn)
using ICP-DRC-MS: 37Cl18O+, 39K16O+, 54Fe1H+ are polyatomic ions created in
the plasma as a result of reactions between elements present in the blood
matrix (Cl, K, and Fe) and the solvent (H2O). Due to the high concentrations of
Cl, K, and Fe in the blood matrix the resulting ion signals of 37Cl18O+, 39K16O+,
and 54Fe1H+ interfere with the measurement of 55Mn+ at m/z 55. The dynamic
reaction cell of the ELAN ICP-DRC-MS is used to reduce ion signals from
polyatomic ions via ion-molecule reaction chemistry[56, 57]. In the reaction
cell, oxygen molecules react with 37Cl18O+, 39K16O+, 54Fe1H+ ions through either
charge transfer reactions or oxygen transfer reactions. The products of the
reactions are either neutral molecules and are not detected (charge transfer),
or a new ions with higher mass (oxygen transfer). In either case, attenuation
of the background ion signal at m/z 55 occurs.
3) Procedures for Collecting, Storing, and Handling Specimens; Criteria for
Specimen Rejection; Specimen Accountability and Tracking
a. Procedures for Collecting, Storing, and Handling Specimens: Specimen handling
conditions, special requirements, and procedures for collection and transport are
discussed in the division (DLS) Policies and Procedures Manual [58]. Copies are
available in branch, laboratory, and special activities specimen-handling offices.
An electronic copy is available at:
http://intranet.nceh.cdc.gov/dls/pdf/policiesprocedures/Policy_and_Procedures_
Manual.DLS.2002mod.pdf. In general,
i. No fasting or special diets are required before collection of blood

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ii. Specimen type – whole blood
iii. Optimal amount of specimen is 1-2 ml. Request a minimum volume of 0.4 ml.
Volume for one analytical measurement is 0.1 ml.
iv. Sample collection devices and containers should be verified to be free of
significant contamination (“pre-screened”) before use.
v. Draw the blood through a stainless steel needle into a pre-screened
vacutainer.
vi. Blood specimens should be transported and stored at  4oC. Once received,
they can be frozen at  -20oC until time for analysis. Specimen stability has
been demonstrated for several months at ≤ -20C.
b. Criteria for Specimen Rejection:
include:

The criteria for an unacceptable specimen

i. Contamination: Improper collection procedures, collection devices, or sample
handling can contaminate the blood through contact with dust, dirt, etc.
Manganese is present in the general environment, found often in combination
with iron, and is present in many alloys (especially stainless steel).
ii. Low Volume: Request a minimum volume of 0.4 ml. Volume for one analytical
measurement is 0.1 ml.
In all cases, a second blood specimen should be requested.
c. Transfer or Referral of Specimens; Procedures for Specimen Accountability and
Tracking: Location, status, and final disposition of the specimens will be tracked
at least by paper document in the “Study Folder” (created before analysts receive
the samples). Apart from this specimen tracking form, this folder will also contain
the paper print outs of results from analysis of the specimens. Maintain records
for a minimum of 3 years. Use only numerical identifiers for samples within the
laboratory (e.g., case ID numbers) in order to safeguard confidentiality. Only the
medical supervisor (MS) or project coordinator (PC) i.e. non CDC personnel
should have access to the personal identifiers.
4) Safety Precautions
a. General Safety
i. Observe all safety regulations as detailed in the Division (DLS) Safety Manual.
Additional information can be found in your lab’s chemical hygiene plan.

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Participate in training regarding blood-borne pathogens prior to performing this
method.
ii. Observe Universal Precautions when working with blood.
iii. Wear appropriate gloves, lab coat, and safety glasses while handling all
solutions.
iv. Special care should be taken when handling and dispensing bases and
concentrated acids. Wear powder free gloves, a lab coat, safety glasses, and
face / neck protection. If TMAH or concentrated hydrochloric acid comes
in contact with any part of the body, quickly wash with copious
quantities of water for at least 15 minutes.
v. Use secondary containment for containers holding biological or corrosive
liquids.
vi. Dispose of all biological samples and diluted specimens in a biohazard
autoclave bag at the end of the analysis according to CDC/DLS guidelines for
disposal of hazardous waste.
vii. The use of the foot pedal on the Digiflex™ is recommended because it
reduces analyst contact with work surfaces that have been in contact with
blood and also keeps the analyst’s hands free to hold the specimen cups and
autosampler tubes and to wipe off the tip of Digiflex™.
viii. Training will be given before operating the ICP-DRC-MS, as there are many
possible hazards including ultraviolet radiation, high voltages, radio-frequency
radiation, and high temperatures. This information is also detailed in the
PerkinElmer ELAN® ICP-DRC-MS System Safety Manual.
ix. Transport and store compressed gas cylinders with proper securing
harnesses. For compressed oxygen gas, use regulators which are oil-free and
are equipped with a flash arrestor.
x. Wipe down all work surfaces at the end of the day with bleach-rite spray or
freshly prepared 10% (v/v) sodium-hypochlorite solution.
b. Waste Disposal: Operators of this method should take the CDC-OHS Hazardous
Chemical Waste Management Course (initial and yearly refreshers).
i. Waste to be Placed Into Biohazard Autoclave Bags & Pans:
1. All biological samples and diluted specimens (after analysis run).

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2. All disposable plastic and paper which contact blood (autosampler tubes,
gloves, etc.).
3. Used non-glass/quartz ICP-MS consumables (i.e. probes, tubing, cones,
ion lenses).
ii. Waste to be Placed Into Sharps Containers: Pipette Tips, broken glass or
quartz instrument consumables (broken spray chambers, torches, nebulizers,
etc. . .). Large broken glass which will not fit in the sharps container should be
placed in a separate autoclave pan from other waste and labeled as “broken
glass” (see the “Autoclaving” section of the CDC safety policies and practices
manual located in the laboratory).
5) Instrument & Material Sources
a. Sources for ICP-MS Instrumentation
i. ICP-MS:
Inductively Coupled Plasma Dynamic Reaction Cell Mass
Spectrometer
(ELAN®
DRC
II)
(PerkinElmer
Norwalk,
CT,
www.perkinelmer.com).
1. DXi-FAST upgrade: Standard peristaltic pump replaced by DXi-FAST
micro-peristaltic pump / FAST actuator and valve combination unit. For
ELAN DRC2, part # DXI-54-P4-F6.
ii. Recirculating chiller / heat exchanger for ICP-MS: Refrigerated chiller
(PolyScience 6105PE for ELAN® 6100 DRCPlus instruments) if unit is to be
placed remotely from ICP-MS or heat exchanger (PolyScience 3370 for ELAN®
DRC II instruments) if unit is to be placed alongside ICP-MS (PerkinElmer
Norwalk, CT, www.perkinelmer.com).
iii. Autosampler:
1. ESI SC4 autosampler: Dual rinse station supplied by two independent
pumps built internal to the autosampler (Elemental Scientific Inc., Omaha,
NE).
2. FAST: Purchase as an option onto the ESI SC4 autosampler (Elemental
Scientific Inc., Omaha, NE).
b. Sources for ICP-MS Parts & Consumables
NOTE: The minimum number of spares recommended before reordering (if
owning one instrument) are listed as “# Spares = ” in the descriptions below.
i. Adapter, PEEK: Securely connects 1.6mm O.D. PFA tubing to 0.03” I.D.
peristaltic tubing. Composed of three PEEK parts.

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1. Female nut for 1.6mm O.D. (1/16”) tubing. Like part P-420 (Upchurch
Scientific, Oak Harbor, WA, www.upchurch.com).
2. PEEK ferrule. Like part P-260x (10pk SuperFlangeless ferrule, Upchurch
Scientific, Oak Harbor, WA, www.upchurch.com).
3. Conical Adapter Body. Like part P-692 (Upchurch Scientific, Oak Harbor,
WA, www.upchurch.com).
ii. Bottles (for rinse solution): Four liter screw-cap polypropylene container with 2
luer connections (like catalog# SC-0305-1, Elemental Scientific Inc., Omaha,
NE., www.elementalscientific.com).
iii. Carboy and cap assembly for waste collection: 10-15L, polypropylene widemouth carboy (100 mm neck size) with handles and no spigot (Like part #
7BE-25126, Lab Safety Supply, Janesville, WI, www.lss.com) with cap
assembly
like
part
#
N0690271
(PerkinElmer
Norwalk,
CT,
www.perkinelmer.com).
iv. Coolant, for Polyscience chiller or heat exchanger: Only PerkinElmer part #
WE01-6558 (PerkinElmer Norwalk, CT, www.perkinelmer.com) is approved for
use by PerkinElmer. # Spares = 6.
v. Cone, sampler (nickel): PerkinElmer part # WE021140 (PerkinElmer Norwalk,
CT, www.perkinelmer.com). Part # SC2011-Ni (Testing has also found
Spectron, Ventura, CA, www.spectronus.com cones to be comparable). #
Spares = 4.
vi. Cone, skimmer (nickel): PerkinElmer part # WE021137 (PerkinElmer Norwalk,
CT, www.perkinelmer.com). Part # SC2012-Ni (Testing has also found
Spectron, Ventura, CA, www.spectronus.com cones to be comparable) #
Spares = 4.
vii. Detector, electron multiplier: Like part # N8125001 (PerkinElmer Norwalk, CT,
www.perkinelmer.com). Available direct from manufacturer (part # 14210,
SGE Incorporated, Austin, Texas, http://www.etpsci.com) or various
distributors. # Spares = 1.
viii. FAST accessories
1. Valve: CTFE High-flow valve head for SC-FAST (uses ¼-28 fittings). Like
part # SC-0599-1010 (Elemental Scientific Inc., Omaha, NE.,
www.elementalscientific.com).
2. Stator: CTFE Stator for 6 port SC-FAST high flow valve (¼-28 fittings).
Like part # SC-0599-1010-01 (Elemental Scientific Inc., Omaha, NE.,
www.elementalscientific.com).
3. Rotor: Composite rotor for 6 port SC-FAST high flow valve (¼-28 fittings).
Like part # SC-0599-1010-05 (Elemental Scientific Inc., Omaha, NE.,
www.elementalscientific.com).

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4. Sample Loop: 1 mL Teflon, white connector-nuts for high flow valve head.
Like part # SC-0315-10 (Elemental Scientific Inc., Omaha, NE.,
www.elementalscientific.com).
5. Probe, Autosampler: Teflon, carbon fiber support, 0.8mm i.d., blue marker,
1/4-28 fittings. Like part number SC-5037-3751 (Elemental Scientific Inc.,
Omaha, NE., www.elementalscientific.com). # Spares = 2.
6. Probe, Carrier Solution: Teflon, carbon fiber support, 0.5mm i.d., orange
marker, 1/4-28 fittings. Like part number SC-5037-3501 (Elemental
Scientific Inc., Omaha, NE., www.elementalscientific.com). # Spares = 2.
7. Tubing, FAST vacuum: Vacuum line for SC-FAST high flow valve,
connects to port #6, black nut for connection to valve head, natural brown
color nut on other end for connection to SC autosampler vacuum port. Like
part
#
SC-0321
(Elemental
Scientific
Inc.,
Omaha,
NE.,
www.elementalscientific.com).
8. Tubing, connects nebulizer to valve: See “Nebulizer, PolyPro-ST micro
flow”
ix. Hose, for connection to chiller: Push on hose. I.D. = ½”, O.D. = ¾”. Use part
# PB-8 (per inch, Georgia Valve and Fitting, Atlanta, GA, www.swagelok.com)
or equivalent. Do not normally need spare hose (unless moving instrument
into a new location).
x. Hose, for exhaust of ELAN: Available as part of ELAN installation kit from
Perkin Elmer (PerkinElmer Norwalk, CT, www.perkinelmer.com). Available
direct from manufacturer as part # S-LP-10 air connector (Thermaflex,
Abbeville, SC, www.thermaflex.net). Equivalent part may be substituted. #
Spares = 10 feet of 4” diameter and 10 feet of 6” diameter hose.
xi. Injector, quartz: I.D. = 2.0 mm. PerkinElmer part # WE023948 (PerkinElmer
Norwalk, CT, www.perkinelmer.com). Available direct from manufacturer as
part
#
400-30
(Precision
Glass
Blowing,
Centennial,
CO,
www.precisionglassblowing.com) or equivalent from various distributors. #
Spares = 2.
xii. Injector support (for pass-through injector): PerkinElmer part # WE023951
(PerkinElmer Norwalk, CT, www.perkinelmer.com). Available direct from
manufacturer as part # 400-37 (Precision Glass Blowing, Centennial, CO,
www.precisionglassblowing.com) or equivalent from various distributors. #
Spares = 2.
xiii. Ion Lens:
PerkinElmer part # WE018034 (PerkinElmer Norwalk, CT,
www.perkinelmer.com). # Spares = 3.
xiv. Nebulizer, PolyPro-ST micro flow: Polypropylene nebulizer with external 1/428 threaded connector for liquid delivery, low pressure version or equivalent.
Like part # ES-4040-7010 (Elemental Scientific Inc., Omaha, NE.,
www.elementalscientific.com). # Spares = 1.
1. Gas connection:

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 19 of 88

a. Teflon tubing: 4mm o.d., 2.4mm i.d. Teflon tubing (like part # ES2502,
Elemental
Scientific
Inc.,
Omaha,
NE.,
www.elementalscientific.com). # Spares = 1.
b. Adapter kit: Plastic adapters to connect Teflon tubing (2.4mm i.d) to
¼” male Swagelok (compression) port on ICP-DRC-MS. Parts can
be obtained as components in a “gas fittings kit for microflow
nebulizer”, kit part # ES-2501-1000, Elemental Scientific Inc.,
Omaha, NE., www.elementalscientific.com). # Spares = 1.
2. Liquid connection: Connects nebulizer to port #3 of high flow FAST valve
head with green, 1/4- 28 fitting. Like part # SC-0317-0250 (Elemental
Scientific Inc., Omaha, NE., www.elementalscientific.com). # Spares = 2.
xv. Nut and Ferrule set, 1/8” Swagelok: Such as part # SS-200-NFSET (stainless
steel) or part # B-200-NFSET (brass) (Georgia Valve and Fitting, Atlanta, GA,
www.swagelok.com) or equivalent. For part numbers listed here a quantity of
1 means 1 nut, 1 front ferrule, and 1 back ferrule. Spares = 20.
xvi. Nut and Ferrule set, 1/4” Swagelok: Such as part # SS-400-NFSET (stainless
steel) or part # B-400-NFSET (brass) (Georgia Valve and Fitting, Atlanta, GA,
www.swagelok.com) or equivalent. For part numbers listed here a quantity of
1 means 1 nut, 1 front ferrule, and 1 back ferrule. Spares = 20.
xvii. Oil, Welch DirecTorr Gold: For roughing pumps. Available direct from
manufacturer as part # 8995G-15 (1 gallon, Welch Rietschle Thomas, Skokie,
IL, www.welchvacuum.com) or from various distributors. Equivalent oil may be
substituted. # Spares = 4.
xviii. O-ring: (for sampler cone) PerkinElmer part # N8120511 (pkg. of 5,
PerkinElmer, Shelton, CT, www.perkinelmer.com) or equivalent. # Spares =
20 o-rings.
xix. O-ring: (for skimmer cone) PerkinElmer part # N8120512 (pkg. of 5,
PerkinElmer, Shelton, CT, www.perkinelmer.com) or equivalent. # Spares =
20 o-rings.
xx. O-ring: (for ELAN DRC II standard injector support).
1. Internal o-rings: ID = ¼”, OD = 3/8”, thickness = 1/16”. Need 2 o-rings per
injector support setup. PerkinElmer part # N8122008 (PerkinElmer,
Shelton, CT, www.perkinelmer.com) or equivalent (such as part # V75-010,
O-rings West, Seattle, WA, www.oringswest.com). # Spares = 20.
2. External o-rings: ID = 3/8”, OD = 1/2”, thickness = 1/16”. Need 2 o-rings
for each injector support setup.
PerkinElmer part # N8122009
(PerkinElmer, Shelton, CT, www.perkinelmer.com) or equivalent (such as
part # V75-012, O-rings West, Seattle, WA, www.oringswest.com). #
Spares = 20.
xxi. O-ring: (for inside of bayonet torch mount): Part # WE017284 (PerkinElmer,
Shelton, CT, www.perkinelmer.com). Do not substitute. The PerkinElmer o-

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 20 of 88

ring is specially metal impregnated to minimize RF leakage though the torch
mount. # Spares = 2.
xxii. Photon Stop: PerkinElmer part # WE018278 (PerkinElmer, Shelton, CT,
www.perkinelmer.com). # Spares = 1.
xxiii. Plugs, Quick Change for Roughing Pump Oil: These plugs will only work on
the Varian roughing pumps which come standard on ELAN DRC II ICPMS
instruments. These plugs will not fit the Leybold pumps which come standard
on the ELAN DRC Plus instruments. Part # W1011013 (PerkinElmer, Shelton,
CT, www.perkinelmer.com). No spares typically needed.
xxiv. RF coil:
PerkinElmer part # WE02-1816 (PerkinElmer, Shelton, CT,
www.perkinelmer.com) or equivalent. # Spares = 2.
xxv. Spray chamber, quartz concentric:
PerkinElmer part # WE025221
(PerkinElmer, Shelton, CT, www.perkinelmer.com) or equivalent. Available
direct from manufacturer as part # 400-20 (Precision Glass Blowing,
Centennial, CO, www.precisionglassblowing.com) or from various distributors.
# Spares = 2.
a. O-ring: (for inside spray chamber at nebulizer port) Such as part #
120-56
(Precision
Glass
Blowing,
Centennial,
CO,
www.precisionglassblowing.com). Additional o-rings can sometimes
be obtained free of charge or at reduced price when acquired while
purchasing spray chambers. # Spares = 20.
xxvi. Torch, quartz: PerkinElmer part # N812-2006 (PerkinElmer, Shelton, CT,
www.perkinelmer.com) or equivalent. Available direct from manufacturer as
part
#
400-10
(Precision
Glass
Blowing,
Centennial,
CO,
www.precisionglassblowing.com) or various distibutors. Damaged torches can
often be repaired for substantially lower cost than purchasing a new one by
companies such as Wilmad LabGlass (Buena, NJ, www.wilmad-labglass.com)
or Precision Glass Blowing (Centennial, CO, www.precisionglassblowing.com).
# New Spares = 2.
xxvii. Tubing, main argon delivery to instrument: I.D. = 1/8”, O.D. = ¼”. Such as
part # C-06500-02 (pkg. of 100ft, polypropylene, Fisher Scientific International,
Hampton, NH, www.fishersci.com) or equivalent. # Spares = 50ft.
xxviii. Tubing, drains waste liquid from spray chamber :
1. PVC 1/8” i.d., 3/16” o.d tubing used to transfer waste liquid between spray
chamber waste port and peristaltic pump waste tubing and between
peristaltic pump waste tubing and liquid waste carboy. Like part # 14-1697A (pkg. of 50ft, Fisher Scientific International, Hampton, NH,
www.fishersci.com) or equivalent. # Spares = 20ft.
2. Connector: Use to connect 1/8” I.D. PVC tubing to 0.125” I.D peristaltic
pump tubing.
Use part # 3140715 (PerkinElmer Norwalk, CT,
www.perkinelmer.com) or equivalent. # Spares = 4.
xxix. Tubing, peristaltic, 0.03” i.d. (sampling/carrier solution):

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 21 of 88

1. Standard PVC, 2-stop (black / black) peristaltic pump tubing, i.d. = 0.03”.
PerkinElmer
part
#
09908587
(PerkinElmer,
Shelton,
CT,
www.perkinelmer.com) or equivalent. # Spares = 6 packs of 12 tubes.
Use this type tubing with standard ELAN peristaltic pump.
2. Standard PVC, 3-stop. (blank / black) peristaltic pump tubing, i.d. 0.76 mm.
Spectron part # SC0056 (Spectron, Ventura, CA, www.spectronus.com) or
equivalent. # Spares = 6 packs of 12 tubes. Use this type tubing with ESI
DXi micro-peristaltic pump.
xxx. Tubing, peristaltic, 0.045” i.d. (spray chamber drain):
1. Standard PVC, 2-stop (red / red) peristaltic pump tubing, i.d. = 0.045”.
PerkinElmer
part
#
N0680375,
(PerkinElmer,
Shelton,
CT,
www.perkinelmer.com) or equivalent. # Spares = 6 packs of 12 tubes.
2. Standard Santoprene, 3-stop (grey / grey / grey) peristaltic pump tubing,
i.d. 1.30 mm.
Spectron part # SC0311 (Spectron, Ventura CA,
www.spectronus.com0 or equivalent. # Spares = 6 packs of 12 tubes.
Use this type tubing with ESI DXi micro-peristaltic pump.
xxxi. Tubing, Stainless Steel, o.d. = 1/8”, wall thickness = 0.028”: Used to connect
DRC gas cylinders to ELAN DRC gas ports. Also can be used to replace
plastic tubing in the DRC gas path within the ELAN to minimize gas
leaks/diffusion into gas stream. Like part # SS-T2-S-028-20 (20ft, Georgia
Valve and Fitting, Atlanta, GA, www.swagelok.com) or equivalent. Spares =
20ft.
xxxii. Tubing, Teflon, corrugated, ¼” o.d.: Connects to the auxiliary and plasma gas
side-arms of the torch. Part # WE015903 (PerkinElmer, Shelton, CT,
www.perkinelmer.com). # Spares = 2.
xxxiii. Union Elbow, PTFE ¼” Swagelok: Connects argon tubing to torch auxiliary
gas sidearm. Like part # T-400-9 (Georgia Valve and Fitting, Atlanta, GA,
www.swagelok.com) or equivalent. Spares = 2.
xxxiv. Union Tee, PTFE, ¼” Swagelok: Connects argon tubing to torch plasma gas
sidearm and holds igniter inside torch sidearm. Like part # T-400-3 (Georgia
Valve and Fitting, Atlanta, GA, www.swagelok.com) or equivalent. Spares =
2.
c. Sources for ICP-MS Maintenance Equipment & Supplies
i. Anemometer: Like digital wind-vane anemometer (Model 840032, SPER
Scientific LTD., Scottsdale, AZ, www.sperscientific.com) or equivalent. Use to
verify adequate exhaust ventilation for ICP-MS (check with hoses fully
disconnected).
ii. Pan, for changing roughing pump oil: Like part # 53216 (United States Plastics
Corporation, Lima, OH, www.usplastic.com) or equivalent. # On hand = 1.

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 22 of 88

Cleaners

(NEYTECH,

d. Sources for General Laboratory Consumable Supplies
i. Bar Code Scanner: Like Code Reader 2.0 (Code Corporation, Draper, UT,
www.codecorp.com) or equivalent. For scanning sample IDs during analysis
setup. Any bar code scanner capable of reading Code 128 encoding at a 3 mil
label density can be substituted.
ii. Carboy (for preparation of blood quality control pool and waste jug for ICPMS
sample introduction system): Polypropylene 10-L carboy (like catalog # 02960-20C, Fisher Scientific, Pittsburgh, PA, www.fischersci.com) or equivalent.
Carboys with spouts are not advised due to potential for leaking.
iii. Containers for diluent and Rinse Solution: Two liter Teflon™ containers (like
catalog# 02-923-30E, Fisher Scientific, Pittsburgh, PA., www.fishersci.com)
and 4L polypropylene jugs (like catalog# 02-960-10A, Fisher Scientific,
Pittsburgh, PA, www.fishersci.com) have both been used. Acid rinse before
use. Equivalent containers may be substituted.
iv. Gloves: Powder-free, low particulate nitrile (like Best CleaN-DEX™ 100%
nitrile gloves,any vendor).
Equivalent nitrile or latex gloves may be
substituted.
v. Paper towels: For general lab use, any low-lint paper wipes such as
KIMWIPES®EX-L Delicate Task Wipers or KAYDRY®EX-L Delicate Task
Wipers (Kimberly-Clark Professional, Atlanta, GA, www.kcprofessional.com).
For sensitive applications in cleanrooms, a wipe designed for cleanroom use
may be desired such as the Econowipe or Wetwipe (Liberty, East Berlin, CT,
www.liberty-ind.com).

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 23 of 88

vi. Pipette (for preparation of blood dilutions to be analyzed): Micromedic DigiflexCX Automatic™ pipette equipped with 10.0-mL dispensing syringe, 2 uL
sampling syringe, 0.75-mm tip, and foot pedal (Titertek, Huntsville, AL,
http://www.titertek.com/).
vii. Pipettes (for preparation of intermediate stock working standards & other
reagents): Like Brinkmann Research Pro Electronic pipettes (Brinkmann
Instruments, Inc., Westbury, NY, http://www.brinkmann.com/home/). 5-100 L
(catalog #4860 000.070), 20-300 L (catalog #4860 000.089), 50-1000 L
(catalog #4860 000.097), 100-5000 L (catalog #4860 000.100). Note: pipette
catalog numbers are without individual chargers. Can purchase individual
chargers (pipette catalog numbers will differ) or a charging stand that will hold
four pipettes (catalog #4860 000.860). When purchasing pipette tips (epTips),
purchase one or more boxes, then “reloads” for those boxes after that: 5-100
L (box catalog # 22 49 133-4, reload catalog # 22 49 153-9), 20-300 L (box
catalog # 22 49 134-2, reload catalog # 22 49 154-7), 50-1000 L (box catalog
# 22 49 135-1, reload catalog # 22 49 155-5), 100-5000 L (box catalog # 22
49 138-5, reload catalog # 22 49 198-9, bulk bag catalog # 22 49 208-0).
Equivalent pipettes and tips can be substituted.
viii. Tubes for sample analysis (for autosampler): Like polypropylene 15-mL
conical tubes, BD Falcon model #352097 (Becton Dickinson Labware, Franklin
Lakes, NJ, www.bd.com). Equivalent tubes may be substituted which are
shown by lot screening to be free of trace metal contamination. Clear plastics
tend to have lowest trace metal contamination. Blue colored caps have also
been used successfully for this method.
ix. Tubes for storage of intermediate working stock standards: Like polypropylene
50-mL conical tubes, BD Falcon model #352098 (Becton Dickinson Labware,
Franklin Lakes, NJ, www.bd.com). For use in storage of intermediate working
stock standards. Equivalent tubes may be substituted which are shown by lot
screening to be free of trace metal contamination. Clear plastics tend to have
lowest trace metal contamination. Blue colored caps have also been used
successfully for this method.
x. Vortexer: Like MV-1 Mini Vortexer (VWR, West Chester, PA, www.vwr.com).
Used for vortexing blood specimens before removing an aliquot for analysis.
Equivalent item can be substituted.
xi. Water purification system: Like NANOpure DIamond Ultrapure Water System
(Barnstead International, Dubuque, Iowa, www.barnstead.com). For ultra-pure
water used in reagent and dilution preparations. An equivalent water
purification unit capable of producing >18 Mega-ohm·cm water may be
substituted.
e. Sources of Chemicals, Gases, and Regulators
i. Acid, Hydrochloric acid: Veritas™ double-distilled grade, 30-35% (GFS
Chemicals Inc. Columbus, OH, www.gfschemicals.com). This is referred to as
“concentrated” hydrochloric acid in this method write-up.
For use in

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 24 of 88

preparation of intermediate working stock standards.
An equivalent
hydrochloric acid product may be substituted, but it must meet or exceed the
purity specifications of this product for trace metals content.
ii. Acid, Nitric acid: Veritas™ double-distilled grade, 68-70% (GFS Chemicals Inc.
Columbus, OH, www.gfschemicals.com). For use in cleaning any bottles,
vials, tubes, and flasks. This is referred to as “concentrated” nitric acid in this
method write-up. An equivalent nitric acid product may be substituted, but it
must meet or exceed the purity specifications of this product for trace metals
content.
iii. Alcohol, Ethyl, USP dehydrated 200 proof (Pharmco Products, Inc.) or
equivalent.
iv. Ammonium pyrrolidine dithiocarbamate, laboratory grade (Fisher Scientific,
Fairlawn, NJ) or equivalent.
v. Argon Gas (for plasma & nebulizer) and Regulator: High purity argon
(>99.999% purity, Specialty Gases Southeast, Atlanta, GA, www.sgsgas.com)
for torch and nebulizer. Minimum tank source is a dewar of liquid argon (180250L). Bulk tank (1500+L is preferred).
1. Regulator for argon (at dewar): Stainless steel, single stage, specially
cleaned regulator with 3000 psig max inlet, 0-100 outlet pressure range,
CGA 580 cylinder connector, and needle valve shutoff on delivery side
terminating in a ¼” Swagelok connector.
Part number
KPRAFPF415A2AG10 (Georgia Valve and Fitting, Atlanta, GA,
www.swagelok.com). An equivalent regulator from an alternate vendor may
be substituted. # Spares = 1.
2. Regulator for argon (between bulk tank and PerkinElmer filter regulator):
Single Stage 316SS Regulator, with 0-300 psi Inlet Gauge, 0-200 psi
Outlet Gauge, Outlet Spring Range, 0-250 psi, ¼” Swagelok Inlet
Connection, ¼ turn Shut off Valve on Outlet with ¼” Swagelok Connection
and Teflon Seals. Part number KPR1GRF412A20000-AR1 (Georgia Valve
and Fitting, Atlanta, GA, www.swagelok.com). An equivalent regulator from
an alternate vendor may be substituted. # Spares = 1.
3. Regulator for argon (PerkinElmer filter regulator on back of ELAN): Argon
regulator filter kit. Catalog number N812-0508 (PerkinElmer, Shelton, CT,
www.perkinelmer.com).
vi. Disinfectant, for work surfaces: Bleach-rite spray (any distributor). On-site
dilutions of bleach (1part bleach + 9 parts water) may be substituted, but must
be re-made daily.
vii. Methane: Methane (Research Grade 5.0, 99.99% purity), for DRC channel A.
Typically purchased in cylinder size 200 (part # ME R200, Airgas South,
Atlanta, GA, www.airgas.com).

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 25 of 88

1. Regulator for methane: A 2-stage, high purity brass regulator with max
rated inlet pressures of 3,000 psi, max outlet pressures of 15-30 psi (with
gauge maximum at 15-30psi). Like part number Y12-N145A350 (Airgas
South, Atlanta, GA, www.airgas.com). An equivalent regulator from an
alternate vendor may be substituted. # Spares = 1.
2. Flash Arrestor: Like part # 6103 (Matheson Tri Gas, Montgomeryville, PA,
www.mathesontrigas.com) or equivalent.
viii. Oxygen: Oxygen (“Research Grade Research Grade 5.0”, 99.9999% purity)
for DRC channel B. Typically purchased in cylinder size 300 (9.5” x 54”)
(Airgas South, Atlanta, GA, www.airgas.com).
1. Regulator for oxygen: High purity brass body with monel trim, two stage
regulator. Stainless steel is not used for this application due to safety
concerns of working with oxygen at high pressure [59]. For one regulator,
order the following parts, and ask that they be tested and assembled
(Engineered Specialty Products, Kennesaw, GA, www.espgauges.com).
a. Tescom part # 44-3410S24-555
Regulator body: Brass bar stock, two stage, Monel trim, TFE seats,
Elgiloy diaphragms, Cv=0.05, 3000 psig max inlet, 1-25 psig outlet
range, 1/4 FNPT inlet / outlet / gauge ports, O2 cleaned to ASTMG93
and CGA4.1.
b. Tescom part # 60500-3000N
Inlet pressure gauge: 2" diameter, 0-3000 psig range , O2 cleaned, ¼”
MNPT bottom, brass.
c. Tescom part # 60500-0015N
Delivery pressure gauge: 2" diameter, 0-15 psig range , O2 cleaned, ¼”
MNPT bottom, brass.
d. Tescom part # 63842-540-B
NPT to CGA Adaptor: ¼” NPT to CGA 540 adapter, brass.
e. Swagelok part # B-200-1-4:
Adapter: Brass male connector, ¼” MNPT to 1/8” Swagelok (Georgia
Valve and Fitting, Atlanta, GA, www.swagelok.com).
An equivalent regulator from an alternate vendor may be substituted.
# Spares = 1.
2. Flash Arrestor (brass):
Like part # 6103 (Matheson Tri Gas,
Montgomeryville, PA, www.mathesontrigas.com) or equivalent.
ix. Standard, Iridium: Like 1,000 mg/L, item #CGIR1-1 (Inorganic Ventures,
Christiansburg, VA http://www.inorganicventures.com). Used as an internal
standard in diluent. Any vendor whose standards are traceable to the National

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016
Institute for Standards and Technology may be substituted.
must have low trace metal contamination.

Page 26 of 88
The standard

x. Standard, Rhodium: Like 1,000 mg/L, item # PLRH3-2Y. (SPEX Industries,
Inc., Edison, NJ, www.spexcsp.com). Used as an internal standard in diluent.
Any vendor whose standards are traceable to the National Institute for
Standards and Technology may be substituted. The standard must have low
trace metal contamination.
xi. Standard, Tellurium: Like 1,000 mg/L, item #CGTE1-1 (Inorganic Ventures,
Christiansburg, VA http://www.inorganicventures.com).Used as an internal
standard in diluent. Any vendor whose standards are traceable to the National
Institute for Standards and Technology may be substituted. The standard
must have low trace metal contamination.
xii. Standard, single element stock standards for preparation of calibrators and
blood quality control pools: National Institute of Standards and Technology
(NIST) Standard Reference Materials (SRMs): 3108 (Cd), 3132 (Mn), 3128
(Pb), 3133 (Hg), 3149 (Se). (Gaithersburg, MD, www.nist.gov). Other sources
of standards can be used if they are NIST traceable.
xiii. Tetramethylammonium hydroxide, 25% w/w, or equivalent (AlfaAesar, 30 Bond
St., Ward Hill, MA 01835)
xiv. Triton X-100™ surfactant: Like “Baker Analyzed” TritonX-100™ (J.T. Baker
Chemical Co., www.jtbaker.com). Another source may be substituted, but it
must be free of trace-metal contamination.
6) Preparation of Reagents and Materials
a. Internal Standard Intermediate Mixture (20 g/mL Rh, Ir and Te):
i. Purpose: Preparation of single intermediate solution containing all internal
standards simplifies the addition of the internal standard(s) into the final diluent
solution. This solution can be purchased rather than prepared.
ii. Preparation: To prepare 50 mL of the intermediate internal standard solution:
1. Partially fill a 50 mL acid-washed volumetric flask (PP, PMP, or Teflon™)
with 1% v/v HNO3 (approximately 25-40 mL).
2. Add 1 mL of 1,000 g/mL Rh standard, 1 mL of 1,000 g/mL Ir standard,
and 1 mL of 1,000 g/mL Te standard. If initial Rh, Ir, or Te standard
concentration is different, adjust volume proportionally.
3. Fill to mark (50 mL) with 1% v/v HNO3 and mix thoroughly.
4. Label appropriately (e.g. “Internal Standard Intermediate Mixture. 20
g/mL Rh, Ir and Te, 1% v/v HNO3”, preparation date, expiration date 1
year from preparation date, and preparer’s initials).

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 27 of 88

b. 20% Triton X-100 intermediate solution:
i. Purpose: Addition to diluent and rinse solutions where Triton X-100 acts as a
surfactant. For ease of daily preparation of the diluent and rinse solutions, first
prepare a 20% Triton X-100 solution.
ii. Preparation: To prepare 1 L of 20% Triton x-100®
1. Add 200 ml of Triton X-100 to a pre-acid washed 1L Teflon container
that is partially filled with 18 M-ohm water.
2. Fill to 1 L with 18 M-ohm water and mix until the Triton X-100 has
completely dissolved into solution (overnight). A magnetic stirring plate
can be used to assist mixing by adding an acid-washed Tefloncoated
stirring bar to the bottle.
c. Sample Diluent
i. Purpose: The diluent used in this method is an aqueous solution of 5 g/L
internal standard mixture (Rh, Ir, Te), in 0.25% v/v tetramethyl ammonia
hydroxide (TMAH), 1% ethyl alcohol, 0.01% APDC, and 0.025% v/v Triton X100. This solution will be used in the preparation of all calibrators and
samples during the dilution process just prior to analysis. It is important that all
samples in a run should be made from the same diluent solution so that the
concentration of the internal standards will be the same among all calibrators
and samples in the run. When using a flow-injection component in the sample
introduction system (i.e. the Elemental Scientific SC4-FAST autosampler), the
‘carrier’ solution should be the same as the diluent used for the method.
Larger volumes of these solutions can be prepared by adjusting component
volumes proportionally.
ii. Preparation:
1. Acid rinse a 2 L Teflon container, and partially fill with 18 M-ohm water.
2. Add 0.2 g of APDC , 5 ml of 25% v/v TMAH, 20 ml of ethyl alcohol, and 5
ml of 20% Triton X-100.
3. Dilute to volume (2L) with 18 M-ohm water
4. Spike 500 l of 20 mg/L Rh, Ir, Te to the final diluent.
5. Invert bottle a few times to insure thorough mixing. Allow to sit for several
hours or overnight before using.
6. Label appropriately (e.g. “5 g/L Rh, Ir and Te”, “0.01% APDC in 0.25% v/v
tetramethyl ammonia hydroxide (TMAH), 1% ethanol, and 0.05% v/v Triton
X-100”, preparation date, expiration date (1 year from prep), and preparer’s
initials).
7. Store at room temperature and prepare as needed.
d. DRC Stability Test Solution

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i. Purpose: The DRC Stability Test Solution is a “dummy” blood matrix sample
analyzed for 1-1.5 hr before the beginning of the analytical run.
ii. Preparation:
1. Fill an acid rinsed 1 L Teflon container with 960 mL of Sample Diluent.
2. Add 20 mL of rejected screened human or bovine blood
3. Add 1.5 mL of Intermediate Stock Calibration Standard
4. Store at 4°C and prepare as needed.
e. Base Blood
i. Purpose: This blood pool material will be mixed with the intermediate working
calibrators just prior to analysis to matrix-match the calibration curve to the
blood matrix of the unknown samples.
ii. Contents: A mixture of multiple blood sources collected from anonymous
donors are used to approximate an average blood matrix.
iii. Preparation & Storage:
1. Purchase several bags of whole blood. Bovine blood or human blood can
be used. Human blood should be screened for infectious diseases such as
Hepatitis B and HIV.
2. Screen each individual bag of blood for concentration of analytes of
interest. See Table 2 in Appendix B for minimum acceptable values
3. Once screened, mix the acceptable blood together in a larger container
(i.e. acid washed polypropylene (PP), polymethylpentene (PMP), or
Teflon™) and stir for 30+ minutes on a large stir plate (acid wash large
Teflon™ stir bar before use).
4. For short term storage, store at 2-4°C. For long-term storage, dispense
into smaller-volume tubes (i.e., 2 mL cryovials) and store at ≤ -20°C.
5. Labels on 2 mL cryovials should be labeled appropriately (e.g. “Base Blood
for Blood metals panel 2, Cd, Hg, Mn, Pb, Se”, dispensed date and vial
number).
f. ICP-DRC-MS Rinse Solution
i. Purpose: The rinse solution used in this method is an aqueous solution of
0.25 % v/v TMAH, 1% ethyl alcohol, 0.01% APDC, and 0.05 % Triton X-100,.
This solution will be pumped through the autosampler rinse station, probe, and
sample loop between sample analyses to prevent carry-over of analytes from
one sample measurement to the next.
ii. Preparation: To Prepare 4 L of the Rinse Solution:
1. Partially fill a 4 L acid-washed bottle (PP, PMP, or Teflon™) with >18
Mega-ohm·cm water (approximately 2-3 L). Use of volumetric flask is not
required.
2. Add 0.4 g of APDC
3. Add 10 ml of TMAH

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4. Add 40 ml of ethyl alcohol,
5. Add 10ml of 20% Triton X-100, (See Section 6.b for details on
preparation)
6. Fill to 4 L using >18 Megaohm·cm water.
7. Store at room temperature and prepare as needed. To prepare volumes
other than specified here, add proportionally larger or smaller volumes of
the solution constituents.
8. Invert bottle a few times to ensure thorough mixing. Allow to sit for several
hours or overnight before using.
9. Label appropriately (e.g. “0.25 % v/v TMAH, 1% ethyl alcohol, and 0.05 %
Triton X-100, 0.01% (w/v) APDC”, preparation date, expiration date one
year from preparation date, and preparer’s initials).
g. Single-Element Stock Standards For Preparation of Intermediate Stock
Calibration Standard
i. Purpose: These single-element standards will be used to prepare the
intermediate stock calibration standard.
ii. Contents: Separate, aqueous single-element standards of Cd, Pb, Hg, Se,
and Mn. Concentrations should be 1,000 mg/L or 10,000 mg/L.
iii. Purchase & Storage:
1. Purchasing from vendors: If the intermediate stock calibration standard is
purchased as a special-mix standard, these single-element stock
standards are not required. Purchase only NIST-traceable single-element
standards at the highest purity (don’t contain other metal impurities).
2. Storage: Store at room temperature.
h. 3% (v/v) HCl Diluent:
i. Purpose: 3% HCl is used to dilute single element stock standards into a single
intermediate stock calibration solution and finally to the intermediate working
calibration standards.
ii. Preparation:
1. In a cleaned 2 L flask, add 1-1.5L >18 Megaohm·cm water.
2. Add 60 mL high purity concentrated HCl.
3. Fill to the mark and mix thoroughly.
4. Label appropriately (e.g. “3 % v/v HCl”, preparation date, expiration date
one year from preparation date, and preparer’s initials).
i.

Intermediate Stock Calibration Standard
i. Purpose: This multi-element solution will be used to prepare the five working
calibration standards.

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ii. Preparation & Storage: This solution may be purchased as a special-mix
standard or prepared in-house from separate single-element stock standards.
1. Purchasing from vendors:
The intermediate stock calibration
standard may be purchased as custom mixture from any vendor
which prepares multi-element solutions that are traceable to the
National Institute for Standards and Technology (NIST) for their
accuracy.
2. In-house Preparation from NIST single element standards: Different
volumes may be prepared by adding proportionally larger or smaller
volumes of solution constituents.
a. Acid-rinse one 100 mL, PP (or PMP) volumetric flask. Mark the flask
according to intended use. Dedicate to purpose.
b. Partially fill the 100 mL flask with the 3% (v/v) HCl diluent (50-75% full).
c. Add necessary volumes of single-element stock standards to achieve
final concentrations listed in Table 3 of Appendix B.
d. Dilute to the volumetric mark with the 3% (v/v) HCl diluent using a
pipette for the final drops. Mix the flask solution thoroughly. Final
concentrations are listed in Table 3 of Appendix B.
e. Once mixed, transfer to an acid-cleaned, labeled, 50-mL container
(PP, PMP, or Teflon™) for storage. Label appropriately (e.g. “Whole
Blood Metals Panel 2 Intermediate Stock Calibration Standard”, “3%
(v/v) HCl”, date of preparation, expiration date (1 year from date of
preparation), initials of preparer, and concentrations for each element).
3. In-house Preparation from other single element standards: Different
volumes may be prepared by adding proportionally larger or smaller
volumes of solution constituents.
a. Acid-rinse one 100 mL, PP (or PMP) volumetric flask. Mark the flask
according to intended use. Dedicate to purpose.
b. Partially fill the 100 mL flask with the 3% (v/v) HCl diluent (50-75% full).
c. Add necessary volumes of single-element stock standards to achieve
final concentrations listed in Table 4 of Appendix B.
d. Dilute to the volumetric mark with the 3% (v/v) HCl diluent using a
pipette for the final drops. Mix the flask solution thoroughly. Final
concentrations are listed in Table 4 of Appendix B.
e. Once mixed, transfer to an acid-cleaned, labeled, 50-mL container
(PP, PMP, or Teflon™) for storage. Label appropriately (e.g. “Whole
Blood Metals Panel 2 Intermediate Stock Calibration Standard”, “3%
(v/v) HCl”, date of preparation, expiration date (1 year from date of
preparation), initials of preparer, and concentrations for each element).

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4. Storage: Store at room temperature. If purchased, label bottle with
additional information such as “store at room temperature”, date received,
date opened, and initials of person to first open.
j. Intermediate Working Calibration Standards
i. Purpose: Used each day of analysis to prepare the final five working
calibrators that will be placed on the autosampler.
ii. Content: Five aqueous dilutions of the intermediate stock calibration standard
solution with a 3% (v/v) hydrochloric acid (HCl) matrix. Final concentrations
are listed in Table 5 of Appendix B.
iii. Preparation & Storage: Different volumes may be prepared by adding
proportionally larger or smaller volumes of solution constituents.
1. Cleaning flasks: Acid-rinse five 100 mL, PP (or PMP) volumetric flasks.
Mark each flask according to intended use. Dedicate to purpose.
2. 3% (v/v) HCl Diluent Preparation: use the same 3% (v/v) HCl prepared in
Section 6.g.
3. Partially fill each 100 mL flask with the 3% (v/v) HCl diluent (50-75% full).
4. Add the correct volume of the Intermediate Stock Standard Calibration
Standard (according to Table 5)
a. If a separate Pb Intermediate Stock Calibrator is used, add the
appropriate volume of this solutions according to Table 5 to the same
flask.
5. Dilute to the volumetric mark with the 3% (v/v) HCl diluent using a pipette
for the final drops. Mix the flask solution thoroughly. Final concentrations
are listed in Table 5 of Appendix B.
6. Once mixed, transfer to acid-cleaned, labeled, 50 mL containers (PP, PMP,
or Teflon™) for storage. Label appropriately (e.g. “Whole Blood Metals
Panel 2 Intermediate Working Calibrators”, “3% (v/v) HCl”, date of
preparation, expiration date (1 year from date of preparation), initials of
preparer, concentration of each element, and Lot # of the stock solution).
7. Pour 10-15 mL of each solution into 15 mL tubes for daily use.
k. Working Calibration Standards
i. Purpose: The working calibration standards will be analyzed in each run to
provide a signal-to-concentration response curve for each analyte in the
method. The concentration of the analyte of interest in a patient blood sample
dilution is determined by comparing the observed signal ratio (element/internal
standard) from the dilution of the patient blood sample to the signal ratio
response curve from the working calibrators.
ii. Content: Dilutions (1:50) of the corresponding five intermediate working
calibration standards. The dilutions are described in Table 6 of Appendix B.

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iii. Preparation & Use: Make immediately prior to analysis when the intermediate
working calibration standards are mixed with base blood (Section 6.d) and
diluent (Section 6.c) using a Digiflex automatic pipette. See Table 6 of
Appendix B and Section 8.b.ii for details of sample preparation.
l.

Single-Element Stock Standards For Preparation of Intermediate Stock
Calibration Verification Standard
i. Purpose: These single-element standards will be used to prepare the
intermediate stock calibration verification standard.
ii. Contents: Separate, aqueous single-element standards of Cd, Pb, Hg, Se,
and Mn. Concentrations should be 1,000 mg/L or 10,000 mg/L.
iii. Purchase & Storage:
1. Purchasing from vendors: If the intermediate stock calibration verification
standard is purchased as a special-mix standard, these single-element
stock standards are not required. Purchase only NIST-traceable singleelement standards at the highest purity (don’t contain other metal
impurities).
2. Storage: Store at room temperature.

m. Intermediate Stock Calibration Verification Standard
i. Purpose: This multi-element solution will be used to prepare the three working
calibration verification standards.
ii. Preparation & Storage: This solution may be purchased as a special-mix
standard or prepared in-house from separate single-element stock standards.
1. Purchasing from vendors:
The intermediate stock calibration
verification standard may be purchased as custom mixture from any
vendor which prepares multi-element solutions that are traceable to
the National Institute for Standards and Technology (NIST) for their
accuracy.
2. In-house Preparation: Different volumes may be prepared by
adding proportionally larger or smaller volumes of solution
constituents.
a. Acid-rinse two 50 mL, PP (or PMP) volumetric flask. Mark the flasks
according to intended use. Dedicate to purpose.
b. Partially fill the 50 mL flasks with the 3% (v/v) HCl diluent (50-75% full).
c. Add necessary volumes of single-element stock standards to achieve
final concentrations listed in Table 7 of Appendix B.
d. Dilute to the volumetric mark with the 3% (v/v) HCl diluent using a
pipette for the final drops. Mix the flask solution thoroughly. Final
concentrations are listed in Table 7 of Appendix B.

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e. Once mixed, transfer to an acid-cleaned, labeled, 50-mL containers
(PP, PMP, or Teflon™) for storage. Label appropriately (e.g. “Whole
Blood Metals Panel 2 Intermediate Stock Calibration Verification
Standard (Cd, Mn, Hg)”, and “Whole Blood Metals Panel 2
Intermediate Stock Calibration Verification Standard (Pb)”, “3% (v/v)
HCl”, date of preparation, expiration date (1 year from date of
preparation), initials of preparer, and concentrations for each element).
3. Storage: Store at room temperature. If purchased, label bottle with
additional information such as “store at room temperature”, date received,
date opened, and initials of person to first open.
n. Intermediate Working Calibration Verification Standards:
i. Purpose: Used as needed to on the day of analysis to prepare the necessary
working calibration verification standard(s) that will be placed on the
autosampler.
ii. Content: Three aqueous dilutions of the intermediate stock calibration
verification standard with a 3% (v/v) hydrochloric acid (HCl) matrix. Final
concentrations are listed in Table 8 of Appendix B.
iii. Preparation & Storage: Different volumes may be prepared by adding
proportionally larger or smaller volumes of solution constituents.
1. Cleaning flasks: Acid-rinse three 100 mL, PP (or PMP) volumetric flasks.
Mark each flask according to intended use. Dedicate to purpose.
2. 3% (v/v) HCl Diluent Preparation: use the same 3% (v/v) HCl prepared in
Section 6.g.
3. Partially fill each 100 mL flask with the 3% (v/v) HCl diluent (50-75% full).
4. Add the correct volume of the Intermediate Stock Calibration Verification
Standard (according to Table 8 in Appendix B).
5. Dilute to the volumetric mark with the 3% (v/v) HCl diluent using a pipette
for the final drops. Mix the flask solution thoroughly. Final concentrations
are listed in Table 8 of Appendix B.
6. Once mixed, transfer to acid-cleaned, labeled, 50 mL containers (PP, PMP,
or Teflon™) for storage. Label appropriately (e.g. “Whole Blood Metals
Panel 2 Intermediate Working Calibration Verification Standard #”, “3%
(v/v) HCl”, date of preparation, expiration date (1 year from date of
preparation), initials of preparer, concentration of each element and Lot #
of the stock solution).
7. Pour 10-15 mL of each solution into 15 mL tubes for as-needed use.
o. Internal Quality Control Materials (“Bench” QC)
i. Purpose: Internal (or “bench”) quality control (QC) materials are used to
evaluate the accuracy and precision of the analysis process, and to determine
if the analytical system is “in control” (is producing results that are acceptably

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accurate and precise). They are included in the beginning and at the end of
each analytical run.
ii. Content: Pooled animal or human blood, and may have been spiked with
NIST-traceable elemental standards to reach desired low-normal and highnormal concentrations.
iii. Preparation & Storage: Quality control materials can be either prepared by
purchased from an external laboratory or prepared within the CDC
laboratories. Quality control must always be traceable to the National Institute
for Standards and Technology (NIST). The CDC laboratory currently prepares
its own bench QC materials using the following procedures:
1. Purchase of whole blood: Bags of human blood can be purchased from
various sources such as American Red Cross of Tennessee Blood
services (http://tennesseebloodservices.com/).
Animal blood may be
available from the Wisconsin State Laboratory of Hygiene (WSLH).
2. Screening blood:
Screen bags of blood for analyte of interest
concentration before mixing together to make 2 separate base blood pools
(for preparing the low and high bench QC materials). Samples can be
screened individually
a. Keep blood refrigerated whenever possible to minimize microbial
growth.
b. Because this is only a quick screen of the analyte of interest
concentration, the number of replicates in the blood method can be
reduced to one in order to reduce analysis time.
c. Analyte concentrations in the final blood pool to be spiked for the low
bench QC pool should be in the low-normal population range. Analyte
concentrations in the final blood pool to be spiked for the high bench
QC pool should be less than some pre-selected target concentration
values in the high normal population range. See Table 2 in Appendix
B for recommended concentration ranges.
3. Combining Collected blood: The goal is for combining samples is to
approach an ‘average’ matrix for each pool.
a. Graduate four acid-washed 10 L carboys (PP or PMP) in 0.5 L
increments (two will be used for decanting into).
b. Combine collected blood samples into two separate acid-washed 10 L
carboys (PP or PMP), according to their concentrations, for the low
bench and high bench QC pools.
c. Mix each blood pool using carboy stirrers and large stir plates. Keep
blood refrigerated whenever possible.
4. Spiking of blood
a. Analyze three samples of each blood pool. Record these results for
future recovery calculations.

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b. Use these results to determine target analyte concentrations possible
for the pools
c. Calculate the volume of single element standards needed to spike
each pool to the desired concentrations. See Table 2 in Appendix B
for recommended concentration ranges.
d. While stirring the pools on large stir plates, spike each pool with
calculated volumes of single element standards (all spiking standards
used must be traceable to NIST).
e. Continue to stir pools overnight after spiking, then reanalyze.
f. Repeat steps 4 and 5 until all analytes reach target concentrations
keeping track of the total volume of spiking solution added to each
blood pool.
5. Dispensing and Storage of blood
a. Container Types: Dispense blood into lot screened containers (i.e. – 2
mL polypropylene tubes). If possible, prepare tubes of QC which have
only enough volume for one typical run + 1 repeat analysis. This
allows for one vial of QC to be used per day of analysis, reducing
chances of contamination of QC materials due to multi-day use.
b. Labels: Place labels on vials after dispensing and capping if the vials
are originally bagged separately from the caps. This minimizes the
chance for contamination during the process. Include at least the
name of QC pool (text and bar code), date of preparation, and a vial
number on the labels.
c. Dispensing: Dispensing can be accomplished most easily using a
Digiflex automatic pipetter in continuous cycling dispense mode. This
process should be done in a clean environment (i.e. a class 100
cleanroom area or hood).
1. Allow blood to reach room temperature before dispensing (to
prevent temperature gradients possibly causing concentration
gradients across the large number of vials being dispensed
and to prevent condensation problems during labeling of
vials). This may require leaving the carboy of blood at room
temperature overnight before dispensing.
2. Replace the tubing attached to the dispensing syringe (left
when looking at front of Digiflex) with a length of clean
Teflon™ tubing long enough to reach into the bottom of the 10
L carboy while it is sitting on the stir plate.
3. Check cleanliness of Digiflex before use by analyzing 1-2%
(v/v) HNO3 which has been flushed through the Digiflex with a
portion of the same solution which has not been through the
Digiflex.

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4. Approximately one hour before dispensing begins,
a. With the large stir plate close to the left side of the Digiflex,
begin stirring the blood pool to be dispensed.
b. Also during this time, flush the Digiflex with blood from the
pool to be dispensed. Place the ends of the tubing
attached to both the sample and dispensing syringes into
the carboy of blood so that blood won’t be used up during
this process. Be sure to secure both ends of tubing in the
carboy with Parafilm so they will not come out during the
flushing process.
5. After dispensing the blood into the vials, cap the vials and
label them. Placing labels on vials after capping minimizes the
chance for contamination during the process.
ii.

Homogeneity Testing: After dispensing, check homogeneity of
analyte concentrations in pool aliquots. Keep samples pulled for
homogeneity analysis in the sequence that they were dispensed
for the purpose of looking for trends in concentrations. Once
dispensed and homogeneity has been shown to be good
throughout the tubes of a pool, store tubes at ≤ -20°C and pull
tubes out as needed for analysis.

iii.

Storage: Blood pools should be stored long term at ≤ -20°C. Short
term storage (several days) at refrigerator temperature (~ 2-4°C).

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a. Port 1: 1mL sample loop (white nut). See “Loop, for FAST valve” in
Section 5.b. for details.
b. Port 2: 0.5 mm ID probe (red nut) for carrier solution. See “Probes” in
Section 5.b. for details.
c. Port 3: nebulizer line (green nut) for transfer of liquid to nebulizer. See
“Nebulizers” in Section 5.b. for details.
d. Port 4: sample loop (white nut). See “Loop” in Section 5.b. for details.
e. Port 5: 0.8 mm ID probe (blue nut) for diluted samples. See “Probes”
in Section 5.b. for details.
f. Port 6: 1/8” i.d. vacuum line (black nut). See “Tubing, FAST vacuum”
in Section 5.b. for details.
2. Tubing connection between autosampler rinse station and rinse solution
reservoir: Tubing of different inner diameters can be obtained from
Elemental Scientific, their distributors, or custom built in the lab to optimize
the rinse station fill rate between samples. Rinse station should not go
empty at any point.
3. Tubing for autosampler rinse station waste removal: Use minimum drain
tubing to make this connection. If this tube is too long, the rinse station will
not drain properly.
4. Rinse solution jug: Leave one of the caps on the top of the rinse jug loose
to allow air venting into the jug as liquid is removed. Otherwise the jug will
collapse on itself as the liquid is removed and a vacuum is created inside.
Use secondary containment tray and label appropriately (see solution
preparation instructions).
5. Waste solution jug: Use secondary containment tray and label
appropriately (see solution preparation instructions).
6. Configuration of tubing and probe for carrier solution: Can use a PEEK
adapter to help with connecting peristaltic tubing to to Teflon tubing.
7. Nebulizer: Changing the nebulizer type will require re-optimization of the
read delay time in the ELAN software. Polypropylene nebulizer type (ESI)
tends to allow for shorter read delay times than quartz concentric
nebulizers.
8. Configuration of tubing for spray chamber waste removal:
a. Chamber-to-peristaltic pump tubing:
i. Spray chambers with threaded connection: Use vendor-supplied
threaded connector on base of chamber, connecting tubing directly
to peristaltic pump tubing through a PEEK adapter or directly.

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ii.

Spray chambers without threaded connection: Use push-on
connectors with Teflon tubing available from various vendors or
connect 1/8” i.d. x ¼” o.d. PVC tubing directly to the waste port on
the spray chamber. Connect other end of PVC tubing to the white
/ black peristaltic pump tubing using a tubing connector
(PerkinElmer item # B3140715).
b. Waste Jug-to-peristaltic pump tubing: Connect 1/8” i.d. x ¼” o.d. PVC
tubing to the white / black peristaltic pump tubing using a tubing
connector (PerkinElmer item # B3140715). Place the free end of the
PVC tubing through the lid of the waste jug (be sure it is secure).
Waste jug should be sitting in a secondary containment tray in case of
overflow.
iii. Cones: Platinum or Nickel cones have been used and tested to be
comparable in performance from either PerkinElmer or Spectron.
iv. Gases & Regulators setup:
1. Argon: Argon stored as liquid in a dewar (180-250 L) or bulk tank.
Gaseous argon used for plasma and nebulizer.
a. Regulator for argon source (if a dewar): Keep the inlet pressure
(headspace pressure of liquid argon dewar) above 100 psi. Set
delivery pressure to 90-100psi to allow for pressure drop across tubing
that stretches to the instrument. See Section 5.e for part numbers and
details.
b. Step down regulator (if source of argon is a bulk tank): Place this single
stage regulator in the lab so that incoming argon pressure can be
monitored and adjusted. Set delivery pressure to approximately 85 100 psig. See Section 5.e for part numbers and details.
c. Regulator at ICP-DRC-MS:
Single stage “argon regulator filter kit”
supplied with the ICP-DRC-MS.
Set the delivery pressure to
approximately 80 psi.
2. Methane (99.99%) gas for DRC channel A
a. Regulator for CH4 gas: Set the delivery pressure = 5-7 psig. See
section 5.e for part numbers and details.
3. Oxygen (99.999+%) gas for DRC channel B.
a. Regulator for O2 gas: Set the delivery pressure = 5-7 psig.
Section 5.e for part numbers and details.

See

b. Flash arrestor: Brass flash arrestor is used on outlet side of regulator.
See Section 5.e for part numbers and details.

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v. Chiller / Heat Exchanger: Refrigerated chiller (for ELAN® 6100 DRCPlus
instruments) or heat exchanger (for ELAN® DRC II instruments). For
refrigerated chiller, set temperature control to 18°C.
vi. Computer: Dell Optiplex GX150, GX270, or GX280 have all been used.
Processors used have included Pentium III (1 GHz) through Pentium IV (2.8
GHz). Recommend 512Mb - 1Gb RAM. External hard disk drive for nightly
backups of data connects via USB port. Software used includes Windows XP
Professional, service pack 2 and ELAN v3.3.
vii. Autosampler: ESI SC-FAST series
b. Parameters for Instrument and Method: See Appendix B Table 1 pp 52-54 for a
complete listing of the instrument and method parameters. Also, see Figures 1a1g in Appendix B for images of the ELAN method screens.
8) Method Procedures
a. Quality Control: Quality control procedures implemented in this method are
defined by the Division Procedures and Practices Guidelines and include two
types of QC systems which are both subjected to the complete analytical
process.
The data from these materials are then used to estimate
methodological imprecision and to assess the magnitude of any time-associated
trends. The concentrations of these materials should cover the expected
concentration range of the analytes for the method. Before QC materials can be
used to judge patient analytical runs, acceptable QC concentration limits must be
calculated from the concentration results observed in at least 20 characterization
runs. During the 20 characterization runs, previously characterized QCs or pools
with target values assigned by outside laboratories should be included to
evaluate the analysis. The process of limits calculation is performed using the
laboratory database and the SAS division QC characterization program.
i. Types of Quality Control:
1. “Bench QC”: The bench QC pools used in this method comprise two levels
of concentration spanning the “low-normal” and “high-normal” ranges of the
analyte of interest. The intent of bench QC is for the analyst to evaluate
the performance of the analytical system on the day of analysis. The
analyst inserts both the “low” and the “high” bench QC specimens two
times in each analytical run (a set of consecutive assays performed without
interruption) so that judgments may be made on the day of analysis. The
first analysis of the two bench QC pools is done after the calibration
standards are analyzed but before any patient samples are analyzed (so
that judgments on the calibration curves may be made before analysis of
patient samples). The second analysis of the two bench QC pools is done
at the end of the run (approximately 20 patient samples total). If more
patient samples are analyzed on the same calibration curve after the
second run of the bench QC, both the low-normal and high-normal bench

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QC must be reanalyzed before and after the additional samples. For
example, the schemes shown in Table 9 p.67 are both acceptable ways to
analyze multiple consecutive “runs”.
2. “Blind QC”: When possible, “blind” QC samples are QC materials placed in
vials, labeled, and processed so that they are indistinguishable from the
subject samples handled by the analyst. Ideally, the supervisor decodes
and reviews the results of the blind specimens without the analyst knowing
of their presence in the runs. When it is not possible to have blind QC
materials processed so that they are indistinguishable by the analyst from
the patient samples, it is acceptable for the analyst to randomly insert into
the run a QC material which only the QC reviewer knows the acceptable
concentration limits for. At least one low-normal concentration and one
high-normal concentration QC material should be kept in the laboratory for
this purpose.
3. External Reference Materials: Materials produced by laboratories outside
of the CDC which have assigned target concentrations can be helpful in
verifying method performance. Samples from previous challenges of
proficiency testing programs (i.e. Centre de Toxicologie du Quebec (CTQ))
can be used. However, only the results for the bench and blind QC
materials are used to determine if the run results can be used.
ii. Calibration Verification:
1. Bi-annual tests as defined in the DLS Policy and Procedures manual:
CLIA requires the verification of accuracy of instrument response to analyte
concentration be completed at least every 6 months. NIST traceable
calibrators are analyzed in each run to define this response up to the
concentration of the highest calibrator in the run. To verify accuracy of
instrument response at concentrations higher than the highest calibrator in
each run, analyze a NIST traceable standard with very high concentrations
(see Table 8 p.66 in the Appendix for concentrations) at least every 6
months. Prepare the Calibration Verification Standard for analysis just as a
working calibrator is prepared. Use the “Blank” as the blank when it is
analyzed. If the observed concentrations for the Calibration Verification
Standard are not within 10% of the target value (see Table 8 p.66 in the
Appendix) the lab supervisor should be notified and the issue should be
investigated. Do not substitute external reference materials (i.e. biological
samples from a PT program) for the Calibration Verification Standard when
performing this. Solutions needed for the Calibration Verification checks
can be purchased from standards vendors (i.e.SPEX, High Purity
Standards, etc . . .) or prepared in-house from NIST traceable single
element standards. Always verify that normal background levels have
been re-achieved through adequate rinse time following analysis of
elevated standards for calibration verification.
a. As-needed confirmations (per supervisor discretion): When a sample
result is greater than the highest calibrator in the run, the supervisor

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may request that the result be confirmed in an analysis run which
includes a standard or external reference material with equivalent
(within 10%) or greater concentration than the sample. In order to
avoid needless contamination of the instrument with high
concentrations of analytes, the analyst should use the lowest
appropriate calibration verification solution concentrations to meet the
need.
For infrequent verification needs, the calibration verification stock
solutions can be used to prepare verification standards to appropriate
concentrations. This will, however, introduce elevated concentrations
of manganese to the sample introduction system.
Frequent
measurement of these very high concentrations can result in high
background levels in the instrument which are difficult to rinse out and
which may limit the ability to measure low concentrations.
For frequent verification needs (i.e. when certain studies have many
elevated results) or when a concentration higher than those shown in
Table 8 p.66 needs to be verified, use NIST-traceable single element
stock standards to prepare single element verification standards. This
will limit the exposure of the instrument to elevated concentrations of
only the elements needing verification.
Always verify that normal background levels have been re-achieved
through adequate rinse time following analysis of elevated standards
for calibration verification. An external reference material (i.e. historical
proficiency testing sample) can be substituted in place of the
Calibration Verification Standard sample in these situations IF:
i.

The target value has been assigned by an external source (i.e.
NIST, or the proficiency testing program).

ii.

The concentration of the external reference material is within 10%
or is higher than the concentration of the material you need it to
confirm.

iii.

There is confidence that there is no contamination of previously
used external reference material.

iv.

A note to file is made that this was done.

If the observed concentrations are not within 10% of the target
value the lab supervisor should be notified and the issue should be
investigated.

b. Daily Analysis of Samples
i. Preparation of the Analytical Equipment
For further details on any part of this description, see the IRAT Daily Startup
SOP for ELAN ICPMS instruments.

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1. Power on the computer, printer, peristaltic pump, and autosampler, and log
into the operating system.
2. Peristaltic pump: Set up the peristaltic pump tubing with proper tension for
the sample rinse station.
3. Software: Start the ESI autosampler and ELAN® ICPMS software from
Windows™.
4. Daily Pre-Ignition Maintenance Checks: Perform daily maintenance checks
as described in the IRAT Daily Startup SOP for ELAN instruments (i.e., Ar
supply pressure, interface component cleanliness and positioning, interface
pump oil condition, vacuum pressure, etc.). Make appropriate notes in the
Daily Maintenance Checklist and Instrument Log Book.
5. Start the Plasma: Press the “Start” button in the software or on the
hardware to ignite the plasma.
a. Start the peristaltic pump: Start the peristaltic pump in the software at
12rpm (using a standard 6 roller peristaltic pump) or 5.4rpm if using a
DXi mini-peristaltic pump. Verify the rotational direction is correct.
6. Aspirate liquid: Place the carrier probe into dilute acid or water.
7. Warm-up time: Allow at least 45 minutes warm-up time for the ICP-DRCMS after igniting the plasma. This warm-up time is for the RF generator.
There will be another “Stability time” for the DRC later in this procedure.
8. Daily Performance Check: After this warm-up time, perform a daily
performance check and any optimizations necessary (as described in the
IRAT Daily Startup SOP for ELANs). Fill in the Daily Maintenance
Checklist according to the optimization procedures performed. Extra detail
than can be documented in the checklist should go into the instrument
logbook.
a. Magnesium (24Mg) may have high RSDs due to the use of Triton-X100
in the rinse solution. Avoid this problem by either temporarily using
non-Triton-containing rinse solution during the daily check, or repeating
the daily check multiple times in succession with no rinse time
between.
b. Saving the Files: Save updated tuning and optimization parameters to
the “default.tun” and “default.dac” files, respectively.
9. Software setup for Analysis:
a. Workspace
(files
&
folders):
Open
the
default
(“CDC_WBMP2_methITB006A_.wrk”) or your own personalized
workspace in the ELAN software. Verify & set up the correct files and
data directories for your analysis (See Table 1 pp 60-62 “File Names &
Directories“).
b. Samples / Batch Window: Update software to reflect the current
sample set. The only fields which need to be filled in include the

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autosampler location, sample identification (id), measurement action,
method, sample flush time, sample flush speed, read delay time, read
delay & analysis speed, wash time, wash speed. Use a bar code
scanner to input data whenever possible. See Table 1 pp 60-62 for
times and speeds. Save the Sample window file and re-use it on other
days by simply replacing the sample IDs for the patient samples.
1. DRC Stability Time: Best analyte-to-internal standard ratio
stability is typically obtained after 1-1.5 hrs of repeated
analysis of blood samples using the DRC method. Analyze
enough “dummy” blood sample dilutions prior to any DRC
analysis run to fill 1-1.5 hours of analysis time. See Table 10
p.68 for example of setup in the Samples / Batch window.
2. Blood vs. Aqueous Method Files:
a. The difference: There are two ELAN method files for this
one method (see Table 1 pp 61-62). It is necessary to use
both to accomplish each run because the current
PerkinElmer software will not allow for more than one blank
per method file. The ONLY DIFFERENCE between these
two files is on the Sampling tab where one lists the
autosampler positions of the blood blank and blood
calibrators (the “bldblk” method file) and the other lists the
autosampler position of the aqueous blank (the “aqblk”
method file).
b. Use: The ONLY TIME when it matters which of these files
is used is when the measurement action includes “Run
blank” or “Run standards”. When the measurement action
is only ‘run sample’, it does not matter whether the “bldblk”
or “aqblk” method file is used. Analysts typically follow the
pattern below, however, for the sake of consistency and as
a reminder of which blank must be used for which type of
sample. See Table 10 p.68.
i. The “bldblk” method file: Use to analyze the initial
blood blank (blank for the calibration curve), the blood
calibrators, and the blood blank checks (WB Blank &
WB Blank 2) at the very beginning of the run. The
blood blank method defines the blood blank in
autosampler location 105 and the blood calibration
standards 1-5 in autosampler locations 106-110,
respectively.
ii. The “aqblk” method file must be used to analyze all QC
materials and patient samples. The aqueous blank
method (set up for a ESI SC4 autosampler) defines the
aqueous blank in autosampler location 113.

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3. Notation of Dilutions: To designate an extra dilution of a
sample, edit the sample ID to reflect the level of dilution being
performed (e.g., A 1:2 dilution of sample 1 would be reflected
in the sample ID “sample 1 (2x dilution)”. This sample ID will
be edited during the data-import process to the database so
that it is recognized as the appropriate sample. Do not use
the ELAN® software to automatically correct for sample
dilutions. Extra dilution is performed on blood samples whose
concentration is greater than the concentrations listed in Table
8 in Appendix B (linearity of the method has been documented
up to these concentrations).
c. Method file modifications: This method can also be used to analyze
whole human blood samples for a subset of the listed samples (i.e. Pb
only). To do this delete the unnecessary elements from the method
windows (bldblk and aqbkl) and save the file with a descriptive name
such
as
“WBMP2_DLS3016_bldblk_Pb_only.mth”
and
“WBMP2_DLS3016_aqblk_Pb_only.mth”.
ii. Preparation of Samples for Analysis (See Table 6 in Appendix B)
1.

Thaw the frozen blood specimens; allow them to reach ambient
temperature.

Prepare enough DRC stability sample to be analyzed for 1-1.5 hr before
the beginning of the run.
This can be prepared using 50 mL
polypropylene tubes or a wide-mouth bottle (which can be put on the
autosampler in place of one of the tube trays).

Set up a series of 15 mL polypropylene tubes corresponding to the
number of blanks, standards, QCs, and patient samples to be analyzed.

Prepare the following solutions in the 15 mL falcon tubes using the
Digiflex™ (see Table 6 p.65 for a summary).
a. Aqueous Blank: Prepare two aqueous blanks consisting of 200 L of
>18 Mega-ohm·cm water and 4800 L of diluent. One will be the
actual aqueous blank and the other will be a backup (“Aqueous Blank
Check”) in case the original aqueous blank gets contaminated.
b. Blood Blank (Std 0): Prepare three blood blank dilutions consisting of
100 L of base blood (same material used to prepare the blood
calibration standards), 100 L of > standard zero (3% (v/v) HCl), and
4800 L of diluent. One of these blood blanks will be the blank for the
calibration standards; the other will be analyzed after standard 5 as
BldBlkChk.
c. Calibrators: Prepare the working calibration standards as 100 L of
the appropriate aqueous intermediate working calibration standard,
100 L of base blood, and 4800 L of diluent.

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d. Patient & QC Samples: Before taking an aliquot for analysis, mix the
sample on the vortex for approximately 15 seconds. Prepare blood
sample dilutions as 4,800 L of diluent and 100 L of the blood sample
and 100 L of >18 Mega-ohm·cm water.
e. Cap all of the blanks, standards, and samples and mix them on the
Vortex for approximately 10 seconds. Uncap them and place them in
the autosampler of the ELAN® ICPMS in the order that was entered in
the Samples / Batch window of the ELAN software.
iii. Specimen Storage and Handling During Testing: Specimens may be left at
room temperature during analysis in case confirmation analyses must be
made. Take stringent precautions to avoid external contamination by the
metals to be determined. Specimens may be stored short term at refrigerated
temperatures, but should be stored long term (>4 weeks) at ≤ -20 °C.
NOTE: Samples must be analyzed within 24 hours of preparation to obtain
valid results for selenium. The method has been validated to produce valid
results for other Pb, Cd, Hg, and Mn even 48 hrs after sample preparation.
See critical parameter test #1 in Appendix A for details.
iv. Starting the Analysis: Begin the analysis using the ELAN software.
v. Monitoring the Analysis: It is preferable to initiate work early enough in the day
to permit the entire run to be monitored. If it is not possible to complete the
analysis by the end of the work day, the run may be left to complete itself
unattended as long as appropriate planning is made for either overnight
operation or Auto Stop (see below).
Monitor the analysis for the following:
1. DRC stability (analyte / internal standard ratio stability)
After the analysis of the DRC stability “dummy” samples, the stability of the
analyte / internal standard ratios across these samples indicates the
instrument stability going into the run.
2. Proper operation of the instrument.
3. Contaminated blanks.
4. Linear calibration curves.
a. Typical correlation coefficients will be 0.999 to 1.000.
b. The ELAN software generates a “simple linear” calibration curve (using
a least squares calculation) for manganese in this method. The curves
are generated using the results from analysis of the blood blank and
the 5 external blood calibrators whose concentrations are defined in
the Calibration tab of the Method file. Specifically, the software plots
the “net intensity” (y-axis) versus the analyte concentration (x-axis).
The “net intensity” is the blank subtracted ratio of the measured

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intensity for the analyte to the measured intensity of the associated
internal standard and is calculated as follows:

net int ensity 

Analyte Meas Intensity sample
Internal Std Meas. Intensity sample



Analyte Meas Intensity Blank
Internal Std Meas Intensity Blank

`
c. Points (1-2) may be removed from the calibration curve if necessary to
provide appropriate correlation coefficients. It is preferable, however,
to re-analyze problematic calibration standards rather than dropping
points. Recurring problems with calibration standards should be
resolved expeditiously.
5. Bench QC results within the acceptable limits.
If an analyte result for the beginning QC material(s) falls outside of the 99%
limits, then the following steps are recommended:
a. If a particular calibration standard is obviously in error, remake a new
dilution at the Digiflex of that working calibrator, reanalyze it, and
reprocess the sample analyses using this new result as part of the
calibration curve.
b. Prepare a fresh dilution of the failing QC material and reanalyze it.
c. Prepare fresh dilutions at the Digiflex of all of the calibration standards
(working blood multi-element standards) and reanalyze the entire
calibration curve using the freshly prepared standards.
If these three steps do not result in correction of the out-of-control values
for QC materials, consult the supervisor for other appropriate corrective
actions. Do not report analytical results for runs that are not in statistical
control.
6. Good precision among replicates.
7. Consistent measured intensities of the internal standards.
Some sample-to-sample variations are to be expected. However the
intensities should be within a few percent of one another, and should
fluctuate around an average value (not drift continuously in one direction).
8. Elevated patient results: Confirmation by repeat measurement will be
required for any result greater than the “1st upper boundary” (see Section
8.b.viii.2). A calibration verification check of equal or greater concentration
must be analyzed in the same run as the elevated study sample result if it
is to be used for reporting (see Section 8.a.ii.2).
vi. Records of Results: Run results will be documented daily in both electronic
and paper form.
1. Electronic Records:

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a. Transfer of Results to the Laboratory Information System / Database:
Transfer data electronically between computers or software to reduce
errors. When keyboard entry must be used, proofread transcribed
data after entry.
b. Long-Term Storage of ELAN software files: Files used and produced
by the ELAN software in analyzing samples will be backed up long
term on compact disk and kept a minimum of three years.
2. Paper Records: The paper copy of the results from the run should be put
into the study folder(s) and should include
a. A summary of the calibration curve statistics.
b. A printout of analysis of each measurement made during the run.
c. On the front sheet of the printed records, write the following
i.

Analyst initials

ii.

Instrument ID

iii.

Date of Analysis

iv.

Run # for the day on this instrument

Study ID and Group Number

vi.

Database batch ID (Not known until the run is imported into the
database)

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c. Enter the appropriate information to identify the instrument, assay,
analysis date & time, run number, analyst, calibrator lot number and
prep date used (use the “IS Lot Number” field) and study. If other than
default values for Method LOD, High Calibrator, Rep Delta Limit, and
units were used in the run, document accordingly.
d. In the “Import Instrument Results” table, correct sample IDs and
document dilution factors if dilution factor notations were added to the
ID in the ELAN software prior to analysis.
e. Once transferred into the database, the data should be evaluated for
QC pass / fail, then appropriate settings entered for QC accept / reject,
final value status, and comments.
viii. Analyst Evaluation of Run Results:
1. Bench Quality Control: After completing a run, and importing the results
into the LIMS, export the QC results to the SAS program where the
analytes in the run will be judged to be in or out of control. The QC limits
are based on the average and standard deviation of the beginning and
ending analyses of each of the bench QC pools, so it will not be possible to
know if the run is officially accepted or rejected until it is completed.
a. Quality Control Rules: The SAS program applies the division QC rules
to the data as follows:
i.

If both QC run means (low & high bench QC) are within 2Sm limits
and individual results are within 2Si limits, then accept the run.

ii.

iii.

If one of the 4 QC individual results is outside a 2Si limit - reject
run if:
1. R 4S Rule – Within-run ranges for all pools in the same run
exceed 4Sw (i.e., 95% range limit)

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Sm = Standard deviation of the run means (the limits are shown on the
chart).
Sw = Within-run standard deviation (the limits are not shown on the
chart).
iv.

Implications of QC Failures: If the division SAS program declares
the run out of control”, then all results from the run are invalid for
reporting from the run. Set all run results as “QC Rejected” in the
database.

2. Patient Results:
a. Elevated Results:
i.

Boundaries Requiring Confirmatory Measurement:
1. Results Greater than the First Upper Boundary (1UB):
Concentrations observed greater than the “first upper
boundary” (defined in the laboratory database as the “1UB”)
should be confirmed by repeat analysis of a new sample
preparation. The concentration assigned to the 1UB for an
element is determined by study protocol but default
concentrations are in Table 11 p.69 in the Appendix. Report
the original result, as long as the confirmation is within 10% of
the original.
Continue repeat analysis until a concentration
can be confirmed.
2. Results Greater Than Highest Calibrator: When a sample
result is greater than the highest calibrator in the run, the
supervisor may request that the result be confirmed in an
analysis run which includes a standard or external reference
material with equivalent (within 10%) or greater concentration
than the sample.
3. Results Greater Than Calibration Verification Standard:
Perform an extra dilution on any blood sample whose
concentration is greater than those listed in Table 8 p.66 in the
Appendix (the linearity of the method has been documented up
to these concentrations). See Table 6 p.65 for description of
sample preparation with extra dilution.

ii.

iii.

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concentrations are in Table 11 of Appendix B. The analyst should
report any patient results confirmed to be greater than the second
upper boundary to the QC reviewer as an “elevated result”. There
is no routine notification for elevated levels for the metals
determined in this method. The protocol for supervisors reporting
elevated results to medical personnel is defined according to the
study protocol.
b. Inadequate Precision Within One Measurement: If the range of the
three replicate readings (maximum replicate concentration value minimum replicate concentration value) for a single sample analysis is
greater than the criteria listed in Table 11 of Appendix B (“>Lim Rep
Delta” in the database) and the range of the three replicate readings is
greater than 10% of the observed concentration, do not use the
measurement for reporting. Repeat the analysis of the sample.
ix. Submitting final work for Review: Once results have been imported, reviewed,
and set as final in the database by the analyst,
1. Submit an email to the QC reviewer informing them of the readiness of the
data for final review. The email should follow requirements specified by the
QC reviewer and will include:
a. Instrument ID, run Date, run number, study ID, group ID.
b. Any bench QC failures (include reasons if known).
c. Any patient sample result greater than the 2UB boundaries (see Table
11 in Appendix B).
d. Anything out of the ordinary about this analytical work which could
have a bearing on the availability (i.e. insufficient sample to analyze),
accuracy, or precision of the results.
2. Include all items called for by the study folder cover sheet in the study
folder (i.e. printouts from the ICP-MS, bench QC evaluation) together in the
study folder before submitting the folder for review when analysis is
complete.
x. Overnight operation or Using Auto Stop: Make every effort to complete
analysis within the work day so that the entire run can be monitored. If it is not
possible to complete the analysis by the end of the work day, the run may be
left to complete itself unattended as long as appropriate planning is made for
either overnight operation or Auto Stop.
1. 24 hrs / day operation in DRC mode:
a. To reduce startup time in the mornings, the analyst is encouraged to
operate the ELAN in DRC mode 24hrs/day during the work week. This
eliminates the need for daily 45 minute RF generator warm-up, and
possibly the need for DRC stability time (if the DRC gas is not off for
extended periods of time before analysis). To maintain the instrument
in DRC mode when not analyzing patient samples, setup multiple

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sample rows in the Samples / Batch window with autosampler position
in zero (rinse station of autosampler) and wash time of 1800s (30
minutes).
Repeat this sample row enough times to keep the
instrument in analysis mode overnight (1 sample with 15 minute wash
will take ~ 25 minutes).
2. AutoStop: If 24 hrs / day ELAN operation is not desired, the instrument
can shut the plasma off unattended after analysis. Setup this as follows:
a. On the “Auto Start / Stop” tab of the Instrument window, enable the
Auto Stop feature.
b. Press the “Change” button within the Auto Stop box and set the
Delayed shutdown time to 5 minutes. This will rinse the sample
introduction system of blood matrix before turning off the plasma.
c. It will be necessary to replace the sample peristaltic pump tubing the
next day since it will have been clamped shut overnight.
c. Equipment Maintenance: Analysts are expected to follow a 4-day analysis / 1day maintenance schedule in the laboratory.
i. ICPMS Maintenance: On the maintenance day, perform all maintenance per
the IRAT ELAN ICP-MS Weekly Maintenance SOP.
All equipment
maintenance should be documented in the instrument checklist and logbook.
ii. Data Backup: Data on the ELAN computer will be backed up via two backup
routines.
1. Daily Backups to External Hard Drive: Automatic backups of the “elandata”
directory and all subdirectories should be programmed to occur each night
onto an external hard disk using a three-file rotating backup scheme.
2. Weekly Backup to CD: Backup all files in the active “elandata” directory
and all subdirectories onto one recordable compact disc during the weekly
maintenance SOP. When the active “elandata” directory on the ICP-DRCMS computer hard drive becomes too large to fit onto a single recordable
compact disk, the oldest data can be removed from the computer to make
it easier to backup the entire directory weekly. This can usually be done
annually.
a. Backup the oldest data on the hard drive to two duplicate compact
disks and verify that the files on the CD are readable
b. Label them with the name of the instrument, the date range of the data,
the current date, your name, and “Copy 1 of 2” or “Copy 2 of 2”
c. After verifying that the CDs are readable, the oldest, backed up data
can be deleted from the ICP-MS computer hard drive.
d. It is best to not store duplicate copies in the same location.

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9) Interpretation of the Results
a. Reportable Range: Whole blood metals values are reportable in the range
between the method LOD (see Section 10.a) and the highest concentration
verified accurate by bi-annual calibration verification tests (see Appendix, Table 8
in Appendix B). For example, if a blood metals concentration is less than the
method LOD, report it as < LOD. Above the highest concentration verified, extra
dilutions are made of the blood sample to bring the concentration within the
verified range.
b. Reference Ranges (Normal Values): See Appendix B, Table 12.
c. Action Levels: Concentrations observed greater than the “second upper
boundary” (defined in the laboratory database as the “2UB”) should be reported
to the QC reviewer as an “elevated result”. The concentration assigned to the
2UB for an element is determined by study protocol but default concentrations
are listed in Table 11 in Appendix B. The analyst should report any patient
results confirmed to be greater than the second upper boundary to the QC
reviewer as an “elevated result”. The protocol for supervisors reporting elevated
results to medical personnel is defined according to the study protocol. Levels of
concern for mercury in blood are >100 g/L for children (6 yr and younger) and
>200 g/L for adults. Levels of concern for lead in blood are 25 g/dL for
children (6yr. and younger) and 40 g/dL for adults. Levels of concern for
cadmium in blood is >5 g/L.
10) Method Calculations
a. Method Limit of Detection (LODs): The method detection limits for elements in
blood specimens are defined as 3 times s0, where s0 is the estimate of the
standard deviation at zero analyte concentration. S0 is taken as the y-intercept of
a linear or 2nd order polynomial regression of standard deviation versus
concentration (4 concentration levels of the analytes in blood each measured 60
times across at least a 2-month timeframe). Method LODs are re-evaluated
periodically.
b. Method Limit of Quantitation (LOQ): The Division of Laboratory Sciences does
not currently utilize limits of quantitation in regards to reporting limits [58].
c. QC Limits: Quality control limits are calculated based on concentration results
obtained in at least 20 separate runs. It is preferable to perform separate
analyses on separate days and using multiple calibrator lot numbers,
instruments, and analysts to best mimic real-life variability. The statistical
calculations are performed using the SAS program developed for the Division of
Laboratory Sciences (DLS_QC_compute_char_stats.sas).
11) Alternate Methods for Performing Test and Storing Specimens If Test System
Fails:
If the analytical system fails, the analysis may be setup on other ELAN DRC
instruments in the laboratory. If no other instrument is available, store the
specimens at 4°C until the analytical system can be restored to functionality. If

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interruption longer than 4 weeks in anticipated, then store blood specimens at ≤ 20°C.

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Appendix A: Critical Parameter Test Results
Critical Parameter Test #1: This test documents that accurate results are attainable if
something prevents a set of prepared samples from being analyzed immediately (per
method). Samples which have been diluted 1+1+48 for analysis up to one (1) day (~29
hours) previously can still be analyzed. Results are presented in Table 1.
Test Details:
Day 1: Prepare a set of dilutions (calibrators, blanks, reference material, fake samples)
for analysis in triplicate (three separate sets of tubes).Analyze the first set
immediately (normal practice). Cap sets #2 and #3 and leave at room
temperature for later analysis.
Day 2: Prepared run set #4 and analyzed it sequentially with run set #2 using normal
method practices.
Day3: Prepared run set #5 and analyzed it sequentially with run set #3 using normal
method practices.

QMEQAS
10B-06*

QMEQAS
07B-03*

HB08708
_WB2

LB08707
_WB2

Table 1. Ruggedness Testing Results: Evaluating the significance of time from preparation
to analysis on sample stability. Test performed on December 6 – 8, 2010 by Deanna Jones.
Results below are the average of the beginning and ending QC results for each analytical
run.
Time, prep to
ID
Hg (µg/L)
Pb (µg /dL)
Cd (µg /L)
Mn (µg /L) Se (µg /L)
analysis
Target Mean
0.585
2.12
0.488
7.98
0.407
–
0.763
2.03
–
2.21
0.398
–
0.578
6.91
– 9.05
and 2SD Range
0.418
2.03
0.399
6.09
0 hr
0.504
1.99
0.419
7.06
24 hr
0.396
2.04
0.509
7.82
48 hr
Target Mean
6.19
10.1
3.14
14.9
5.89 – 6.48
9.84 – 10.3
2.94 – 3.34
13.5 – 16.4
and 2SD Range
5.86
10.0
3.03
12.5
0 hr
5.46
9.5
2.85
13.6
24 hr
2.64
9.2
2.79
13.5
48 hr
Target Mean
228
213 - 243
and 2SD Range
192
0 hr
202
24 hr
56
48 hr
Target Mean
239
223 - 255
and 2SD Range
212
0 hr
221
24 hr
62
48 hr
*samples purchase from Le centre de toxicology du Quebec (Quebec, Canada)

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 55 of 88

Appendix A: Critical Parameter Test Results (Continued)
Critical Parameter Test #2: This test evaluated the significance of the RF Power setting
of the ICP when analyzing blood samples for whole blood metals. The RF Power
setting per method is 1450W. The reduced and elevated settings tested are 1150W
and 1600W, respectively. Results are presented in Table 2.
Test Details:
1. Prepare a set of dilutions (calibrators, blanks, reference material, dummy samples)
for analysis in triplicate (three separate sets of tubes).
2. Analyze them in three separate runs on the same day, same instrument.
3. Change the RF Power across the runs
4. Allow 15 minutes equilibration time between runs for RF Power to stabilize

QMEQAS08
B-08*

QMEQAS08
B-02*

HB08708_W
B2

LB08707_W
B2

Table 2. Ruggedness Testing Results: Evaluating the significance of RF Power setting on
sample stability. Test performed on December 6 and December 10, 2010 by Deanna Jones.
Results below are the average of the beginning and ending QC results for each analytical run.
ID
RF Power (W)
Hg (µg /L)
Pb (µg /dL)
Cd (µg /L)
Mn (µg /L)
Se (µg /L)
Target Mean
0.585
2.12
0.488
7.98
and 2SD Range 0.407 – 0.763 2.03 – 2.21 0.398 – 0.578 6.91 – 9.05
0.517
2.09
0.432
7.35
1150 W
1450 W
0.512
2.03
0.369
6.76
(per method)
0.529
2.02
0.418
7.17
1600 W
Target Mean
6.19
10.1
3.14
14.9
5.89 – 6.48
9.84 – 10.3
2.94 – 3.34
13.5 – 16.4
and 2SD Range
5.90
10.0
2.93
13.7
1150 W
1450 W
6.23
10.2
2.90
12.8
(per method)
5.99
10.1
3.07
13.3
1600 W
Target Mean
293
273
- 313
and 2SD Range
269
1150 W
1450 W
288
(per method)
314
1600 W
Target Mean
165
154 - 176
and 2SD Range
179
1150 W
1450 W
147
(per method)
146
1600 W
*samples purchase from Le centre de toxicology du Quebec (Quebec, Canada)

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 56 of 88

Appendix A: Critical Parameter Test Results (Continued)
Critical Parameter Test #3: This test evaluated the significance of the dynamic reaction
cell gas flow rate of the reaction gas (oxygen and methane) while analyzing blood
samples for elements analyzed in DRC mode (Hg, Mn, and Se). The cell gas flow rate
for Mn and Hg is methane (CH4) and the per method setting is 1.2 mL/min. The cell gas
flow rate for Se is oxygen (O2) and the per method setting is 0.84 mL/min. The reduced
and elevated settings for O2 are 0.96 mL/min and 1.44 mL/min, respectively. The
reduced and elevated settings for CH4 are 0.7 mL/min and 1.0 mL/min, respectively.
The Results are presented in Tables 3 and 4.
Test Details:
1. Prepare a set of dilutions (calibrators, blanks, reference material, dummy samples)
for analysis in triplicate (three separate sets of tubes).
2. Analyze them in three separate runs on the same day using the same instrument.
3. Change the cell gas flow rate.

HB08708_WB2

LB08707_WB2

Table 3. Ruggedness Testing Results: Evaluating the significance of dynamic reaction cell
gas flow rate on sample stability. Test performed on December 6, 2010 and January 4, 2010
by Deanna Jones. Results below are the average of the beginning and ending QC results for
each analytical run.
Cell Gas
ID
Hg (µg /L)
Pb (µg /dL)
Cd (µg /L)
Mn (µg /L) Se (µg /L)
Flow Rate
Target Mean
0.585
2.12
0.488
7.98
0.407
–
0.763
2.03
–
2.21
0.398
–
0.578
6.91
– 9.05
and 2SD Range
0.96 mL/min O2;
0.457
2.10
0.471
8.49
0.7 mL/min CH4
1.2 mL/min O2;
0.479
2.10
0.438
8.15
0.84 mL/min CH4
1.44 mL/min O2;
0.555
2.11
0.457
8.12
1.0 mL/min CH4
See
Table 4
Target Mean
6.19
10.1
3.14
14.9
5.89 – 6.48
9.84 – 10.3
2.94 – 3.34
13.5 – 16.4
and 2SD Range
0.96 mL/min O2;
4.71
10.0
3.19
14.4
0.7 mL/min CH4
1.2 mL/min O2;
5.45
10.1
2.92
15.2
0.84 mL/min CH4
1.44 mL/min O2;
5.34
10.3
3.04
14.6
1.0 mL/min CH4

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 57 of 88

Appendix A: Critical Parameter Test Results (Continued)

QMEQAS08B-02*

QMEQAS07B-09*

Table 4. Ruggedness Testing Results: Evaluating the significance of dynamic reaction cell
gas flow rate on sample stability. Test performed on December 6, 2010 and January 4, 2010
by Deanna Jones. Results below are the average of the beginning and ending QC results for
each analytical run.
Cell Gas
ID
Hg (µg /L)
Pb (µg /dL)
Cd (µg /L)
Mn (µg /L) Se (µg /L)
Flow Rate
Target Mean
157
146
- 168
and 2SD Range
0.96 mL/min O2;
187
0.7 mL/min CH4
1.2 mL/min O2;
186
0.84 mL/min CH4
1.44 mL/min O2;
191
1.0 mL/min CH4
See
Table 3
Target Mean
293
273 - 313
and 2SD Range
0.96 mL/min O2;
328
0.7 mL/min CH4
1.2 mL/min O2;
334
0.84 mL/min CH4
1.44 mL/min O2;
339
1.0 mL/min CH4
*samples purchase from Le centre de toxicology du Quebec (Quebec, Canada)

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 58 of 88

Appendix A: Critical Parameter Test Results (Continued)
Critical Parameter Test #4: This test evaluated the significance of the RPq value while
analyzing blood samples for Se, Mn and Hg. The RPq value setting per method for Mn
and Hg is 0.6, and for Se it is 0.65. The reduced and elevated RPq values for Mn an Hg
are 0.48 and 0.72, respectively. The reduced and elevated RPq values for Se are 0.52
and 0.78, respectively. The results are presented in Tables 5 and 6.
Test Details:
1. Prepare a set of dilutions (calibrators, blanks, reference material, fake samples) for
analysis in triplicate (three separate sets of tubes).
2. Analyze them in three separate runs on the same day, using the same instrument.
3. Change the RPq value.
Table 5. Ruggedness Testing Results: Evaluating the significance of RPq value on sample
stability. Test performed on December 21, 2010 by Deanna Jones. Results below are the
average of the beginning and ending QC results for each analytical run.

HB08708_WB2

LB08707_WB2

RPq

Hg (µg /L)

Pb (µg /dL)

Cd (µg /L)

Mn (µg /L)

Target Mean
and 2SD Range
0.48 Mn and Hg;
0.52 Se
0.6 Mn and Hg;
0.7 Se
0.72 Mn and Hg;
0.78 Se
Target Mean
and 2SD Range
0.48 Mn and Hg;
0.52 Se
0.6 Mn and Hg;
0.7 Se
0.72 Mn and Hg;
0.78 Se

0.585
0.407 – 0.763

2.12
2.03 – 2.21

0.488
0.398 – 0.578

7.98
6.91 – 9.05

0.455

1.97

0.361

7.86

0.418

2.03

0.399

6.09

0.402

2.07

0.402

7.99

6.19
5.89 – 6.48

10.1
9.84 – 10.3

3.14
2.94 – 3.34

14.9
13.5 – 16.4

5.54

9.4

2.79

14.4

5.86

10.0

3.03

12.5

5.53

9.7

2.88

14.9

Se (µg /L)

See
Table 6

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 59 of 88

Table 6. Ruggedness Testing Results: Evaluating the significance of RPq value on sample
stability. Test performed on December 21, 2010 by Deanna Jones. Results below are the
average of the beginning and ending QC results for each analytical run.
ID

RPq

Hg (µg /L)

Pb (µg /dL)

Cd (µg /L)

Mn (µg /L)

QMEQAS08B-02*

QMEQAS07B-09*

Target Mean
and 2SD Range
0.48 Mn and Hg;
0.52 Se
0.6 Mn and Hg;
0.7 Se
0.72 Mn and Hg;
0.78 Se
See
Table 5
Target Mean
and 2SD Range
0.48 Mn and Hg;
0.52 Se
0.6 Mn and Hg;
0.7 Se
0.72 Mn and Hg;
0.78 Se
*samples purchase from Le centre de toxicology du Quebec (Quebec, Canada)

Se (µg /L)
293
273 – 313
262
250
277
361
337 - 385
347
349
364

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 60 of 88

Appendix A: Critical Parameter Test Results (Continued)
Critical Parameter Test #5: This test evaluated the significance of the Axial Field
Voltage (AFT) while analyzing blood samples for whole blood metals. The Axial Field
Volatge may vary on each instrument. The Axial Field Voltage was increased and
decreased by 20%. The results are presented in Table 7.
Test Details:
1. Prepare a set of dilutions (calibrators, blanks, reference materials, fake samples) for
analysis in triplicate (three separate sets of tubes).
2. Analyze them in three separate runs on the same day, same instrument.
3. Change the AFV value +/- 100 V.

QMEQAS0
9B-08*

QMEQAS
07B-09*

HB08708_
WB2

LB08707_
WB2

Table 7. Ruggedness Testing Results: Evaluating the significance of Axial Field Voltage on
sample stability. Test performed on December 20, 2010 by Deanna Jones. Results below are
the average of the beginning and ending QC results for each analytical run.
Axial Field
ID
Hg (µg /L)
Pb (µg /dL)
Cd (µg /L)
Mn (µg /L) Se (µg /L)
Voltage
Target Mean
0.585
2.12
0.488
7.98
0.407 – 0.763 2.03 – 2.21 0.398 – 0.578 6.91 – 9.05
and 2SD Range
0.511
2.00
40.415
7.77
(decreased)
0.461
2.04
0.394
6.36
(per method)
0.414
2.01
0.376
6.95
(increased)
Target Mean
6.19
10.1
3.14
14.9
5.89 – 6.48
9.84 – 10.3
2.94 – 3.34
13.5 – 16.4
and 2SD Range
5.50
9.8
2.91
14.3
(decreased)
5.62
9.8
2.84
12.0
(per method)
5.75
10.1
2.99
12.8
(increased)
Target Mean
157
146 – 168
and 2SD Range
139
(decreased)
147
(per method)
138
(increased)
Target Mean
548
511 - 585
and 2SD Range
501
(decreased)
556
(per method)
532
(increased)
*samples purchase from Le centre de toxicology du Quebec (Quebec, Canada)

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 61 of 88

Appendix B
Table 1. Instrument and Method Parameters.
Instrument: PerkinElmer ELAN DRC II ICP-MS
ESI SC4 autosampler with (optional) PC3 Peltier cooled spray chamber
Optimization Window Parameters
RF power 1.45 KW
Plasma Gas Flow (Ar) 15 L/min
Auxiliary Gas Flow (Ar) 1.2 L/min
Nebulizer Gas Flow (Ar) ~0.90 – 1.0 L/min (optimized as needed for sensitivity)
Ion Lens Voltage(s) AutoLens (optimized as needed for sensitivity)
QRO, CRO, CPV, Optimized per instrument by service engineer, or advanced
Discriminator Threshold user.
Parameters of x-y alignment, nebulizer gas flow, AutoLens voltages, mass calibration,
and detector voltages are optimized regularly. Optimization file name = default.dac.
Configurations Window Parameters
Cell Gas Changes Pressurize Delay (From Standard to DRC mode) = 60
Pause Times Exhaust Delay (From DRC to Standard mode) = 30
Flow Delay (Gas changes while in DRC mode) = 30
Channel Delay (Gas channel change in DRC mode) = 30
File Names & Directories
Method file names calibration curve (programmed for blood blank)
WBMP2_DLS3016_bldblk.mth
For QC & patient sample analysis
(programmed for aqueous blank)
WBMP2_DLS3016_aqblk.mth
Dataset Create a new dataset subfolder each day. Name as “20110820” for all work done on August 20, 2011
Sample File Create for each day’s work
Report file name For sample results printouts
cdc_quant comprehensive.rop

Tuning
Optimization
Calibration
Polyatomic
Report Options
Template (transferring
results to the database)

For calibration curve information
CDC_Quant Comprehensive (calib curve info).rop
Default.tun
Default.dac
N/A
elan.ply
CDC_Database Output.rop
Report Format Options: select only “Use Separator”
File Write Option: Append
Report File name: include date, instrument, and group
being analyzed in file name (i.e. 2005-0311b_DRC2A_HM0364.txt)

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 62 of 88

Table 1. Instrument and Method Parameters.
Method Parameters
Method Parameters:
Sweeps/reading
Readings/replicate
Replicates
Enable QC Checking
Isotopes Monitored
and Internal Standard
Associations
(Exact Mass)
Dwell Times

Timing Page (see Figures 1a, 2a and 2d in Appendix B)
30
1
3
Off
use 103Rh, 130Te, 193Ir as internal standards
103
Rh (102.905): 55Mn (54.93805)
130
Te(129.907): 202Hg (201.971), 80Se(79.9165)
193Ir(192.963):208Pb(207.977), 114Cd(113.904)
100 ms for 55Mn,202Hg, 80Se, 208Pb, and 114Cd
50 ms for 130Te,103Rh, and 193Ir

Scan Mode Peak Hopping for all isotopes (1 MCA channel)
DRC channel A Gas 99.999% Methane (5-7 psig delivery pressure)
Flow Rate typically 0.84 L/min *
*optimized per instrument, and periodically verified
DRC channel B Gas 99.99% Oxygen (5-7 psig delivery pressure)
Flow Rate typically 1.2 L/min *
*optimized per instrument, and periodically verified
RPa 0 for all isotopes
Typically*
0.6 for 103Rh,55Mn,130Te, and 202Hg.
0.65 for 130Te and 80Se.
RPq
0.25 for 193Ir, 208Pb, and 114Cd
Use the same RPQ for each analyte and its IS.
(* Optimize per instrument, and periodically verified)
Method Parameters: Processing Page (see Figures 1b in Appendix B)
Detector mode Pulse
Process Spectral Peak N/A
AutoLens On
Isotope Ratio Mode Off
Enable Short Settling Off
Time
Blank subtraction After internal standard
Measurement units Cps
Process Signal Profile N/A
Method Parameters: Equations Page (see Figures 1c in Appendix B)
Equations None
Method Parameters: Calibration Page (see Figures 1d in Appendix B)
Calibration Type External Std.
Curve type Simple Linear

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 63 of 88

Table 1. Instrument and Method Parameters.
Sample units “g/L” or “ppb”
Calibration Standard Mn: 1.5, 4.5, 10.5, 15, 30
Concentrations (g/L) Cd, Hg: 0.5, 1.5, 3.5, 5, 10
Pb (µg /dL): 1, 3, 7, 10, 20
Se: 30, 90, 210, 300, 600
Method Parameters: Sampling Page (see Figures 1e and 1f in Appendix B)
“Peristaltic Pump Under On
Computer Control”
Sample Flush 6s at 1.5 rpm
Read Delay 60s at 1.5 rpm
Wash 40s at 1.5 rpm
Autosampler Locations For calibration curve (points to blood blank)
of Blanks and Standards WBMP2_DLS3016_bldblk.mth
Blood Blank and Calibration Stds 1 – 5 in autosampler
positions 105 - 110.
For QC & patient sample analysis (points to aqueous blank)
WBMP2_DLS3016_aqblk.mthn
Aqueous Blank in autosampler position 112 and 113.
Table 2. Suggested maximum analyte concentrations for base blood and Quality
control material
Analyte
Maximum Base Blood
Low QC Spiking
High QC Spiking
Range ( µg /L)
Range ( µg /L)
Concentration (µg/L)
Mn
<8
6 – 10
15 - 20
Hg
<0.5
0.5 – 0.75
5–7
Se
<200
125 – 175
225 – 275
Cd
<0.5
0.4 – 0.5
2.5 – 3.5
Pb ( µg /dL)
<2
1–2
9 - 11
Table 3. Preparation of Intermediate Stock Calibration Solution from NIST primary
standards
Analyte
Cd
Pb
Hg
Mn
Se
Certified value (mg/g)
10.005*
9.987#
9.954^
10.00+
10.11%
Target mass to add (g)
0.0101
0.201
0.0101
0.03
0.594
Target final weight (g)
100.00
Expected final
1.0105
20.0739
1.0054
3.000
60.0534
concentration (µg /g)
* certified value for lot # 060531
#
certified value for lot # 030721
^
certified value for lot # 061204
+
certified value for lot # 050429
%
certified value for lot # 992106

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 64 of 88

Appendix B (continued)
Table 4. Preparation of Intermediate Stock Calibration Solution from single
element stock calibrator solutions without Pb
Analyte
Cd
Se
Hg
Mn
Stock concentration (mg/L)
1000
1000
1000
1000
Spike volume (mL)
0.100
6.00
0.100
0.300
Final Volume (mL)
100
Final concentration ( µg /L)
1.00
60.0
1.00
3.00

Table 5. Preparation of Intermediate Working Standards
Standard #
Volume of Flask
(mL)
Volume Spike of Int.
Stock Std. (mL)

100

0.05

0.15

0.35

0.50

1.00

Concentrations ( µg /L)
1.5
3.5
5
1.5
3.5
5
4.5
10.5
15
3
7
10
90
210
300

10
10
30
20
600

Cd
0.5
Hg#
0.5
Mn*
1.5
Pb ( µg /dL)+
1
#
Se
30
+ 193
Ir used as internal standard
#
130Te used as internal standard
* Rh-103 used as internal standard

Appendix B (continued)

Table 6. Preparation of samples, working standards, and QC materials for
analysis
Total volume of prepared sample may be changed, from what is presented here.
However, volumes for each component should be adjusted proportionally.
Dilution ID

Water (L)

Base
Blood
(L)

AQ
Intermedi
ate
Working
Standard

Patient or
QC blood
sample
(L)

Diluent
(L)

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 65 of 88
(L)
50

AQ Blank
100
2400 *
Blood Blank and BldBlkChk
50
50
2400 *
Working Calibration Standards
50
2400 *
Patient blood or
50
50
2400 *
Blood-Based QC
Patient Blood
150
50
4800
2x Dilution H
* 2400 L diluent is best dispensed from the Digiflex™ as 2 1200-L portions (i.e.- When preparing
a Working Calibration Standard dilution, dispense 1200 L diluent + 50 L standard in one cycle of
Digiflex™, then 1200 L diluent + 50 L base blood in the next cycle of the Digiflex™ to prepare a
2.5 mL total volume dilution.)
H
Extra dilution is performed on blood samples whose concentration is greater than the
concentrations listed in Table 8 in Appendix B (linearity of the method has been documented up to
these concentrations). Any extra level of dilution can be prepared as long as the 4.8:5 ratio of
diluent to total dilution volume is maintained. Use of the lowest possible dilution level is preferred
because matrix differences may lead to different observed concentration results as the sample
dilution becomes greater (i.e. 2x dilution is preferred over 10x if 2x is sufficient to dilute analyte into
the documented linearity range).

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 66 of 88

Appendix B (continued)
Table 7. Preparation and Final Concentrations of Intermediate Stock
Calibration Verification Standards. *
Flask # 1
Flask #2
Analytes
Cd
Hg
Mn
Analyte
Pb
Stock
Stock Concentration
Concentration
1000
1000
1000
9.987
(NIST 3128) (mg/g)
(mg/L)
Spike volume
1
1
3
Target Mass (g)
10
(mL)
Final volume
100
100
100
Final volume (mL)
100
(mL)
Final
Final concentration
concentration
10
10
30
999
(mg/L)
(mg/L)
* If standards are made gravimetrically the final concentrations will not exactly
match these and the QC ID used in the laboratory database will need to change to
maintain proper record keeping of analysis result to target concentration.
Table 8. Preparation and Final Concentrations of Intermediate Working
Calibrator Verification Standards
Calibration Verification Standard #
CV1
CV2
CV3
Volume of Flask
100
100
100
Volume of Cd, Hg, Mn, Intermediate Stock
0.25
0.5
1
Calibration Verification Standard (mL)
Volume of Pb Intermediate Stock Calibration
0.1
0.2
0.4
Verification Standard (mL)
Volume of 1000 mg/L Se Stock Calibration
0.05
0.1
0.2
Solution
Final Volume (mL)
100
100
100
Final Concentrations *
CV1
CV2
CV3
Cd ( µg /L)
25
50
100
Hg ( µg /L)
25
50
100
Mn ( µg /L)
75
150
300
Pb ( µg /dL)
100
200
400
Se ( µg /L)
500
1000
2000
* If standards are made gravimetrically the final concentrations will not exactly match
these and the QC ID used in the laboratory database will need to change to maintain
proper record keeping of analysis result to target concentration.

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 67 of 88

Appendix B (continued)
Table 9. Acceptable ways to perform two consecutive analytical runs,
bracketing with bench quality control samples.
Setup 2 (typical)
Setup 1
Run #1
Run #1
Calibration Standards
Calibration Standards
Low Bench QC
Low Bench QC
High Bench QC
High Bench QC
patient samples
patient samples
Low Bench QC
Low Bench QC
High Bench QC
High Bench QC
Run #2
Low Bench QC
High Bench QC
patient samples
Low Bench QC
High Bench QC

Run #2
Calibration Standards
Low Bench QC
High Bench QC
patient samples
Low Bench QC
High Bench QC

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 68 of 88

Appendix B (continued)
Table 10. A typical SAMPLE/BATCH window.
AS
Sample ID
Measurements Action
Method
Location*
233
DRCstability1 Run sample
…DLS3016_bldblk.mth
233
DRCstability2 Run sample
…DLS3016_bldblk.mth
233
DRCstability3 Run sample
…DLS3016_bldblk.mth
233
DRCstability4 Run sample
…DLS3016_bldblk.mth
Continue DRC stability samples . . .
233
DRCstability9 Run sample
…DLS3016_bldblk.mth
233
DRCstability10 Run sample
…DLS3016_bldblk.mth
103
Aq blank
Run sample
…DLS3016_bldblk.mth
104
WB Blank_chk Run sample
…DLS3016_bldblk.mth
111
WB Blank
Run blank, standards, and sample ** …DLS3016_bldblk.mth
112
WB Blank2
Run sample
…DLS3016_bldblk.mth
103
Aq blank
Run blank and sample ¥
…DLS3016_aqblk.mth
113
L Bench QC
Run sample
…DLS3016_aqblk.mth
114
H Bench QC Run sample
…DLS3016_aqblk.mth
301
Sample 1
Run sample
…DLS3016_aqblk.mth
302
Sample 2
Run sample
…DLS3016_aqblk.mth
303
Sample 3
Run sample
…DLS3016_aqblk.mth
115
L Bench QC
Run sample
…DLS3016_aqblk.mth
116
H Bench QC Run sample
…DLS3016_aqblk.mth
* The exact autosampler positions of QCs and patient samples do not have to be those
shown above, but the order in which these are run should be as shown above.
** When executing this row, the ELAN will first analyze the blood blank at AS position 105,
then standards 1-5 at autosampler positions 106-110, then the “WB Blank” sample at A/S
position 111. The sampling information about AS positions 105-110 are stored in the “bldblk”
method file.
¥ When executing this row, the ELAN will first analyze the aqueous blank at AS position
112, then the “Aq blank ” at AS position 103. The sampling information about AS positions
112 is stored in the “aqblk” method file.

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 69 of 88

Appendix B (continued)
Table 11. Boundary Concentrations for Whole Blood Concentrations (/L).
1st Upper
2nd Upper
Range
Analyte
Boundary
Boundary
Maximum
(“Lim Rep Delta”) †
(“1UB”) *
(“2UB”) **
Mn
20
35
2.0
Pb
10.0
10.0
1.0
Cd
5.0
5.0
1.0
Hg
10.0
10.0
1.0
Se
400
400
20
st
th
* Typically, the 1 upper boundary (1UB) is the 99 percentile of non-weighted,
corrected concentration results from the NHANES 1999-2000 subset groups.
Concentrations observed greater than the “first upper boundary” (defined in the
laboratory database as the “1UB”) should be confirmed by repeat analysis of a new
sample preparation. The concentration assigned to the 1UB for an element is
determined by study protocol but default concentrations are listed in this table. Report
the original result, as long as the confirmation is within 10% of the original. Continue
repeat analysis until a concentration can be confirmed.
** Typically the 2nd upper boundary (2UB) is set to 2x the 1UB. At the discretion of the
supervisor, the 1UB may vary per study according to the concerns of the study.
Regardless of the study, report patient results confirmed to be greater than the 2UB to
the QC reviewer as an “elevated result”.
† Range maximum is the range of the three replicate readings for a single sample
analysis. This value is also called the “Lim RepDelta” in the database which handles
data for the Inorganic Radiation and Analytical Toxicology Branch. If the range of
replicate readings is greater than the range maximum, and represents greater than a
10% relative standard deviation for the measurement, do not use the measurement for
reporting.

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 70 of 88

Appendix B (continued)
Table 12. Reference Ranges for Blood Concentrations.
Analyte
Survey Geometric
50th
75th
90th
95th
N
(units)
Years
Mean
99-00
0.412
0.300
0.600
1.00
1.30
7970
Cd (g/L)*
01-02
**
0.300
0.400
0.900
1.30
8945
99-00
0.343
0.300
0.500
1.40
2.30
705
Hg (g/L)*
01-02
0.318
0.300
0.700
1.20
1.90
872
(1-5 yrs)
Hg (g/L)*
99-00
1.02
0.900
2.00
4.90
7.10
1709
(16-49 yrs,
01-02
0.833
0.700
1.70
3.00
4.60
1928
female)
99-00
1.66
1.60
2.40
3.80
4.90
7970
Pb (g/dL)*
01-02
1.45
1.40
2.20
3.40
4.40
8945
Se (g/L) † 157 – 265 g/L [60]
Non-exposed 4 – 14 ( µg /L) [41]
Exposed workers (adults) 3.2 – 101 µg /L [23]
Children receiving long term parenteral nutrition 33.8 – 101 µg /L [61]
Ohio adults (N=49) residing near a refinery (possible Mn emission):
Mean (range) 9.4 (4.2-21.7) µg/L [25]
Mexican infants
Mn ( µg /L)†
Age 1, mean (SD) = 24.3 (4.5) µg/L, median = 23.7 µg/L, N=270
Age 2, mean (SD) = 21.1 (6.2) µg/L, median = 20.3 µg/L, N=430 [62]
Japanese women (N = 1420)
GM 13.2 µg/L overall,
Range of median (max) across 8 regions 12.0-14.3 (25.0-33.4) µg/L [63]
South African children, ages 8-10 years old (n = 49)
Mean (SD) 8.48 (2.45) µg/L, range 4.58-18.20 µg/L. [29]
* From the Third National Report on Exposure to Environmental Chemicals [64]
** Not calculated. Proportion of results below limit of detection (0.3) was too high to
provide a valid result.
†
Blood Se and Mn were not included in the Third National Report on Exposure to
Environmental Chemicals.

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 71 of 88

Figure 1a. ELAN ICP-DC-MS Method Screen Shots (timing page).

Optimize
Per Instrument

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 72 of 88

Appendix B (continued).
Figure 1b. ELAN ICP-DC-MS Method Screen Shots (processing page).

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Appendix B (continued).
Figure 1c. ELAN ICP-DC-MS Method Screen Shots (equation page).

Page 73 of 88

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 74 of 88

Appendix B (continued).
Figure 1d. ELAN ICP-DC-MS Method Screen Shots (calibration page).

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 75 of 88

Appendix B (continued).
Figure 1e. ELAN ICP-DC-MS Method Screen Shots (sampling page, AqBlank
method).

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 76 of 88

Appendix B (continued).
Figure 1f. ELAN ICP-DC-MS Method Screen Shots (sampling page, BldBlank
method).

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Appendix B (continued).
Figure 1g. ELAN ICP-DC-MS Method Screen Shots (report page).

Page 77 of 88

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 78 of 88

Appendix B (continued).
Figure 2a. ESI SC4 Autosampler Screen Shots (Main page). Additional flush times
and “Max Rinse Time” are approximate. Optimize these for best reduction of elemental
carry-over between samples. Tray types can be changed to allow for different volumes
of diluted sample digests. ‘FAST control’ must be enabled before start of method, but
does not need to be used in instrument optimization (pre-analysis) steps. Rinse and
additional flush times for eliminating carry-over from one sample to the next while using
the minimum amount of rinse solution.
A rinse time of -1 causes the rinse station to be skipped.
A rinse time of 0 causes the probe to only dip into the station, but spends no time there.
Additional flush times can be optimized to keep the rinse station full while not using too
much rinse solution. The inner diameter size of the tubing providing the rinse solution to
the rinse station determines how quickly the station will fill. Various sizes are available
for purchase or can be made in the laboratory.

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 79 of 88

Appendix B (continued).
Figure 2b. ESI SC4 Autosampler Screen Shots (“Configure” page). “High Speed”
option is to only be used for ‘High Speed’ models of the SC4 (look for “HS” in serial
number). Speeds and accel / decel values can be optimized per analyst preference and
to minimize droplet splatter off of probe.

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 80 of 88

Figure 2c . ESI SC4 Autosampler Screen Shots (“Communication” page).
Communication ports will differ depending on available ports on instrument control
computer.

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 81 of 88

Appendix B (continued).
Figure 2d . ESI SC4 Autosampler Screen Shots (“FAST” page). Timer A can be
optimized to achieve proper filling of loop with diluted sample digestate. Timers B, C, D,
E, and F control rinsing the loop after analysis and can be optimized for eliminating
carry-over from one sample to the next while using the minimum amount of rinse
solution. File should be saved with the name “Blood Clotted
PbCdHgSe_ITB004A_2008-March-1_SCFAST.txt”. It can be found in the directory
C:\Program Files\ESI\ESI-SC\.
Manually clicking the “Load” button prior to starting analysis will ensure the position of
the actuator is always the same at the beginning of the analysis.
Manually clicking the “Vacuum On” button prior to starting the analysis will help initial
sample uptake to be consistent (the vacuum pump may be slow to start for the first
sample if this is not done, possibly resulting in loop filling inconsistencies).

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 82 of 88

Appendix B (continued).
Figure 2e. ESI SC4 Autosampler Screen Shots (5x12 Rack Setup window).
Settings are approximate. To be sure the loop is filled, the probe should go down close
to the bottom of the cup, but not touch. Optimize retraction speed for least droplet
splatter.

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 83 of 88

Figure 2f. ESI SC4 Autosampler Screen Shots (50mL Tube Rack Setup window).
Settings are approximate. To be sure the loop is filled, the probe should go down close
to the bottom of the cup, but not touch. Optimize retraction speed for least droplet
splatter.

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 84 of 88

Appendix B (continued).
Figure 2g. ESI SC4 Autosampler Screen Shots (Rinse Station Rack Setup
Window). Settings are approximate. Optimize down height for best probe cleaning,
and retraction speed for least droplet splatter.

Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016

Page 85 of 88

References
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Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016
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Roels, H.A., et al., Assessment of the Permissible Exposure Level to Manganese
in Workers Exposed to Manganese-Dioxide Dust. British Journal of Industrial
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Gennart, J.P., et al., Fertility of Male Workers Exposed to Cadmium, Lead, or
Manganese. American Journal of Epidemiology, 1992. 135(11): p. 1208-1219.
Bader, M., et al., Biomonitoring of manganese in blood, urine and axillary hair
following low-dose exposure during the manufacture of dry cell batteries.
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Lauwerys, R., et al., Fertility of Male Workers Exposed to Mercury-Vapor or to
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Standridge, J.S., et al., Effect of Chronic Low Level Manganese Exposure on
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Woolf, A., et al., A child with chronic manganese exposure from drinking water.
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Wasserman, G.A., et al., Water manganese exposure and children's intellectual
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Bazzi, A., J.O. Nriagu, and A.M. Linder, Determination of toxic and essential
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Rollin, H., et al., Blood manganese concentrations among first-grade
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Aschner, M., Manganese: Brain transport and emerging research needs.
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Davis, J.M., Methylcyclopentadienyl manganese tricarbonyl: Health risk
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Blood Metals Panel 2 (BMP2) by ICP-DRC-MS
IRAT-DLS Method Code: 3016
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Jarvisalo, J., et al., URINARY AND BLOOD MANGANESE IN
OCCUPATIONALLY NONEXPOSED POPULATIONS AND IN MANUAL METAL
ARC WELDERS OF MILD-STEEL. International archives of occupational and
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Klaassen, C., BILIARY-EXCRETION OF MANGANESE IN RATS, RABBITS,
AND DOGS. Toxicology and applied pharmacology, 1974. 29(3): p. 458-468.
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Agency for Toxic Substances and Disease Registry (ATSDR). 2000.
Toxicological profile for Manganese. Atlanta, G.U.S.D.o.H.a.H.S., Public Health
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IRAT-DLS Method Code: 3016
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Exposure to Environmental Chemicals, http://www.cdc.gov/exposurereport, 2005.

Division of Laboratory Sciences
Laboratory Protocol
Analyte:
Matrix:
Method:

Method Code:
Branch:

Prepared By:

Inorganic Hg, methyl Hg, ethyl Hg
Blood
Blood mercury speciation performed by SSID-GC-ICP-DRC-MS
(Species-Specific Isotope Dilution Gas Chromatography Inductively
Coupled Plasma Dynamic Reaction Cell Mass Spectrometry)

DLS-3020
Inorganic and Radiation Analytical Toxicology

__________________

______________________

Printed Name

Supervisor:

Signature

__________________

Branch Chief:

Signature

__________________

Signature

_________
Date

______________________

Printed Name

Adopted:

Date

______________________

Printed Name

_________

_________
Date

__________________
Date

Updated:

__________________
Date

Director's Signature Block:
Reviewed: __________________________________
Signature

Reviewed: __________________________________
Signature

_____________
Date

This page is intentionally left blank.

Modifications/Changes:
see Procedure Change Log STARLIMS

iii

This page is intentionally left blank.

Laboratory Procedure Manual
Analyte:
Matrix:
Method:

Method No:

Inorganic mercury, Methyl mercury, Ethyl mercury
Blood
Blood mercury speciation SSID-GC-ICP-DRC-MS
(Species-Specific Isotope Dilution Gas
Chromatography- Inductively Coupled Plasma
Dynamic Reaction Cell Mass Spectrometry)
DLS-3020

Adopted:
Revised:
As performed by:

Contact:

Inorganic and Radiation Analytical Toxicology Branch
Division of Laboratory Sciences
National Center for Environmental Health
Dr. Robert L. Jones
Phone: 770-488-7991
Fax:
770-488-4097
Email:
[email protected]
James L. Pirkle, M.D., Ph.D.
Director, Division of Laboratory Sciences

Important Information for Users
CDC periodically refines these laboratory methods. It is the responsibility of the user to contact the
person listed on the title page of each write-up before using the analytical method to find out whether
any changes have been made and what revisions, if any, have been incorporated.

Blood mercury species SSID-GC-ICP-DRC-MS

IRAT-DLS

DLS Method Code: 3020

Page 2 of 51
This page is intentionally left blank.

Blood mercury species SSID-GC-ICP-DRC-MS

IRAT-DLS

DLS Method Code: 3020

Page 3 of 51

Table of Contents
I.

Clinical Relevance and Test Principle ________________________________________ 7
A.

Clinical Relevance ___________________________________________________________7

B. Test Principle _________________________________________________________________7

II.

Safety Precautions _____________________________________________________ 8

III.

Data System Management _______________________________________________ 9

Data Entry and Transfer _______________________________________________________9

B. Routine Computer Hard Drive Maintenance ________________________________________9
C. Electronic Data Backup _________________________________________________________9
1.
2.

Schedule of Data Backups____________________________________________________________ 9
Backup Procedures _________________________________________________________________ 9

1.
2.

Computer Maintenance: ____________________________________________________________ 10
Instrument Maintenance:____________________________________________________________ 10

Documentation of System Maintenance _______________________________________ 10

IV.

Collecting, Storing, and Handling Specimens; Criteria for Rejecting Specimens ___ 10

Specimen Type ____________________________________________________________ 10

B. Specimen Collection, Handling and Storage ______________________________________ 10
C. Criteria for an Unacceptable Specimen __________________________________________ 11

Procedures for Microscopic Examinations _________________________________ 11

VI.

Chemicals, Standards, and Quality Control Material _________________________ 11

Chemicals ________________________________________________________________ 11

B. Isotope Dilution Standards ____________________________________________________ 12
C. Quality Control Material _______________________________________________________ 12

VII.

Instrumentation, Equipment, Software and Supplies _________________________ 12

Instrumentation ___________________________________________________________ 12
1.
2.
3.
4.
5.
6.

Gas Chromatography System ________________________________________________________ 12
ICP-DRC-MS System ________________________________________________________________ 12
Robotic Sample Processing Station ___________________________________________________ 13
Equipment ________________________________________________________________________ 13
Computer Software_________________________________________________________________ 14
Supplies _________________________________________________________________________ 14

VIII.

Standard Procedure __________________________________________________ 15

Preparation of Stock Solutions _______________________________________________ 15

B. Preparation of Working Spike Solution ___________________________________________ 16
1.

Preparation of Working Triple-Spiked Standards Solution __________________________________ 16

C. Preparation of Quality Control Material __________________________________________ 17

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2.

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Base pool preparation: ______________________________________________________________ 17
Low and High pool preparation: _______________________________________________________ 17

Sample preparation ________________________________________________________ 17
Preparation of blood samples for digestion: _________________________________________________ 17
2. Digestion: ________________________________________________________________________ 17
3. Derivatization of the digested samples: ________________________________________________ 17

Requirements for Batch Analysis of Samples and QC Material _______________________ 17

GC Instrument Program _______________________________________________________ 18

CombiPal Autosampler Program ______________________________________________ 19

ICP-DRC-MS Instrument Setup _______________________________________________ 21
1.
2.
3.

Programming the DRC Gas Flow Delay Parameter ________________________________________ 21
Programming the ELAN ".mth" file _____________________________________________________ 22
Creating the ELAN Sample Table ".sam" file _____________________________________________ 24

ICP-DRC-MS Performance Checks ______________________________________________ 25
The following performance checks should be recorded in an instruments log book. ________________ 25
1. Daily Performance Check ____________________________________________________________ 25
2. Weekly Performance Check __________________________________________________________ 25
3. Monthly Performance Check _________________________________________________________ 27

ICP-DRC-MS Warm Up ________________________________________________________ 28

K. GC-ICP-DRCII-MS System Startup _______________________________________________ 29
1.

L.
M.

IX.
A.

Entering Sample Names into the ELAN Sample Table _____________________________________ 29

Starting the Run _____________________________________________________________ 30
Instrument Shut Down ______________________________________________________ 32

Post-Run Data Analysis _________________________________________________ 32
Configuration of TotalChrom Integration Method_________________________________ 32

B. Configuration of ELAN ChromLink™ _____________________________________________ 36
C. Data Processing and Analysis __________________________________________________ 37

Recording of Sample and QC Data________________________________________ 42
A.

Transferring the Data to the Branch Database __________________________________ 42

B. QC Data____________________________________________________________________ 43

XI.
A.

Final Data Review _____________________________________________________ 43
Analysis Printouts and Analyst Run Report______________________________________ 43

B. Plotting QC Results __________________________________________________________ 43
C. Supervisor Review ___________________________________________________________ 43

XII.

Replacement and Periodic Maintenance of Key Components __________________ 43

ICP-MS Maintenance _______________________________________________________ 43

B. GC Maintenance ____________________________________________________________ 44

XIII.

Limits of Detection ___________________________________________________ 44

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XIV.

Reportable Range of Results __________________________________________ 45

XV.

Special Procedure Notes - CDC Modifications _______________________________ 45

XVI.

Quality Control Procedures ____________________________________________ 45

Establish QC limits for each QC pool. ________________________________________ 4645

B. Precision and Accuracy _______________________________________________________ 46
C. Remedial Action If Calibration or QC Systems Fail to Meet Acceptable Criteria __________ 46

XVII.

Reference Ranges ___________________________________________________ 46

XVIII.

Action-Level Results __________________________________________________ 47

XIX.

Specimen Storage and Handling During Testing ___________________________ 47

XX.

Alternate Methods for Performing Test and Storing Specimens If Test System Fails 47

XXI.

Test-Result Reporting System; Protocol for Reporting Critical Calls (If Applicable) 47

XXII.
Transfer or Referral of Specimens; Procedures for Specimen Accountability and
Tracking __________________________________________________________________ 48
XXIII.

References _________________________________________________________ 48

XXIV.

Appendix ___________________________________________________________ 48

Appendix A. Critical Parameters Testing Results. ________________________________ 48
1. Critical Parameter Test #1: Evaluate the significance of the GC injector temperature __________ 48
2. Critical Parameter Test #2: Evaluate the significance of the Combipal-agitator
equilibration/extraction temperature ______________________________________________________ 49
3. Critical Parameter Test #3: Evaluate the significance of the length of extraction of sample from
SPME fiber in GC injector ________________________________________________________________ 49
4. Critical Parameter Test #4: Evaluate the significance of changing GC split ratio. ______________ 50
5. Critical Parameter Test #5: Evaluate the significance of changing GC carrier gas flow rate. ______ 51

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List of Tables
TABLE 8-1: Dilutions Factors for Preparing Working Triple-Spike Standards ________ 1716
TABLE 8-2: GC Settings ___________________________________________________ 1918
TABLE 8-3: CombiPAL Autosampler Settings _________________________________ 2019
TABLE 8-4: Chronos Settings (Baking) __________________________________________ 20
TABLE 8-5: Chronos Settings (Conditioning) __________________________________ 2120
TABLE 8-6: Chronos Parameter Settings (for Run) _____________________________ 2120
TABLE 8-7 ELAN Timing Parameters ________________________________________ 2221
TABLE 8-8: ELAN Analyte Parameters _______________________________________ 2322
TABLE 8-9: ELAN Processing Parameters ____________________________________ 2322
TABLE 8-10: ELAN Sampling Parameters ____________________________________ 2322
TABLE 8-11: ELAN Report Parameters ______________________________________ 2423
TABLE 8-12: Sample Template Data ________________________________________ 2524
TABLE 8-13: ELAN Optimization Parameters _________________________________ 2726
TABLE 8-14: ELAN Samples Table __________________________________________ 2928
TABLE 8-15 Chronos Samples Table ________________________________________ 3130
TABLE 9-1: Integration ___________________________________________________ 3332
TABLE 9-2: Baseline Timed Events _________________________________________ 3433
TABLE 9-3: Replot _______________________________________________________ 3433
TABLE 9-4: Global Information _____________________________________________ 3534
TABLE 9-5: Method Editor - Components Settings _____________________________ 3534
TABLE 9-6: Components Defaults - Identification ______________________________ 3635
TABLE 9-7: Components Defaults - Calibration ________________________________ 3635
TABLE 9-8: TotalChrom™ Navigator - Reprocess Batch ________________________ 4140
TABLE 17-1: Reference ranges for Mercury Species ___________________________ 4746

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CLINICAL RELEVANCE AND TEST PRINCIPLE
A. Clinical Relevance
Mercury (Hg) is widespread in the environment and found in its elemental form (Hg0), inorganic forms
such as mercurous (Hg+), and mercuric (Hg2+) and various organic forms such as methyl mercury (MeHg),
ethyl mercury (EtHg), phenyl mercury (PhHg), and others. The health effects of mercury are diverse and
depend on the form of mercury encountered and the severity and length of exposure. The relative order of
increasing toxicity is: Hg0 < Hg2+ << CH3Hg+ [1]. With large acute exposures to elemental mercury vapor,
the lungs may be injured. At levels below those that can cause lung injury, low-dose or chronic inhalation may
affect the nervous system. Symptoms include weakness, fatigue, loss of weight (with anorexia),
gastrointestinal disturbances, salivation, tremors, and behavioral and personality changes, including
depression and emotional instability [2]. Exposure to inorganic mercury usually occurs by ingestion. The most
significant effect is on the kidneys, where mercury accumulates, leading to tubular necrosis. In addition, there
may be an irritant or corrosive effect on the gastrointestinal tract involving stomatitis, ulceration, diarrhea,
vomiting, and bleeding. Psychomotor and neuromuscular effects also may occur [3].
Methyl mercury is more toxic than inorganic mercury. The effects of methyl mercury include changes in
vision, sensory disturbances in the arms and legs, cognitive disturbances, dermatitis, and muscle wasting.
The critical organ for methyl mercury is the brain. Methyl mercury readily crosses the blood-brain barrier due
to its lipid solubility and accumulates in the brain where it is slowly converted to inorganic mercury. Whether
CNS damage is due to methyl mercury or inorganic mercury, or both, is still controversial [4]. Ethyl mercury is
another organic form of mercury. Very little is actually known about ethyl mercury metabolism in humans,
including whether it has the same potency as a neurotoxin, whether the blood concentration is ever
significant, and even whether it crosses the blood-brain barrier. But the use of thimerosal, which metabolizes
to ethyl mercury and thiosalicylate, as a vaccine preservative makes this subject very important.
In the general population, total blood mercury is due mostly to the dietary intake of organic forms, particularly
methyl mercury and ranges from 0.2 to 5.8µg/L [5]. Urinary mercury mainly comprises inorganic mercury due
generally to dental amalgam containing elemental mercury and occupational exposure and ranges from 0.2
to 10µg/L [5]
The method described in this manual assesses mercury exposure, as defined by exposure to individual
mercury species, by analyzing blood through the use of Solid Phase Micro Extraction (SPME) fiber for
delivering sample to gas chromatography (GC) coupled to inductively coupled plasma-dynamic reaction cellmass spectrometry (ICP-DRC-MS). Blood is chosen as a matrix because it might contain various organic
mercury species as well as inorganic mercury while urine contains mostly inorganic mercury. This hyphenated
method will provide accurate quantification of three mercury blood species: inorganic mercury (Hg2+), methyl
mercury (MeHg), and ethyl mercury (EtHg).
Species Name

Abbreviation

Molecular Structure

Inorganic mercury

InHg

Hg2+

Methyl mercury

MeHg

CH3Hg+

Ethyl mercury

EtHg

C2H5Hg+

B. Test Principle
The quantification of InHg, MeHg, and EtHg is determined by using species-specific isotope dilution
(SSID) method employing gas chromatography (GC) to separate the species followed by introduction into an
ICP-DRC-MS for detection. SSID is a specialized extension of the Isotope Dilution (ID) technique. SSID
measures individual chemical species (inorganic, methyl and ethyl mercury species) in samples using ID
principles. The blood sample is spiked with known amounts of each Hg species that have been enriched with
isotopic variants of the target element of interest.

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The first step of this method involves the addition ("spiking") of enriched isotopes (199Hg2+, CH3200Hg+,
and C2H5200Hg+) to the blood sample. Each Hg species spike is labeled with an enriched Hg isotope such that
its isotopic pattern is unique to the species' chemical identity, i.e., the manner of isotope spiking is "species
specific". Next, the spiked sample is digested in tetramethylammonium hydroxide (TMAH) which disassociates
bound mercury species from proteins, polypeptides and other biomolecules. The digested blood sample with
freed mercury species is chemically reacted ("derivatized") with a reagent* that adds 3-carbon chains (npropyl groups) to the mercury atom of each species molecule without compromising species identity. This
type of chemical derivatization results in loss of ionic charge and reduced polarity; the net effect is to make
each mercury species molecule volatile so it can escape the liquid phase and accumulate in the gas phase
("headspace") directly above the sample. Derivatization is performed inside a partially filled vial sealed with a
rubber septa cap that can be penetrated by a needle.
Solid Phase Microextraction (SPME) is a sampling technique that uses a thin polymer fiber with a
hydrophobic coating; the method described here uses a SPME fiber with a 100 µm coating of
polydimethylsiloxane (PDMS). The SPME assembly consists of the fiber inserted through the inside a 22
gauge stainless steel needle. A key design feature is the fiber can be mechanically withdrawn into the needle
during vial septum penetration and then pushed out to expose the fiber to the headspace. During headspace
exposure (the "extraction" step), the gaseous derivatized Hg species adsorb onto the PDMS coating of the
SPME fiber; when other factors held are constant, the adsorbed mass increases as a function of sample
concentration. After a predetermined time, the SPME fiber is retracted into the injection needle; the needle is
withdrawn from sample vial; it moves to the injector port of the programmable temperature gradient gas
chromatograph (GC) and, on programmatic command, performs a programmed temperature ramp injection
sequence. This action transfers the propylated inorganic, methyl and ethyl Hg species to the head of a 30 m
capillary GC column which, using He as the carrier gas, ramps the column temperature to 280 °C. The order
of chromatographic separation of the Hg species is based on increasing molecular weight:
methylpropylmercury (derivatized methyl Hg), ethylpropylmercury (derivatized ethyl Hg), last peak is
dipropylmercury (derivatized inorganic Hg). Hg species exiting the GC column are seen as chromatographic
peaks detected using an inductively-couple argon plasma (ICP) as the ion source and a quadrupole mass
spectrometer (Q-MS) for mass specific quantification. Species identification is based on chromatographic
retention time; species specific isotope ratios are calculated from integrated peak areas derived from m/z
signals corresponding to 199Hg, 200Hg, 201Hg and 202Hg isotopes. The ICP-MS is equipped with a Dynamic
Reaction Cell (DRC(tm)) for minimizing polyatomic interferences. Operating the ICP-MS in DRC mode has an
added benefit of enhancing Hg signal strength through an effect known as "collisional focusing" [6,7].

II.

SAFETY PRECAUTIONS
Important
Precautionary information that is important to protecting personnel and safeguarding equipment will be
presented inside a box, like this one, throughout the procedure where appropriate.
Follow universal precautions when handling blood samples. Wear gloves, a lab coat, and safety glasses
while handling human blood, plasma, serum, urine, or other bodily fluid or tissue. Place disposable plastic,
glass, and paper (e.g., pipette tips, autosampler tubes, and gloves) that come in contact with human
biological fluids, such as blood, in a biohazard autoclave bag. Keep these bags in appropriate containers until
they are sealed and autoclaved. When work is finished, wipe down all work surfaces where human biological
fluid was handled with a 10% (v/v) sodium hypochlorite solution (or equivalent). Dispose of all biological
samples and diluted specimens in a biohazard autoclave bag at the end of the analysis according to
CDC/DLS guidelines for disposal of hazardous waste.
PerkinElmer provides safety information that should be read before operating the instrument. This
information is found in the PerkinElmer ELAN® 6100 ICP-DRC-MS System Safety Manual. Possible hazards
include ultraviolet radiation, high voltages, radio-frequency radiation, and high temperatures.

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Caution!
Exercise caution when handling and dispensing concentrated nitric and hydrochloric acid. Always
remember to add acid to water. Nitric and hydrochloric acid are caustic chemicals that are capable of
severe eye and skin damage. Wear powder-free gloves, a lab coat, and safety glasses. If nitric or
hydrochloric acid comes in contact with any part of the body, quickly wash the exposed area with copious
quantities of water for at least 15 minutes.

III.

DATA SYSTEM MANAGEMENT
To maintain the integrity of specimen and analytical data generated by this method, eliminate hand entry
of specimen identifiers or analytical results whenever possible, proofread all transcribed data, and regularly
defragment and back up the ICP-MS computer's hard drive.

A. Data Entry and Transfer
Whenever possible, use bar code scanners to enter sample identifiers into the GC-ICP-DRC-MS computer
software to avoid errors associated with the keyboard-entry process and to speed up sample processing.
When bar code scanners cannot be used, proofread transcribed data after entry. Handle or transfer data
electronically when reporting or moving data to other computerized data-handling software. In the Inorganic
and Radiation Analytical Toxicology Branch sample analysis results generated by this method are stored for
long periods in Microsoft Access™ or MS SQL Server (Frontends) database software. The results should
include at least the analysis date, analytical run number, quality-control (QC) results for the run, results of
specimen analysis by specimen identification (ID), and method identifier.

B. Routine Computer Hard Drive Maintenance
Defragment the computer hard drive regularly by using software such as Microsoft Windows® Disk
Defragmenter (located in Start > Programs > Accessories > System Tools) or an equivalent defragmentation
program to maximize computer performance and maintain data integrity for files on the hard drive. An entry
will automatically be made in the Windows™ system event log when this process is done and will provide
documentation of this step.

C. Electronic Data Backup
1. Schedule of Data Backups


Weekly: Full data backups onto one or more recordable compact discs (CD-R) or digital
video discs (DVD).



Daily: Full data backups onto an secondary hard drive.

2. Backup Procedures
Whenever making a backup (daily or weekly) include the directories and subdirectories :


C:\elandata (include all subdirectories)



C:\gc (must include subdirectories "data" and "methods", also include other relevant
directories)

Before making weekly backups, saving a copy of the Windows™ event log in the active
"elandata" directory will ensure archiving of all recent software system events (including

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communications between ICP-DRC-MS and ELAN software, as well as times of hard drive
defragmentation, and other Windows™ system events).
b) Secondary Hard Disk Backups



If available, use the computer’s secondary hard disk to store backup files.



Configure Microsoft Windows® Backup™ (Start > Programs > Accessories > System
Tools) program to do a daily backup of the computer's data directories (see Backup
Procedures)

Compact Disc (CD) Backups


Use the CD writing program installed on the computer to create CD backups (e.g., "Easy
CD Creator"™ by Adaptec, or equivalent software). Select the option that “closes” the CD
at the end of the writing session so the CD cannot be accidentally over-written.



Use CD-R disks only (recordable compact disks), not CD-RW disks (rewritable compact
disks).

d) Backup of Sensitive Data


Make a backup for sensitive data on duplicate, recordable compact disk. Store the two
CD-R disks in two different buildings.

D. Documentation of System Maintenance
1. Computer Maintenance: Record any maintenance of computer hardware, GC or ICP-DRC-MS
software in the instrument logbook. Place other electronic records relating to integrity of the data
and hard drive in the Windows™ event log. Back up the event log on a regular basis by saving a copy
in the active "elandata" directory. The event log will then be backed up along with the ELAN data
when backup CD-R disks and tapes are made.
2. Instrument Maintenance: Document system maintenance in hard copies of data records (i.e., daily
maintenance checklists, PerkinElmer service records, and instrument log book) as well as in
electronic records relating to instrument optimization (default.dac) and tuning (default.tun).

IV.

COLLECTING, STORING, AND HANDLING SPECIMENS; CRITERIA FOR
REJECTING SPECIMENS
A. Specimen Type
Specimen type is whole blood. No special instructions for fasting or special diets are required of patient or
study subjects.

B. Specimen Collection, Handling and Storage
1) The preferred volume of blood specimen is ≥0.5 mL; the minimum volume is 0.25 mL.
2) Acceptable containers for specimen acquisition include pre-screened polyethylene vials and
pre-screened blood collection tubes.
3) Samples should be stored in the dark at –20°C or lower temperature. Long term storage at
–70°C or less is preferred.
4) Specimen handling conditions are outlined in the Division protocol for blood collection and
handling (copies available in Branch, laboratory and Special Activities specimen handling

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offices). Collection, transport, and special requirements are discussed. If more than one
blood collection tube is used to draw blood from a subject, the blood tube for trace metals
tube should be drawn last. Draw the blood through a stainless steel needle into a prescreened blood collection tube. Blood specimens should be transported and stored at ≤
4°C. Once received, they can be frozen at ≤ –20°C until time for analysis. Portions of the
sample that remain after analytical aliquots are withdrawn can be refrozen at ≤ –20°C.
Thawing and refreezing of samples before analysis is discouraged.

C. Criteria for an Unacceptable Specimen
The criteria for an unacceptable specimen are either a low volume (< 0.25 mL) or suspected
contamination due to improper collection procedures or collection devices. Specimen contact with dust
or dirt may compromise test results. In all cases, request a second blood specimen.

PROCEDURES FOR MICROSCOPIC EXAMINATIONS
Not applicable for this procedure.

VI.

CHEMICALS, STANDARDS, AND QUALITY CONTROL MATERIAL
A. Chemicals
1) Deionized (DI) water, ≥18 MΩ cm resistivity.
2) Sodium acetate anhydrous, (CAS# 127-09-3), (C2H3NaO2--MW 82.04), (Sigma-Aldrich,
Milwaukee, WI) or equivalent vendor.
3) Glacial acetic acid, (CAS# 64-19-7), (CH3COOH--MW 60.05), reagent ACS grade (GFS
Chemicals, Powell, OH) or equivalent vendor.
4) Double-distilled Hydrochloric Acid, (CAS# 7647-01-0), (HCl-MW 36.461--37.0%--12.1M) (GFS
Chemicals Inc., Columbus, OH) or equivalent vendor.
5) Double-distilled Nitric Acid (CAS# 7697-37-2), (HNO3-MW 63.013-70.0%--15.8M) (GFS
Chemicals Inc., Columbus, OH) or equivalent vendor
6) Tetramethylammonium hydroxide, 25% w/w in methanol (CAS# 75-59-2), (C4H13NO-MW
91.15), (Alfa Aesar, Ward Hill, MA) or equivalent vendor.
7) Sodium tetra(n-propyl)borate, (CAS# 45067-99-0), (NaPr4B -MW 206.16), (ABCR, Germany)
or equivalent vendor.
8) Bleach (10% sodium hypochlorite solution) from any vendor.
9) Base whole blood, donated or purchased.
10) Inorganic mercury, 1000 mg/L in 10% nitric acid (SPEX, CertiPrep) or equivalent.
11) Methyl mercury chloride, standard solution 1000 mg/L (AlfaAesar) or equivalent.
12) Ethyl mercury chloride, powder (AlfaAesar) or equivalent

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B. Isotope Dilution Standards
1)

199Hg-Isotopically-Enriched Inorganic Mercury (199HgCl2), (Applied Isotope Technologies, Inc. Sunnyvale, CA) P/N 30530 or equivalent.

200Hg-Isotopically-Enriched Methylmercury (CH3200HgCl), (Applied Isotope Technologies, Inc. Sunnyvale, CA) P/N 30521 or equivalent.

201Hg-Isotopically-Enriched

Ethylmercury (CH3CH2HgCl), (Applied Isotope Technologies, Inc. Sunnyvale, CA) P/N 35025 or equivalent.

C. Quality Control Material
Quality control (QC) materials are made from pools of whole blood obtained from a donor source or
purchased from the vendor. See the Preparation of Quality Control Material section for details of preparation.
The control "base" blood and two QC blood pools intended for the mercury speciation assay are designated
as:
QC level

QC Designation ID

Base pool

BB-yy###

Low pool

LB-yy###

High pool

HB-yy###

Where substitutions are: yy = the last two digits of production year and ### = assigned pool identification
number. QC material intended for bench quality control purposes needs to be "characterized" as described in
the section Establish QC limits for each QC pool.

VII.

INSTRUMENTATION, EQUIPMENT, SOFTWARE AND SUPPLIES
A. Instrumentation
1. Gas Chromatography System
1) Gas Chromatograph: Perkin Elmer® Clarus 500, or equivalent system.
2) GC capillary column: Perkin Elmer® Elite-5 30m (meter), 0.25mmID, 0.25µm df (Catalog #
N9316076, Shelton, CT), or equivalent.
3) GC Transfer Line, noncoated capillary column: Perkin Elmer® Fused Silica Tubing 5m,
0.25mmID (Catalog # N9301356, S/N 920620, Shelton, CT) ), or equivalent.
4) GC-ICP-MS Heated Transfer Line accessory: Redshift® (P/N N0777440 for 115V or P/N
N40777361 for 230V, Italy), or equivalent.
5) Solid Phase Micro Extraction (SPME) Fiber Assembly, specifically, Supelco Analytical 100µm
Polydimethylsiloxane (PDMS) Coating (Red - Pack of 3), (Catalog # 57301, Supelco
Analytical, Bellefonte, PA), or equivalent.
2. ICP-DRC-MS System
1) Inductively-coupled plasma mass spectrometer, specifically, the ELAN™ DRCII (PerkinElmer
Instruments, Shelton CT), or equivalent.
2) Platinum (Pt) cones (Catalogue # SC2013-Pt and SC2014-Pt Perkin Elmer Instruments,
Shelton, CT) or equivalent.

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3) Perkin Elmer ICP-MS injector (Perkin Elmer Instruments, Shelton, CT), or equivalent.
3. Robotic Sample Processing Station
1) Robotic Liquid Sample Processing Workstation: LEAP® Technologies CombiPal® twin head
system, or equivalent system (Figure 1).
SPME
Head #1

SPME
Head #2

RAIL #1

RAIL #2

(2) Sample
Tray Holders
Controller
32 Vial SampleTrays
GC Injection Port
Heated
Sample
Agitator
#1

GAS CHROMATOGRAPH

Heated
Sample
Agitator
#2

Figure 1: Twin Head SPME Processing Workstation (LEAP Technologies, Inc.)
The Twin Head SPME CombiPAL (LEAP Technology, Inc.) sample processing workstation, mounted on
top of the GC, has a unique design: features two (2) computer-controlled SPME fiber injection heads
that run on a dual rail system. The two SPME Heads are independently-controlled and perform SPME
fiber equilibration/injection operations in tandem. This configuration allows for increased SPME fiber
equilibration time up to 20 minutes per SPME head while maintaining a sample-to-sample injection cycle
time of 10 minutes. The result is improved SPME efficiency and faster duty rates (6 injections per hour).
4. Equipment
1) Water purification system for providing ultrapure water with a resistivity ≥18 MΩ cm.
2) High-precision analytical balance capable of accurately weighing milligram amounts of
material to the tenth of a milligram or better.
3) A pH meter with one hundredth's of a pH unit readout or better, fitted with glass electrode
(pH probe). Temperature compensation probe for pH meter.

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4) Calibrated hand-held adjustable pipettors that cover the range of accurate liquid delivery
from 5 µL to 5000 µL. Research Pro™ Eppendorf® electronic programmable pipettors
(distributed by Brinkmann Instruments, Westbury NY) or equivalent.
5) Gas regulators for argon, helium and xenon (Airgas, Atlanta, GA) or equivalent.
6) Conventional oven (FREAS Model 605, Thermo scientific, Catalog No. 3166188), or
equivalent.
7) Plastic tent (Captair Pyramid, Erlab, North Andover, MA) or equivalent for preparation of
sodium tetra(n-propyl)borate solution.
5. Computer Software
1) The ELAN Instrument Control version 3.4 with Service Pack 2 (or later version) should be
installed on the computer controlling the ELAN DRC II™.
2) Chromatography data handling software, specifically, TotalChrom™ Workstation, version
6.3.1 or later (PerkinElmer® Instruments, Shelton CT). Install PerkinElmer's TotalChrom
Workstation package on the same computer containing the ELAN Instrument Control
software*. Contact PerkinElmer for installation and configuration of TotalChrom. Or consult
theTotalChrom Workstation User's Guide. This method assumes that version 6.3.1 of
TotalChrom Workstation package is installed.
3) Operating software for CombiPal autosampler, specifically, Chronos (2007-2010 Axel
Semrau GmbH & Co KG)
4) ChromLink™ 2.1 software (PerkinElmer Instruments). The installation of ChromLink™ is
straightforward when using the supplied installation utility.
5) pdFactory Pro (FinePrint Software, LLC, www.fineprint.com) or equivalent software. This
product is used for creating electronic Portable Document Files (pdf) directly from any
Windows® compatible application print dialog box.
6) A custom Microsoft Excel® macro procedure named "Extract TC Data". See Appendix B for
description and macro code.
6. Supplies
1) 200 µL pipette tips, 960 tips per case (Eppendorf® catalogue # 2235137-1, distributed by
Brinkmann Instruments, Westbury NY), or equivalent.
2) 300 µL pipette tips, 960 tips per case (Eppendorf® catalogue # 2235144-3, distributed by
Brinkmann Instruments, Westbury NY), or equivalent.
3) 1000 µL pipette tips, 960 tips per case (Eppendorf® catalogue # 2249044-3, distributed by
Brinkmann Instruments, Westbury NY), or equivalent.
4) 5 mL pipette tips, 500 tips per case (Eppendorf® catalogue # 2235081-1, distributed by
Brinkmann Instruments, Westbury NY), or equivalent.
5) 50-mL acid-cleaned volumetric flasks for triple spiked solution preparation (polypropylene or
Teflon flasks preferred). To acid-wash flasks, rinse with 1.2M hydrochloric acid followed by
rigorous rinsing with DI water. Repeat this process several times depending on prior use of
the containers.
6) 250-mL acid-cleaned volumetric flasks for derivitaizing reagent (NaPr4B) preparation
(polypropylene or Teflon flasks preferred). To acid-wash flasks, rinse with 1.2M hydrochloric
acid followed by rigorous rinsing with DI water. Repeat this process several times depending
on prior use of the containers.

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7) Acid-cleaned 1L PE bottles for buffer solution preparation. To acid-wash flasks, rinse with
1.2M hydrochloric acid followed by rigorous rinsing with DI water. Repeat this process
several times depending on prior use of the containers.
8) 50 mL polypropylene tubes pre-screened for Hg (Becton Dickinson) or equivalent.
9) 15 mL polypropylene tubes pre-screened for Hg (Becton Dickinson) or equivalent.
10) 1.5 mL polypropylene (PP) microcentrifuge tubes (Eppendorf® catalogue # 2236380-8,
distributed by Brinkmann Instruments, Westbury NY), or equivalent.
11) Tube racks for 1.5 mL microcentrifuge tubes (Eppendorf® catalogue # 2236422-7,
distributed by Brinkmann Instruments, Westbury NY), or equivalent.
12) Six or more PAL Tray20mL (part# 65487454, CTC Analytics, PAL system accesories)
13) CombiPal autosampler vials 20 mL, glass, (Microliter Analytical Supplies, Inc. product #162000) or equivalent.
14) Head space caps for 20mL glass vials (Microliter Analytical Supplies, Inc. product #160050M) or equivalent
15) Kay-Dry™ paper towels and Kim-Wipe™ tissues (Kimberly-Clark Corp., Roswell GA, or
equivalent vendor).
16) Teflon™-coated magnetic stirs bars (4). (Catalog Number 58948-974 or equivalent), VWR
Scientific Products, Buffalo Grove, IL.
17) Teflon™-coated magnetic stirs bars. (Catalog Number 58947-140 or equivalent, VWR
Scientific Products, Buffalo Grove, IL).
18) Cotton swabs (Hardwood Products Co. ME, or equivalent vendor).
19) Nitrile, powder-free examination gloves (N-Dex®, Best Manufacturing Co., Menlo, GA, or
equivalent vendor).
20) Biohazard autoclave bags (Curtin-Matheson Scientific, Inc., Florence, KY, or equivalent
vendor).

VIII. STANDARD PROCEDURE
A. Preparation of Stock Solutions
a) NaOAc Buffer Solution (0.1 M sodium acetate anhydrous)
1) Dissolve 16.41g Sodium acetate anhydrous into approximately 1900 mL of DI water in a
2000 mL polypropylene vessel with a magnetic stir bar on a magnetic stir plate, and mix
thoroughly.
2) Measure pH and adjust pH to 4.75 with glacial acetic acid. Make final volume adjustment to
2000mL with DI water.
b) Sodium tetra(n-propyl)borate (NaPr4B, 2% w/v)
1) Place one unopened vial containing 5.00 g of sodium tetra(n-propyl)borate, a squirt bottle of
DI water, and a clean 50 mL conical centrifuge tube inside a glove box or tent. Fully purge
glove box or tent with 100% nitrogen for 5 minutes to remove oxygen.
2) Open the vial containing sodium tetra(n-propyl)borate and add 10-20 mL of DI water with
constant swirling.

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3) Pour the dissolved contents into the clean 50 mL conical centrifuge tube. Wash the reagent
vial with additional DI water to dissolve remaining solids and quantitatively transfer
remaining reagent to the 50 mL tube. Cap the 50mL tube and remove from tent. Once
dissolved, the NaPr4B solution may now be safely used in normal atmosphere.
4) Inside a chemical fume hood, quantitatively transfer reagent solution containing 5.00 g of
NaPr4B to a 250 mL volumetric flask. Dilute to the final volume with DI water. Makes a 2%
(w/v) NaPr4B solution. Store at 4°C.

B. Preparation of Working Spike Solution
Caution!
Mercury compounds are toxic! Take extra care to avoid accidental dermal contact, ingestion or inhalation
of these materials. Wear appropriate personal protective equipment. Above all, wear a laboratory coat and
latex or nitrile gloves. Clean up any spill that might occur according to applicable hazardous material spill
procedures.
Exercise the utmost care in executing all measurements precisely to obtain the best accuracy for the
final concentrations. Use only pipettes, disposable tips and volumetric flasks that have been tested for
accuracy and will deliver liquid volumes with a precision of ±1% or better.
1. Preparation of Working Triple-Spiked Standards Solution
On the day samples will be digested, prepare fresh working spike solution. Take out of the 4°C
refrigerator bottles of vendor-supplied standard solutions of the following: In199Hg, Me200Hg , and
Et201Hg. Allow bottles to come to room temperature. The following intermediate and working
solutions should be prepared by weighing, thus the final concentrations of “spike” material in
working solution should be determined by weight.
a) Intermediate standard solution
1) Pipette 50 µL of In199Hg and Me200Hg standards into separately labeled 1.5 mL centrifuge
tubes. Add 1200 µL of DI water to each and mix thoroughly.
2) Pipette 50 µL of Et201Hg standards into a third 1.5 mL centrifuge tube. Add 150 µL of DI
water to each and mix thoroughly.
b) Working solution (containing mixture of 3 isotope standards):
1) Fill the 50 mL volumetric flask approximately half full with DI water.
2) Pipette 100 µL of each intermediate standard solution (In199Hg, Me200Hg, and Et201Hg) into
the flask. Complete the volume to the 50 mL mark with DI water.
3) The concentration of each isotope standard should be within a range of 0.7–1.3 µg Hg/L.
Note that units of concentration are expressed in terms of elemental Hg (not molecular
mass). Calculate the exact concentration of each isotope standard contained in the Working
Triple-Spike Standard Solution by dividing the starting concentrations (indicated by the
isotope standard’s certificate of analysis) by the dilution factors shown in Table 8-1 (dilution
factors may be adjusted to produce isotope standard concentrations in the range of 0.7–1.3
µg Hg/L) . Make appropriate adjustments to the calculations if volumes are deviate from the
prescribed amounts.

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TABLE 8-1: Dilutions Factors for Preparing Working Triple-Spike Standards
Mercury Species

Calculation (units = mL)

Dilution Factor

In199Hg

1.2 ÷ 0.05 × 50 ÷ 0.1 =

12000

Me200Hg

1.2 ÷ 0.05 × 50 ÷ 0.1 =

12000

Et201Hg

0.15 ÷ 0.05 × 50 ÷ 0.1 =

1500

C. Preparation of Quality Control Material
Order bovine blood from a proper vendor and characterize for total Hg (use DLS Method ITB001A 3001
"Whole Blood Hg/Pb/Cd" or an equivalent method).
1. Base pool preparation: Bovine blood with the lowest mercury content should be used. Distribute
base blood into pre-labeled 2 ml vials by 1.5 ml aliquots.
2. Low and High pool preparation: Analyze base blood for InHg, MeHg, and EtHg species. On the basis
of this calculation, spike add with each specie to the desiredappropriate volumes of InHg, MeHg, and
EtHg (not enriched) species values to obtain "low" and "high" pools. While maintaining constant
stirring of each pool, aliquot 0.5 mL of blood into a sufficient number of pre-labeled 2 mL vials to
provide QC material for 1000 or more runs. Store aliquoted QC material at a temperature of –70°C
or colder.

D. Sample preparation
Caution!
Work with open vials containing biological samples inside of a biological safety cabinet (BSC). Recap vials
before removing them from BSC. Wear appropriate personal protective equipment (lab coat, safety glasses
and gloves).
Preparation of blood samples for digestion:
1) Pipette 100 µL of blood samples into pre-labeled 1.5 mL tubes.
2) Add 100 µL of Triple Spiked Standards Solution. Recap and vortex each tube before
continuing to the next tube.
3) Add 500 µL of TMAH to all tubes. Cap and vortex.
2. Digestion:
1) Place rack containing capped tubes in an incubator or oven set to 80 ± 3°C for ≥ 20 hours.
3. Derivatization of the digested samples:
1) Aliquot 200 µL of digested samples into pre-labeled 20 mL SPME vials containing a “mini”
stir bar in each vial.
2) Add 7.7 mL of NaOAc Buffer Solution and 250 µL of NaPr4B Derivatization Reagent. Cap the
vial immediately and mix.

E. Requirements for Batch Analysis of Samples and QC Material
Process a predetermined set of samples and QC material for batch analysis. One “batch” run is defined
as the analysis of a contiguous set of samples (typically 20, may be more) bracketed by “Bench QC” material

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at the beginning and end of the set. Each Bench QC level (typically, a “high” and a “low”) should be analyzed
at the beginning and again at the end of the batch run. QC needs to be treated like the unknown samples,
i.e., each QC sample is individually prepared and goes through all the steps as is done for unknown samples.
It is not appropriate to report the QC results coming from the split analysis of a single QC sample if it has
already been processed (i.e., diluted, centrifuged, filtered, digested, derivatized, etc.). Note that this limitation
does not apply to the duplicate use QC material originating from the same original vial as long as they both
are processed identically like unknown samples and on the same day. It is permissible to "piggyback" two
runs in succession that are separated by at least two blanks during a single autosampler load (such as for an
overnight analysis), as long as each run of samples is bracketed by their own uniquely co-prepared bench QC
material.
1) Identify, gather, and thaw a predetermined number of sample tubes/vials containing the
blood samples to be analyzed in a batch run.
2) For each batch of samples run, thaw one tube of low and high bench QC (often identified as
"LB-yyxxx" and "HB-yyxxx"; for explanation of nomenclature, see Quality Control Material
section).
3) Label the necessary number of 1.5 mL microcentrifuge vials with appropriate identification
to ensure that they will be matched to their corresponding unknown samples and QC.
Similarly, label an equal number of 20 mL SPME vials.
4) Use the TMAH digestion procedure for blood samples and QC material.
5) Use the preparation of digested samples for analysis procedure to prepare samples and QC
material.
6) Cap all autosampler vials with the proper fitting septum caps.
The use of a barcode scanning device to electronically record sample identification from barcodes
printed on vial labels should be utilized if available.

F. GC Instrument Program
1) Set the GC analytical run parameters according to Table 8-2. Refer to the Clarus 500 User
Guide for programming specifics.
2) One or more parameters specified in Table 8-2 may be changed, if determined necessary, to
meet analytical performance goals.

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TABLE 8-2: GC Settings
Parameter

Setting

GC injector Temperature

Step #1: 1 min @ 220°C
Step #2: linear ramp to 280°C, hold

Carrier gas flow rate (Helium)

2 mL/min

Oven Temperature

Step #1: 1 min @ 75°C
Step #2: linear ramp to 250°C (at
rate = 45 °C/min)

Total flow ratio

28:1 @ 0.25 min

Transfer line Temperature (Aux auxiliary zone)

250°C

(Aux - auxiliary pneumatics)

OFF (all)

G. CombiPal Autosampler Program
1) CombiPal is initially set up and optimized by LEAP technology service engineers. The settings
can be optimized according to analyst needs (refer to PAL system user manual). The
following procedures present the key steps that are taken to set up the CombiPal
autosampler system for this method.
(a) Using PAL control terminal start at a window displaying "JOB QUEUE". Press the
ESCape key to return to the previous menu. Press function key F1 - "MENU". Rotate
the outer knob to scroll through items in a menu list. To select a highlighted item
press the central knob (ENTER button). Then use the outer knob to scroll through
available options for that item or to change a numeric value. Then press the inner
knob again to ENTER the displayed option.
(b) In the "MENU" window select "Utilities" followed by "Tray" then select "Agitator" and
enter parameters in Table 8-3. Then press "ESC" to get back to "Tray" option scroll
to the right select "Tray" (this "tray" is an option for first "Tray" selection) - enter
parameters
(c) In the "Utilities" window select "Injector" and select "GCInj1" - enter parameters. Also
in the "Utilities" window select "Vial" and select "Standard" - enter parameters in
Table 8-3.

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TABLE 8-3: CombiPAL Autosampler Settings
Parameter

Setting

Tray (Agitator)
Needle Penetration

20.0 mm

Tray type

SMMTray

Offset X, Y, Z

0, 0, 55.5 mm

Stand by Temp

OFF

Actual Temp

25.7°C (changes)

Speed

750 RPM

Agitation Time ON

Agitation Time OFF

Tray (Tray)
Needle Penetration

12.0 mm

Tray type

VT32-20

Offset X, Y, Z

-55.3, -188.1, -2.0 mm

Injector (GCInj)
Needle Penetration

35.0 mm

Vial (Standard)
Needle Penetration

43.0 mm

2) CombiPal is operated by Chronos software. Click on Chronos icon on the operating computer
desktop - Chronos method files used for this method can be accessed through clicking on
"Method Editor" on the Main Menu of Chronos. In the right corner click on tab "Load", which
allows to upload needed method file. There are five method files used in this method: SPME
bakeout LEFT.cam, SPME bakeout RIGHT.cam, SPME conditioning LEFT.cam, SPME
conditioning RIGHT.cam, TWIN SPME_cdc.cam. (The settings can be changed by the analyst
for better separation and analytical response.)
(a) To clean SPME fibers every day before starting a run - SPME bakeout LEFT.cam and
SPME bakeout RIGHT.cam are used. The settings should be as shown in Table 8-4.

TABLE 8-4: Chronos Settings (Baking)
Parameter

Setting

Bakeout Time (s)

420

GC Runtime + Cooling Down (s)

120

Needle Penetration Vial (mm)

Fiber Exposure (mm)

Needle Penetration (mm)

(b) To condition new SPME fibers use - SPME conditioning LEFT.cam and SPME
conditioning RIGHT.cam. The settings should be as shown in Table 8-5.

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TABLE 8-5: Chronos Settings (Conditioning)
Parameter

Setting

Bakeout Time (s)

1800

GC Runtime + Cooling Down (s)

120

Needle Penetration Vial (mm)

Fiber Exposure (mm)

Needle Penetration(mm)

TABLE 8-6: Chronos Parameter Settings (for Run)
Parameter

Setting

Source Tray

Tray1,1

Time

Enrichment Time

1200 s

Desorption Time

420 s

GC Cooling Down

180 s

Needle Penetration Vial

34 mm

Fiber Exposure

12 mm

Needle Penetration Inlet

35 mm

H. ICP-DRC-MS Instrument Setup
To improve workflow efficiency, do the programming steps described in this section before the day of
analysis.
1. Programming the DRC Gas Flow Delay Parameter
A special ELAN DRC™ setting, called "Flow Delay", needs to be changed from its default setting to
avoid the problem of the ELAN software forcing a time delay of several seconds before collecting
data at the start of a chromatographic run when in DRC mode. This setting can only be changed by
entering the ELAN software's Service Mode. This change only needs to be done once per software
installation or upgrade, or if the setting was deliberately changed by a field service engineer. It is a
good idea to inform the service engineer who intends to perform work on the instrument of the
importance of returning the "Flow Delay" to the non-default value of 1.

Important!
While in Service Mode, DO NOT make changes to any setting except for the one change described below.
1) From within the ELAN program and in the window entitled "Instrument Control Session",
choose menu item "Options" > "Service Mode." You will be prompted to enter a Service
Mode password. Enter the password "Elan6000" (omit the quotes and pay attention to

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capitalization) and click OK. If this password is not accepted, you will have to contact a
supervisor or a PerkinElmer service technician.

2) You will be presented with a new tab called "Service" within the Instrument window.
Maximize the window. At the bottom will be a row of tabs, click on "Gas". Look for the
parameter called "Flow Delay (Gas changes while in DRC Mode)". If its setting is a value
other than "1", click on the "Set Pauses..." button. Change the value in the field named
"Flow Change" to 1. Click the "Apply" button then click the "Close" button. Choose menu
item "Options" > "Exit Service Mode."
2. Programming the ELAN ".mth" file
1) If it is not already open, launch the ELAN program and in the window entitled "Instrument
Control Session", choose menu item "File" > "Review Files". Click the "Load" button for
"Method", the first item on the list. Navigate to the folder "C:\elandata\Method" and click on
"GC_Hg.mth" file then click the "Open" button.
2) If the "GC_Hg" file cannot be found, or it has been changed or corrupted in a manner that
makes its use questionable, then cancel the open file dialog box and close the Review Files
window by clicking the "Done" button. Do the following steps; otherwise, proceed to step 3:
(a) Make the active method file the active window (do this by clicking on the tool bar
icon that looks like a notepad with a "Cu" on it). Then click "File" > "New" on the
menu bar and then choose "Data Only" in the New Method window that appears.
Click "OK" then maximize the window. Complete this window with the information in
the Table 8-7.

TABLE 8-7 ELAN Timing Parameters
Parameter

Setting

Sweeps/Reading:

Readings/Replicate:

2725

Number of Replicates:

Tuning File:

C:\elandata\Tuning\default.tun

Optimization File:

C:\elandata\Optimization\gc_xe_drc.dac

(b) On the first line of the worksheet-like table, click in the cell of row 1 of the "Analyte
(*)" column. Type "Hg" then press "Enter" key. The row will suddenly be filled-in with
mercury's "Begin Mass (amu)" of 201.971 (or something close) and several default
parameters. Right click on "Hg" and a periodic table will appear. Select five Hg
isotopes from mass ~197.9670 to ~201.9710 and click "OK". All five isotopes
should be seen in the "Analyte (*)" column. Tab to next cells and fill in the
information shown in Table 8-8.

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TABLE 8-8: ELAN Analyte Parameters
Parameter

Setting

Analyte:

Begin Mass (amu):

197.9670* (first row, then proceed to the 4 other
isotopes)

End Mass:

Scan Mode:

Peak Hopping

MCA Channels:

Dwell Time:

Integration Time:
68125 (automatically determined by software)
*Actual mass may differ by a few hundredth of amu.
(c) Click on the "Processing" tab and enter the following information:

TABLE 8-9: ELAN Processing Parameters
Parameter

Setting

Detector:

Dual

Measurement Unit:

Cps

Process Spectral Peak:

Average

Process Signal Profile:

Average

Apply Smoothing:

Checked

Factor:

Auto Lens:

Off

Isotope Ratio Mode:

Off

(d) Skip the "Equation" tab. Click on "Sampling" tab and enter the following information:

TABLE 8-10: ELAN Sampling Parameters
Parameter

Setting

Peristaltic Pump Under Computer Control:

Unchecked

Sampling:

External

(e) (e) Click on the "Report" tab and enter the information in Table 8-11.

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TABLE 8-11: ELAN Report Parameters
Parameter

Setting

Report View | Send to
Printer:

Unchecked

Report Options
Template:

Automatically Generate
NetCDF File:

Checked
C:\elandata\reportoutput\

Report to File | Send to
File:

Unchecked

Report Options
Template:

Report File Name:

Report Format:

File Write Option

*Content of these fields is not important since Send To Printer/File is unchecked.

(f) Choose menu item "File" > "Save As" and navigate to "C:\elandata\Methods\"
folder. Enter "GC_Hg" as the name of the method file and click the "Save" button.
3) The ELAN method "GC_Hg" is now loaded into memory.
3. Creating the ELAN Sample Table ".sam" file
1) If it is not already open, launch the ELAN program and in the window entitled "Instrument
Control Session", choose menu item "File" > "Review Files". Click the "New" button for
"Dataset", the second item on the list. Navigate to the folder "C:\gc\data\" and enter the file
name "Hg" (where yy = last 2 digits of current year, mm = this month, and dd =
date of run, for example, Hg110421 denotes Mercury Speciation run Apr.21, 2011, then
click the "Open" button. The new dataset folder has been created and is now active. Click
on the "DONE" button in the Review Files Window.
2) Clicking on the tool bar icon that looks like three Erlenmeyer flasks. Then choose "File" >
'"New" on the menu bar. A new window will appear entitled "Samples - [Untitled]". Click the
"Batch" tab then click on the "Sample Template" button. A dialog box entitled "Sample
Template Data" will appear. Enter the following information:

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TABLE 8-12: Sample Template Data
Parameter

Setting

Sample ID:

001_

Measurement Action (*):

Run Sample

Method:

GC_Hg.mth

Sample type:

Sample

Wash Override (sec)

3) Parameters not mention in Table 8-12 can be left blank. Since the CombiPal autosampler
can analyze 64 samples the sample ID can range from" 001_ " to "064_
" .
4) From the menu bar, choose "File" > "Save As" and save the file in the directory "C:\gc\data\"
using the name "Hg.sam" (where yy = last 2 digits of current year, mm = this
month, and dd = date of run).
5) It is a good idea to save a copy of this file as a template, thereby avoiding the need to recreate it every time.

I. ICP-DRC-MS Performance Checks
The following performance checks should be recorded in an instruments log book.
1. Daily Performance Check
1) Daily before samples are analyzed, Aqueous Blanks and 10X dilution of Low Bench QC
should be analyzed to ensure that the instrument is functioning properly. Prepare all the
following using the same technique and supplies as samples, unless stated otherwise.
(a) Aqueous Blank. To prepare, add 200 µL of TMAH into a 20 mL vial. Insert a stir bar
in each tube. To each tube add 7.7 mL of NaOAc Buffer solution and 250 µL of
derivatization reagent (NaPr4B). Cap the vial immediately and gently mix it.
Aqueous Blanks are ready to be analyzed.
(b) 10X Diluted Low Bench QC. To prepare mix 0.1mL of Low Bench QC (LB) in 0.9 mL
NIST 955C L1. This sample is digested and then analyzed exactly the same way as
sample.
2) After SPME-GC-ICP-MS analysis of Aqueous Blank and 10X Diluted Low Bench QC are
complete, open the Aqueous Blank and 10X Diluted Low Bench QC RAW files in
TotalChrome. Examine the Aqueous Blank chromatograph for possible indicators of
contamination. Then look at the 10X Diluted Low Bench QC chromatogram. This
chromatogram should have visible peaks for the mass 202 isotopes of MeHg, EtHg, and
InHg. Their intensities should be three times (3X) greater than the baseline RMS (root mean
square) noise level, otherwise there may not be sufficient sensitivity and the instrument may
need to be optimized (see weekly/monthly performance check section).
2. Weekly Performance Check
1) Visual check of Torch, injector, RF coil, and Cones:
(a) Slide the vacuum chamber and interface away from the torchbox, and visually
check the cleanliness of these components. Notate any cleaning / replacing done

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in the "Daily Maintenance / Performance Checklist". For details on cleaning
procedures refer to IRAT Weekly Maintenance SOP.
(b) Injector: There should not be deposits on the inside of the injector. If there is,
remove and clean with 1-5 % v/v ultrapure nitric acid, a cotton swab. Alternatively,
replace injector with spare and clean dirty one in an overnight soak in 5% v/v nitric
acid (can be ultrasonicated, but is not typically necessary).
(c) Torch: Check for melting or cracking, and cleanliness. If necessary, replace with
spare and soak overnight in 5% v/v nitric acid bath. If torch is only dirty,
replacement / cleaning can be deferred to the regular weekly maintenance day.
(d) RF coil: Check for excessive corrosion (flaking). Replace if necessary.
(e) Sampler cone: Check for excessive buildup of matrix, cracking, or pitting. If
necessary, replace dirty cones with clean spare cones and clean.
2) Replace GC injector septum.
3) Visually check level and condition of oil in roughing pumps. Appropriate level for oil is ~¾
full. If color indicates the need to change oil soon, do so at the next weekly maintenance. Oil
is clear yellow when new. Light brown or “tea colored” is ok to use. Dark brown or “coffee
colored” indicates need to replace.
4) Weekly GC-ICP-MS system is optimized with Xe gas because of its similarities in ionization
potential to Hg.
(a) Start Plasma, soon after the plasma ignites set flow rate of Xe gas mixture (0.1% Xe
in Ar) at 0.01 - 0.25 mL/min on the GC main display to achieve Xe intensity >
350,000 cps (counts per second). In ELAN upload "gc_xe_daily_drc.mth". After gas
signal stabilizes (30-60min) in the "Sample" window press "Analyze Sample" in
"manual" mode. If Xe intensity > 350,000 cps, the instrument is optimized. If not
proceed with the following steps to optimize this instrumental setup. (The proffered
number of counts varies with the instrument.)
(b) In ELAN upload "gc_xe_xy_drc.mth". Also in ELAN program and in the window
entitled "Instrument Control Session", choose menu item "File" > "Review Files".
Click the "Load" button for "Optimization", the sixth item on the list. Navigate to the
folder "C:\elandata\Optimize" and click on "gc_xe_drc.dac" file then click the "Open"
button.
(c) SmartTune™ is used for DRC Mode optimization. Optimization parameters can be
selected from "edit list". Appropriate method file in "method" section to be selected.
The optimization for maximum Xe intensity should be performed (see IRAT
Optimization SOP).
(d) After recommended parameters were optimized save the optimized file as
"gc_xe_drc.dac". The current optimized values will appear automatically and will be
similar to the ones in Table 8-13.
(e) After optimization for maximum Xe intensity navigate to the folder
"C:\elandata\Method" and click on "gc_xe_daily_drc.mth" file then click the "Open"
button. Record this data.

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TABLE 8-13: ELAN Optimization Parameters
Parameter

Setting

Parameter

Setting

Nebulizer Gas Flow
(NEB):

1.5

Cell Path Voltage Std

- 14†

Auxiliary Gas Flow:

1.2

Rpa

Plasma Gas Flow:

Rpq

.5†

Lens Voltage:

4.75*

Cell Gas A

0.3

ICP RF Power:

1450

Cell Gas B

Analog Stage Voltage:

-1950*

DRC Mode NEB

1.5

Pulse Stage Voltage:

950*

DRC Mode QRO

- 16.90†

Quadrupole Rod Offset
Std

DRC Mode CRO

- 1.90†

Cell Rode Offset Std

DRC Mode CPV

- 51†

Discriminator Threshold

Axial Field Voltage

150†

†Suggested starting values only. Optimum parameters will depend on the outcome of
the optimization procedure.

3. Monthly Performance Check
1) Before starting make sure the plasma is turned off and transfer line temperature (AUX
button on GC main menu) should be lowered to 25°C (this is the lowest temperature the
set-up allows for). Disconnect the transfer line from ICP-MS.
2) Carefully examine the end of capillary in transfer line: make sure the end is open, no
melting, cracks, smooth surface, and no condensation. Replace if not in a good condition.
Additionally if the chromatographic peaks are progressively broadening may consider
replacing GC capillary. If this does not solve the problem, consider replacing GC column.
3) Connect spray chamber with the nebulizer to ICP-MS. Place the probe into the rinse solution.
Clamp the sample tubing in the peristaltic pump. Start the peristaltic pump at a low to midrange speed (i.e. 7 - 24 rpm). Allow at least 45 minutes warm-up time for the ICP-MS after
igniting the plasma. This warm-up time is for the RF generator.
4) After this warm-up time, perform a "daily performance check". Note: for this method the
standard IRAT Daily Startup SOP for ELANs is used monthly (since it requires changing
instrument set up). This monthly procedures makes sure that ICP-MS part of this
hyphenated method is functioning properly.
(a) Daily Performance Test
 Open your daily performance workspace, which should contain the following
files.
 Method file: Daily Performance_cdc.mth (the same as the Perkin Elmer file, with
the addition of the analyte Be at mass 9, and with the autosampler setup under
the sampling page).
 Dataset: "Daily Performance"
 Sample file: "daily_performance_cdc.sam"
 Report Template: "daily.rop"

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 Tuning File: default.tun
 Optimization file: default.dac
 Calibration file: none needed
 Polyatomic file: "elan.ply"
(b) Place a tube of "Daily Performance Testing Solution" into the row specified in the
sample window (batch tab). Select row one in the sample window (batch tab), and
click "Analyze Batch".
(c) Inspect the daily performance report as follows:
 Intensities and Oxides: Intensities should be above the Perkin Elmer
specification at 3 % oxides. Intensities are in the "Meas. Intens. Mean" column.
Oxides are given as a fraction under the "Net Intens. Mean" column on the "CeO"
row.

Instrument

DRC II (per 1 ppb)

>6,000cps

In
> 30,000cps

U
>20,000cps

Oxides
0.03 (3%)

 Compare against historical information in the instrument log.
Precision ("Net Intens. RSD"):
6100 : < 2-3% for Be*, Mg, Rh, and Pb.
DRC Plus and DRC II instruments: < 2-3% for Be*, Mg, In, and U.
Background counts:
DRC II : < 2 cps for masses 8.5 and 220
Doubly Charged species:
Typical "Meas. Intens. Mean" for Ba++ is ? < 0.03.
(d) If the results of the daily performance test fail to meet the PerkinElmer criteria,
optimization tests will need to be run (see SOP "ICP-MS Optimization")

J. ICP-DRC-MS Warm Up
1) Launch the ELAN ICP-DRC-MS Software and note whether all graphical indicators of
instrument readiness are green. If not, take the appropriate actions described in the
instrument's software and hardware manual.
2) Perform necessary maintenance checks as described in Chapter 5 of the ELAN 6100
Hardware Guide (e.g., argon supply, interface components, cleanliness, positioning, and
interface pump oil condition). Note the base vacuum pressure in the INSTRUMENT window
of the software. (Before igniting the plasma, the vacuum is typically about 8 x 10 -6 torr.)
Keep a record any maintenance procedures along with the base vacuum pressure in the
Daily Maintenance Checklist notebook.
3) In the INSTRUMENT window of the ELAN software, click the "Front Panel" tab and click the
plasma "Start" button to ignite the plasma. In the same window, the ignition sequence bar
(blue progress bar) will start to expand from the right, indicating the approximate time
before plasma ignition. The plasma may at first flicker but it should establish a more or less
steady intensity after 5-10 seconds.
On a rare occasion, the plasma may ignite emitting an orange, violently flickering light,
and electrical discharge noises will be heard. In this case, immediately shut off the
plasma by pressing the yellow "Stop" button on the ICP-DRC-MS instrument's front

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control panel. Wait 30 seconds then investigate the cause of the plasma misfire. A more
common occurrence is that the plasma may extinguish itself a few seconds after
ignition. Promptly reignite by pressing the "Start" button on the ICP-DRC-MS
instrument's front control panel. Usually, the plasma will stay lit after the second try. If
not, investigate the cause of this instability (refer to the ELAN DRC II Hardware Guide).

4) Soon after the plasma ignites perform daily performance.
5) Fill in the Daily Maintenance Checklist Book according to the completed optimization
procedures. If a tuning (mass-calibration) procedure was done, save it to the file
"default.tun," and also in a separate file containing the analysis date "default_MMDDYY.tun"
(where MM=month, DD=day, and YY=year).

K. GC-ICP-DRCII-MS System Startup
1. Entering Sample Names into the ELAN Sample Table
1) Click on the tool bar icon that looks like three Erlenmeyer flasks. If the current Samples
window is not this run's sample file, then choose "File" > "Open" on the menu bar and
navigate to and open this run's current data folder in "C:\gc\data\". Click on the file named
"Hg.sam" (yy = year, mm = digit month, dd = date) and open it. The Samples
window will be the one created in the section Creating the ELAN Sample Table ".sam" file.
2) Fill in the name of each sample by double-clicking after the "_" (underscore) in the cell
"sample ID". Type in the sample name and press "Enter" on the keyboard. In this manner,
enter the name of blanks, quality control, and sample that will analyzed in the run. If
barcodes are used on the sample labels, use the barcode scanner attached to the ICP-DRCMS computer to scan the sample ID from the barcode on each sample before placing it into
position in CombiPal autosampler tray.
3) Filling out the Samples table (Table 8-14).

TABLE 8-14: ELAN Samples Table
A/S Loc.

Batch ID

Sample ID

Measurement
Action

Method

…

Wash Speed
(+/- rpm)

001_ Aq.Blk

Run Sample

002_ Aq.Blk

Run Sample

003_ LB 10X

Run Sample

004_ LB 10X

Run Sample

005_ LB-yyxxx

Run Sample

006_ HB-yyxxx

Run Sample

007_ Sample

Run Sample

008_ Sample

Run Sample

…rows for 18 samples and SRM material were omitted for brevity
16

031_LB-yyxxx

Run Sample

032_HB-yyxxx

Run Sample

In the example table above, a run of 20 samples is shown so the last vial ends up being placed
in A/S Location #16 (this location corresponds to autosampler location which is defined in
Chronos, since there are two autosampler trays two #16 are used - "Left" and "Right". It is not
necessary to put A/S loc in ELAN - only for analysts benefit).

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The numbers preceding the underscore character correspond to the order of injection. These
numbers will later help the analyst find individual chromatograms based on injection number
instead looking for specific sample names during post-run data processing in TotalChrom.
4) When the sample table entries are verified to be correct choose "File" > "Save".
5) Print the ELAN Sample table by choosing the "File" > "Print Setup" > "Reports". In the
ensuing dialog box, select the preferred printer and click "OK". Next, choose "File" > "Print"
and then click the "Print" button. Refer to the printout of the ELAN Sample table for the
correct vial positions when loading samples into the CombiPal autosampler tray.

L. Starting the Run
1) Create daily data folder in C:\GC\Data under the name Hgyymmdd (i.e., Hg110422)
2) Launch ELAN Instrument Control program if it is not already up. Do not launch or start any
other programs at this time.
3) Check that the correct ELAN method is loaded and active in the window "Instrument Control
Session". If it is not correct, load the correct Method file. Check under the Sampling tab that
"Peristaltic pump under computer control" is unchecked, and the pull-down menu
"Sampling" indicates "External".
4) Check that the correct Sample file in the window "Instrument Control Session" is active. If it
is not correct, load the correct Sample file.
5) Load created dataset file "Hgyymmdd"
6) Check that the GC methods are correctly programmed.
7) Check that CombiPal autosampler methods are correctly programmed.
8) This step offers the advantage that the ELAN data files will be converted in real time to
TotalChom™ ".raw" files that have names containing a date-time stamp corresponding to
actual time of injection.
(a) Launch TotalChrom Navigator. In the resulting TotalChrom Navigator window,
choose menu item "Apps" > "ChromLink" (alternatively, you may launch ChromLink™
from the operating system "Start" > "Programs" menu).
(b) In the ChromLink™ program window, choose the menu item "Configuration" > "Mass
Details" and check the Nominal Name and Mass for mercury isotopes. If it is
missing or the ELAN tune ("default.tun") file was re-optimized earlier then
ChromLink™ needs to be configured (see Configuration of ELAN ChromLink™ on
page 35 for details). To save time, the analyst may choose to close the TotalChom™
Navigator and ChromLink™ windows and skip step 6 in its entirety. Data file
conversion via ChromLink™ can easily be done during post-run data reprocessing.
(c) In the ChromLink program window, click on the "Browse" button just right of the
"ELAN ChromLink file location" field. Navigate to the current working folder, doubleclick on it then click the "OK" button so that ChromLink knows where to save its
processed files.
(d) Otherwise, refer to step (b) of Data Processing and Analysis on page 37 for details
on proper setting of the ELAN ChromLink™ window's parameter fields. In the ELAN
ChromLink window, click the button "Start Processing ELAN Data Files" to put
ChromLink in watch mode so it will process each data for each injection in real
time. A new dialog box will open and indicate it is ready to convert data and waiting
for the first file.

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9) Launch Chronos program, which communicates with CombiPal autosampler and ELAN
software.
(a) In the main menu click on "Sample List" then upload analysis method for SPME
fiber cleaning "baking".
(b) Upload "SPME bakeout LEFT.cam" (to clean the left fiber) then select "Create
Schedule". The Schedule window will appear with time intervals required for the
measurement. Click "RUN" on the main menu, the run window appears and click
"run" again to proceed with the fiber cleaning.
(c) Repeat step (b) to clean right fiber and use "SPME bakeout RIGHT.cam"
(d) After fiber cleaning and daily performance check the QC and samples are ready for
analysis using SPME fibers. On the "Main Menu" select "Sample List" then click
"Load List". Upload Dual SPME .csl file. This file communicates with Left and Right
fibers. The table similar to the one below (Table 8-15) will appear. The table should
have Analysis Method "C:\Chronos\Methods\TWIN SPME_cdec.CAM" uploaded.

TABLE 8-15 Chronos Samples Table
Analysis Method

Source
Vial

Incubation
Time (s)

Enrichment
Time (s)

Desorption
Time (s)

GC cooling
down (s)

C:\Chronos\Metho
ds\TWIN
SPME_cdec.CAM

1200

420

180

1200

420

180

1200

420

180

1200

420

180

Rows for source vials 3 to 31 are omitted..
32

1200

420

180

1200

420

180

(e) "Source Vial" column shows which autosampler position will be first analyzed.
There are two racks (left and right) with vial position 1 to 32. Left rack gets
analyzed by Left SPME fiber and Right rack gets analyzed by Right SPME fiber.
There are two numbers "1" in the source vial column. First one always corresponds
to Left rack/Left fiber.
(f) Chronos communicates to autosmpler though "source vial" position, the analyst has
to make sure sample in ELAN correspond to location seen by Chronos. Example: if
the analyst wants to analyze slots 1-5 on both racks (total of 10 samples) the rows
below 10 should be deleted. To delete select the rows and click "Remove
Samples".
10) Check that the DRC gas is indeed flowing by making the ELAN's Instrument window active
and clicking on the Diagnostics tab. Inspect the Cell Gas A or B, its value should be
fluctuating at the current value ± 0.01 mL/min. If it is not, see section Turning on the
Reaction Cell Gas for details to turn on the DRC gas flow.
11) Check that all blanks, QC, and sample vials are loaded into their correct positions in the
CombiPal autosampler tray as designated by the ELAN Sample window (or its printout) and
in position seen by Chronos.

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12) Before samples are analyzed, daily performance check should be analyzed to ensure that
the instrument is functioning properly.
(a) Click on the ELAN "Instrument Control Session" window to make it active. Highlight
the samples and click the "Analyze Batch" button. A Run Progress box will appear
indicating that the ELAN software is now waiting for a signal from the Chronos that
indicates the occurrence of an injection.
(b) In Chronos: to analyze sample select "Create Schedule" then press "RUN" on main
menu and "RUN" again on the run window.
(c) View the chromatograms in TotalChrom and in the real time window of ELAN to
ensure that there are no problems with the analysis. After the Aqueous Blanks and
10X LB been analyzed the run can be started.
13) Open the ELAN "Instrument Control Session" Real-Time window by clicking the tool bar
button that looks like a Gaussian distribution (the blue chromatographic peak). After the
Real-Time window opens, click on the drop-down menu and select "Signal". Real-time data
will now be displayed.
14) CombiPal autosampler will seek the first vial and make an injection. A blue bar in the
ELAN's progress box will now indicate that data is being collected. The system can now run
unattended.
15) Check the progress of the run after 2 or 3 injections. Note the chromatograms appearing in
the ELAN's Real Time window. Adjust the signal scale in the Real Time window, as
necessary. Compare the positions and peak heights of each mercury species. It helps to
visually compare it to a printed reference chromatogram. If abnormalities in retention time,
peak height, or peak shape are readily apparent, the analyst may need to stop the
autosampler and abort the run in the Chrionos program and then ELAN. Correct the
problem(s) and restart the run.

Important
Remember to disable the ELAN's Auto Stop feature before re-enabling it otherwise the ELAN may perform
an auto shutoff prematurely.

M. Instrument Shut Down
1) The autosampler will stop after all samples have been analyzed.
2) Shut off ICP-DRC-MS plasma.
3) At the controller computer, visit the ELAN Instrument Control Session application and open
the "Dataset" window. Confirm that all samples ran successfully and that the corresponding
data for each sample is listed in this window.
4) Remove the QC and sample vials from the GC tray. Discard them according to CDC
biohazard waste disposal guidelines.

IX.

POST-RUN DATA ANALYSIS
A. Configuration of TotalChrom Integration Method
The following information is presented as a starting point to help the analyst develop robust integration
method parameters that will work best for most chromatography data. Many of these parameters will work

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just fine as presented below. However, the separation chemistry of GC columns can vary due to frequency of
use, column replacement, or because of individual sample "oddities". Some parameters may need to be
adjusted from time to time to maximize the ability of TotalChom™ to properly integrate peaks and identify
components with minimum operator intervention. Therefore, the analyst should pay particular attention to the
chromatograms produced in every run and make necessary adjustments as warranted. The analyst should be
familiar with the TotalChrom's frequently used integration functions, which are described in Chapter 18 of
TotalChrom Workstation User's Guide: Volume II.
1) The creation of a new method file in TotalChrom is done the first time TotalChrom is setup,
or it will need to be recreated if the file "Hg.mth" cannot be found or has been corrupted. In
the TotalChrom Navigator window, choose the menu item "Build" > "Method." In the next
dialog box, click the "Create a new method" radio button and click "OK." The default method
will load into the method editor.
2) Click on the "Components" item in the menu bar in Method Editor. If the menu item "Delete
All Components" is not grayed out, select it and click "OK" when prompted to "Delete all
components, calibration levels, and calibration replicates".
3) Choose the menu Item "Process" > "Integration". Click on the "Integration" tab in the
"Process" window. Enter the information shown in Table 9-1. These values are to be used
as a starting point, but the analyst may make appropriate changes to one or more of the
integration parameters as necessary.

TABLE 9-1: Integration
Basic Parameters

Advanced Parameters
Value

Value

Bunching Factor :

Peak Separation Criteria

Noise Threshold :

115

Width ratio :

0.2

Area Threshold :

577

Valley to peak ratio :

0.01

Exponential Skim Criteria
Peak height ratio :

5.000

Adjusted height ratio :

4.000

Valley height ratio :

3.000

4) Click on the "Baseline Timed Events" tab. Enter the information shown below in Table 9-2.
The analyst may make appropriate changes to one or more of the Baseline Timed Events as
may prove necessary.

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TABLE 9-2: Baseline Timed Events
Defined Events
Time

Event

Value

Code

0.000

Set Area Threshold

125

0.000

Set Noise Threshold

0.000

Smooth Peak Ends On

+SM

2.222

Set Bunching Factor

Level

5) Click on the "Optional Reports" tab. Uncheck the box for "Keep temporary files".
6) Click on the "Replot" tab. Enter the information shown in Table 9-3. The analyst may make
appropriate changes to one or more of the Replot parameters as may prove necessary.

TABLE 9-3: Replot
Plots

Miscellaneous

Generate a separate
replot :

checked

Start plot at end of
delay :

checked

Number of pages:

Gradient overlay :

not checked*

Retention Labels :

Top of Plot*

Draw baselines :

checked*

Component Labels :

Actual time

Timed Events :

checked*

Scaling Type :

Absolute
Scaling

Plot Title :

Chromatogram

X axis label :

Time [min]

Scaling Parameters

Y axis label :
Scale Factor :
1.000000*
*These parameters maybe altered to suit the analyst.

Response [mV]

7) It is unnecessary to click on the "User Programs" tab because it is not used. Close the
Process window by clicking on the "OK" button.
8) In the Method Editor window, choose the menu item "Components" > "Global Information."
Click on the "Integration" tab in the "Process" window. Enter the information shown in Table
9-4:

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TABLE 9-4: Global Information
Volume units :

µL

Unidentified Peak
Quant.

Quantitation units :

Calibration factor :

1.000e+06

Sample Volume :

1.000

Always use calib. Factor
:

selected

Void time (min) :

0.000

Calibration

RRT Calculation

External Standard

selected

Reject outliers during
calibration :

not checked

Use first peak in run as
RRT reference:

selected

Sample Amount
Options
Correct amounts for
calibration standards :

not checked

Convert unknown
samples to
concentration units:

checked

9) The "LIMS Results" tab is not used. Click the "OK" button to close the window. The
parameters in Table 9-4 are starting points. The analyst may make appropriate changes to
one or more of the Global Information parameters as may prove necessary.
10) In the Method Editor window, choose the menu item "Components" > "New Component." The
white list box in the left portion of the window will be empty. Click in the empty field labeled
"Name" and type "Hg0". Press the tab key and enter "1.614" in the field labeled "Retention
time". Select the radio button labeled "Peak" if it is not already selected. Leave the other
fields and check boxes unaltered. Click the "New Component" button. Enter each of the
component names and parameters listed in TABLE 9-5.

TABLE 9-5: Method Editor - Components Settings
Retention
Time

Absolute
window

Relative
window

Find tallest peak in window

Hg0

1.614

InHg

3.470

MeHg

2.640

Yes

Ethg

3.176

Yes

Name

11) Click the "New Component" button before starting a new component. After entering the last
component, click the "OK" button. The values for Retention Time, Absolute Window and
Relative Window serve as starting points. The analyst may alter these values as actual
chromatographic results may dictate.

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12) In the Method Editor window, choose the menu item "Components" > "Defaults." Click on
the "Identification" tab". Enter the information shown in Table 9-6.

TABLE 9-6: Components Defaults – Identification
Parameter

Setting

Parameter

Setting

Component Type :

Peak

Reference :

blank

Absolute window :

Internal Standard :

blank

Relative window :

Find tallest peak :

Not checked

13) Click on the "Calibration" tab in Components Defaults Window. Enter the information shown
in Table 9-7.

TABLE 9-7: Components Defaults – Calibration
Parameter

Setting

Amount

Calibration Type :

Use Calibration Factor

1.0000E-6

Scaling :

None

Weighing :

None

Response :

Area

14) The "User Values/LIMS" tab is not used. Close the "Components Defaults" window by clicking
the "OK" button.
15) In the Method Editor window, Choose "File" > "Save As." A window appears inviting you to
enter any information pertinent to this method which will be saved with the method. Enter
your name and the date this method was created. Click "OK" and a "TotalChrom File-SaveAs" dialog box opens. Navigate the directory tree to get to the folder C:\GC\Methods ).
Double-click on this folder. In the "File name:" field, enter "Hg-Template.mth". If there is
already a file in that folder with the same name, highlight that file and right-click the mouse.
Choose "Rename" and give the file a new name (e.g. add "backup" to the name). Now, you
can click the "Save" button. Close the "Method Editor" window.

B. Configuration of ELAN ChromLink™
ELAN ChromLink™ should be configured after initial installation of the program or when the ELAN
tune ("default.tun") file is re-optimized, and when there is available at least one recent ELAN NetCDF
file (with the ".nc" extension) containing data for the mass of interest that was collected since last
update of the "default.tun" file.
1) Launch TotalChrom Navigator. In the resulting TotalChrom Navigator window, choose menu
item "Apps" > "ChromLink" (alternatively, you may launch ChromLink™ from the operating
system Start > Programs menu).
(a) Inside the ELAN ChromLink window, click on menu item "Configuration" > "Default
TotalChrom Method". Click on the "Browse..." button and navigate to the directory
C:\gc\methods\. Select "Hg-TC.mth" and click the "Open" button.
"C:\gc\methods\Hg-TC.mth" will now be the ChromLink™ default method. Click "OK"
to close the "Default TotalChrom Method" window.

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(b) Inside the ELAN ChromLink window, click on the "Set" button. A window entitled
"Operating Mode" will open. Inside the ELAN ChromLink window, click on the "Set"
button. A window entitled "Operating Mode" will open. Click on the "Automatic process all ELAN NetCDF files in specified location" radio button. The lower radio
buttons will gray out. Click the "OK" button to close the window.
(c) Click on the "Browse" button by the "ELAN NetCDF file - location/file to be
converted" space. Choose the path "C:\Elandata|reportOutput". The location/file
to be converted will now read "C:\Elandata|reportOutput\*.nc".
(d) Click on the "Browse..." button for "ELAN NetCDF Chromlink file location (sequence
and raw files generated by Chromlink)" field. An open file dialog box will open;
choose the file to which all the data should be place (usually the data file that was
created that day).
(e) Click on the "Start Processing ELAN Data Files" button. A window entitled
"Processing ELAN Data" will appear.
2) At this time, ChromLink™ may be closed by selecting "File" > "Exit." Click "OK" at the dialog
box asking if you want to quit ChromLink™.
3) In addition to configuring ChromLink™ itself, it is necessary to alter one value in the "seed"
method file that ChromLink™ uses to set a select number of parameters to certain default
values. This step only needs to be done once following the installation of ChromLink™.
(a) In the TotalChrom Navigator window, choose the menu item "Build" > "Sequence"
and a dialog box called "Startup" will appear. Click on the radio button labeled "Load
sequence stored on disk" then click the "OK" button. Navigate to the folder on the C
drive that contains the ChromLink™ program file (usually in C:\PenExe\ChromLink
but if it is not there, check under the C:\Program Files directory). Click on the
sequence file "seed.seq" to highlight it (if this file is missing, reinstall ChromLink™).
Click the "Open" button. A spreadsheet style sequence table will present itself in a
window called "Sequence Information - Channel A". There will be a minimized
window for channel B data; ignore this window.
(b) Choose menu item "File" > "Save." Close the Sequence Editor window by choosing
"File" > "Exit" from the menu bar.

C. Data Processing and Analysis
Refer to Figure 1 "Post-Run Data Processing Work Flow Diagram" (page 38) for a summary
representation of the important aspects of post-run data processing.
1) Open Microsoft Windows® File Explorer and open the current working GC data directory
(e.g., C:\GC\Data\). Select all files ending with the .rst and .idx and
"delete" them to the Microsoft Windows® Recycling Bin.
2) If it is not already open, launch TotalChrom.
3) If ChromLink was not run in real-time data collection mode during the run as described in
step 6 under Starting the Run (see page 29), then do the following:
(a) In the TotalChrom Navigator window, choose menu item "App" > "ChromLink."
Choose the menu item "Configuration" > "Mass Details" and check the Nominal
Name and Mass for mercury. If it is missing or altered then ChromLink™ needs to
be configured (see Configuration of ELAN ChromLink™ on page 32 for details).
(b) Check that the Mode field indicates "Automatic - Process all NetCDF files in
specified location". If it does not, click the Set button to the right of this field and in

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the resulting "Operating Mode" dialog box, click the "Automatic - process all ELAN
NetCDF files in specified location" radio button, then click the OK button. Next,
check that the Field labeled "ELAN NetCDF file - location/file to be converted"
indicates the correct data folder. This should be "C:\elandata\Reportoutput\*.nc". If
it is not, click the Browse button to the right of it and navigate to that folder. Doubleclick on that folder then click the "OK" button to close the front most dialog box.
Last, click the Browse button to the right of the field labeled "ELAN ChromLink file
location..." In the dialog box "Select TotalChrom Data Location", navigate to the
folder containing the run data and double-click on it. Click the OK button to close
that dialog box. In the ELAN ChromLink window, click the button "Start Processing
ELAN Data Files" to start processing of the run data. A new dialog box will open and
provide current information on the status of the data conversion.
(c) When data conversion by ChromLink is completed within a minute or two, a
message in the step field will indicate "Successfully Finished". Click the Close
button. At this point, you may close the ELAN ChromLink application by choosing
"File" > "Exit" or clicking on the window "x" box. In the resulting "OK to quit?"
confirmation dialog box, click the "OK" button.
4) In the TotalChrom Navigator window, choose the menu item "Build" > "Method." Click the
"Load method stored on disk" radio button and click "OK." In the TotalChrom File-Open"
dialog box, find C:\GC\Methods folder and open the "GC_Hg.mth" file. The template method
file should now be loaded.
If, instead of loading the method file, an error message appears stating that the file is
unavailable because it is in use and asks if you would like to open it in Read-Only mode,
click the "No" button. Cancel the Open-File dialog box. Exit the Graphic Method Editor. In the
Navigator window, choose menu item "Admin" > "CAM Administrator." A window will appear
with two panes. In the left pane, click on the "+" sign in front of "TotalChrom Servers" to
expand it. Click on the computer icon on the next line that just appeared to highlight it. In the
right pane, under the heading "Resource/Instrument", select the first item. If there is more
than one item, select every item by shift-clicking on each item. Every item should now be
highlighted. Choose "Edit" > "Remove Locks" (or press the Delete key on the keyboard). Next,
click on the "+" sign in front of "Users" to expand it. Click to highlight your TotalChrom user
name that appeared. In the right pane, under the heading "Resource/Instrument", select
every item and Choose "Edit" > "Remove Locks." This action serves to unlock files and make
them available for editing. If in the future, TotalChrome™ complains that files cannot be
edited because they are locked, use CAM Administrator to unlock them. Choose "File" >
"Exit" to quit CAM Administrator. Start again at the beginning of this step to open the Method
Editor.

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Figure 1: Post-Run Data Processing Work Flow Diagram

5) Choose "File" > "Save As." At the next window you will be invited to enter information about
the method. You may enter pertinent information but this is optional. Click the "OK" button
and a TotalChrom File-Save-As" dialog box opens. Navigate the directory tree to get to the
folder that contains the ELAN data files for this run (typically in the folder C:\GC\Data\ ).
Double-click on this folder. In the "File name:" field, enter the same name as it exactly
appears for the folder that will contain it (i.e. Hg convention where yy=2-digit
year, mm=2-digit month, dd=2-digit date). Click the "Save" button then close the "Method
Editor" window.
6) In the TotalChrom Navigator window, choose the menu item "Build" > "Graphic Edit." A
TotalChrom File-Open" dialog box appears, but click on cancel to close it. On the Graphic
Method Editor's menu bar, choose "File" > "Open" and navigate the file-open dialog box to
the folder containing the method file created in the preceding step. Click on that file and
click the "Open" button. Return to the Graphic Method Editor's menu bar and choose "File" >
"New Data File." Navigate to C:\GC\Data\ and double-click on the folder containing the run
data. Find and click on a data file (indicated by the ".raw" extension) that corresponds to the

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"-LB0972". When this file appears in the File Name field, click the "Open" button. If a
message box appears with the warning "Unable to open this file: default.mth", click "OK" to
clear the message (you do not have to go to CAM Administration to unlock it). Do the same if
another message warning box appears (i.e. click "OK" again to clear it). You should be in the
"Graphic Method Editor - " window and see a chromatogram.
7) Under the menu item "Calibration" > "Show Windows" there should be a check mark beside
"Show Windows." Retention window bars (looks like "H" style error bars) will be present
when it is checked. Each retention time window bar should be located above the
chromatographic baseline and contain an identified peak within its bounds. If there are any
bars at the bottom of the chromatogram located below the baseline, choose menu item
"Calibration" > "Edit Components." Click on the first mercury species peak that falls outside
its retention time window to select it. In the group of data fields located on the right side of
the window, click on the "Name" dropdown arrow (located on the right side of the data entry
field) and choose the appropriate species by nameIt is usually not necessary to alter the
retention time window's "Absolute" and "Relative" window parameters, but you may do so if
experience dictates that a change will be beneficial. Click the "Next" or "Prev" button. Repeat
these steps for each mercury species peak that was not properly identified because it was
outside its retention time window. When the editing of peak retention time windows is
complete, click on the menu bar item "Return". Next, choose "File" > "Save" followed by "File
> Exit".
8) "In the TotalChrom Navigator window, choose the menu item "Build" > "Sequence" and a
dialog box called "Startup" will appear. Click on the radio button labeled "Load sequence
stored on disk" then click the "OK" button. The following steps should be used to create the
sequence used for reprocessing.
(a) Navigate to the folder containing the run data and click on the sequence file (ends
with ".seq") corresponding to the run (named "Hgyymmdd.seq" where where yy=2digit year, mm=2-digit month, dd=2-digit date ). Click the "Open" button. A
spreadsheet style sequence table will present itself in a window called "Sequence
Information - Channel A". There will be a minimized window for channel B data,
ignore this window. Look for the "Method" column and click on the first cell in row 1
in this column. Right click the mouse and a contextual menu will appear; choose
"Browse". In the resulting File-Select dialog box, navigate and choose the method
file (ending in ".mth") created earlier. Click on the Select button. The path and name
of the new method file will replace the default information in this cell. Right click
this cell again and choose Fill Down. The new file name information will fill down to
every cell in the "Method" column. Look for the "Rpt Fmt File" column and follow the
same process that was followed with the "Method" column. Instead of choosing the
method file (ending in ".mth") choose the report file (ending in ".rpt). If the report
file is not listed, click on the "default.rpt" file. In the bar that contains the file's path,
change the "default" to "Hgyymmdd." Right click this cell again and choose Fill
Down. The new file name information will fill down to every cell in the "Rpt Fmt File"
column.
9) In the TotalChrom Navigator window, choose the menu item "Reprocess" > "Batch." A new
window appears entitled "Batch Reprocessing". Choose menu item "File" > "Sequence" and
another window appears entitled "From Sequence". Locate the top field labeled "Sequence
file" and look for a button with an open folder icon immediately to the right of the field. Click
this button and navigate, if necessary, to the folder containing the run's sequence files. Click
on the sequence file and click the "Open button." Upon return to the previous window, set
Start Analysis to "Peak Detection" and End Analysis to "Quantitiation". Set Batch Printer to
"pdfFactory Pro". Change Batch Execution to "ndlb-168462" or anything other than
"Interactive". Check "overwrite existing result files" and select "Update existing raw file
header with new sequence". All other parameters should remain unchanged. The
parameters are shown in TABLE 9-8.

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10) Click the "OK" button. Reprocessing of the chromatographic raw data will commence. The
bottom panel in the window will update with each file's name as it is processed. When
processing is done, this panel will be clear of files. Close this window.

TABLE 9-8: TotalChrom™ Navigator - Reprocess Batch
Parameter Name

Parameter Setting

Starting Row

Ending Row

Channel A

Checked

Channel B

Not Checked

Start Analysis

Peak Detection

End Analysis

Quantitation

Batch Execution

”ndlab-168462” or equivalent

Batch Printer

pdfFactoryPro

Batch Plotter

None

Enable Optional Reports in Method
Use Method is Result File

Not Checked
Grayed Out

Overwrite Existing Result Files

Checked

Raw File Treatment

Update existing raw file header with new
sequence

11) In the TotalChrom Navigator window, choose the menu item "Reprocess" > "Results." A new
window should open called "Reprocess Results". If you get an error message telling you that
you can only open this in read-only mode, then unlock the files (follow the procedure
described in step 4 of this section). Select from the menu "File" > "Open." In the open file
dialog box, click on the "Files of type:" dropdown menu and select "IDX files (*.idx)". Navigate
to the folder containing this run's data and click on the newest file (in the format of
"Hg--"). Click "Open." In the Reprocess
Results window will be presented a chromatogram for the first sample in the sequence.
Carefully inspect the chromatogram one peak at a time for correct peak identification and
accurate baseline. If you are satisfied that there are no integration problems, proceed to the
next sample's chromatogram by selecting "File" > "Next File" from the menu bar or pressing
"F3." Examine all chromatograms in this manner and make corrections in peak identity and
integration as necessary. Make notes concerning issues encountered with individual
chromatograms and changes that were made. If a chromatogram is changed or edited in
any way, be sure to select "File" > "Save" to save your changes. See the chapter entitled
"Developing Processing Parameters in the Method" in the PerkinElmer TotalChrom
Workstation Users Guide for a detailed explanation on how to use integration events to
optimize the integration of a chromatogram. After review of each and every chromatogram,
select "File" > "Exit" from the Reprocess Results menu bar.
12) Repeat step 9 except set both Start and End Analysis to "Report Generation". Set Batch
Printer to "pdfFactory Pro". A new window will open. When reprocessing has completed, click
the "Save" button on the pdFactory Pro window. In the Save As dialog box that appears,
navigate to the run's data folder and create a new pdf file named "Hg report". Be
sure to include a space between "report" and the first word of the new file. Click the "Save"
button. This pdf file is to be kept and backed up, for archival purposes, in the same folder

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with all the other chromatographic data files for this run. Click the "Close" button to close
pdFactory Pro window.
13) Open Microsoft Excel and choose "GCICPMS" > "Extract TC Data" from the menu. In the
open file dialog box, navigate to the folder containing the run's data files. A box titled
"Choose a Sequence File" should appear. Navigate to the folder that contains the sequence
file that you are working with. Double click on the sequence file. Immediately a macro will
run that will extract the data and put it into a format that is easily exported into the
database. Just before the macro finishes, a "Save As" dialog box will open giving you the
opportunity to save the file as an Excel workbook. Give the file a name as follows:
"Hg results". The multi-tabbed Excel workbook contains a worksheet suitable for
data exportation to the Frontends (MS SQL Server 7™) database.
14) Calculated “spike” concentrations for enriched InHg, MeHg and EtHg in Triple-spiked
standard solution should be entered in the column “ppb Spike” in Microsoft Excel. Then, in
Microsoft Excel toolbar choose "GCICPMS" > "Deconvolute" from the menu, sample
concentration values will be calculated and appear in the column “ppb Result”.
15) The data processing portion on the instrument controller computer is now complete. At this
point you may close Microsoft Excel® and TotalChrom Navigator.
16) Throughout the sample analysis it is important to ensure that three mercury species peaks
(InHg, MeHg, and EtHg) are well resolved. Thus, the following criterion is followed to
accept/reject the sample analysis. The Microsoft Excel document containing concentration
results displays retention times for each mercury species in the column labeled “RT”.
Mercury species elute from a chromatographic column in the following order MeHg, EtHg,
InHg with relative retention times tr1, tr2, tr3, respectfully. For the purpose of establishing
relevant criterion, relative retention time for InHg (tr3) is set to 1. Then the ratio (tr2/tr1)
=0.91 ± 0.13 and (tr3/tr1)=0.77 ± 0.12 (± represents three times standard deviation). If the
relative retention time ratios fall outside the allowable error- the samples should be
repeated.

RECORDING OF SAMPLE AND QC DATA
A. Transferring the Data to the Branch Database
1) Transfer the "Hg results".xls file via recordable media (e.g., USB flash drive) to the
appropriate subdirectory on the network drive where exported data are stored. (Note that
directories are named according to instrument/year/month/ and study name or ID, for
example,
\\cdc\project\CCEHIP_NCEH_DLS_IRATB_COMMON\Nutritional\Instruments\ELAN
_DRCM/2011/04/Hg110423 Results - Study 2006-09".)
2) From a computer that has access to Frontends database used for tracking data, log in using
your user ID and password. After you log into the database, open the FrontEndSet folder in
the GO TO window.
3) Click the "Add Sample Results to Database" button. New buttons will appear. Click the
"Import Instrument Data File" button. For "Instrument", choose "ELAN-DRC2M" (or the
appropriate instrument). For "Assay", choose "HgSpecGC". Enter the appropriate Analyst and
Study for these two fields. It is not necessary to fill-in the "IS Lot Number" Field. Select the
location of the data file on the network drive and press the "Open" button.

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4) In the "Imported Results" table, pressing the "Find X's" button will show only those samples
whose sample ID is not recognized as a valid QC pool ID or sample ID for this study.
(Sample IDs are set up when the study is logged into the database.) If necessary,
corrections to sample IDs and dilution factors can be made in this table (e.g., correction of
transcription errors and adjustment for level of dilution). If samples were diluted for
analysis, both the sample ID and the dilution factor need to be edited in this table before the
values are transferred to the database. First, change the dilution factor to reflect the way
that the sample was analyzed then edit the sample ID to remove any comments about the
level of dilution at which the sample was analyzed. (The replace command is useful here.)
5) When corrections to sample IDs are made, press the "Recheck" button to evaluate the
sample IDs. Any sample or analyte row marked "Not Recognized" will not be transferred to
the database when the "Transfer" button is pressed.

B. QC Data
Store the results of the QC samples analyzed in each run in the Frontends database when all other data
for the run is imported from the ELAN software. Refer to "Recording of Data" described above for how to
import data into the Frontends database. The database allows for the printing of several types of QC reports.

XI.

FINAL DATA REVIEW
A. Analysis Printouts and Analyst Run Report
If the samples analyzed are part of a study that has an associated study folder place the analysis
printouts in the study folder(s). Store the results of the patient samples analyzed in each run in the
Frontends database when all other data for the run is imported from the ELAN software. Refer to "Recording
of Data" described above for how to import data into the Frontends database. The database allows for the
printing of a run summary report that indicates whether any particular patient-sample results are outside of
the normal concentration reference range or whether any measurement failed precision limits.

B. Plotting QC Results
When the Frontends database is used QC plots are updated automatically when the data are imported
into the database. Monitor these plots regularly for any trends in the bench QC results. If trends are
observed, contact the laboratory supervisor.

C. Supervisor Review
The Frontends database allows the supervisor to review the QC and sample results directly in the
database. The data from each analytical run is stored in pdf format on the CDC shared drive under the
instrument that the analysis was performed on.

XII.

REPLACEMENT AND PERIODIC MAINTENANCE OF KEY COMPONENTS
A. ICP-MS Maintenance
Part numbers listed below are PerkinElmer part numbers from their 2010-2011 Consumables Catalog.
1) Cross-Flow II Replacement Liquid and Gas Tip Ferrules (part #09920518 and #09920515,
respectively). Keep at least two spares on hand.

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2) Injector Support/Torch Base (part #N812-0116). Keep one spare on hand.
3) Torch O-Ring Kit (packages of four, part #N812-0100). Keep four spare packages on hand.
4) Quartz torch. At least one spare torch should be on hand (part #N812-2006).
5) 0.8-mm i.d. sample injector (part #N8126039). At least one spare injector should be on
hand.
6) RF coil (part #WE02-1816). One spare should be on hand.
7) Platinum (Pt) Skimmer (part WE027803) and sampler cones (part WE027802). Keep at
least two spares of each on hand.
8) Skimmer and sampler cone O-rings (part #N812-0512 and #N812-0511, respectively).
Keep at least 10 spares of each on hand.
9) Series II replacement Ion lens (part #WE018034). Keep two spares on hand.
10) Pump oil for the roughing pump (part #N812-2004). Keep two bottles on hand in general
lab storage.

B. GC Maintenance
1) Septa (part # 20654, Supelco, Bellefonte,PA) in the GC injector should be changed weekly.
2) Glass inlet (part # 2631405, Supelco) in the GC injector should be checked weekly, if
condensation or any particulates are found (often parts of septa) should be replaced.
3) GC column (Catalog # N9316076) should be replaced when the quality of chromatographs
decreases.
4) GC Transfer Line noncoated column (Catalog # N9301356, S/N 920620,) should be
changed when the quality of chromatographs worsens.
5) Graphite ferrules (part # 09903700, Perkin Elmer Instruments, Shelton, CT).
6) Vu-Union Vespel (part # 20428, Rescek).

XIII. LIMITS OF DETECTION
The limit of detection (LOD) for mercury species in blood specimens are calculated using data from ≥60
separate analytical runs. To determine this method’s LOD’s, prepared blood samples were used containing
known amounts of inorganic, methyl and ethyl mercury at four different concentrations (levels #1–4). Level
#1 had concentrations of each mercury species that were close to the anticipated LOD; Level #4 had
concentrations ~10X greater than those of Level #1. Upon final completion of this study’s analytical phase,
the LOD for each species was obtained from summary statistics of the pooled results. The standard deviation
of pooled results at each level was plotted against its concentration and the points fitted by least squares
linear regression. The Y-axis intercept (standard deviation), which is in concentration units, is multiplied by 3.
It is this value that is defined as the LOD. For reporting purposes, results below the LOD are reported as “<
LOD”. The LOD should be reevaluated every two years.

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TABLE 13-1: Limits of Detection (LOD)
Species
Chemical Name

Abbreviated Name

Limit of Detection,
g/L

Inorganic Mercury

BHGI

0.27

Methyl Mercury

BHGM

0.12

Ethyl Mercury

BHGE

0.16

XIV. REPORTABLE RANGE OF RESULTS
Only analyte results having values within the analytical concentration range may be reported without
requiring dilution of the sample. The analytical concentration range for this method is LOD to 10 µg/L.
Samples having results greater than the upper limit of the analytical range must be diluted to bring the
analyte within the analytical concentration range and reanalyzed using this method. The final result is the
diluted sample result arithmetically corrected for the dilution. Total mercury concentration values produced
by DLS 3001 method will be available for the analyst to predict needed dilution factors prior the analysis of
blood samples by this method (DLS 3020).

XV.

SPECIAL PROCEDURE NOTES - CDC MODIFICATIONS
None applicable for this operation.

XVI. QUALITY CONTROL PROCEDURES
The Inorganic Toxicology and Nutrition Branch uses the method described in this protocol for
environmental and occupational health screening studies.
This analytical method uses two types of Quality Control (QC) systems: With one type of the QC system,
the analyst inserts bench QC specimens two times in each analytical run (a set of consecutive assays
performed without interruption) so that judgments may be made on the day of analysis. With the other type of
QC system, "blind" QC samples are placed in vials, labeled, and processed so that they are indistinguishable
from the subject samples (as many as possible). The supervisor decodes and reviews the results of the blind
specimens. With both systems, taking these samples through the complete analytical process assesses all
levels of the analyte concentrations. The data from these materials are then used to estimate methodological
imprecision and to assess the magnitude of any time-associated trends. The bench QC pools used in this
method comprise two levels of concentration spanning the "low-normal" and "high-normal" ranges for each
mercury species. Both of these pools are analyzed after the calibration standards, but before any patient
samples are analyzed so that judgments on the mercury species calibration curves may be made before
analysis of patient samples. These bench QCs should be analyzed again at the end of the run (no more than
20 samples). If a second run of = 20 samples are analyzed using the same calibration curve as the first run,
the QC results obtained from the second run's own bench QC samples need to be analyzed and treated
independent of the first run.

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A. Establish QC limits for each QC pool.
Perform an analysis of the mean and standard deviation for each pool from the concentration results
observed in at least 20 characterization runs. During the 20 characterization runs, analyze samples from
previously characterized QCs or pools with target values assigned by outside laboratories to evaluate each
run's QC. In addition to providing QC limits, the characterization runs also serve to establish homogeneity of
the pools. After the homogeneity of the bench materials is established, analysis by another independent
reference method (e.g., isotope dilution mass spectroscopy) is useful.

B. Precision and Accuracy
QC Results Evaluation. After completing a run, consult the QC limits to determine whether the run is "in
control." The QC rules apply to the average of the beginning and ending analyses of each of the bench QC
pools. The QC rules are as follows:
1) If both the low-and the high-QC results are within the 2s limits, accept the run.
2) If one of two QC results is outside the 2s limits, apply the rules below and reject the run if
any condition is met.
13s - Average of both low QCs OR average of both high QCs is outside of a 3s limit.
22s - Average of both low QCs AND average of both high QCs is outside of 2s limit on the
same side of the mean.
R4s sequential - Average of both low QCs AND average of both high QCs is outside of 2s
limit on opposite sides of the mean.
10x sequential - The previous nine average QCs results (for the previous nine runs) were
on the same side of the mean for either the low OR high QC.
If the run is declared "out of control," the analysis results for all patient samples analyzed during that run
are invalid for reporting.

C. Remedial Action If Calibration or QC Systems Fail to Meet Acceptable Criteria
If an analyte result for a QC material falls outside of the limits for mean or range, the following steps
should be taken, if possible:



Check the chromatograms for each blank, QC, and sample for proper peak integration and
identification. Change integration parameters or manually reintegrate peaks, if necessary, and
reprocess the run in TotalChom™.
Setup a new run for the reanalysis of the patient samples affected by the previous failed run. Be sure
to use freshly prepared QC material.

If these steps do not result in correction of the out-of-control values for QC materials, consult the
supervisor for other appropriate corrective actions. No analytical results should be reported for runs that are
not in statistical control.

XVII. REFERENCE RANGES
The reference range for each mercury species (see TABLE 17-1) is based on literature reports and from
periodic review of accumulated data collected during the analysis of blood samples representing a normal,
healthy population believed to be free of unusual exposure to mercury. Where data is absent or scant,
references ranges are based on the scientific literature, if available.

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TABLE 17-1: Reference ranges for Mercury Species
Species Chemical Name

Reference Range1, µg/L

Inorganic Hg
Methyl Hg
Ethyl

< 10 µg/L (within the established linearity for the method)

Hg2

Above ranges are estimates based on data from the Fourth National Report on Exposure to
Environmental Chemicals.
2 There are no established reference ranges for EtHg.
1

XVIII. ACTION-LEVEL RESULTS
The analyst should report any patient results confirmed to be greater than the second upper boundary
(defined in the laboratory database as the "2UB" and currently 5.8 µg/L) to the QC reviewer as an "elevated
result". The protocol for supervisors reporting elevated results to medical personnel is defined according to
the study protocol. Levels of concern for mercury in blood are >100 µg/L for children (6 years and younger)
and >200 µg/L for adults. These values are based on total mercury results.

XIX. SPECIMEN STORAGE AND HANDLING DURING TESTING
Specimens may reach and maintain ambient temperature during analysis. Take stringent precautions to
avoid external contamination. After the samples are analyzed, return them to ≤ –20°C freezer storage as
soon as possible.

XX.

ALTERNATE METHODS FOR PERFORMING TEST AND STORING
SPECIMENS IF TEST SYSTEM FAILS
If the analytical system fails, freezer storage (≤–20°C) is recommended until the analytical system is
restored functionality.

XXI. TEST-RESULT REPORTING SYSTEM; PROTOCOL FOR REPORTING
CRITICAL CALLS (IF APPLICABLE)
For critical calls, the supervisor should notify the supervising physician or principal investigator as soon
as possible. The most expeditious means should be used (e.g., telephone, FAX, or E-mail).

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XXII. TRANSFER OR REFERRAL OF SPECIMENS; PROCEDURES FOR SPECIMEN
ACCOUNTABILITY AND TRACKING
The analyst who receives specimens or samples delivered to Inorganic and Radiation Analytical
Toxicology Branch sets up a "Specimen Folder." Fill out a tracking form and place it in the folder to be given
to the analyst performing the analysis. The form tracks location, status, and final disposition of the
specimens.
Use standard electronic record keeping means (e.g., Microsoft Access™, optical disk, or tape backup) to
track specimens. Maintain records, including related quality assurance (QA) and QC data, for 3 years or
longer. Keep duplicate records (off site, if sensitive or critical) in electronic or hardcopy format. Use only
numerical identifiers (e.g., case ID numbers); all personal identifiers are available only to the medical
supervisor or project coordinator to safeguard confidentiality.

XXIII. REFERENCES
1. Tietz Textbook of Clinical Chemistry, edited by Carl A. Burtis, Edward R. Ashwood, Third Edition, 1999,
992-993.
2. Agency for Toxic substances and Disease Registry (ATSDR). Toxicological profile for mercury. Atlanta, GA:
Public Health service, 2000.
3. Third National Report on Human Exposure to Environmental Chemicals: Atlanta (GA): CDC, 2005.
4. Toxicological Effect of Methylmercury, National Research Council, National Academy Press, 2001, 54-59.
5. NHANES 1999-2002, Update on Hg, Mahaffey, K.R., Fish Forum-2005, US EPA, Washington, D.C.,
September 2005
6. Baranov VI, Tanner SD. A dynamic reaction cell for inductively coupled plasma mass spectrometry (ICPDRC-MS). Part 1. The rf-field energy contribution in thermodynamics of ion-molecule reactions. J. Anal.
At. Spectrom. 1999;14:1133-1142.
7. Tanner S, Baranov VI, Vollkopf U. A dynamic reaction cell for inductively coupled plasma mass
spectroscopy (ICP-DRC-MS). Part III. Optimization and analytical performance. J. Anal. At. Spectrom.
2000;15:1261-1269.

XXIV. APPENDIX
A. Appendix A. Critical Parameters Testing Results.
1. Critical Parameter Test #1: Evaluate the significance of the GC injector temperature
a) Test Details (to be repeated for each temperature):
1) Set up the analysis in accordance with the prescribed method.
2) Set the injector temperature to 200°C (reduced temperature).
3) Digest blood QC according to the prescribed method.
4) Analyze all digested samples together in one run.
5) Repeat the analysis using a column oven temperature of 220°C (the method prescribed
temperature).

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6) Repeat the analysis using a column oven temperature of 230°C (increased temperature).
Critical Parameter Test 1 Results. Temperature of the GC injector. One quality control pool was tested. Tests
performed 8/5/10 by Mark Fresquez. Results below are the averages of observed results at each condition along with
the 95th percent confidence intervals (in parentheses).
QC
Pool ID

GC injector Temperature
(°C )
200°C (Reduced)

QC Pool
LB07292

220°C (Per Method)
230°C (Increased)

InHg (µg/L)

MeHg (µg/L)

EtHg (µg/L)

1.27
(1.17, 1.38)
1.28
(1.00, 1.55)
1.39
(1.27, 1.51)

1.04
(0.99, 1.09)
1.08
(1.00, 1.16)
1.09
(1.02, 1.17)

1.22
(1.14, 1.30)
1.32
(1.27, 1.36)
1.20
(1.06, 1.35)

Expected Range ( ± 2 SD)*

*Expected range is a determined range calculated from the mean ± 2 SD of a 22 run characterization.

2. Critical Parameter Test #2: Evaluate the significance of the Combipal-agitator
equilibration/extraction temperature
a) Test Details (to be repeated for each temperature):
1) Set up the analysis in accordance with the prescribed method.
2) Set the agitator equilibration/extraction temperature to 40°C (elevated temperature).
3) Digest blood QC according to the prescribed method.
4) Analyze all digested samples together in one run.
5) Repeat the analysis using an equilibration/extraction temperature at room temperatures ~
25°C (the method prescribed temperature).
6) Repeat the analysis using an equilibration/extraction temperature of 50°C (increased
temperature).
Critical Parameter Test 2 Results. Temperature of the Combipal – agitator equilibration/extraction. One quality
control pool was tested. Tests performed 8/17/10 by Mark Fresquez. Results below are the averages of observed
results at each condition along with the 95th percent confidence intervals (in parentheses).
QC
Pool ID

Equilibration/Extraction
Temperature (°C)
~ 25°C (Per Method)

QC Pool
LB07292

40°C (Increased)
50°C (Increased)

InHg (µg/L)

MeHg (µg/L)

EtHg (µg/L)

1.42
(1.20, 1.65)
1.33
(1.27, 1.39)
1.25
(1.08, 1.42)

1.05
(1.03, 1.07)
1.05
(1.01, 1.09)
1.07
(1.05, 1.10)

1.17
(1.07, 1.27)
1.17
(1.11, 1.23)
1.19
(1.06, 1.32)

Expected Range ( ± 2 SD)*

3. Critical Parameter Test #3: Evaluate the significance of the length of extraction of sample from
SPME fiber in GC injector
a) Test Details:

Blood mercury species SSID-GC-ICP-DRC-MS

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Page 50 of 51

1) Set up the analysis in accordance with the prescribed method.
2) Set the extraction time to 10 minutes (reduced time).
3) Digest blood QC according to the prescribed method.
4) Analyze all digested samples together in one run.
5) Repeat the analysis using an extraction time of 15 minutes (reduced time).
6) Repeat the analysis using an extraction time of 20 minutes (the method prescribed time).
Critical Parameter Test 3. Length of extraction time. Test performed 8/18/10 by Mark Fresquez. Results
below are the averages of observed results at each condition along with the 95th percent confidence
intervals (in parentheses).
QC
Pool ID

Sample extraction time
(minutes)
10 min. (Reduced)

QC Pool
LB07292

15 min. (Reduced)
20 min. (Per Method)

InHg (µg/L)

MeHg (µg/L)

EtHg (µg/L)

1.69
(1.58, 1.80)
1.34
(1.32, 1.38)
1.33
(1.32, 1.37)

1.02
(0.97, 1.08)
1.04
(1.02, 1.05)
1.05
(1.05, 1.09)

1.16
(1.09, 1.22)
1.20
(1.18, 1.22)
1.17
(1.14, 1.18)

Expected Range ( ± 2 SD)*

4. Critical Parameter Test #4: Evaluate the significance of changing GC split ratio.
a) Test Details:
1) Set up the analysis in accordance with the prescribed method.
2) Set the split ratio to 20:1 (reduced ratio).
3) Digest blood QC according to the prescribed method.
4) Analyze all digested samples together in one run.
5) Repeat the analysis using split ratio of 28:1 (the method prescribed).
6) Repeat the analysis using split ratio of 35:1 (increased ratio).
Critical Parameter Test 4. GC split ratio. Test performed 8/24/10 by Mark Fresquez. Results below are
the averages of observed results at each condition along with the 95th percent confidence intervals (in
parentheses).
QC
Pool ID

QC Pool
LB07292

GC split ratio

InHg (µg/L)

MeHg (µg/L)

EtHg (µg/L)

20:1 (Reduced)

1.31
(1.26, 1.36)
1.34
(1.29, 1.39)
1.34
(1.27, 1.41)

1.06
(1.02, 1.10)
1.04
(1.03, 1.05)
1.06
(1.04, 1.08)

1.17
(1.14, 1.19)
1.15
(1.13, 1.19)
1.17
(1.15, 1.20)

28:1 (Per Method)
35:1 (Increased)

Expected Range ( ± 2 SD)*

Blood mercury species SSID-GC-ICP-DRC-MS

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Page 51 of 51

5. Critical Parameter Test #5: Evaluate the significance of changing GC carrier gas flow rate.
a) Test Details:
1) Set up the analysis in accordance with the prescribed method.
2) Set the flow rate 1.5 mL/min (reduced).
3) Digest blood QC according to the prescribed method.
4) Analyze all digested samples together in one run.
5) Repeat the analysis using a flow rate of 2.0 mL/min (the method prescribed).
6) Repeat the analysis using a flow rate of 2.5 mL/min (increased).
Critical Parameter Test 5. GC carrier gas flow rate. Test performed 8/24/10 by Mark Fresquez. Results
below are the averages of observed results at each condition along with the 95th percent confidence
intervals (in parentheses).
QC
Pool ID

GC carrier gas flow rate
(mL/min)

1.5 mL/min (Reduced)
QC Pool
LB07292

2.0 mL/min (Per Method)
2.5 mL/min (Increased)

Expected Range ( ± 2 SD)*

InHg (µg/L)

MeHg (µg/L)

EtHg (µg/L)

1.36
(1.32, 1.39)
1.31
(1.25, 1.37)
1.35
(1.31, 1.39)

1.07
(1.04, 1.10)
1.07
(1.05, 1.08)
1.05
(1.03, 1.07)

1.16
(1.14, 1.18)
1.16
(1.13, 1.19)
1.17
(1.12, 1.21)

Division of Laboratory Sciences
Laboratory Protocol
Analyte:

Matrix:
Method:

Method Code:
Branch:

arsenobetaine, arsenocholine, trimethylarsine oxide,
monomethylarsonic acid, dimethylarsinic acid, arsenous (III)
acid, arsenic (V) acid
Urine
Urine arsenic speciation HPLCICPDRCMS (Renamed from:
Inductively Coupled Plasma Dynamic Reaction Cell Mass
Spectrometry (HPLC-ICP-DRC-MS))
3000.2 (Renamed from 0161A/01-OD)
Inorganic and Radiation Analytical Toxicology

Prepared By:

Carl P. Verdon, PhD
author’s name

Supervisor:

date

signature

date

signature

date

Kathleen L. Caldwell, PhD
Supervisor’s name

Branch Chief:

signature

Robert L. Jones PhD
Branch Chief’s name

Adopted:
date

Updated:

August 08, 2010
date

Director's Signature Block:
Reviewed:
signature

date

signature

date

signature

date

Urine arsenic species HPLCICPDRCMS (Formerly Arsnic species in Urine)
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DLS Method Code: 3000.2 (formerly 016A/01-OD)
Modifications/Changes: see Procedure Change Log STARLIMS

IRAT-DLS

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DLS Method Code: 3000.2 (formerly 016A/01-OD)

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IRAT-DLS

Laboratory Procedure Manual
Analyte:

Matrix:
Method:

arsenobetaine, arsenocholine,
trimethylarsine oxide,
monomethylarsonic acid, dimethylarsinic
acid, arsenous (III) acid, arsenic (V) acid
Urine
Urine arsenic speciation
HPLCICPDRCMS
(Renamed from High Performance Liquid
Chromatography Inductively Coupled Plasma Dynamic
Reaction Cell Mass Spectrometry (HPLC-ICP-DRC-MS))

Method No:

3000.2 (Formerly 0161A/01-OD)

Revised:

November 16, 2011

as performed by:
Inorganic and Radiation Analytical Toxicology Branch
Division of Laboratory Sciences
National Center for Environmental Health
contact:
Dr. Robert L. Jones
Phone: 770-488-7991
Fax:
770-488-4097
Email: [email protected]
James L. Pirkle, M.D., Ph.D.
Director, Division of Laboratory Sciences

Important Information for Users
CDC periodically refines these laboratory methods. It is the responsibility of the user to contact
the person listed on the title page of each write-up before using the analytical method to find out
whether any changes have been made and what revisions, if any, have been incorporated.

Urine arsenic species HPLCICPDRCMS (Formerly Arsenic species in Urine)
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Table of Contents
1.

CLINICAL RELEVANCE AND TEST PRINCIPLE .................................................................... 1
a.

Clinical Relevance

Test Principle

SAFETY PRECAUTIONS ........................................................................................................... 3

DATA SYSTEM MANAGEMENT ................................................................................................ 3
a.

Data Entry and Transfer

Routine Computer Hard Drive Maintenance

Data Backup

(1) Schedule of Data Backups
(2) Backup Procedures

4
4

Documentation of System Maintenance

4.
COLLECTING, STORING, AND HANDLING SPECIMENS; CRITERIA FOR
REJECTING SPECIMENS.......................................................................................................... 5
a.

Specimen Type

Specimen Collection, Handling, and Storage

Criteria for an Unacceptable Specimen

PROCEDURES FOR MICROSCOPIC EXAMINATIONS ......................................................... 6

CHEMICALS, STANDARDS, AND QUALITY CONTROL MATERIAL.................................... 6

Chemicals and Standards

Standards

Quality Control Material

INSTRUMENTATION, EQUIPMENT, SOFTWARE, AND SUPPLIES ...................................... 8
a.

Instrumentation
(1) HPLC System
(2) ICP-DRC-MS System

8
8
9

Equipment

Computer Software

Supplies

INSTRUMENT SETUP AND CONFIGURATION ..................................................................... 12
a.

HPLC Hardware Setup

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Software Installation

Peristaltic Pump Setup

Electrical Connections

Six-Port Switching Valves

STANDARD PROCEDURE ...................................................................................................... 18
a.

Preparation of Stock Solutions

Preparation of Matrix-Matched Material (“Base Urine”)

Preparation of Working Solutions

Preparation of Stock Standards (Concentrated)

Preparation of Intermediate Standards

Microwave Digestion for Conversion to Arsenate (AsV)

Method of Standard Additions (MSA)

Determining the Total Arsenic Concentration

Assessment of Purity by HPLC-DRC-ICP-MS

Preparation of Concentrated Stock Internal Standard

Preparation of Working Calibrators

(1) Preparation of Intermediate “Mixed Species” Calibration
Solutions
(2) Preparation of Working Calibrator Series

27
27

Preparation of Quality Control Material

Processing of Urine Samples and QC Material

HPLC Instrument Setup

(1) Programming the HPLC Pump Methods
(2) Programming the HPLC Autosampler
o.

ICP-DRC-MS Instrument Setup
(1)
(2)
(3)
(4)

Programming the DRC Gas Flow Delay Parameter
Programming the ELAN “.mth” file
Programming the ELAN “.dac” file
Creating the ELAN Sample Table “.sam” file

HPLC–ICP-DRC-MS System Connection and Startup
(1) Interfacing the HPLC Column to the ICP-DRC-MS
Nebulizer
(2) Priming the HPLC Pump
(3) Adjusting the External Peristaltic Pump

31
34
35
35
35
38
38
40
40
41
42

ICP-DRC-MS Warm Up and Performance Check

Turning on the Reaction Cell Gas

Entering Sample Names into the ELAN Sample Table

Urine arsenic species HPLCICPDRCMS (Formerly Arsenic species in Urine)

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10.

11.

12.

13.

Starting the Run

Instrument Shut Down

POST-RUN DATA ANALYSIS ................................................................................................... 50
a.

Configuration of TotalChrom™ Integration Method

Configuration of ELAN ChromLink™

Data Processing and Analysis

RECORDING OF SAMPLE AND QC DATA ............................................................................ 65
a.

Transferring the Data to the Central Database

QC Data

FINAL REVIEW OF THE DATA ................................................................................................ 66
a.

Analysis Printouts and Analyst Run Report

Plotting QC Results

Supervisor Review

REPLACEMENT AND PERIODIC MAINTENANCE OF KEY COMPONENTS .................... 67
a.

ICP-MS Maintenance

14.

LIMIT OF DETECTION AND LINEAR RANGE TESTED ....................................................... 68

15.

REPORTABLE RANGE OF RESULTS.................................................................................... 68

16.

SPECIAL PROCEDURE NOTES – CDC MODIFICATIONS .................................................. 69

17.

QUALITY CONTROL PROCEDURES ..................................................................................... 69
a.

Establish QC limits for each QC pool.

Precision and Accuracy

Remedial Action If Calibration or QC Systems Fail to Meet Acceptable
Criteria

18.

LIMITATIONS OF METHOD; INTERFERING SUBSTANCES AND CONDITIONS.............. 71

19.

REFERENCE RANGES ............................................................................................................ 71

20.

ACTION-LEVEL RESULTS ...................................................................................................... 71

21.

SPECIMEN STORAGE AND HANDLING DURING TESTING .............................................. 71

22.

ALTERNATE METHODS FOR PERFORMING TEST AND STORING SPECIMENS
IF TEST SYSTEM FAILS .......................................................................................................... 72

23.

TEST-RESULT REPORTING SYSTEM; PROTOCOL FOR REPORTING CRITICAL
CALLS (IF APPLICABLE) ........................................................................................................ 72

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24.

TRANSFER OR REFERRAL OF SPECIMENS; PROCEDURES FOR SPECIMEN
ACCOUNTABILITY AND TRACKING ..................................................................................... 72

25.

BI-ANNUAL EXTENDED LINEAR RANGE VERIFICATION STUDY.................................... 72

26.

BI-ANNUAL INSTRUMENT-TO-INSTRUMENT COMPARISON ........................................... 72

27.

REFERENCES........................................................................................................................... 73

28.

APPENDIX ................................................................................................................................. 76
a.

Macro Procedure “Extract TC Data”

Sample Chromatographic Report

76
106

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List of Tables
TABLE 1-1: Species of Arsenic ........................................................................................... 2
TABLE 9-1: Preparation of Stock Standards .................................................................... 21
TABLE 9-2: Formula Weights of Arsenic Species ............................................................ 22
TABLE 9-4: METHOD OF STANDARD ADDITIONS TABLE 1 ........................................ 24
TABLE 9-5: METHOD OF STANDARD ADDITIONS TABLE 2 ........................................ 25
TABLE 9-3: Gilson 402™ Settings for Making Diluted Series .......................................... 28
TABLE 9-4: HPLC Pump Program Settings ..................................................................... 32
TABLE 9-5: HPLC Pump Column Wash Program ............................................................ 33
TABLE 9-6: ELAN® Timing Parameters ............................................................................ 36
TABLE 9-7: ELAN® Analyte Parameters........................................................................... 36
TABLE 9-8: ELAN® Processing Parameters ..................................................................... 37
TABLE 9-9: ELAN® Sampling Parameters........................................................................ 37
TABLE 9-10: ELAN® Report Parameters .......................................................................... 37
TABLE 9-11: ELAN® Optimization Parameters................................................................. 38
TABLE 9-12: Sample Template Data................................................................................ 39
TABLE 9-13: Sample Template Data................................................................................ 39
TABLE 9-14: ELAN Samples Table .................................................................................. 46
TABLE 10-1: Integration.................................................................................................... 51
TABLE 10-2: Baseline Timed Events................................................................................ 51
TABLE 10-3: Replot .......................................................................................................... 52
TABLE 10-5: Method Editor – Components Settings ....................................................... 53
TABLE 10-6: Components Defaults — IDENTIFICATION ............................................... 54
TABLE 10-7: Components Defaults — CALIBRATION .................................................... 54
Figure 1: Post-Run Data Processing Work Flow Diagram................................................ 58

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TABLE 10-8: TotalChrom™ Navigator – Reprocess Batch ............................................. 62
TABLE 14-1: Limits of Detection (LOD) and Linear Range Tested (LRT) for As Species 68
TABLE 19-1: Reference ranges for Arsenic Species ........................................................ 71

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CLINICAL RELEVANCE AND TEST PRINCIPLE
a. Clinical Relevance

People encounter arsenic in many chemical forms that vary in toxicity. The most toxic of the
naturally-occurring arsenic compounds are inorganic forms of arsenic and their monomethylated
metabolites (1). Less toxic are the organic arsenic compounds (2-5). Exposure to inorganic
arsenic can result in a variety of adverse health effects, such as skin disorders, nerve impairment,
cancer of the liver, bladder, kidneys, prostate, and lungs, and even death from large doses (6, 7).
People may be exposed to inorganic arsenic through activities such as drinking water
contaminated from geological sources (8-14) or because of occupational exposure (15-19),
especially breathing air contaminated with sawdust or smoke from wood treated with chromated
copper arsenic preservatives (20-25). Organic arsenic compounds are generally less toxic and
may be encountered by ingesting various types of fish, shellfish, or seaweed (26-31).
The method described in this manual assesses arsenic exposure, as defined by exposure to
individual arsenic species by analyzing urine through the use of high performance liquid
chromatography (HPLC) coupled to inductively coupled-plasma dynamic reaction cell-mass
spectrometry (ICP-DRC-MS). Urine is analyzed because urinary excretion is the major pathway
for eliminating arsenic from the mammalian body (32-34). This hyphenated method will provide
accurate quantification of seven urinary arsenic species: arsenite (valence III) and arsenate
(valence V), organic forms of arsenic to include monomethylarsonic acid (MMA) and
dimethylarsinic acid (DMA), trimethylarsine oxide (TMAO), arsenocholine (AsCo), arsenobetaine
(AsB) (see TABLE 1-1).

b. Test Principle
The concentrations of arsenate [As(V)], arsenite [As(III)], MMA, DMA, TMAO, AC, and AB are
determined by using high performance liquid chromatography (HPLC) to separate the species
coupled to an ICP-DRC-MS to detect the arsenic species. This analytical technique is based on
separation by anion-exchange chromatography (IC) followed by detection using quadrupole ICPMS technology and includes DRC™ technology (35), which minimizes or eliminates many argonbased polyatomic interferences (36). Column separation is largely achieved due to differences in
charge-charge interactions of each negatively-charged arsenic component in the mobile phase
with the positively-charged quaternary ammonium groups bound at the column’s solid-liquid
interface. Upon exit from the column, the chromatographic eluent goes through a nebulizer where
it is converted into an aerosol upon entering the spray chamber. Carried by a stream of argon
gas, a portion of the aerosol is transported through the spray chamber and then through the
central channel of the plasma, where it is heated to temperatures of 6000-8000° K. This thermal
energy atomizes and ionizes the sample. The ions and the argon enter the mass spectrometer
through an interface that separates the ICP, which is operating at atmospheric pressure
(approximately 760 torr), from the mass spectrometer, which is operating at approximately 10-5
torr. The mass spectrometer permits detection of ions at each mass-to-charge ratio in rapid
sequence, which allows the determination of individual isotopes of an element. Once inside the
mass spectrometer, the ions pass through the ion optics, then through the DRC™, and finally
through the mass-analyzing quadrupole before being detected as they strike the surface of the
detector. The ion optics uses an electrical field to focus the ion beam into the DRC™. The DRC™
component is pressurized with an appropriate reaction gas and contains a quadrupole. In the

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DRC™, elimination or reduction of argon-based polyatomic interferences takes place through the
interaction of the reaction gas with the interfering polyatomic species in the incoming ion beam.
The quadrupole in the DRC™ allows elimination of unwanted reaction by-products that would
otherwise react to form new interferences.
TABLE 1-1: SPECIES OF ARSENIC
Name

Arsenobetaine

Abbreviation

Structural Formula
CH3

AB
H3C

pKa

CH 2 COOH

CH3

Arsenocholine

CH3

H3C

CH 2CH 2 OH

CH3

Monomethyl arsonic acid

MMA

OH
H3C

(V)

4.1, 8.7

OH
O

Dimethylarsinic acid

DMA

CH3

H3C

(V)

6.2

Trimethylarsine oxide

TMAO

CH3

H3C

(V)

CH3

Arsenic (V) acid
(arsenate)

As(V)

OH
(V)

Arsenous (III) acid
(arsenite)

2.2, 7.0, 11.5

As(III)

9.2, 12.1, 13.4

(III)

As
HO

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Page 3 of 107

SAFETY PRECAUTIONS

Precautionary information that is important to protecting personnel and safeguarding
equipment will be presented inside a box, such as this one, throughout the procedure
where appropriate.
Follow universal precautions. Wear gloves, a lab coat, and safety glasses while handling human
blood, plasma, serum, urine or other bodily fluid or tissue. Place disposable plastic, glass, and
paper (e.g., pipette tips, autosampler tubes, and gloves) that come in contact with human
biological fluids, such as urine, in a biohazard autoclave bag. Keep these bags in appropriate
containers until they are sealed and autoclaved. When work is finished, wipe down all work
surfaces where human biological fluid was handled with a 10% (v/v) sodium hypochlorite solution.
Dispose of all biological samples and diluted specimens in a biohazard autoclave bag at the end
of the analysis according to CDC/DLS guidelines for disposal of hazardous waste.
PerkinElmer provides safety information that should be read before operating the instrument.
This information is found in the PerkinElmer ELAN® 6100 ICP-DRC-MS System Safety Manual.
Possible hazards include ultraviolet radiation, high voltages, radio-frequency radiation, and high
temperatures.

Caution!
Exercise caution when handling and dispensing concentrated nitric acid. Always
remember to add acid to water. Nitric acid is a caustic chemical that is capable of severe
eye and skin damage. Wear powder-free gloves, a lab coat, and safety glasses. If nitric
acid comes in contact with any part of the body, quickly wash the exposed area with
copious quantities of water for at least 15 minutes.

DATA SYSTEM MANAGEMENT

To maintain the integrity of specimen and analytical data generated by this method, eliminate
hand entry of specimen identifiers or analytical results whenever possible, proofread all
transcribed data, and regularly back up the ICP-MS computer’s hard drive. It is recommended
that a defragmentation program be run on the computer’s hard drive on a periodic basis.

a. Data Entry and Transfer
Whenever possible, use bar code scanners to enter sample identifiers into the ICP-DRC-MS
computer software to avoid errors associated with the keyboard-entry process and to speed up
sample processing. When bar code scanners cannot be used, proofread transcribed data after
entry. Handle or transfer data electronically when reporting or moving data to other computerized
data-handling software. In the Inorganic and Radiation Analytical Toxicology Branch, sample
analysis results generated by this method are stored for long periods in Microsoft Access™ or MS
SQL Server 7™ database software. The results should include at least the analysis date,
analytical run number, quality-control (QC) results for the run, results of specimen analysis by
specimen identification (ID), and method identifier.

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b. Routine Computer Hard Drive Maintenance
Defragment the computer hard drive by using software such as Microsoft Windows® Disk
Defragmenter (located in Start > Programs > Accessories > System Tools) or an equivalent
program to maximize computer performance and maintain data integrity for files on the hard
drive. An entry will automatically be made in the Windows™ system event log when this process
is done providing documentation of this step.

c. Data Backup
(1) Schedule of Data Backups
Weekly. Full data backups onto one or more recordable compact discs (CD-R) or digital
video discs (DVD).
Daily. Full data backups onto an external hard drive.

(2) Backup Procedures
Whenever making a backup (daily or weekly) include the directories and subdirectories:


C:\elandata (include all subdirectories)



C:\hplc (include subdirectories “data” and “methods as well as other relevant
directories)



Other relevant folders.

Connect the ELAN data system computer to an external hard disk with
sufficient storage capacity to store several copies of the backup files (≥18
gigabytes).



Configure Microsoft Windows® Backup™ (located in Start > Programs >
Accessories > System Tools) program to do a daily backup of the ELAN data
system computer’s data directories (see Backup Procedures)

(b) Compact Disc Backups


Use CD-R disks only (recordable compact disks), not CD-RW disks
(rewritable compact disks) so that after creation the recordable compact disk
cannot be over-written.



Use Adaptec “Easy CD Creator”™ or equivalent software to backup.

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(c) Removing Data from the ICP-DRC-MS Computer Hard Drive
When the active “elandata” and “hplc” directories on the ICP-DRC-MS computer
hard drive becomes too large to fit onto a single CD-R, remove the oldest data on
the hard drive so that a regular backup can be done onto a single CD-R.
Usually, this procedure can be done annually.







Back up the oldest data on the hard drive in duplicate onto two CD-R disks.
Manually select each dataset folder (subdirectories under
“C:\elandata\dataset” and “C:\hplc\data”) and other relevant files (i.e.,
optimization, tuning, and sample files) that are to be included on these
backups.
Verify that backup CD-R disks operate correctly before deleting any data
from the hard drive. To verify the operation of a CD-R disk, open any file on
the disk by using the appropriate computer software (ICP-DRC-MS
software).
After verifying that all backups are operational, delete the original data from
the hard drive.
Keep one copy of the CD-R disk in a building other than the laboratory (in
case of fire). Keep the other near the ICP-MS laboratory.

(d) Backup of Sensitive Data
Make a backup for sensitive data on duplicate, recordable compact disk. Store
the two CD-R disks in two different buildings.

d. Documentation of System Maintenance
Computer Maintenance: Record any maintenance of computer hardware, HPLC or
ICP-DRC-MS software in the instrument logbook. Place other electronic records relating
to integrity of the data and hard drive in the Windows™ event log. Back up the event log
on a regular basis by saving a copy in the active “elandata” directory. The event log will
then be backed up along with the ELAN data when backup CD-R disks and tapes are
made.
Instrument Maintenance: Document system maintenance in hard copies of data records
(i.e., daily maintenance checklists, PerkinElmer service records, and instrument log book)
as well as in electronic records relating to instrument optimization (*dac) and tuning
(default.tun).

COLLECTING, STORING, AND HANDLING SPECIMENS; CRITERIA
FOR REJECTING SPECIMENS
a. Specimen Type

Specimen type is human urine. No special instructions for fasting or special diets are required of
patient or study subjects.

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b. Specimen Collection, Handling, and Storage
Optimal amount of specimen is 0.5 mL; the minimum is 0.25 mL. Use sterile specimen containers
for specimen acquisition. Acceptable containers for allotment of urine for this method include 5.0mL polypropylene cryogenic vials (e.g., Nalgene, Item # 5000-0050). Screen lots of specimen
collection cups, containers, and sample tubes for total arsenic contamination before use.
Specimen handling conditions are outlined in the Division of Laboratory Science’s protocol for
urine collection and handling. To prevent inter-conversion of arsenic species, immediately store
or transport urine specimens at  -20C. Upon receipt, they must remain frozen at  -20°C until
time for analysis. Refreeze at  -20C portions of the sample that remain after analytical aliquots
are withdrawn. Samples thawed and refrozen several times may be compromised.

c. Criteria for an Unacceptable Specimen
The criteria for an unacceptable specimen are low volume sample volumes (< 0.25 mL),
suspected contamination due to improper collection procedures or collection devices, and/or
contamination during sample preparation/analysis. Specimen contact with dust or dirt may
compromise test results. In all cases, request a second urine specimen, if possible.

PROCEDURES FOR MICROSCOPIC EXAMINATIONS

Not applicable for this procedure.

CHEMICALS, STANDARDS, AND QUALITY CONTROL MATERIAL
a. Chemicals and Standards
1. Water, high purity ( ≥ 18 Mcm resistivity).
2. Ammonium carbonate, (CAS# 506-87-6), MW 96.09, GFS Chemicals, Item
#839-12471, or equivalent.
3. Tris(hydroxymethyl)aminomethane, (CAS# 77-86-1), MW 121.14, Bio-refined,
GFS Chemicals, Item # 1948-75811, or equivalent.
4. Ammonium Sulfate, (CAS# 7783-20-2), MW 132.13, GFS Chemicals, Item #
1906-13341, or equivalent.
5. Ammonium Acetate, (CAS# 631-61-8), MW 77.08, GFS Chemicals, Item #
547-12401, or equivalent.
6. Acetic Acid, Glacial (CAS# 64-19-7) M.W. 60.05, GFS Chemicals Inc., Item #
624, or equivalent.
7. Ammonium Hydroxide, (CAS# 1336-21-6) M.W. 35.05, Fisher Scientific, Item
# A470500, or equivalent.
8. Methanol (CAS# 67-56-1) M.W. 32.04, GFS Chemicals, Item # 2483-50441,
or equivalent.
9. 10% hydrogen in argon gas mixture, ≥ 99.999% purity, Matheson Tri-Gas

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Products, San Jose, California, or equivalent.
10. Double-distilled nitric acid, GFS Chemicals, or equivalent.
11. 1,000 mg/L Gallium, SPEX CertiPrep, Item # PLGA2-2Y, or any equivalent
traceable to the National Institute for Standards and Technology.
12. Certified pH 7 and pH 10 calibration solutions.
13. Liquid argon.
14. Acetonitrile, HPLC or Spectrophotometer grade, GFS Chemicals, or
equivalent.
15. Bleach (10% sodium hypochlorite solution) from any vendor.
16. Base urine pooled from anonymous donors or purchased from a vendor.
17. Potassium Persulfate, purified GFS Chemicals, Item #61712, or equivalent.

b. Standards
The following is a list of possible sources of material for the seven arsenic species. A
mixture of the sources is used at any given time to provide calibration material that is from
different sources. Other sources can be used, and please note that the availability of the
sources listed is subject to change without notice.
1. Arsenic (III) oxide, As2O3, CAS 1327-53-3, MW 197.84, Sigma-Aldrich, Item #
202673, or equivalent.
2. Arsenic (III) speciation standard in 2%HCl, Spex CertiPrep, Item # SPECAS3, or equivalent.
3. Arsenic (V) oxide hydrate, As2O5·xH2O, CAS 12044-50-7, MW 229.84, SigmaAldrich, Item # 363456, or equivalent source or vendor.
4. Arsenic (V) speciation standard in water, Spex CertiPrep, Item # SPEC-AS5,
or equivalent.
5. Arsenobetaine, (CH3)3AsCH2COOH, CAS 64436-13-1, MW 178.06, SigmaAldrich, Item # 11093, or equivalent.
6. Arsenobetaine, (CH3)3AsCH2COOH, CAS 64436-13-1, MW 178.06, Wako
USA, Item # 321-34911, or equivalent.
7. Arsenobetaine, (CH3)3AsCH2COOH, CAS 64436-13-1, MW 178.06, Argus,
Vernio, Italy, Item # AR60008, or equivalent.
8. Arsenocholine bromide, (CH3)3AsCH2CH2OH·Br, MW 244.99, Argus, Vernio,
Italy, Item # AR60010, or equivalent.
9. Arsenocholine bromide, (CH3)3AsCH2CH2OH·Br, MW 244.99, Wako USA,
Item # 328-34921, or equivalent.
10. Dimethylarsinic acid, (CH3)2As(OH)2, MW 138.01, Sigma-Aldrich, Item # PS51, or equivalent.
11. Cacodylic Acid, (CH3)2As(O)OH, CAS 75-60-5, MW 138.00, Sigma-Aldrich,
Item # 20835-10G-F, or equivalent.
12. Disodium methyl arsenate, CH3AsO3·6H2O, CAS 144-21-8, MW 291.9,
Sigma-Aldrich, Item # PS-281, or equivalent.

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13. Monosodium acid methane arsonate sesquihydrate, CAS 2163-80-6, SigmaAldrich, Item #, PS-429, or equivalent.
14. Trimethylarsine oxide, (CH3)3AsO, CAS 4964-14-1, MW 136.03, Argus
Chemicals, Vernio, Italy, Item # AR60011, or equivalent.
15. Trimethylarsine oxide, (CH3)3AsO, CAS 4964-14-1, MW 136.02, Wako USA,
Item # 321-34891, or equivalent.

c. Quality Control Material
Quality control (QC) material is made from pools of human urine collected from several
anonymous donors. See the “Error! Reference source not found. section of this method for
details of preparation. The two urine QC pools made for arsenic speciation are designated as:
QC level

QC Designation ID

low pool

LU-yy###

high pool

HU-yy###

Where yy is the last two digits of production year and ### is the assigned pool identification
number.
QC material that is to be used for bench quality control purposes will need to be “characterized”
as described in the section Establish QC limits for each QC pool.

INSTRUMENTATION, EQUIPMENT, SOFTWARE, AND SUPPLIES
a. Instrumentation
(1) HPLC System
1. HPLC Pump, specifically, PerkinElmer® Series 200™ Pump, made with
biocompatible materials consisting of polyethylethylketone (PEEK) and other
polymers in the fluid path (PerkinElmer LAS), or equivalent.
2. HPLC Autosampler, specifically, PerkinElmer® Series 200™ Autosampler,
made with biocompatible materials consisting of PEEK and other polymers in
the fluid path (PerkinElmer LAS), or equivalent.
3. Autosampler temperature cooling tray for 100 samples, specifically,
PerkinElmer® Series 200™ Peltier Cooling Tray and Assembly, or equivalent.
4. Column oven, specifically, PerkinElmer® Series 200™ Column Oven, or
equivalent.
5. Chromatography data handling software, specifically, TotalChrom™
Workstation, version 6.0.2 or later (PerkinElmer LAS), or equivalent.
6. Anion-exchange HPLC column, specifically, PRP-X100™, 4.6 X 150 mm
dimensions, 5 µm particle size in PEEK hardware, Hamilton Company, Item #
79174, or equivalent.
7. Autosampler injection needle, stainless steel, PerkinElmer LAS, Item #

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N2930023, or equivalent.
8. Autosampler injector loop, 20 µL, IDEX Health & Science, Item # 9055-022, or
equivalent.
9. Autosampler injector loop, 200 µL, IDEX Health & Science, Item # 9055-025,
or equivalent.
10. Electrically-activated 6-port switching valve, IDEX Health & Science, Item #
EV750-100-S2, or equivalent. An additional switching valve may also be used
as demonstrated in Section 8. Instrument Setup and Configuration.

(2) ICP-DRC-MS System
1. Inductively-coupled plasma mass spectrometer, specifically, the ELAN™ DRC
II with Dynamic Reaction Cell (DRC™) capability, PerkinElmer LAS, or
equivalent.
2. ELAN instrument control and data handling software, version 3.0 or greater,
PerkinElmer LAS.
3. Cyclonic spray chamber, PerkinElmer LAS, or equivalent.
4. Concentric glass nebulizer, Precision Glassblowing, Item # 500-70QQDAC, or
equivalent.
5. External peristaltic 4-channel peristaltic pump, “Minipuls 3”, Gilson Inc., , or
equivalent.

b. Equipment
1. Water purification system for providing ultrapure water with a resistivity ≥18
Mcm.
2. Eppendorf® Model 5417R refrigerated centrifuge fitted with FA45-24-11 fixed
angle rotor or equivalent refrigerated centrifuge capable of ≥18,000 rcf for
centrifugation of 1.5 mL capacity microcentrifuge tubes.
3. High-precision analytical balance capable of accurately weighing milligram
amounts of material to the tenth of a milligram or better.
4. Analytical balance for routine weighing of material to the nearest hundredth of
a gram and with a loading capacity of at least 200 g.
5. pH meter with one hundredths of a pH unit readout or better, fitted with glass
electrode (pH probe).
6. Temperature compensation probe for pH meter.
7. Gilson 402™ Programmable Diluter-Dispenser (or equivalent) equipped with
10.0-mL dispensing syringe and a 2-mL sampling syringe.
8. Calibrated hand-held adjustable pipetters that cover the range of accurate
liquid delivery from 50 µL to 5000 µL. Research Pro™ Eppendorf® electronic
programmable pipetters (Fisher Scientific), or equivalent.
9. “Repeater Plus” Pipetter, Fisher Scientific, Item # 2226020-1, , or equivalent
pipetting device(s) capable of accurately dispensing multiple microliter
aliquots of liquid.

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10. Gas regulator for 10% DRC gas, Matheson Tri-Gas Products, or equivalent.
11. Gas regulator for argon gas, Matheson Tri-Gas Products, or equivalent.
12. Ethos EZ microwave Digestion Labstation with PRO-24 High Throughput
Rotor, Milestone, Inc., or equivalent.

c. Computer Software
1. pdFactory Pro 1.52 or later version, FinePrint Software, LLC,
www.fineprint.com or equivalent. This product is used for creating electronic
Portable Document Files (pdf) directly from Microsoft® Windows print dialog
box.
2. A custom Microsoft Excel® macro procedure named “Extract TC Data”. See
Macro Procedure “Extract TC Data” section in the Appendix for description
and macro code.

d. Supplies
1. 2-200 L pipette tips, 960 tips per case, Fisher Scientific, Item # 05-403-66,
or equivalent.
2. 20-300 L pipette tips, 960 tips per case, Fisher Scientific, Item # 05-403-67,
or equivalent.
3. 50-1000 L pipette tips, 960 tips per case, Fisher Scientific, Item # 05-403-68,
or equivalent.
4. 5 mL pipette tips, 500 tips per case, Fisher Scientific, Item # 05-403-117, or
equivalent.
5. Acid-cleaned 2 liter polyethylene (PE) bottles. To acid-wash containers, rinse
with 10% (v/v) reagent-grade nitric acid, followed by rigorous rinsing with 18
Mcm water. Repeat this process several times depending on prior use of
the containers.
6. Acid-cleaned 1 liter PE bottles. To acid-wash containers, rinse with 10% (v/v)
reagent-grade nitric acid followed by rigorous rinsing with 18 Mcm water.
Repeat this process several times depending on prior use of the containers.
7. Four acid-cleaned 500 mL PE bottles. To acid-wash containers, rinse with
10% (v/v) reagent-grade nitric acid followed by rigorous rinsing with 18 Mcm
water. Repeat this process several times depending on prior use of the
containers.
8. Acid-cleaned 100 mL PE bottles. To acid-wash containers, rinse with 10%
(v/v) reagent-grade nitric acid followed by rigorous rinsing with 18 Mcm
water. Repeat this process several times depending on prior use of the
containers.
9. 1.5 mL polypropylene (PP) microcentrifuge tubes, Fisher Scientific, Catalogue
# 05-402, or equivalent.
10. Tube racks for 1.5 mL microcentrifuge tubes (approximately 6), Fisher
Scientific, 05-405-3, or equivalent.
11. Vial rack for HPLC autosampler vials, 50-position, Fisher Scientific, Item # 03-

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375-9, or equivalent.
12. Five or more flangeless ferrules, “blue” for 1/16” O.D. tubing, ChromTech or
IDEX Health & Science, Item # P-200X, or equivalent.
13. Five or more “FingerTight III” HPLC “Tefzel” fittings, 10-32 threading, for 1/16”
O.D. tubing, ChromTech or IDEX Health & Science Item # F-300X, or
equivalent.
14. Repeater pipetter tips, “Combitips Plus” 5 mL, Fisher Scientific, Item # 21-381330, or equivalent.
15. HPLC tubing, 0.007” I.D. X 1/16” O.D., polyethylethylketone (PEEK), 5 feet
length.
16. Peek sample uptake fitting, Analytical West, Item #500-QD-PEEK, or
equivalent
17. Tubing, 0.03” I.D. X 1/16” O.D., polypropylene (PP), 5 feet length.
18. Four “end-of-tubing” prefilters (10 µm) for each HPLC reservoir bottle,
(ChromTech or IDEX Health and Science, Item # A-438, or equivalent.
19. In-line HPLC post-pump filter (2 µm), ChromTech or IDEX Health and
Science, Item # A-430, or equivalent.
20. Luer fitting plastic syringe, 10 mL or larger.
21. Kay-Dry™ paper towels and Kim-Wipe™ tissues (Kimberly-Clark Corp.,
Roswell GA, or equivalent vendor).
22. Teflon™-coated magnetic stirs bars (2), VWR, Item #58948-974, or
equivalent.
23. Cotton swabs (Hardwood Products Co. ME), or equivalent.
24. Nitrile, powder-free examination gloves (N-Dex®, Best Manufacturing Co.,
Menlo, GA), or equivalent.
25. Biohazard autoclave bags (Curtin-Matheson Scientific, Inc., Florence, KY), or
equivalent.
26. 15 mL 15 ml (# 352097) and 50 ml (#352098) polypropylene centrifuge tubes
or equivalent: Becton Dickinson Labware or equivalent.

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INSTRUMENT SETUP AND CONFIGURATION
a. HPLC Hardware Setup

A PerkinElmer representative should perform the first installation of the Series 200™ HPLC
system. If the instrument has been moved, laboratory personnel can assemble and configure the
system according to the PerkinElmer supplied user manuals for the Series 200 LC Pump and the
Series 200 Autosampler. The following should be considered in planning a new location:
1. Two 6-outlet A/C power strips rated for 15-amp duty will be required to plug-in
all HPLC equipment and its associated components.
2. The HPLC equipment needs to be placed adjacent to the right side of the ICPDRC-MS instrument. Allow for approximately 4 feet of bench space for the
HPLC equipment and data processing computer. A bench height of 32–34
inches is optimal.
3. HPLC components need to be arranged, from left to right, in the following
order:
(a) Six-port switching valve “#1” (EV750-100)
(b) Six-port switching valve “#2” (EV750-100-S2). This switching valve must
have a serial port for connection to an external desktop computer.
(c) Column oven
(d) Pump with autosampler. Set the autosampler on top of the pump.
4. Replace the existing injection sample loop with a 20 µL loop.
5. Replace autosampler needle with one made of stainless steel (PerkinElmer
P/N N293-0023), if it does not already have one. Titanium needles are
unsuitable due to their potential to be a source of arsenic contamination. Refer
to the PerkinElmer Series 200 LC Autosampler user manual for instructions
on needle replacement.
6. Replace all stainless steel HPLC tubing following the HPLC pump’s purge port
with “yellow” PEEK tubing, 0.007” I.D. X 1/16” O.D. Use “yellow” PEEK tubing
for the remainder of the fluid path. Keep tubing lengths short but long enough
to allow the connected components to be moved if necessary.
7. Install a PEEK construction in-line filter (10 µm) between the HPLC pump and
the autosampler.
8. Do not use the column oven’s stainless steel tubing to preheat the mobile
phase before it enters the column. The preheating is unnecessary and the
extra tubing length will not help the quality of the chromatography.
9. Attach via a plastic tie clamp (or equivalent) one end of a given length of ¼”
diameter Tygon™ tubing to the end of the bench holding the HPLC
equipment. Position the other end of this Tygon™ tubing to empty into a large
waste jug. Position this tubing close enough to the 6-port switching valves so
that HPLC drain tubing from ports 5 and 6 on switching valves #1 and #2,
respectively, will easily fit into the Tygon tubing. Ports 5 and 6, discussed in
“e. Six-port Switching Valves”, are dedicated for effluent waste.

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b. Software Installation
The ELAN Instrument Control version 3.0 or greater should be already installed on the computer
controlling the ELAN DRC II™. If it is not, contact a PerkinElmer service representative to get the
latest version of the software.
It is preferable to install PerkinElmer’s TotalChrom™ Workstation package on the same computer
that contains the ELAN Instrument Control software*. It is advised that a PerkinElmer
representative install and initially configure TotalChrom™. Alternatively, laboratory personnel can
install and configure TotalChrom themselves by following instructions contained in TotalChrom
Workstation User’s Guide, or by getting help from PerkinElmer Technical Services. As part of
installing TotalChrom™, create a folder named “hplc” on the root level of the C drive. Create the
following subfolders inside the “hplc” folder: “methods”, “data”, “optimization”, and “reports”. Feel
free to create additional subfolders as the need arises. This method assumes that version 6.2.0 of
TotalChrom™ Workstation package is installed.
ChromLink™ 2.0 or a greater version (PerkinElmer LAS) is installed into its own program folder.
The installation of ChromLink™ is quick and straightforward when using the supplied installation
utility.
pdFactory Pro 1.52 or a greater version (FinePrint Software, LLC) is software used for creating
electronic Portable Document Files (PDF). Documents are “printed” from the Microsoft® Windows
print dialog box, but instead of printing on paper, the document appears on the screen in preview
*

Raw signal versus time data is collected by the ELAN software and stored on ELAN controller computer’s
hard drive as a “NetCDF” file before it is read by the TotalChrom Workstation software. For the purposes of
this arsenic speciation method, TotalChrom Workstation is used only for post-run data processing and is not
used to operate the HPLC or the ELAN DRC.

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form. The document can then be either printed in hardcopy or saved to a hard drive or disk as a
PDF file. Because of the “trial and error” nature of solving chromatographic integration
challenges, frequent reprocessing (and reprinting) may be required. In these instances,
generating PDF documents with the option to print a hardcopy becomes an indispensable tool.

c. Peristaltic Pump Setup
An external peristaltic pump offers a number of advantages over the peristaltic pump built into the
ICP-DRC-MS. It can be started, stopped and its speed set independent of the ICP-DRC-MS
control software. This “feature” is important when one considers that the HPLC pump “knows
nothing” about the state of affairs beyond the HPLC system, and it will continue to pump mobile
phase regardless of whether the spray chamber is being drained or not. Likewise, the built-in
peristaltic pump is under ELAN software control only and cannot be operated in manual mode.
While it is feasible to configure the ELAN software to force the built-in peristaltic pump to keep
emptying the spray chamber after completing an analysis, it is nonetheless easy to make a
mistake during the ELAN program setup. The built-in pump’s timing and speed is set in the
ELAN’s sample file and not the method file. The sample file is created each time before a batch
run posing the risk that the built-in peristaltic pump’s timing and speed could be set up incorrectly
or forgotten by the analyst. If this happens, the software will stop the built-in peristaltic pump well
before the HPLC pump stops, causing the spray chamber to flood and the plasma to be
extinguished by mobile phase. It is quite possible for the HPLC to pump mobile phase for an
extended time before it auto-stops causing extensive flooding of the torch box and the ICP-DRCMS which can result in damage to the instrument electronics which can only be stopped manually
because it is not under instrument control. Set up a 4-channel peristaltic pump (Gilson “Minipuls
3” or equivalent) on the ICP-DRC-MS spray chamber shelf behind the spray chamber.
1. Do not connect the peristaltic pump to the control computer; connect the
pump to A/C power only. Run the pump in manual mode only.
2. Designate one clamp area of this pump for nebulizer waste tubing and a
separate clamp area of this pump for post-column internal standard tubing.

d. Electrical Connections
Make the necessary electrical connections between specific I/O terminals of the HPLC
autosampler, the pump, the ICP-DRC-MS, and the 6-port switching valves. The event I/O
terminals are prominently labeled and are located on the right side of each instrument. For the 6port switching valves, the terminals are located on the back of each unit.
1. Between the HPLC pump and the autosampler, use two 12 inch long common
telephone wires with RJ-11 connectors to connect:
(a) The HPLC pump’s terminal labeled “RDY” to the HPLC autosampler’s
terminal labeled “RDY IN”, and
(b) The HPLC pump’s terminal labeled “EXT RUN” to the HPLC
autosampler’s terminal labeled “INJ 1”.
2. Use the supplied cable (PerkinElmer LAS Item # B3001203) to connect the
Pump’s “RUN OUT” terminal to the ICP-DRC-MS’s “AUXILLARY I/O”.
3. Use the supplied cable (PerkinElmer LAS Item # N2600418) to connect the
Pump’s “TE 1” terminal to the electrically-activated terminal block I/O on the
back of 6-port switching valve #1.

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4. Use a standard serial cable to connect an external desktop computer to the
back of 6-port switching valve #2.

e. Six-Port Switching Valves
Each 6-port switching valve’s DIP switches need to be configured upon initial installation. If the
switching valves are not put into “pulse mode”, the units will not respond to the ~1 second contact
closure from the PerkinElmer Series 200 HPLC autosampler. This only needs to be done once for
each new unit. (Note that settings of the front panel buttons should be checked before the start of
each chromatographic run.) For each 6-port switching valve:
1. Turn the switching valve unit upside down and remove the 4 screws using a
Philips head screwdriver. Remove the metal cover and look for DIP switch
“SW3” on the printed circuit board. Use a small flat-head screw driver or the
tip of a pen to adjust the position of each switch so it matches the following
illustrations. These illustrations apply to both EV750-100 and EV750-100-S2.

Rheodyne® EV750-100 DIP Switch “SW3”

DIP switch #

Position

down

2. Screw the cover back on the switching valve unit. For switching valve #1,
connect the timed event wire leads that come from the “TE1” timed event
output on the Series 200 HPLC pump to I/O junctions #4 and GND on the
back of the EV750-100 (order of connection of colored wires does not matter).

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3. For switching valve #2, connect a standard serial cable from a desktop
computer to the serial port on the back of the EV750-100-S2. Aside from the
power cord, this is the only cable which will be attached to switching valve #2.
4. Position both 6-port switching valves close to and on the left side of the HPLC
Series 200 Column Oven, adhering to the order listed in Section 8.a.3. Using
supplied HPLC pressure fittings, make HPLC tubing connections to the
appropriate ports as described in TABLE 8-1-A and TABLE 8-1-B.
TABLE 8-1-A: TUBING CONNECTIONS ON EV750-100
Valve
Port

Flow Direction
From

Tubing
Description

Valve Port #1

Sample Loop

PEEK (200 µL loop)

Valve Port #2 on
EV750-100-S2

Valve Port #2

HPLC tubing, 0.007” i.d.,
1/16” O.D., “yellow” PEEK

Valve Port #3

Nebulizer

PEEK Sample Uptake
Fitting

Sample Loop

Valve Port #4

PEEK (200 µL loop)

Valve Port #5

Waste

PP or PFA Tubing,
0.03” i.d., 1/16” o.d.,

Internal Standard
(via external
peristaltic pump)

Valve Port #6

PP or PFA Tubing,
0.03” i.d., 1/16” o.d.,

TABLE 8-1-B: TUBING CONNECTIONS ON EV750-100-S2
Valve
Port

Flow Direction

Tubing
Description

From

HPLC column

Valve Port #1

HPLC tubing, 0.007” i.d.,
1/16” O.D., “yellow” PEEK

Valve Port #2

Valve Port #2 on
EV750-100

HPLC tubing, 0.007” i.d.,
1/16” O.D., “yellow” PEEK

Line-In for
Various Solutions
(via ICP-MS
peristaltic pump)

Valve Port #3

PP or PFA Tubing,
0.03” i.d., 1/16” o.d.,

Valve Port #4

bridge

HPLC tubing, 0.007” i.d.,
1/16” O.D., “yellow” PEEK

bridge

Valve Port #5

HPLC tubing, 0.007” i.d.,
1/16” O.D., “yellow” PEEK

Valve Port #6

Tygon™ Waste
Line

PP or PFA Tubing,
0.03” i.d., 1/16” o.d.,

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5. Plug-in the A/C power adapter to the back of each unit and into a regular
110V outlet. The switching valve will automatically come on and one of the
diode indicators will light up. Press the “Local/Remote” button on the front of
the unit so that the top yellow indicator light is on.
(a) For Switching Valve #1: Press the left arrow button until the LCD display
indicates “1”. Press the “Local/Remote” button again so that the bottom
green indicator light is now on, representing “remote.”
(b) For Switching Valve #2: Press the left arrow button until the LCD display
indicates “2.” Ensure that the top yellow indicator light is on,
representing “local.”
Note: These LCD positions are for a typical HPLC-ICP-DRC-MS analysis
run. Users may toggle between “local” and “remote” and positions “1” and
“2”, for instrument optimization or other tasks.
6. The switching valves are now ready for operation.

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STANDARD PROCEDURE
a. Preparation of Stock Solutions
1. 0.5 M Ammonium Acetate, pH 5. Dissolve 27.2 g of ammonium acetate and
8.0 mL of glacial acetic acid into approximately 950 mL of 18 Mcm water.
Adjust pH to 5.0 by adding concentrated glacial acetic acid drop-wise.
Complete volume to 1000 mL with 18 Mcm water. Mix thoroughly. Expires
in 1 year. Prepare ahead of time or as needed.
2. 0.1 M Ammonium Acetate, pH 5. Dissolve 5.44 g of ammonium acetate and
1.68 mL of concentrated glacial acetic acid into approximately 950 mL of 18
Mcm water. Adjust pH to 5.0 using drop wise additions of either 10%
ammonium hydroxide or glacial acetic acid. Complete volume to 1000 mL with
18 Mcm water. Mix thoroughly. Expires in 1 year. Prepare ahead of time or
as needed. Note: This solution is not prepared as a dilution of 0.5 ammonium
acetate, pH 5.
3. 0.5 M Ammonium Carbonate. Dissolve 48.05 g of ammonium carbonate into
approximately 900 mL of 18 Mcm water. Complete volume to 1000 mL with
18 Mcm water. Mix thoroughly. Expires in 1 year. Prepare ahead of time or
as needed.
4. 0.5 M TRIS Buffer. Dissolve 60.57 g of tris(hydroxymethyl)aminomethane in
approximately 900 mL of 18 Mcm water. Complete to 1000 mL with 18
Mcm water. Mix thoroughly. Expires in 1 year. Prepare ahead of time or as
needed.
5. 0.5 M Ammonium Sulfate. Dissolve 66.07 g of ammonium sulfate in
approximately 900 mL of 18 Mcm water. Complete to 1000 mL with 18
Mcm water. Mix thoroughly. Expires in 1 year. Prepare ahead of time or as
needed.
6. 5% Acetonitrile. To make autosampler rinse solution, add 50 mL of
acetonitrile (HPLC or Spectrophotometer grade) to 950 mL of 18 Mcm
water. Mix thoroughly. Expires in 1 year. Prepare ahead of time or as needed.

b. Preparation of Matrix-Matched Material (“Base Urine”)
Collect human urine from anonymous donors following the same collection procedure
used for the preparation of QC material. Assay each donation for, if possible, speciated
arsenic or, alternatively, for total arsenic. Exclude urine donations that exceed 10 µg/L for
each arsenic species or 25 µg/L total arsenic. Pool a sufficient number of urine donations
to make a volume greater than 2 liters. Divide the pool into 40 mL aliquots and save at 70°C in 50 mL centrifuge tubes labeled “Base Urine – Speciated Arsenic”.

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c. Preparation of Working Solutions
1. 25% (v/v) Base Urine Pool in 0.075 M Ammonium Acetate, pH 5. May be
prepared ahead of time before the day of analysis. Add 30 mL of 0.5 M
ammonium acetate pH 5 to a clean 250 mL PP bottle. Add 50 mL of “Base
Urine – Speciated Arsenic”. Add 120 mL of 18 Mcm water. Mix thoroughly.
Prepare as needed. Store refrigerated at 4°C. Expiration date is 1 day from
the date made.
2. HPLC Buffer A Preparation. May be prepared ahead of time before the day
of analysis. To a clean 2 liter or greater capacity beaker containing a clean
magnetic stir bar add approximately 1.9 L of 18 Mcm water (≥ 18 M·cm).
Add the following:
(a) 40.0 mL of 0.50 M ammonium carbonate
(b) 40.0 mL of 0.50 M TRIS buffer
(c) 10 mL of methanol.
Ensure that the temperature-adjust mode is enabled on the pH meter to be
used. Be sure solution is being mixed on a magnetic stir plate. Using a
hydrogen glass electrode (pH probe), monitor pH while slowly adding either
glacial acetic acid (to lower pH) or 10% ammonium hydroxide (to increase
pH) drop wise to bring the pH to 8.60 ± 0.05. In this case, an Eppendorf®
Repeater Pipetter fitted with a 500 µL syringe tip works well for adjusting pH.
After complete mixing, transfer beaker’s contents to a 2 L graduated cylinder.
Complete volume to 2000 mL with 18 Mcm water. Transfer entire contents
to HPLC “Bottle A”, cap, and mix thoroughly. Label bottle “10 mM Amm.
Carbonate / 10 mM TRIS / 0.5% MeOH / pH 8.6” (or using other appropriate
notation to indicate contents). Prepare as needed. Expiration date is 2
weeks from the date made.
3. HPLC Buffer B Preparation. May be prepared ahead of time before the day
of analysis. To a clean 2 liter or greater capacity beaker containing a clean
magnetic stir bar, add approximately 1.85 L of 18 Mcm water (≥ 18 M·cm).
Add the following:
(a) 40.0 mL of 0.50 M ammonium carbonate
(b) 40.0 mL of 0.50 M TRIS buffer
(c) 10 mL of methanol.
(d) 60.0 mL of 0.50 M ammonium sulfate
Ensure that the temperature-adjust mode is enabled on the pH meter to be
used. Be sure solution is being mixed on a magnetic stir plate. Using a
hydrogen glass electrode (pH probe), monitor pH while slowly adding either
glacial acetic acid (to lower pH) or 10% ammonium hydroxide (to increase
pH) drop wise to bring the pH to 8.0 ± 0.1. After complete mixing, transfer
beaker’s contents to a 2 L graduated cylinder and add 18 Mcm water to
complete volume to 2000 mL. Transfer entire contents to HPLC “Bottle B”,
cap and mix thoroughly. Label bottle “15 mM Amm. Sulfate / 10 mM Amm.
Carbonate / 10 mM TRIS / 0.5% MeOH / pH 8.0” (or using other appropriate
notation to indicate contents). Prepare as needed. Expiration date is 2
weeks from the date made.

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4. HPLC Buffer D Preparation. 5% (v/v) acetonitrile (HPLC grade) in 18 Mcm
water. Mix thoroughly. Prepare as needed. Expiration date is 1 year from the
date made.
5. HPLC Buffer E Preparation. 5% (v/v) acetonitrile (HPLC grade) in 18 Mcm
water. Mix thoroughly. Prepare as needed. Expiration date is 1 year from the
date made.
6. Internal Standard Preparation. May be prepared ahead of time before the
day of analysis. To an empty 1 liter PP bottle labeled “Internal Standard”, add
5.0 mL of methanol. Next, add 50 µL of 50 mg/L trimethylarsine oxide (TMAO
Internal Standard Stock Solution). Fill with 18 Mcm water to the 1000 mL
mark. Mix thoroughly. Final concentration is 2.5 µg/L TMAO. Prepare as
needed. Expiration date is 1 year from the date made

d. Preparation of Stock Standards (Concentrated)
CAUTION!
Arsenic compounds are toxic! Take extra care to avoid accidental ingestion or inhalation of these
materials. Wear appropriate personal protective gear. At a minimum, wear a laboratory coat
and latex or nitrile gloves. Clean up any spill that might occur according to applicable
hazardous material spill procedures.
Note 1: All preparations should be performed gravimetrically (wt/wt), unless
otherwise noted. All gravimetric measurements should assume the density of
water equal to 1g/cm3.
Note 2: The steps outlined in Section 9.b – 9.h may be optionally outsourced
under contract to a partner facility.
Definitions:




Stock Standard: Initial solution of one of seven arsenic analytes prepared by
dissolving solid or liquid standard material into aqueous or acidic solution.
Intermediate Standard: A 10,000ppb solution prepared from dilution of a Stock
Standard.
Working Calibrator: A dilution of the Intermediate Standards prepared in urine
and ammonium acetate buffer. A Working Calibrator is used in urine arsenic
species HPLC-ICP-DRC-MS analysis to build a calibration curve.

Use a high precision analytical balance capable of accurately weighing milligram
amounts of material to the tenth of a milligram or better. It is important to use the
balance in a vibration-free room that is free of air drafts and away from direct sun
light, to the fullest extent possible.
1. Using a clean Teflon-coated spatula, prepare the arsenic Stock Standards
described in TABLE 9-1 into 50.0 mL centrifuge tubes or other suitable
storage vessels. Record the weights of the initial solid arsenic material for
each species and all final weights of the corresponding arsenic solutions after
dissolution.

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TABLE 9-1: PREPARATION OF STOCK STANDARDS
Arsenic Species

Range to
Weigh (g)

Solvent Used to Dissolve

Final Weight (g) After
Dissolution

Arsenobetaine

0.02–0.03

Water (18 Mcm)

10.00

Arsenocholine bromide (or
other salt equivalent)
Disodium methyl arsenate

0.20–0.25

Water (18 Mcm)

50.00

0.20–0.25

Water (18 Mcm)

50.00

Trimethylarsine oxide

0.10–0.125

Water (18 Mcm)

50.00

Dimethylarsinic acid

0.10–0.125

Water (18 Mcm)

50.00

Arsenic (V) oxide hydrate

0.10–0.125

Water (18 Mcm)

50.00

Arsenic (III) oxide

0.10–0.125

Dissolve in 1.5 ml of 6N HCl with
mild heating. Add water (18
Mcm) to complete volume

50.00

2. Tightly cap the storage vessels for future use. Expiration date is 1 year from
the date weighed.
3. Calculate the concentration of each Stock Standard using the recorded
weights for each species. The resulting units of concentration are milligrams
per liter (mg/L). Record these values in a laboratory notebook.
4. Calculate the “Arsenic (As) atomic equivalent” concentration of each arsenic
species concentrated standard using Equation 1.
Equation 1
“As atom
equivalent”
conc. in mg/L

concentration of concentrated
stock standard in g/L



# As atoms per
species molecule

F.W. of As species



74.92 atomic wt. As

-3

10 g/mg

Insert into the equation the appropriate values for the formula weights (F.W.) and
number of arsenic atoms per molecule for each species. A list of commonly
used formula weights for each arsenic species is shown in TABLE 9-2;
however, formular weights provided by the chemical manufacturers, if
different, supersede the values presented in the table and should be used
instead.

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TABLE 9-2: FORMULA WEIGHTS OF ARSENIC SPECIES
Formula Weight

Number of Arsenic
Atoms per
Molecule

Formula

Arsenobetaine

178.06

(CH3)3AsCH2COOH

Arsenocholine bromide

244.99

(CH3)3AsCH2CH2OH·Br

Disodium methyl arsenate

291.9

CH3AsO3· 6H2O

Trimethylarsine oxide

136.03

(CH3)3AsO

Dimethylarsinic acid

138.01

(CH3)2As(OH)2

Arsenic (V) oxide hydrate

229.84

As2O5· xH2O

Arsenic (III) oxide

197.84

As2O3

Arsenic Species

Note: All arsenic solutions from this point forward are referenced in terms of arsenic
concentration.
5. Record the “arsenic (As) atomic equivalent” concentration value on each
arsenic stock standard storage vessel.

e. Preparation of Intermediate Standards
Into separate 50 mL centrifuge tubes, gravimetrically prepare 10,000 ppb
solutions of each stock standard. Use the arsenic atomic equivalent for each
species to determine the appropriate dilution needed to obtain the expected
concentration of approximately 10,000 ppb. Record all weights, and calculate
the expected concentration of each solution marking these values in a laboratory
notebook. Note: Exact arsenic concentrations will be determined by DRC-ICPMS in subsequent steps.

f. Microwave Digestion for Conversion to Arsenate (AsV)
Even at the same arsenic concentration, different arsenic species can produce different
instrument responses during analysis. Therefore, it is important to chemically convert
each arsenic species to one common chemical form prior to analysis for total arsenic.
Digestion of the arsenic species by microwave-assisted oxidation to arsenate (AsV)
allows for analysis with an instrument calibrated using aqueous arsenate calibrators.
This prevents introduction of systematic errors that otherwise might be caused by the
determination of undigested arsenic species concentrations calculated from an inorganic
arsenate calibration curve.
1. Prepare 100 mL of 3% potassium persulfate (K2S2O8) in 18-MΩ deionized
water. Expiration date is 1 day from the date made. This solution may be
prepared by weight/volume.
2. For each arsenic species analyte, do this step in triplicate: For each of the
Intermediate Standard solutions, gravimetrically transfer 0.5 g into a

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microwave Teflon vessel and record the weight to three significant digits. Add
10 mL of 3% K2S2O8 to each vessel. (A volumetric measure is sufficient here,
as the final solution, post-microwave assisted digestion, will be gravimetrically
brought to 50.0 g total weight). Additionally, triplicate blanks (0.5 g water +
10.0 mL K2S2O8) and triplicate certified AsV solution are desired for quality
control if space permits in the microwave.
3. Perform microwave-assisted digestion of these solutions using the microwave
program “10 ml K2S2O8” or a suitable program that allows for the following
parameters listed in TABLE 9-3:
TABLE 9-3: MICROWAVE DIGESTION OVEN PARAMETERS

t(min.)

E [W]

T1 [°C]

00:10:00

1000 max

140°C

00:05:00

1000 max

200°C

00:15:00

1000 max

200°C

Ventilation 10 min.

1000 max

4. Once the microwave-assisted digestion is complete, it is important to allow
each vessel to cool to room temperature before opening to avoid possible loss
of volatile arsenic. For safety purposes, open all vessels underneath a
chemical fume hood (or equivalent) to avoid inhalation of toxic fumes.
5. Using the analytical balance, once digestion vessels are at room temperature,
quantitatively transfer (with 18-MΩ de-ionized water) each digested solution
into separate labeled 50 mL Falcon tubes. Bring the final weight to 50.0 g
with 18-MΩ deionized water, and record the total mass to three significant
digits. The expected arsenic concentration of each solution should be
approximately 100 ppb as arsenate (AsV).
6. To determine if all arsenic species have been converted to AsV, measure an
aliquot of each species by the current CLIA urine arsenic species HPLC-DRCICP-MS method. Since this step is performed solely to confirm that the
species are no longer present in their original forms, this may be performed in
a qualitative manner.

g. Method of Standard Additions (MSA)
Note: It is imperative that the total arsenic concentration of each solution be determined
by the Method of Standard Additions (MSA), and that all solutions are prepared
gravimetrically (wt/wt) unless otherwise noted.
1. Prepare 1000 mL of 2 ppb gallium in deionized water by diluting 2 mL of 1000
µg/L stock Ga in 1000 mL of deionized water . This solution will be used to
dilute post-microwave digested solutions. This solution does not need to be
prepared gravimetrically.
2. Prepare a 5 ppm AsV “spiking solution” in deionized water. Weigh 0.25 g of
1,000 µg/L certified arsenate solution and bring to a final weight of 50.0 g

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using deionized water Record all weights, and calculate the exact
concentration of this solution. This concentration will be used in steps e.4.ii,
iii, and iv.
3. Using the 2 ppb Ga solution prepared in Step 1, dilute each of the microwavedigested solutions 1:2 into new 50 mL centrifuge tubes by weight, with a final
weight of 50.0 g. (For example, dilute 25.0 g of each microwave digested
solution to 50.0 g total weight using the 2 ppb Ga solution). Record all
weights. Final concentrations should be approximately 50ppb.
4. For each new 50ppb microwave-digested solution, label four new 15mL
centrifuge tubes, incorporating the analyte name, replicate, and MSA spike
concentration. An example (demonstrating only one replicate for one
microwave-digested solution) is shown below in TABLE 9-4. Each tube will be
used to prepare new solutions for MSA in subsequent steps.
Note: A spike of 0 ppb corresponds to an unspiked sample for which a value
will be determined in subsequent steps via total arsenic analysis.
TABLE 9-4: METHOD OF STANDARD ADDITIONS TABLE 1

Tube #
1
2
3
4

Analyte
AB
AB
AB
AB

Labels
Replicate
1
1
1
1

Spike in pbb
0
25
50
100

5. The solutions mentioned in Step 4 and outlined in TABLE 9-4 must be
prepared. For accuracy, it is important to prepare each solution in the
following order:
For each 50 ppb microwave-digested solution:
(i) Into the tube labeled 0 ppb transfer by weight 10.0 g of the
approximately the approximately 50 ppb diluted microwave-digested
solution prepared in Step 9.3. Record the weight.
(ii) Into the tube labeled 25 ppb, transfer by weight approximately 0.05 g
of the approximately 5 ppm spiking solution prepared in step 9.g.2.
Additionally, transfer by weight 10.0 g of the approximately 50 ppb
diluted microwave-digested solution prepared in Step 9.3. Record all
weights.
(iii) Into the tube labeled 50 ppb, transfer by weight approximately 0.10 g
of the approximately 5 ppm spiking solution prepared in step 9.g.2.
Additionally, transfer by weight 10.0 g of the approximately 50 ppb
diluted microwave-digested solution prepared in Step 9.3. Record all
weights.
(iv) Into the tube labeled 100 ppb, transfer by weight approximately 0.20
g of the approximately 5 ppm spiking solution prepared in step 9.g.2.
Additionally, transfer by weight 10.0 g of the approximately 50 ppb
diluted microwave-digested solution prepared in Step 9.3. Record all
weights. TABLE 9-5: MSA TABLE 2 shows an example of the
information that needs to be recorded for each analyte and
corresponding tube.

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TABLE 9-5: METHOD OF STANDARD ADDITIONS TABLE 2

Tube
AB, Replicate 1, 0ppb
AB, Replicate 1, 25ppb
AB, Replicate 1, 50ppb
AB, Replicate 1, 100ppb

Target
Weight,
Spiking
Solution
n/a
0.05g
0.10g
0.20g

Measured
Weight,
Spiking Solution
n/a
0.0517g
0.0997g
0.2051g

Target
Weight,
Final
Solution
10.0g
10.0g
10.0g
10.0g

Measured Weight,
Final Solution
9.8973g
9.8269g
9.8738g
9.8571g

h. Determining the Total Arsenic Concentration
1. Analyze each MSA solution for total arsenic content using a validated ICP-MS
method. Ensure that the method uses Ga as an internal standard.
2. After the analysis is complete, prepare an MSA calibration curve for each
solution, and calculate the concentration of the unknown sample by dividing
the y-intercept by the slope.
3. Using the recorded weights for each MSA solution, calculate the exact
concentrations of arsenic in each tube.
4. Once the arsenic concentration of each MSA solution has been determined,
perform blank subtraction, and then use all recorded weights for each
replicate to back-calculate the exact 10,000 ppb concentration of the
Intermediate Standard solutions prepared in section 9.e. An example of the
necessary calculation is given below:

Intermediate Standard Concentration Determination
calculation M1MSA=
V2MSA*M2MSA/V1MSA

M1MW=
V2MW*M2MW/V1MW

Key:
V2MSA
M2MSA
V1MSA
M1MSA

Final Wt (g) of solution prepared in Step 9.g.3
Measured concentration (ppb) as determined from MSA
Initial Wt (g) of solution prepared in Step 9.g.3
Exact concentration (ppb) of microwave‐digested solution (Step 9.f.2)

V2MW
M2MW
V1MW
M1MW

Final wt (g) of solution prepared in Step 9.f.5
The M1MSA concentration (ppb)
Added amount (g) of Intermediate Standard from step 9.f.2
Exact concentration (ppb) of Intermediate standard

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5. After the concentration for each replicate of the Intermediate Standards has
been determined, calculate the average concentration of all Intermediate
Standard replicates per analyte.
6. Additionally, calculate the average concentration of all microwave blanks
prepared in Step 9.f.2. Subtract the average blank value from all Intermediate
Standard concentrations.

Assessment of Purity by HPLC-DRC-ICP-MS
1. From each 10,000 ppb solution, prepare a 10 ppb solution.
2. Using the CLIA “Urine arsenic species HPLC-ICP-DRC-MS” method, analyze
each arsenic species for the presence of significant levels (>2%) of arsenic
species impurities which are defined as the presence of any other arsenic
species that are included in Table 1-1 of this method.
3. Tabulate all impurities for each solution. If significant levels of impurities are
found, consider purchasing additional standard solid material from an
alternative source and remake the stock standard. If the total amount of
impurities is small (<2%), it is permissible to calculate a correction factor
which will be used to adjust the volumes in the next dilution step (i.e.,
preparation of Working Calibrators) so that the final concentration reaches the
intended value.
4. Correct the measured Intermediate Standard concentrations based on their
purities. For example, if the Intermediate Standard solution of arsenobetaine
(AB) was found to have 2% total impurities, the measured value should be
multiplied by 98% to obtain the pure value of this Intermediate Standard.
5. For AB, AC, DMA, MMA, AsIII, or AsV solutions that are found to have
impurities, be sure to add the concentrations of the impurities to the value of
the species because once combined in the mixed calibrators, the impurities
will contribute to the final value for that species. Impurities in the TMAO
solution will not be used in the calculation because TMAO will not be
combined with the other species.

Preparation of Concentrated Stock Internal Standard
Prepare ahead of time before the day of analysis. Expiration is 1 year from date of
preparation.
1. Prepare concentrated Stock Internal Standard, 50 mg/L trimethylarsine oxide
(TMAO). Dilute a calculated volume (µL) of trimethylarsine oxide (TMAO)
stock standard in 18 Mcm water to make a total volume of 20 mL. Calculate
the number of µL of stock TMAO standard to add using Equation 2:

Equation 2
µL of stock TMAO
standard to add

50 ppm  20 mL  1000 µL/mL
actual ppm of stock TMAO standard

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k. Preparation of Working Calibrators
At any given time, it is necessary to have at least two sets of Working Calibrators in
storage from two independent preparations of Stock Standards. The original material
must be from different lots, preferably from alternate vendors. Arsenic speciation analysts
should alternate between both sets of working calibrators from run to run.

(1) Preparation of Intermediate “Mixed Species” Calibration Solutions
Prepare the intermediate mixed species calibration solutions on the day of analysis.
1. Add 7.5 mL of 0.5 M ammonium acetate (pH 5) to a clean 50 mL volumetric
flask labeled “Mixed As Species 250 µg/L” or something similar. Likewise,
add 7.5 mL of ammonium acetate pH 5 to another dedicated 50 mL volumetric
flask labeled “TMAO 250 µg/L” or something similar.
2. To each flask, add 12.5 mL of Base Urine.
3. Based on the pure measured value of each Intermediate Standard (which
takes into consideration the addition of any impurities per analyte) prepare the
250µg/L mixed as species solution. To the flask labeled “Mixed As Species
250 µg/L”, add each of the following Intermediate Standards: AC, AB, DMA,
MMA, As(III) and As(V) (six solutions in all so that each analyte’s final
concentration will be 250 µg/L at a volume of 50.0 mL). Use of the dilution
equation C1*V1 = C2*V2 is helpful, where C1 is the pure measured value of
an analyte + the sum of any impurities in the form of that analyte from the
other Intermediate Standards and V1 is the weight of each Intermediate
Standard for which to solve. Do not account for any impurities from TMAO in
this step, as TMAO will be diluted in a separate flask and not mixed with the
other arsenic species.
4. Repeat Step 3 for the TMAO Intermediate Standard. Using the flask labeled
“TMAO 250 µg/L”, based on the pure measured value, weigh the appropriate
amount of TMAO Intermediate Standard to obtain a 250 µg/L solution based
on a final volume of 50.0 mL.
5. Bring both flasks to a final volume of 50.0 mL using 18 Mcm water. Mix the
contents of each flask thoroughly. Transfer each solution to an appropriately
labeled 50 mL centrifuge tube or equivalent container. This solution expires in
8 hours unless it is frozen. If the solution is frozen, the expiration date is 1
year from the date made.

(2) Preparation of Working Calibrator Series
1. Label six clean 15 mL PP screw-top Falcon (or equivalent) tubes with caps
as follows: “Mix S0”, “Mix S1”, “Mix S2”, “Mix S3”, “Mix S4”, and “Mix S7”.
Label an additional five 15 mL PP screw-top tubes as follows: “TMAO T0”,
“TMAO T1”, “TMAO T2”, “TMAO T3”, and “TMAO T4”. Arrange these tubes in
order and place in a test tube rack.
2. Inspect a Gilson 402™ Dilutor/Dispenser to ensure it has a clean 10 mL
dispensing syringe and a clean 500 µL sample syringe. It is helpful to
examine the rubber gasket for precipitate or dirt. If precipitate or dirt is found
on either syringe, replace that syringe with a new one. If possible, it helps to
have syringes dedicated to calibrator preparation.

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3. Place the tubing that will draw diluent into the dilution syringe into a buffered
solution consisting of 25% Base Urine in 0.075 M ammonium acetate (pH 5).
Place a waste beaker in position to collect the effluent liquid from the tip of the
sample/dispense tubing. Next, thoroughly rinse the liquid path by pressing the
“Prime” button on the Gilson and allow the syringes to cycle 3 to 4 times
before stopping the prime function with a press of the “Start/Stop” button.
4. To make the “S0” calibrator, set the diluent syringe volume to “10000” and the
sample syringe volume to “0”. Press the “Start/Stop” button once to draw 10
mL of diluent solution (25% urine in 0.075 M ammonium acetate pH 5) into the
diluent syringe then press again to dispense 10 mL to the “Mix S0” tube.
5. To make the calibrator levels “S1” to “S4” and “S7”, set the diluent syringe
volume to the appropriate values indicated in TABLE 9-3 for the diluent and
sample syringe volumes. Submerge the tip of the Gilson 402™
sample/dispense tubing into the “Mixed As Species 250 µg/L” intermediate
solution and press the “Start/Stop” button on the Gilson. A volume in µL
(indicated in the appropriate row in the third column of TABLE 9-3) will be
aspirated. Place the appropriately labeled receiving tube (“S1”, “S2”…”S4”
and “S7”) under the sample/dispense tubing and press the “Start/Stop” button.
The indicated volume of diluent and sample will be dispensed to make a total
volume of 10 mL of diluted calibrator in each tube. If necessary, repeat the
dispense step into the same tube to make a total dispensed volume equal to
10 mL.

TABLE 9-3: GILSON 402™ SETTINGS FOR MAKING DILUTED SERIES
Calibrator
Level

Gilson Volume
Setting, µL
Diluent
Syringe

Sample
Syringe

S0 / T0

10000

S1 / T1

9980

S2 / T2

Target Arsenic Concentration
(Nominal Conc. entered into HPLC
software)

(0)

0.50

(2)

9900

100

2.50

(10)

S3 / T3

9500

500

12.5

(50)

S4 / T4

8500

1500

37.5

(150)

10000

250.0

(1000)

6. Repeat the preceding step for each calibrator level indicated in TABLE 9-3.
Between calibrators, rinse the tip of the sample/dispense tubing with 18
Mcm water from a squeeze bottle.
7. Repeat the previous two steps, this time using “TMAO 250 µg/L” to make “T0”
through “T4”.
8. Per CLIA requirements, twice per year, an extended-range linear verification
analysis has to be completed for each CLIA method. Therefore, S5, S6, and
T5, T6, and T7 calibrators are also made during this preparation step. The
concentrations of each are as follows:
S5/T5 are 250 ppb, S6/T6 are 500 ppb, and S7/T7 are 1000 ppb.

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9. Cap all tubes. Mix them thoroughly by vortexing and/or inverting repeatedly.
10. Divide each calibrator into twenty (or less) 0.5 mL aliquots contained in
correspondingly labeled clean HPLC autosampler vials. Cap each with a
“snap-cap” septum cap. Store at ≤ -70°C. Expires in 9 months.
11. At a later date, thaw one set of calibrators (S0 – S4 and T0 – T4) as needed
for future calibrations. An S7 will be thawed, if necessary, for extended
calibration range verification.

Preparation of Quality Control Material
Collect human urine from anonymous donors in clean, trace metals-free urine cups.
Refrigerate urine donations at ≤ 4°C as soon as possible for periods of 2 days or less.
For longer periods, freeze the urine donations until needed. Assay each donation for, if
possible, speciated arsenic or, alternatively, for total arsenic. Assign urine donations to a
“low” pool or to a “high” pool according to whether its arsenic concentration exceeds a
predetermined threshold value (i.e. 15 µg/L). Do not pool urine donations with an arsenic
concentration that exceeds the threshold by more than a factor of 10. After pooling urine
donations into their respective pools, clarify each pool by centrifugation in acid-washed
250 mL centrifuge bottles (30 minutes at 4000 rpm in a preparative table-top centrifuge).
Pour off the supernatant and dispose of the pellets. To each pool, add a calculated
volume of the chosen arsenic species standard solution to raise the concentration of that
arsenic species to the desired value. While maintaining constant stirring of each pool,
aliquot 1.0 mL (or more) of urine into a sufficient number of pre-labeled 2 mL vials to
provide QC material for 1000 or more runs. Store aliquotted QC material at ≤ 70°C.

m. Processing of Urine Samples and QC Material
Process a chosen number of urine samples and QC material on the day of analysis.
One run is defined as the analysis of a contiguous set of samples (typically 20)
bracketed by bench QC material at the beginning and end of the set. Each bench
QC level needs to be analyzed at the beginning and end of a run in separate
tubes/vials. Sharing of even a single QC tube or vial for more than a one QC
determination is disallowed. It is permissible to “piggyback” two runs in succession
following a single calibration done during a single autosampler load (such as for an
overnight analysis), as long as each run of samples is bracketed by its own uniquely
co-prepared bench QC material. The number of samples per run can exceed 20 as
long as the total analysis time does not exceed 24 hours.
1. Identify, gather, and thaw the necessary specimen tubes containing the urine
samples for the batch (“run”) to be analyzed.
2. Likewise, for each batch run, thaw one tube each of low and high bench QC
samples “LU-yyxxx” and “HU-yyxxx” (for explanation of nomenclature, see
Quality Control Material above).
3. Label the required number of Eppendorf® (or equivalent) 1.5 mL
microcentrifuge tubes corresponding to the samples and bench QC samples
to be run. Label a pair of microcentrifuge tubes for each bench QC, since
each bench QC will be injected at the beginning and end of each batch run

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and need to be contained in separate tubes. Likewise label an equal number
of HPLC autosampler vials and set these aside for later use. It is helpful to
use preprinted barcode labels to improve efficiency and reduce the chance of
labeling errors.
4. Attach a bottle of 0.1M ammonium acetate solution to a Gilson 402™
Dilutor/Dispenser diluent draw-line. In order to minimize evaporation, it is
helpful to use a capped bottle with a small hole in the cap that is slightly larger
than the outer diameter of the draw-line. Insert the draw-line through the hole
and assure that the end of the draw line is completely submersed in
ammonium acetate solution. It is important that the end of the line remain
submersed throughout sample preparation in order to prevent air bubbles.
5. Using the “Prime” function on the Gilson 402™ Dilutor/Dispenser, flush lines
with 0.1M ammonium acetate solution and empty into a small waste container.
6. If not done so at a previous date, create a sample preparation method on the
Gilson 402™ Dilutor/Dispenser that will in a step-wise, sequential, and usercontrolled fashion:
(a) Uptake 10 µL of air into sample draw-line.
(b) Uptake 200 µL of sample (urine) into sample draw-line.
(c) Uptake 1600 µL of 0.1M ammonium acetate solution into the diluent
draw-line.
(d) Dispense 800 µL of sample + diluent mix (200 µL sample + 600 µL 0.1M
ammonium acetate solution).
(e) Dispense 1000 µL of diluent (for flushing).
(f) Repeat steps (a) – (e) until the program is stopped.
The method should be set up such that no step shall execute until the user
has pressed the black button on the black dispenser control device.
Name and save this method into memory.
For detailed instructions on programming, please consult the Gilson 402™
Dilutor/Dispenser instrument manual.
CAUTION!
Work with open vials or tubes containing biological samples in a biological safety cabinet (BSC).
Wear appropriate personal protective equipment (gloves, lab coat and safety glasses).
7. Recall the sample preparation method created in step 6. Using this method,
follow its steps (including the 10 µL air uptake) to mix 200 µL of sample from
each specimen tube with 600 µL 0.1M ammonium acetate solution and to
transfer the mix to each sample’s respective 1.5 mL microcentrifuge tube.
Note that calibrators, which have been pre-made, do not undergo this type of
sample preparation.
8. After each sample/diluent mix is transferred, dispense 1000 µL of 0.1M
ammonium acetate solution (pH 5) into a waste container and repeat the
process for each remaining sample.

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9. After capping all 1.5 mL microcentrifuge tubes containing sample/diluent mix,
vortex each for 3-5 seconds. Next, centrifuge tubes for 5 minutes at 14,000
rpm in a refrigerated centrifuge pre-cooled to ≤ 4°C.
10. Following centrifugation, transfer approximately 0.6 mL of the supernatant to
the appropriately pre-labeled HPLC autosampler vials. Be careful not to
disturb any pellet that might be present at the bottom of the microcentrifuge
tube during transfer.
11. Cap all autosampler vials with the proper fitting “snap-cap” septum caps.
12. Thaw one “set” of calibrators, including S0 – S4 and T0 – T4, at room
temperature. Vortex each thawed calibrator for 3-5 seconds.
13. To autosampler vials labeled “Bk” (which stands for “Blank”), transfer 0.5 mL
of 0.075M ammonium acetate (dilute from stock solution). The number of Bk
vials will be dependent upon the total number of samples to be analyzed.
14. For each run, one extra sample of 200 µL LU-xxxx + 600 µL 0.1M ammonium
acetate solution is needed. This sample is used for instrument equilibration
and conditioning only, so an LU-xxxx vial from a previous day’s run containing
leftover sample will suffice. If there are no leftover samples, one may be
made according to step 7 of this section.
15. If barcode labels have been affixed to the vials, at the appropriate time, use
the barcode scanner attached to the instrument computer to scan the sample
ID from the barcode label on each sample and QC vial before placing it into
position in the HPLC autosampler tray.

n. HPLC Instrument Setup
To improve work flow, instrument setup described in this section may be completed before the
day of analysis.

(1) Programming the HPLC Pump Methods
1. On the PerkinElmer Series 200 Pump control panel, press the Quit button to
bring the pump controller to the starting screen.
2. If the method number for the correct stored pump program is known, that
method can be called up into active memory. Press the function key F6
(labeled as “DIR” on screen). The screen changes and presents a table with
column headers “Method”, “Name” and “Last Modified”. Press function key F4
(“RCL”) and you will be prompted to enter a 2-digit number. Press the number
for the correct method (e.g., “1”) followed by the “enter” key. The stored
method will be loaded into memory and becomes the active pump program.
The method can be inspected by pressing the function key F2 (“PUMP”) and
using the up or down arrow keys to scroll the program steps.
3. If the correct pump program cannot be found, then the pump program will
have to be reentered. To do this, execute the following steps:
(a) Press the Quit button, then the function key F6 (labeled as “DIR” on
screen). The screen changes and presents a table with column headers
“Method”, “Name” and “Last Modified”. Press function key F4 (“RCL”)
and you will be prompted to enter a 2-digit number. Press “0” followed by
the “enter” key. The “DEFAULT” method will now be loaded.

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(b) Press the function key F2 (“PUMP”). A new screen presents a table with
column headers “Step”, “Time”, “Flow”, “%A”, “%B”, “%C”, “%D” and
“Curve”. The default method has only one line, Step “0”, with the “>”
marker just left of it. The highlighted data field is “Time” and contains
“10.0”. Press “6.5” on the keypad followed by the right arrow key to
replace the previous number. The highlight will advance right to “Flow”.
Press 1 then the right arrow key. The next field is “%A”; enter 100 then
press the right arrow key. Repeat this for fields “%B”, “%C” and “%D”,
entering 0 for each. Press the right arrow key again to return to “Time”.
(c) Press the “insert” key on the pump controller keypad. A new step, Step
“1”, is added and is a replicate of the step before it. Input the data
indicated in the table below. Notice that the last column “CURV” in step
1 calls for the input of a number. Likewise, create Step “2”, using the
data from the table below. Note that you can move back and forth
between fields, and up and down from one step to another, by using the
left, right, up and down arrow keys on the keypad.
TABLE 9-4: HPLC PUMP PROGRAM SETTINGS
STEP

TIME

FLOW

CURV

0
1
2

6.5
5.0
4.5

1.00
1.00
1.00

100
0.0
0.0

0.0
100
100

0.0
0.0
0.0

(i) Press the function key F6 (“STOR”). You will be prompted to enter a
method number, press “1” then the “enter” key. If message comes up
asking if you want to overwrite the existing method stored at that
location, press the “1” key for “Yes”. Next you will be prompted to
name the method, press the “0” key for “No”. (You may enter “Yes”
and create a name for the method but this is optional).
(ii) Press the function key F3 (“T.E.”). This is for entering timed events.
Since an electrical-activated external switching valve is going to be
used, the timing of brief ~1 second contact closures needs to be
programmed.
EVENT
1
2

TIME

T.E.1

T.E.2

0.5
1.0

YES
YES

(iii) Enter the parameters from the above table using the same technique
as was used to create the pump method. Note that to successfully
input the minutes for each event, you need to press “enter” after the
inputting the number, instead of using the right arrow key. Timed
Event #1 (“T.E.1”) is turned on by pressing “1” (“Yes”) on the keypad.
Store a “ready” time (the number of minutes the pump waits during
re-equilibration before allowing the next injection) by pressing
function key F8 (“RDY”) and input “30” followed by the “enter” key.
Press function key F6 (“STOR”) and respond to the prompt for a

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method number by inputting the same method number (e.g., “1”)
used to store the pump program. Respond to the next two prompts
with a “yes” then a “no”.
4. Press the function key F4 (“PRESS”). Press function key F4 (“MAX”) then
input “3000” followed by the “enter” key. Press function key F3 (“MIN”) then
input “100” followed by the “enter” key. Press function key F6 (“STOR”) and
respond to the prompt for a method number by inputting the same method
number used to store the pump program. Respond to the next two prompts
with a “yes” then a “no”.
5. Create a separate HPLC pump method (e.g. method “19”) which will serve as
a column wash method after the completion of a batch run:
(a) Follow step 3 above to input the following in TABLE 9-5:
TABLE 9-5: HPLC PUMP COLUMN WASH PROGRAM
STEP

TIME

FLOW

CURV

0
1
2

0
20
HALT

1.00
1.00
0

100
0.0
-

0.0
0.0
-

0.0
100
-

(b) No Timed Events need to be programmed.
6. Store this method using a number of your choosing (e.g. “19”).
7. To link the pump methods into a sequence, do the following:
(a) Press softkey F5 (“SEQ”) on the HPLC pump. Press F8 (“DELS”) to
clear any preexisting sequence. The following fields will be shown:
“SET”, “METHOD”, “FIRST”, “LAST” and “INJ”. The “METHOD” field will
be active highlighted field.
(b) Input “1” then press the “enter” key. The next highlighted field will be
“FIRST”; input “100” then press the “enter” key. The “LAST” field will
automatically change to 100. Press the “enter” key again. The last field
“INJ” will now be highlighted; input “5” then press the “enter” key.
(c) A second line for set 2 will automatically be created. Press the “enter”
key to advance to the “FIRST” field and input “1” followed by the “enter”
key. In the “LAST” field, enter the number of samples to be analyzed. If
you do not yet know this number, leave the default number alone (it can
be changed later). Advance to the “INJ” field by pressing the “enter” key
and input “1” followed by the “enter” key.
(d) A third line for set 3 will automatically be created. Change the
“METHOD” field to a value corresponding to the method number for the
“column wash” pump method (e.g., 19), then change the “FIRST” and
“LAST” fields to 99. Leave the “INJ” field set to “1”.
(e) Do not press the softkey F6 (“LINK”). This will be done later before the
start of a batch run.
8. The HPLC pump is now programmed.

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(2) Programming the HPLC Autosampler
1. On the Series 200 Autosampler, press the “quit” key. This brings up the
“READY” screen. Next, press function key F6 (“DIR”) to show the method
names directory. Press function key F4 (“RCL”) and input “0” followed by the
“enter” key. Answer the next prompt with “yes” and you will be returned to the
starting screen.
2. Press function key F2 (“METH”). A new screen presents a table with column
headers “First”, “Last”, “Volume”, “Replicates” and “Time”. The highlighted
data field is “First” and contains a value of “1”. Key in “100”. Complete the
entry by pressing the “enter” key. It will automatically advance the highlight to
the data field “Last” which already contains a value of “100”. Since this
method will make only make injections from position “100”, press “enter”. The
next highlighted field is “VOLUME”. Input a value that is 2.5X the size of the
autosampler’s injection loop. For instance, if the autosampler’s injection valve
has a 20 µL loop installed, input 50 for “VOLUME”. Enter 5 as the number of
“REPLICATES” so that five replicate injections will be made from the vial in
position 100. Leave the default value for “TIME” unchanged. Press the
function key F6 (“STOR”), key in “1” then “enter”. Respond to the next two
prompts with a “yes” then a “no”.
3. To program the second method, it is not necessary to exit the existing one.
Since the method just programmed was stored, you can edit the existing
method in memory and save it to a different location. Using the arrow keys on
the autosampler panel, highlight data field “FIRST” which will contain a value
of “100”. Input the starting vial position from which the autosampler is to make
its first injection, which is usually “1”. Press the “enter” key. Enter the number
for the last vial position for this sample set, and then press the “enter” key.
The highlighted field is “VOLUME” which is still the volume programmed for
Method 1. Press the “enter” key to advance to “REPLICATES”. Replace the
existing value by keying in 1. Press “enter”. Leave the default value for
“TIME” unchanged. Press the function key F6 (“STOR”). Key in “2” then
“enter”. Respond to the next two prompts with a “yes” then a “no”.
4. Program a third method that will be used in conjunction with the column wash
pump program. This method will do an injection for a blank or empty vial in
autosampler tray position 99. Set “First” and “Last” vial position fields to “99”.
Set the “Replicate” field to “1”. While the “Volume” field can be set to any
value, a value of “1” is preferred. Press the function key F6 (“STOR”), key in a
number of your choosing (e.g., “19”) then press “enter”. Respond to the next
two prompts with a “yes”. Input a name for this method (“i.e., “WASH”) then
press “enter”.
If necessary, additional methods may be created following the steps in this
section.
5. At the “READY” screen, press function key F5 (“SEQ”). Press F8 (“DELS”) to
delete previous sequences, if present. Three columns will be shown: “SET”,
“METHOD”, and “INJECTIONS”. The “METHOD” column will be highlighted;
key in “1” followed by the “enter” key. The table will automatically create and
jump to a line for set 2. Enter “2” then press “enter”. You will again be
prompted to enter a method number for set 3. Enter “3” then press “enter”.
You will be prompted to enter a fourth set, but that will not be necessary
unless more than three methods need to be linked together. You can scroll
the sets by using the up and down arrow keys. If you inadvertently created

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too many sets, position the “>” symbol to point at the unwanted set and press
the “delete” key. The “INJECTIONS” column, which shows the number of
injections programmed for each method, cannot be edited.
6. Link the methods together by pressing the F6 function key (“LINK”).
7. The HPLC autosampler is now programmed.

o. ICP-DRC-MS Instrument Setup
To improve workflow, complete the programming steps described in this section before the day of
analysis.

(1) Programming the DRC Gas Flow Delay Parameter
A special ELAN® DRC™ setting, called “Flow Delay”, needs to be changed from its default setting
to avoid the problem of the ELAN software forcing a time delay of several seconds before
collecting data at the start of a chromatographic run in DRC mode. This change only needs to be
done once per software installation or upgrade, or if the setting was deliberately changed by a
field service engineer. It is a good idea to inform the service engineer who intends to perform
work on the instrument of the importance of returning the “Flow Delay” to the non-default value of
1.

Important!
While in Service Mode, DO NOT make changes to any setting except for the one change
described below.

1. From within the ELAN program and in the window entitled “Instrument Control
Session”, choose menu item Options > Service Mode. You will be prompted
to enter a Service Mode password. Enter the password “Elan6000” (omit the
quotes and pay attention to capitalization) and click OK. If this password is
not accepted, you will have to contact a supervisor or a PerkinElmer service
technician.
2. You will be presented with a new tab called “Service” within the Instrument
window. Maximize the window. Click on “Gas” in the row of tabs at the
bottom of the window. Look for the parameter called “Flow Delay” (Gas
changes while in DRC Mode). If its setting is a value other than “1”, click on
the “Set Pauses…” button. Change the value in the field named “Flow
Change” to “1”. Click the “Apply” button then click the “Close” button. Choose
menu item Options > Exit Service Mode.

(2) Programming the ELAN “.mth” file
1. If it is not already open, launch the ELAN program and in the “Instrument
Control Session” window, choose menu item File > Review Files. Click the
“Load” button for “Method”, the first item on the list. Navigate to the folder
“C:\elandata\Method” and click on “As_HPLC-1_drc.mth” file† then click the
“Open” button.
2. Proceed to step three unless, the “As_HPLC-1_drc.mth” file cannot be found,
or it has been changed or corrupted in a manner that makes its use
†

Actual file names may differ from those presented throughout this document.

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questionable. If this is the case, cancel the open file dialog box and close the
Review Files window by clicking “Done”. Perform the following steps:
(a) Make the active method file the active window. Do this by clicking on the
tool bar icon that looks like a notepad with a “Cu” on it. Click File > New
on the menu bar and then choose “Data Only” in the New Method
window that appears. Click OK then maximize the window. Enter the
information in TABLE 9-6 into this window :
TABLE 9-6: ELAN® TIMING PARAMETERS
Parameter

Sweeps/Reading:
Readings/Replicate:
Number of Replicates:
Tuning File:
Optimization File:
Enable Short Settling Time:

Setting

1
1403
1
C:\elandata\Tuning\default.tun
C:\elandata\Optimization\as_hplc_drc.dac
Unchecked

(b) On the first line of the worksheet-like table, click in the cell of row 1 of the
“Analyte (*)” column. Type “As” then the enter key. The row will
suddenly be filled-in with arsenic’s “Begin Mass (amu)” of 74.92 (or
something close) and several default parameters. Tab from cell to cell to
fill in the information shown in TABLE 9-7.
TABLE 9-7: ELAN® ANALYTE PARAMETERS
Parameter

Analyte:
Begin Mass (amu):
End Mass:
Scan Mode:
MCA Channels:
Dwell Time:
Integration Time:

Setting

As
74.9216 (or something close will be automatically entered
by software)

Peak Hopping
1
488
(automatically determined by software)

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TABLE 9-8: ELAN® PROCESSING PARAMETERS
Parameter

Detector:
Measurement Unit:
Process Spectral Peak:
Process Signal Profile:
Apply Smoothing:
Factor:
Auto Lens:
Isotope Ratio Mode:

Setting

Pulse
Cps
Average
Average
Checked
5
Off
Off

(d) Skip the “Equation” tab. Click on the “Sampling” tab and enter the
following information:
TABLE 9-9: ELAN® SAMPLING PARAMETERS
Parameter

Peristaltic Pump Under
Computer Control:
Sampling:

Setting

Unchecked
External

(e) Click on the “Report” tab and enter the following information:
TABLE 9-10: ELAN® REPORT PARAMETERS
Parameter

Report View | Send to
Printer:
Report Options Template:
Automatically Generate
NetCDF File:
Report to File | Send to File:
Report Options Template:
Report File Name:
Report Format:
File Write Option

Setting

Unchecked
*
C:\elandata\reportoutput\
Unchecked
*
*
*
*

* Content of these fields is not important since Send To Printer/File is unchecked.

(f) Choose menu item File > Save As and navigate to
“C:\elandata\Methods\” folder. Enter “As_HPLC-1_drc.mth” as the name
of the method file and click the “Save” button.
3. The ELAN method “As_HPLC-1_drc.mth” is now loaded into memory.

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(3) Programming the ELAN “.dac” file
1. If it is not already open, launch the ELAN program and in the “Instrument
Control Session” window, choose menu item File > Review Files. Click the
“Load” button for “Optimization”, the sixth item on the list. Navigate to the
folder “C:\elandata\Optimize” and click on the “as_hplc_drc.dac” file then
“Open”.
2. If the “as_hplc_drc.dac” file cannot be found, or it has been changed or
corrupted in a manner that makes its use questionable, cancel the open file
dialog box and close the Review Files window by clicking the “Done” button.
Do the following steps; otherwise, proceed to step 3:
(a) Make the active method file the active window (do this by clicking on the
tool bar icon that looks like a peak with a red arrow on crest of it). Then
click File > Open on the menu bar navigate to the folder
“C:\elandata\optimize\”. Click on the most current “default.dac” file then
click the OK button. Complete the “Current Value” column with the
information in TABLE 9-11. Note: Values in TABLE 9-11 are suggested
starting values. Instruments vary in their optimal parameter values, and
analysts should use their discretion.
TABLE 9-11: ELAN® OPTIMIZATION PARAMETERS
Parameter

Nebulizer Gas Flow (NEB):
Auxiliary Gas Flow:
Plasma Gas Flow:
Lens Voltage:
ICP RF Power:
Analog Stage Voltage:
Pulse Stage Voltage:
Quadrupole Rod Offset Std
Cell Rode Offset Std
Discriminator Threshold

Setting

0.9*
1.2
15
7.5*
1450
- 1750*
1000*
0*
- 8*
70*

Parameter

Cell Path Voltage Std
Rpa
Rpq
Cell Gas A
Cell Gas B
DRC Mode NEB
DRC Mode QRO
DRC Mode CRO
DRC Mode CPV

Setting

- 16*
0
0.6
0.6*
0
0.9*
- 10.5*
- 2*
- 15*

*Suggested starting values only. Optimum parameters will depend on outcome of the optimization
procedure (see Error! Reference source not found.).

(b) Choose menu item File > Save As in the “ELAN Instrument Control
Session” window menu bar and navigate to “C:\elandata\Optimization\”
folder. Enter “as_hplc_drc.dac” as the name of the optimization file and
click the “Save” button.
3. The ELAN method “as_hplc_drc.dac” is now loaded into memory.

(4) Creating the ELAN Sample Table “.sam” file
1. If it is not already open, launch the ELAN program and in the “Instrument
Control Session” window, choose menu item File > Review Files. Click the
“New” button for “Dataset”, the second item on the list. Navigate to the folder
“C:\hplc\data\” and enter the file name “As” (where yy = last 2 digits
of current year, mm = month, and dd = date of run, for example, As110201

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denotes a Arsenic Speciation run on Feb 1, 2011) and click the “Open” button.
The new dataset folder has been created and is now active. Click on the
“DONE” button in >Review Files Window.
2. Click on the tool bar icon that looks like three Erlenmeyer flasks. Choose File
> New on the menu bar. A new window will appear entitled “Samples –
[Untitled]”. Click the “Batch” tab then click on the “Sample Template…”
button. A dialog box entitled “Sample Template Data” will appear. Enter the
following information:
TABLE 9-12: SAMPLE TEMPLATE DATA
Parameter

Sample ID Prefix:
Sample ID Number:
Sample ID Suffix:
Increment:
Autosampler Position – Number:
Autosampler Position – Increment:
Range – Start Row:
Range – End Row:

Setting

1
101
_
1
100
0
1
5

Click the “Generate” button.
3. Again, click on the “Sample Template…” button. The same dialog box entitled
“Sample Template Data” will appear. Enter this information:
TABLE 9-13: SAMPLE TEMPLATE DATA
Parameter

Sample ID Prefix:
Sample ID Number:
Sample ID Suffix:
Increment:
Autosampler Position – Number:
Autosampler Position – Increment:
Range – Start Row:
Range – End Row:

Setting

001
_
1
1
1
6
105

Click the “Generate” button.
4. Scroll the sample table to the right using the horizontal scroll bar until the
columns “Sample Flush” through “Wash Speed” are showing. Highlight the
cell in the first row of the “Sample Flush” column. Enter “0” then tab to the
next cell to the right, enter “0” again, tab again…i.e. enter “0” for all cells in the
first row of columns “Sample Flush” through “Wash Speed”. Next, click on the
column header for “Sample Flush” to select the entire column and drag right to
select all the columns right of and including the “Sample Flush” column. While
these six columns are highlighted (i.e. darkened), go to the menu bar and

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choose Edit > Fill Down. Zeros will fill down to replace every value with a zero.
Scroll left to the first leftmost cell and click on it to select it.
5. From the menu bar, choose File > Save As and save the file in the directory
“C:\hplc\data\” using the name “As.sam” (where yy = last 2 digits of
the current year, mm = month, and dd = date of run).
6. It is a good idea to save a copy of this file as a template, thereby avoiding the
need to re-create it every time.

p. HPLC–ICP-DRC-MS System Connection and Startup
(1) Interfacing the HPLC Column to the ICP-DRC-MS Nebulizer
1. Turn off the ICP-MS plasma if it is on.
2. Remove any non-HPLC tubing that may have been installed in the nebulizer.
3. Connect the HPLC column effluent tubing (coming from port #3 of switching
valve #1) to the ICP-DRC-MS nebulizer/spray chamber assembly as shown
below. Note that this tubing is the “Peek Sample Uptake Fitting (Analytical
West item name "500-QD-PEEK") or equivalent”.

4. The PEEK Sample Uptake Fitting is a prefabricated piece of yellow PEEK
tubing attached to a white nebulizer-connector piece. When inserting the
connector into the nebulizer, ensure that it is pushed in as far as it will go and
is secure.

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Important!
Inspect the tubing-nebulizer interface. I t is important that there is no gap between the end of the
HPLC tubing and the portion of the nebulizer where it abruptly narrows to a capillary tube. Small
gaps can contribute significantly to chromatographic peak broadening and tailing.

(2) Priming the HPLC Pump
1. Turn on the HPLC Series 200 Vacuum Degasser (switch is on back of unit).
2. If it has not already been done, place each mobile phase reservoir tubing into
the correct reservoir bottles, i.e. place end of tubing “A” into the bottle
containing HPLC Buffer A, and end of tubing “B” into the bottle containing
HPLC Buffer B. Reservoir tubing “D” and “E” are placed into bottles
containing 5% acetonitrile in water. Press the “rinse” key on the autosampler
to prime and rinse the autosampler fluid path.
3. On the HPLC pump, open the door that accesses the pump head. Attach a 30
mL or larger plastic syringe to the purge port. Open purge port by turning its
knob counterclockwise ~¼ to ½ turn.

Important!
Be sure the HPLC pump’s purge port is open and that a syringe is attached before
completing the next step. While there is no specific danger to the analyst, an over-pressure
situation can occur that, under certain circumstances, could damage the HPLC pump, column or
other components.
4. At the HPLC pump control panel, press the “Purge” button. Press F4 (“%A”)
followed by F3 (“FLOW”). Key in 10 then press the “enter” key. The pump will
immediately start and quickly ramp up to a flow rate of 10 mL/min.
5. Allow the pump to fill the syringe with about 15 mL of buffer A. During this
time, watch the reservoir tubing for air bubbles which should be flushed out. If

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there are “stuck” bubbles adhering to the inside wall the tubing, strike the
tubing sharply with several firm snaps from your finger to jar and free the
bubble(s). The tubing should be bubble-free after ~10 mL of buffer has been
pumped through.
6. Press F5 (“%B”). The pump will now switch to reservoir B. Again, watch for
bubbles and make sure they are flushed out of the tubing. Allow another 15
mL of buffer to be pumped or until the syringe is almost full, then press the
“stop” key to stop flow.
7. Close the purge port (turn knob clockwise), remove syringe, and close door.
Dispose of syringe contents to waste or in a sink.

(3) Adjusting the External Peristaltic Pump
1. Check the external peristaltic pump’s tubing for signs of wear which will be
evident by flattening of the tubing and pinch-roller marks. Excessively worn
tubing should be replaced.
2. If necessary, install new large diameter (“white-black”) peristaltic tubing on the
bottom channel of the external peristaltic pump. Connect the left end of the
“white-black” to the tygon “waste line” that leads to the large liquid waste
carboy jug. Connect the right end of the “white-black” to the tubing that
empties the ICP-DRC-MS’s spray chamber. Close the bottom channel clamp.
Do a preliminary tightening of the peristaltic pump bottom channel’s tension
clamps on the “white-black” pump tubing. Later, when you are able to observe
liquid actively draining from the spray chamber, you will make further
adjustments to the tension clamps so that the spray chamber will properly
drain without applying excessive pressure on the tubing. Close the remaining
clamps of the other channels except the top channel.
3. In the peristaltic pump’s top channel, install new small diameter “black-black”
peristaltic tubing on the top channel and close its clamp. Note that the
peristaltic pump will rotate counterclockwise. Into the right end of the “blackblack” peristaltic tubing, insert the free end of the tubing that will draw Internal
Standard solution (i.e. the one that will come from the Internal Standard
bottle). Into the left end of the “black-black” peristaltic tubing, insert the tubing
that will carry Internal Standard to Port #6 of switching valve #1 (see TABLE
8-1-A).

q. ICP-DRC-MS Warm Up and Performance Check
1. Perform a pre-ignition check of the ICP-DRC-MS according to PE
recommendations specified in the manual.
2. Ensure that the digit “1” is displayed on switching valve #1. If it is not, press
the “local/remote” button until a yellow light indicates local mode. Then,
toggle the arrows to display a value of “1” in the digital window. Additionally,
ensure that a value of “1” is in the display on switching valve #2.
3. Launch the ELAN® ICP-DRC-MS program and note whether all graphical
indicators of instrument readiness are green. If not, take the appropriate
actions described in the instrument’s software and hardware manual.
4. Perform necessary daily maintenance checks as described in Chapter 5 of the
ELAN® 6100 Hardware Guide (e.g., argon supply, interface components,
cleanliness, positioning, and interface pump oil condition). Note the base

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vacuum pressure in the INSTRUMENT window of the software. (Before
igniting the plasma, the vacuum is typically between 8 x 10 -7 and 1.8 x 10 -6
torr). Keep a record any maintenance procedures along with the base
vacuum pressure in the Daily Maintenance Checklist logbook.
5. Start the peristaltic pump by pressing the appropriate arrow on the peristaltic
pump control panel. Press either the up or down arrow keys to adjust the
peristaltic pump speed to “6”. Ensure that the direction of rotation is correct so
that the spray chamber is being drained and that waste liquid will go to the
waste carboy jug.
6. In the INSTRUMENT window of the ELAN software, click the “Front Panel” tab
and click the plasma “Start” button to ignite the plasma. In the same window,
the ignition sequence bar (blue progress bar) will start to expand to the right,
indicating the approximate time before plasma ignition. Before the bar
reaches its end, look at the spray chamber on the ICP-DRC-MS and watch for
plasma ignition. Proper ignition will occur suddenly and with a single audible
“pop”. A bright white light will emanate from the injector assembly that
connects to the spray chamber. The light may at first flicker, but it should
establish a more or less steady intensity after 5–10 seconds.
On a rare occasion, the plasma may ignite emitting an orange, violently flickering light, and
electrical discharge noises will be heard. In this case, immediately shut off the plasma by
pressing the yellow “Stop” button on the ICP-DRC-MS instrument’s front control panel. Wait
30 seconds then investigate the cause of the plasma misfire. A more common occurrence is that
the plasma may extinguish itself a few seconds after ignition. Promptly reignite by pressing the
“Start” button on the ICP-DRC-MS instrument’s front control panel. Usually, the plasma will stay
lit after the second try. If not, investigate the cause of this instability (refer to the ELAN DRC II
Hardware Guide).
7. Soon after the plasma ignites, place the sample probe (the one connected to
the peristaltic pump’s “black-black” tubing, PerkinElmer P/N B300-0161,
normally used for the ELAN® autosampler) into 5% nitric acid rinse solution or
the daily performance check solution. Set the speed on the external peristaltic
pump to “20”. Watch the tubing that drains the spray chamber for a half
minute or so. If the tubing is filling with liquid and you do not see bubbles
being carried away from the spray chamber drain (and especially if you see
liquid starting to rise within the spray chamber) immediately remove the
sample probe from the rinse solution. Check that the peristaltic pump is
rotating in the proper direction so that the spray chamber is draining. If not,
immediately correct the direction of rotation on the peristaltic pump. Next,
tighten the thumb screw on the bottom tension clamp of the peristaltic pump
about ¼ turn. Examine smoothness of flow of liquid draining from the spray
chamber. If there is no liquid flow or if it continually “starts and stops”, tighten
the thumb screw again. Keep tightening the thumb screw until large bubbles
flow through the drain line at a consistent pace. Now, slowly loosen the
thumb screw until the flow stops or becomes hesitant. Make one final
adjustment by tightening the thumb screw ½ turn. At this point, the tension on
the peristaltic pump tubing should be correct. Re-insert the sample probe into
the rinse solution.
8. Repeat the preceding steps for adjusting the tension clamp for the “blackblack” tubing in the top channel.
9. Let the ICP-DRC-MS warm up for 30-45 minutes.

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10. The following step is for the initial method setup only:
(a) While the instrument is warming up, in the ELAN program window
entitled “Instrument Control Session”, choose menu item File > Review
Files. Click the “Load” button for “Optimization”, the sixth item on the list.
Navigate to the folder “C:\elandata\Optimize” and click on
“as_hplc_drc.dac” file then click the “Open” button. Return to File >
Review Files and click the “Load” button for “Method”, the first item on
the list. Navigate to the folder “C:\elandata\Method” and click on “Daily
Performance.mth” file then click the “Open” button. Add a new line for
arsenic “As” in the Quantitative Analysis Method window. Set the Dwell
Time to 50. Do a File > Save and save the edited method as
“As_HPLC_daily.mth”. Click on the Sampling tab and uncheck
“Peristaltic Pump under Computer Control”. Return to the Timing tab.
11. After warm-up, complete the appropriate daily optimization procedures as
described in Chapter 3 of the ELAN® 6100 DRC Software Guide. Include
beryllium (m/z 9) in the mass calibration, and be sure to use mass calibration
solution containing 1 g/L beryllium. Do the autolens optimization and daily
performance check by using a 1 g/L multielement solution that includes 1
g/L of arsenic. Instrument response for 1 g/L arsenic should give counts
>2000 cps (in Standard Mode). Fill in the Daily Maintenance Checklist in the
instrument logbook according to the completed optimization procedures. If a
tuning (mass-calibration) procedure was done, save it to the file “default.tun,”
and also in a separate file containing the analysis date “default_MMDDYY.tun”
(where MM=month, DD=day, and YY=year). Save the new optimization
parameters (i.e., detector voltages, autolens values and nebulizer gas flow
rate) to the file “As_HPLC_std.dac”. Save it again to another new file named
“default_” (where yy=year, mm=month, and dd=day; do not
include the brackets in the file name).
If an HPLC analysis is to be run the same day, you may leave the plasma on
until it is time to convert the nebulizer to interface with the HPLC. If not, press
“Stop” on the ELAN control panel to turn off the plasma.

r. Turning on the Reaction Cell Gas
1. Start the flow of the reaction-cell gas (10% hydrogen, 90% argon) and allow
the cell conditions to equilibrate. Make sure the regulator on the reaction-cell
gas cylinder is set to approximately 7 psi.
2. Click on the “Manual Adjust” tab of the “Optimization” window and enter a
value of “0” in the appropriate cell-gas field (cell-gas A or B, depending on
how the instrument is set up). Then enter a value of 0.6* in the same field. A
clicking should be heard from the ICP-DRC-MS cell-gas solenoid as the flow
turns on.
3. Monitor the flow on the mass-flow controller by clicking on the “Diagnostics”
tab of the INSTRUMENT window of the ELAN program and look for a field
labeled “Cell Gas A”. The flow should reach approximately “0.6*” within 10–15
seconds.
4. Flush the cell gas for 30 seconds by lifting the flush level at the front of the
instrument. (The flush step may not be necessary if this same gas cell was
used recently and no gas tubing has since been disconnected.) If possible,

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allow 30 minutes for the cell to equilibrate before beginning analysis, with the
cell gas flowing at 0.6* mL/min. Note: The cell gas will automatically turn off
after 45 minutes if the analysis has not begun.
*Or the DRC gas value that is found to be optimal
5. Once the cell gas has warmed up, perform a DRC neb gas optimization and a
lens voltage optimization. Update the values if needed. Perform a DRC
Mode Daily Optimization Check and record the results in the Daily
Maintenance Checklist.
6. After the analysis of the DRC mode Daily performance check is complete and
deemed satisfactory, change the selections on the 6-port switching valves #1
and #2 to match the values described below:
(a) Switching Valve #1: Remote, “1”
(b) Switching Valve #2: Local, “2”
7. Place the free end of the tubing that will carry the internal standard into a
bottle containing 1 liter of the Internal Standard solution.

s. Entering Sample Names into the ELAN Sample Table
1. Click on the tool bar icon that looks like three Erlenmeyer flasks. If the current
Samples window is not this run’s sample file, then choose File > Open on the
menu bar and navigate to and open this run’s current data folder in
“C:\hplc\data\”. Click on the file named “As.sam” (yy = year, mm =
digit month, dd = date) and open it. The Samples window will be the one
created in the Creating the ELAN Sample Table “.sam” file section.
2. Fill in the name of each sample by double-clicking after the “_” (underscore) in
the cell matching its “A/S Loc”. Type in the sample name and press “Enter” on
the keyboard. In this manner, enter the name of every blank, calibrator, quality
control, and sample that will analyzed in the run. If barcodes are used on the
sample labels, use the barcode scanner attached to the ICP-DRC-MS
computer to scan the sample ID from the barcode on each sample before
placing it into position in HPLC autosampler tray.
3. Keep the following in mind while filling out the Samples table.






Autosampler tray position 100 will contain the vial containing excess low or
high QC sample, called “EQ”, which will be injected with five replicates during
the initial system equilibration period that occurs before the start of
calibration. “EQ” is not used for QC but is strictly for equilibrating the HPLC
and conditioning the ICP-DRC-MS.
Autosampler tray position 99 needs to contain a blank vial (even an empty
vial will do).
Insert a “Bk” between the equilibrators. Also insert blank checks throughout
the run as needed to show that carryover is not occurring.
No more than 24 hours should lapse between the time that the actual
analytical run starts (the analysis of the “Bk” in austoampler location 1) and
the analysis of the last vial is complete. Keep this in mind when determining
how many samples will be analyzed.

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TABLE 9-14: ELAN SAMPLES TABLE
A/S
Loc.

100
100
100
100
100
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
…
36
37
38

Batch ID

Sample ID

Measurement
Action

101_EQ
Run Sample
102_EQ
Run Sample
103_EQ
Run Sample
104_EQ
Run Sample
105_EQ
Run Sample
001_Bk
Run Sample
002_S0
Run Sample
003_S1
Run Sample
004_S2
Run Sample
005_S3
Run Sample
006_S4
Run Sample
007_T0
Run Sample
008_T1
Run Sample
009_T2
Run Sample
010_T3
Run Sample
011_T4
Run Sample
012_Bk check
Run Sample
013_LU-XXXX
Run Sample
014_HU-XXXX
Run Sample
015_Bk check
Run Sample
…rows for 20 samples omitted for brevity
036_Bk check
Run Sample
037_LU-XXXX
Run Sample
038_HU-XXXX
Run Sample

Method

…

Wash Speed (+/- rpm)

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
…

…
0
0
0

The sample names will resemble those typed in bold in the example table above. In
the example table above, a run of 20 samples is shown so the last vial ends up being
placed in A/S Location #38. Of course, the actual position of the last sample depends
on the total number of vials in the autosampler tray. Note that, if more than one
group of samples is to be analyzed, each group shall be bracketed by its own QC. In
some instances, on the sample table this rule will result in four QC samples being run
in succession (for instance, LU-xxxx, HU-xxxx, LU-xxxx, HU-xxxx). Be sure to delete
all unused rows after the last vial in the ELAN Samples window, i.e. clear all rows
after the last row by selecting them and press Ctrl-Delete.
The numbers preceding the underscore character (with the exception of sample
numbers “101” through “105”) correspond to the order of injection. These numbers
will later help the analyst find individual chromatograms based on injection number
rather than being forced to scroll long lists of alphabetically-sorted file names in
Windows Open File dialog boxes looking for specific sample names during post-run
data processing in TotalChrom™.

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4. When satisfied that the Sample table entries are correct, choose File > Save.
5. Analysts may print the ELAN Sample table by choosing the File > Print Setup
> Reports command. In the ensuing dialog box, select the preferred printer
and click OK. Next, choose File > Print and then click the Print button.
Printouts may be helpful for the correct vial positions when loading samples
into the HPLC autosampler tray.

t. Starting the Run
1. Restart the ELAN controller computer by going to the Windows Start button
and choosing “Restart”. This serves to purge the computer of possible
memory/register conflicts and will give the system and ELAN software a fresh
start. It is unnecessary to shutoff the plasma as the ELAN instrument will
sustain it while the computer does a restart.
2. Check the waste carboy. If more than two-thirds full, empty it.
3. Check that the tubing that draws internal standard is inserted into the bottle
containing Internal Standard and that there is sufficient quantity of Internal
Standard.
4. Check that there is sufficient mobile phase to last the entire run. In addition,
be sure that Bottle D and the HPLC Autosampler’s wash bottle contains
sufficient amount of 5% (v/v) acetonitrile. It is very important that line D does
not become filled with air bubbles at any point during analysis.
5. Set the HPLC Series 200 Column Oven to 35°C if it is not already at that
temperature.
6. Check for stray “.nc” files by using Microsoft Windows® File Explorer to look
inside the C:\elandata\ReportOutput folder. Move any existing files that end
with “.nc” extension to another folder so that the C:\elandata\ReportOutput
folder is empty of “.nc” files. Close Windows File Explorer.
7. Launch ELAN Instrument Control program if it is not already. Do not launch or
start any other programs at this time.
8. Check that the correct Sample file in the window “Instrument Control Session”
is active. If it is not correct, load the correct Sample file. In this window, note
the injection number of the last vial as indicated by the numbered prefix
leading the first underscore (“_”) character (e.g., 42 from “042_samplename”).
9. Check that the HPLC pump methods are correctly programmed according to
Programming the HPLC Pump Methods. On the HPLC pump, press the
softkey F5 (“SEQ”). Confirm that there are just three lines indicating sets 1, 2
and 3. Check that sets 1 and 3 are configured properly. Press softkey “SET”,
input 2 then press the “enter” key. Press softkey F4 (“LAST”) and input the
injection number of the last vial noted in the preceding step. Press the “enter”
key. Press softkey F6 (“LINK”) then press the “return” key. The display will
return to the top level. Confirm that the top line in the pump control panel
display displays “METHOD01 STORD SHTDN Q01.100.00” indicating that the
pump methods are now linked. Do not press the softkey F8 (“STRT”) at this
time.
10. Check that the HPLC autosampler methods are correctly programmed
according to Programming the HPLC Autosampler. On the HPLC
autosampler, press the softkey F6 (“DIR”), followed by softkey F4 (“RCL”).

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Input 2 then press “enter” followed by “yes”. Next, press F2 (“METH”). A new
screen presents a table with column headers “First”, “Last”, “Volume”,
“Replicates” and “Time”. Using the arrow keys on the autosampler panel,
highlight data field “LAST” and input the injection number of the last vial noted
in the preceding two steps. Press the “enter” key. Next, press F6 (“STOR”),
input 2 then press the “enter” key. Respond to the next two prompts with a
“yes” then a “no”. Press the “return” key. If the word “LINKED” does not
appear on the autosampler display, then link the methods together by
pressing the F6 function key (“LINK”).
11. Check that the correct ELAN method is loaded and active in the window
“Instrument Control Session”. If it is not correct, load the correct Method file.
Check under the Sampling tab that “Peristaltic pump under computer control”
is unchecked, and the pull-down menu “Sampling” indicates “External”.
12. Check that the DRC gas is indeed flowing by making the ELAN’s Instrument
window active and clicking on the Diagnostics tab. Inspect the Cell Gas A or
B, its value should be fluctuating at 0.6 (or other optimal value) ± 0.01 mL/min.
If it is not, see section Turning on the Reaction Cell Gas for details to turn on
the DRC gas flow. Make the optimization window active and Choose File >
Save to save the method file.
13. Choose File > Review Files in the “Instrument Control Session” menu bar. In
the next window, click the “Load” button for “Dataset” (second item) and
navigate to this run’s data folder, double-click on it and click on the “OK”
button. The correct Dataset path should now be indicated. Click the “Done”
button.
14. Check that all blanks, calibrators, QC and sample vials are loaded into their
correct positions in the HPLC autosampler tray, as designated by the ELAN
Sample window (or its printout).
15. Press function key F5 (“SEQ”) on the Autosampler and check that the total
number of injections (i.e., the sum of all injections for each listed method)
agrees with the number of vials + 4 (accounting for the extra 4 injections of
the equilibrator vial in position #100) in the autosampler tray. Press the
“return” key to get back to the main screen. See section Programming the
HPLC Autosampler for details on how to program the autosampler. Check
that the HPLC autosampler’s methods are linked and that the word “LINKED”
appears in the autosampler’s information screen.
16. This step is optional but offers the advantage that the ELAN data files will be
converted in real time to TotalChom™ “.raw” files that have names containing
a date-time stamp corresponding to actual time of injection.
(a) Launch TotalChrom™ Navigator. In the resulting TotalChrom™
Navigator window, choose menu item Apps > ChromLink (alternatively,
you may launch ChromLink™ from the operating system Start >
Programs menu).
(b) In the ChromLink™ program window, choose the menu item
Configuration > Mass Details and check the Nominal Name and Mass for
arsenic. If it is missing or the ELAN tune (“default.tun”) file was reoptimized earlier then ChromLink™ needs to be configured (see
Configuration of ELAN ChromLink™ on page 55 for details). To save
time, the analyst may choose to close the TotalChom™ Navigator and
ChromLink™ windows and skip step 16 in its entirety. Data file

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conversion via ChromLink™ can easily be done during post-run data
reprocessing.
(c) In the ChromLink program window, click on the “Browse” button to the
right of the “ELAN ChromLink file location” field. Navigate to the current
working folder, double-click on it then click the “OK” button so that
ChromLink knows where to save its processed files.
(d) Otherwise, refer to step (b) of Data Processing and Analysis for details
on proper setting of the ELAN ChromLink™ window’s parameter fields.
In the ELAN ChromLink window, click the button “Start Processing ELAN
Data Files” to put ChromLink in watch mode so it will process each data
for each injection in real time. A new dialog box will open and indicate it
is ready to convert data and waiting for the first file.
17. Click on the ELAN “Instrument Control Session” window to make it active,
then click the mouse in the Samples window on the corner rectangle of the
sample table (where row headers intersect column names). The entire
sample table will become highlighted (dark background). Click the “Analyze
Batch” button. A Run Progress box will appear indicating that the ELAN
software is now waiting for a signal from the HPLC that indicates the
occurrence of an injection.
18. If the HPLC pump is not already pumping, press function key F8 (“STRT”).
This will start the flow of Buffer A and put the pump into a “wait for injection”
mode.
19. On the HPLC autosampler, press the “start” key. If the equilibration wait time
has been reached, the autosampler will immediately begin its injection
sequence. Otherwise, it will respond with the message ‘WAITING FOR
EXTERNAL READY” and wait for the equilibration wait time to complete.
20. In the Instrument window, click the “Auto Start/Stop” tab. It is important to note
that if the “Enable” radio button is already selected and an ELAN run was
cancelled by the analyst, you will need to select the “Disable” radio button to
reset the Auto Stop timer. Forgetting to do this will result in premature shutoff
of the plasma. Following this action, click the “Enable” radio button. Next, click
on the “Change” button and set the “Delayed Shutdown Time” to 30 minutes.
Click Okay.
21. Open the ELAN “Instrument Control Session” Real-Time window by clicking
the tool bar button that looks like a Gaussian distribution (or a blue
chromatographic peak, if you prefer). After the Real-Time window opens,
click on the drop-down menu and select “Signal”. Real-time data will now be
displayed.
When the HPLC pump’s equilibration time has been reached, the autosampler will
seek the first vial and make an injection. A blue bar in the ELAN’s progress box will
now indicate that data is being collected. The system can now run unattended.
Check the progress of the run after 2 or 3 injections. Note the chromatograms
appearing in the ELAN’s Real Time window. Adjust the signal scale in the Real Time
window, as necessary. Compare the positions and peak heights of each arsenic
species relative to the internal standard. It helps to visually compare it to a printed
reference chromatogram. If abnormalities in retention time, peak height or shape are
readily apparent, the analyst may need to stop the autosampler and pump and abort

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the run in the ELAN program. The HPLC pump and autosampler are stopped by
pressing the “stop” buttons on their respective control panels. Correct the problem(s)
and restart the run.

Important
Remember to disable the ELAN’s Auto Stop feature before re-enabling it. Otherwise, the ELAN
may perform an auto shutoff prematurely.

u. Instrument Shut Down
1. Shut off ICP-DRC-MS plasma if it has not already been done. Stop all
peristaltic pumps and loosen tensioning bars and tubing.
2. Check that the HPLC autosampler controller readout indicates that the
sequence was successfully completed. If not, note the message and
investigate the reason for the message, for example, if a sample vial is
missing.
3. At the controller computer, visit the ELAN Instrument Control Session
application and open the “Dataset” window. Confirm that all samples were
analyzed.
4. Remove the calibrator, QC and sample vials from the HPLC tray. Discard
them according to CDC biohazard waste disposal guidelines.

10. POST-RUN DATA ANALYSIS
a. Configuration of TotalChrom™ Integration Method
The following information is presented as a starting point to help the analyst develop
robust integration method parameters that will work best for most chromatography
data. Many of these parameters will work just fine as presented below. However,
the separation chemistry of HPLC columns can vary due to frequency of use, column
replacement, or because of individual sample “oddities”. Some parameters may
need to be adjusted from time to time to maximize the ability of TotalChom™ to
properly integrate peaks and identify components with minimum operator
intervention. Therefore, the analyst should pay particular attention to the
chromatograms produced in every run and make necessary adjustments as
warranted. The analyst should be familiar with TotalChrom™ ‘s frequently used
integration functions which are described in Chapter 18 of TotalChrom Workstation
User’s Guide: Volume II.
1. The creation of a new method file in TotalChrom™ is done the first time
TotalChrom™ is setup, or it will need to be recreated if the file “Arsenic1.mth”
cannot be found or has been corrupted. In the TotalChrom™ Navigator
window, choose the menu item Build > Method. In the next dialog box, click
the “Create a new method” radio button and click OK. The default method will
load into the method editor.

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2. Choose the menu item Process > Integration. Click on the “Integration” tab in
the “Process” window. Enter the information shown in TABLE 10-1.

TABLE 10-1: INTEGRATION
Basic Parameters

Advanced Parameters

Bunching Factor :

Peak Separation Criteria

Noise Threshold :

Width ratio :

0.2

Area Threshold :

Valley to peak ratio :

0.01

Exponential Skim Criteria
Peak height ratio :

Adjusted height ratio :

Valley height ratio :

The analyst may make appropriate changes to one or more of the Integration
parameters in TABLE 10-1 if necessary.
Click on the “Baseline Timed Events” tab. As a guideline, enter the information shown
in TABLE 10-2 or other parameters as determined to be appropriate.
TABLE 10-2: BASELINE TIMED EVENTS
Defined Events
Time

Event

Value

Code

0.000

Smooth Peak Ends On

+SM

0.000

Locate Maximum On

0.000

Set Bunching Factor

0.000

Disable Peak Detection

LM
3

BF
-P

0.350

Enable Peak Detection

0.500

End Peak Detection Inhibit

1.000

End Peak Detection Enable

-I

1.000

Locate Maximum Off

1.000

Set Bunching Factor

1.200

Common Baseline On

2.200

Set Bunching Factor

2.250

Common Baseline Off

6.250
6.250
8.750
10.80

Set Noise Threshold
Set Area Threshold
Peak End Detection Inhibit
Peak End Detection Enable

-LM
1

+CB
- CB
3
15

NT
AT
+I
-I

Level

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Be sure there is no checkmark in the box for “Correct actual times of all baseline
events based on actual RT of nearest reference peak”. The parameters in TABLE
10-2 are starting points. The analyst may make appropriate changes to one or more
of the Baseline Timed Events if necessary.
3. Click on the “Optional Reports” tab. Uncheck the box for “Keep temporary
files”.
4. Click on the “Replot” tab. Enter the information shown in TABLE 10-3.

TABLE 10-3: REPLOT
Plots

Miscellaneous

Generate a separate replot :

not checked

Start plot at end of delay :

Retention Labels :

Peak crests*

Gradient overlay :

Actual time

Draw baselines :

checked*

Timed Events :

checked*

Component Labels :
Scaling Type :

Autozero offset

Scaling Parameters
Full scale (mV) :

2.000*

checked
not checked*

X axis label :

Time [min]

Y axis label :

Intensity [cps]

*These parameters maybe altered to suit the analyst.

It is unnecessary to click on the “User Programs” tab because it is not used. Close
the Process window by clicking on the “OK” button. The parameters in TABLE 10-3
are starting points. The analyst may make appropriate changes to one or more of the
Replot parameters if necessary. In the Method Editor window, choose the menu item
Components > Global Information. Click on the “Integration” tab in the “Process”
window. Enter the information shown in TABLE 10-4:

TABLE 10-4: Global Information
Volume units :
Quantitation units :

µL
µg/L

Unidentified Peak Quant.
Calibration factor :

1.000e+99

Sample Volume :

1.000

Always use calib. Factor :

selected

Void time (min) :

0.000

RRT Calculation
Use first peak in run as RRT
reference:

selected

Calibration
Internal Standard
Reject outliers during
calibration :
Sample Amount Options
Correct amounts for calibration
standards :
Convert unknown samples to
concentration units:

selected

Not checked

Not checked
Not checked

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The “LIMS Results” tab is not used. Click the “OK” button to close the window. The
parameters in TABLE 10-4 are starting points. The analyst may make appropriate
changes to one or more of the Global Information parameters if necessary.
6. In the Method Editor window, choose the menu item Components > New
Component. The white list box in the left portion of the window will be empty.
Click in the empty field labeled “Name” and type “IS”. Press the tab key and
enter “0.6” in the field labeled “Retention time”. Put a checkmark in the box
labeled “This component is an internal standard”. Select the radio button
labeled “Peak” if it is not already selected. Leave the other fields and check
boxes unaltered. Click the “New Component” button. Enter each of the
component names and parameters listed in TABLE 10-7.

Relative window

Find tallest peak

Is a retention
reference?

1.70

Use as a RRT
reference?

Absolute window

0.6

This Component is
an Int. Std?

Retention Time

Yes

No
No

Internal Standard

Name

TABLE 10-5: METHOD EDITOR – COMPONENTS SETTINGS

2.00

TMAO

2.10

AsIII

2.50

DMA

4.00

MMA

9.70

AsV

11.90

Click the “New Component” button before starting a new component. After
entering the last component, click the “OK” button. The values for Retention
Time, Absolute Window and Relative Window serve as starting points. The
analyst may alter these values as actual chromatographic results may
dictate.
7. In the Method Editor window, choose the menu item Components > Defaults.
Click on the “Identification” tab”. Enter the information shown in TABLE 10-6.
8. Click on the “Calibration” tab in Components Defaults Window. Enter the
information shown in TABLE 10-7.

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TABLE 10-6: COMPONENTS DEFAULTS — IDENTIFICATION
Component Type :

Peak

Reference :

Absolute window :

Internal Standard :

Relative window :

Find tallest peak :

blank
IS
Not checked

TABLE 10-7: COMPONENTS DEFAULTS — CALIBRATION
Calibration Type :
Curve Type :
Scaling :
Weighing :
Purity (%) :
Response :
Origin Treatment – Include :
Origin Treatment – Force :

Use Curve
1st Order
None
None
100
Area
Not checked
Not checked

Level
S0
S1
S2
S3
S4

Amount
1.0000E-6
2
10
50
150

9. The “User Values/LIMS” tab is not used. Close the “Components Defaults”
window by clicking the “OK” button.
10. In the Method Editor window, click on “Components” in the menu bar. If the
menu item “Delete All Components” is not grayed out, select it and click the
“OK” button when prompted to “Delete all components, calibration levels, and
calibration replicates”. Click the “OK” button.
11. In the Method Editor window, choose the menu item Components > New
Component. The white list box in the left portion of the window will be empty.
Click in the empty field labeled “Name” and type “IS”. Press the tab key and
enter “0.6” in the field labeled “Retention time”. Put a checkmark in the box
labeled “This component is an internal standard”. Select the radio button
labeled “Peak” if it is not already selected. Leave the other fields and check
boxes unaltered. Click the “New Component” button. Enter each of the
component names and parameters listed in TABLE 10-7.
12. In the Method Editor window, choose the menu item Components > Edit
Component then click the “Calibration” tab. Click to highlight “TMAO” in the
component list box. Click on the cell containing “S0” and type “T0” (capital
“T”) on the keyboard followed by the Enter key. In this manner, replace “S0”
through “S4” with “T0” through “T4”, respectively. Click the “OK” button when
finished.

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13. In the Method Editor window, choose the menu item File > Description, and
type in your name and date this method was created. Add any other pertinent
information at this time. Click the OK button to close window.
14. In the Method Editor window, Choose File > Save As. A window appears
inviting you to enter any information pertinent to this method, which will be
saved with the method. Enter your name and the date this method was
created. Click “OK” and a “TotalChrom™ File-Save-As” dialog box will open.
Navigate the directory tree to get to the folder C:\HPLC\Methods. Doubleclick on this folder. In the “File name:” field, enter “Arsenic1.mth”. If there is
already a file in that folder with the same name, highlight that file and rightclick the mouse. Choose “Rename” and give the file a new name (e.g. add
“backup” to the name). Click “Save’ and close the Method Editor window.

b. Configuration of ELAN ChromLink™
ELAN ChromLink™ should be configured after initial installation of the program or
when the ELAN tune (“default.tun”) file is re-optimized. At least one recent ELAN
NetCDF file (with the “.nc” extension) containing data for the mass of interest that
was collected since the last update of the “default.tun” file will need to be available in
order to complete this step.
1. Launch TotalChrom™ Navigator. In the TotalChrom™ Navigator window that
appears, choose the menu item Apps > ChromLink (alternatively, you may
launch ChromLink™ from the operating system Start > Programs menu).
(a) Inside the ELAN ChromLink window, click on Configuration > Default
TotalChrom Method. Click on the “Browse…” button and navigate to the
directory C:\hplc\methods\. Select “Arsenic1.mth” and click the “Open”
button. “C:\hplc\methods\arsenic1.mth” will now be the ChromLink™
default method. Click “OK” to close the “Default TotalChrom Method”
window.
(b) Inside the ELAN ChromLink window, click on “Set”. The “Operating
Mode” window will open. Click on the “Manual – process single ELAN
NetCDF file” radio button then click the “Review ELAN NetCDF mass file
components before processing” radio button. Click “OK” to close the
window. Click on the “Browse…” button for “ELAN NetCDF file –
location/file to be converted” field. An open file dialog box will open,
prompting you to choose a file. Navigate to the latest working HPLC
Data folder and choose any file with the “.nc” extension (perhaps one of
the calibrators). Click “Open”. The dialog box will disappear and you will
be returned to the “ELAN ChromLink” window. The path and file to be
converted will now be shown in the field called “ELAN ChromLink file –
location/file to be converted”.
(c) Click on the “Start Processing ELAN Data Files” button. The “Processing
ELAN Data” window will briefly open, followed by a window called “Mass
Components in ELAN Data File”. The file name being processed will
appear in the ELAN File Contents panel along with its “Mass” and
“Nominal Name”. Write down the mass value. The next panel called
Configured Mass Components will show the mass and nominal name of
the mass components that will be identified from the configured list and

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be processed as separate TotalChrom RAW files. If the fields for “Mass”
and “Nominal Name” are empty, or are different compared to the
corresponding fields for Configured Mass Components, then click the
“Edit Configured Mass Components” button. A new window called “List
of Mass Components” will open. Click on the cell in the table at the top
of the window containing the mass that you wrote down earlier. Next,
Click on the field named “Nominal Names” in the “Names” panel and
enter “As”. Be sure the field named “ELAN Name (mass)” contains the
mass value that you wrote down earlier. Leave the field “Expression”
unchanged. Next, click on the “Browse…” button and navigate to
“C:\hplc\methods\arsenic1.mth” then click “OK”. Put a checkmark in the
box for “Process this mass component to produce its own TotalChrom
RAW file”. Make sure that the box for “Process this mass component as
part of an expression” is unchecked. Now, click the “Update Selected
Mass Component” button. One line should now show the following
information:
Nom.
Name

Mass

74.92†

†

Expression

raw?

In
expr?

TotalChrom Method File

C:\HPLC\Methods\Arsenic1.mth

Or some value close to the atomic weight of arsenic.

If not, repeat the above steps, except this time click the “Add as a new
mass component” button. Delete unnecessary lines by clicking on the
line then clicking the “Delete selected mass component” button. When
satisfied that the List of Mass Components window is properly
configured, click the “Close” button. Next, click the “Close” button to
close the “Mass Components in ELAN Data File” window. Close the
“Processing ELAN Data” window by clicking its “Close” button.
(d) Inside the ELAN ChromLink window, click on the “Set” button. A window
entitled “Operating Mode” will open. Click on the “Automatic – process all
ELAN NetCDF files in specified location” radio button. The lower radio
buttons will gray out. Click “OK” to close the window.
2. At this time, ChromLink™ may be closed by selecting File > Exit. Click “OK”
at the dialog box asking if you want to quit ChromLink™.
3. In addition to configuring ChromLink™ itself, it is necessary to alter one value
in the “seed” method file that ChromLink™ uses to set a select number of
parameters to certain default values. This step only needs to be done once
following the installation of ChromLink™.
(a) In the TotalChrom™ Navigator window, choose the menu item Build >
Sequence and a dialog box called “Startup” will appear. Click on the
radio button labeled “Load sequence stored on disk” then click the OK
button. Navigate to the folder on the C drive that contains the
ChromLink™ program file (usually in C:\PenExe\ChromLink but if it is not
there, check under the C:\Program Files directory). Click on the
sequence file “seed.seq” to highlight it. If this file is missing, reinstall
ChromLink™. Click “Open”. A spreadsheet style sequence table will
present itself in a window called “Sequence Information – Channel A”.
There will be a minimized window for channel B data, ignore this window.

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Scroll across to the “Int Std Amt” column and click on the first cell in row
1 of this column. Replace the existing value with the concentration of
working Internal Standard which is 2.5 µg/L Error! Reference source
not found..
(b) Choose menu item File > Save. Close the Sequence Editor window by
choosing File > Exit from the menu bar.

c. Data Processing and Analysis
Refer to Figure 1 “Post-Run Data Processing Work Flow Diagram” (page 58) for a
summary representation of the important aspects of post-run data processing.
1. Open Microsoft Windows® File Explorer and open the current working HPLC
data directory (e.g., C:\HPLC\Data\). Select all files
ending with the .rst and .idx and “delete” them.
2. If it is not already open, launch TotalChrom™.
3. If ChromLink was not run in real-time data collection mode during the run as
described in step 16 under Starting the Run (see page 48), do the following:
(a) In the TotalChrom™ Navigator window, choose menu item Apps >
ChromLink. Choose the menu item Configuration > Mass Details and
check the Nominal Name and Mass for arsenic. If it is missing or altered
then ChromLink™ needs to be configured (see Configuration of ELAN
ChromLink™ on page 55 for details).
(b) Check that the Mode field indicates “Automatic – Process all NetCDF
files in specified location”. If it does not, click the “Set” button to the right
of this field and in the resulting “Operating Mode” dialog box click the
“Automatic – process all ELAN NetCDF files in specified location” radio
button. Click “OK”. Next, check that the Field labeled “ELAN NetCDF
file – location/file to be converted” indicates the correct data folder. This
should be “C:\elandata\Reportoutput\*.nc”. If it is not, click the Browse
button to the right of it, and in resulting dialog box, navigate to that folder.
Double-click on that folder then click “OK” to close the front most dialog
box. Click the Browse button to the right of the field labeled “ELAN
ChromLink file location…”. In the dialog box “Select TotalChrom™ Data
Location”. Navigate to the folder containing the run data and doubleclick on it. Click “OK” to close that dialog box. In the ELAN ChromLink
window, click the button “Start Processing ELAN Data Files” to start
processing of the run data. A new dialog box will open and provide
current information on the status of the data conversion.
(c) When data conversion by ChromLink is completed within a minute or
two, a message in the Step field will indicate “Successfully Finished”.
Click “Close”. At this point, you may close the ELAN ChromLink
application by choosing File > Exit or clicking on the window “x” box. In
the resulting “OK to quit?” confirmation dialog box, click “OK”.
4. In the TotalChrom™ Navigator window, choose the menu item Build >
Method. Click the “Load method stored on disk” radio button and click “OK”.
In the TotalChrom™ File-Open” dialog box, find C:\HPLC\Methods folder and
open “Arsenic1.mth” file. The template method file should now be loaded.

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If instead of loading the method file an error message says the file is
unavailable because it is in use and asks if you would like to open it in ReadOnly mode, click “No”. Cancel the Open-File dialog box, and exit the Graphic
Method Editor. In the Navigator window, choose menu item Admin > CAM
Administrator. A window will appear with two panes. In the left pane, click
on the “+” sign in front of “TotalChrom Servers” to expand it. Click on the
computer icon on the next line that just appeared to highlight it. In the right
pane, under the heading “Resource/Instrument”, select the first item. If there
is more than one item, select every item by shift-clicking on each item. Every
item should now be highlighted. Choose Edit > Remove Locks (or press the
Delete key on the keyboard). Next, click on the “+” sign in front of “Users” to
expand it. Click to highlight your TotalChrom™ user name that appeared. In
the right pane, under the heading “Resource/Instrument”, select every item
and Choose Edit > Remove Locks. This action serves to unlock files and
make them available for editing. If in the future, TotalChrom™ complains
that files cannot be edited because they are locked, use CAM Administrator
to unlock them. Choose File > Exit to quit CAM Administrator. Start again at
the beginning of this step to open the Method Editor.
5. Choose File > Save As. At the next window you will be given the option to
enter information about the method which can be done at your discretion.
Click “OK” and a TotalChrom™ File-Save-As” dialog box opens. Navigate the
directory tree to get to the folder that contains the ELAN data files for this run
(typically in the folder C:\HPLC\Data\). Double-click on this folder. In the “File
name:” field, enter the same name as it exactly appears for the folder that will
contain it (i.e. As convention where yy = last two digits of the year,
mm = two digit month, dd = two digit date). Click “Save” then close the
“Method Editor” window.
6. In the TotalChrom™ Navigator window, choose the menu item Build >
Graphic Edit. Choose File > Open from the menu bar and navigate the fileopen dialog box to the folder containing the method file created in the
preceding step. Click on that file and then click “Open”. Return to Graphic
Method Editor’s menu bar and choose File > New Data File. Navigate to
C:\HPLC\Data\ and double-click on the folder containing the run data. Find
and click on a data file (indicated by the “.raw” extension) that corresponds to
the “S4” calibrator run. When this file appears in the File Name field, click the
Open button. In the File-Open dialog box that appears, click “Cancel”. If a
message box appears with the warning “Unable to open this file: default.mth”,
click OK to clear the message (you do not have to go to CAM Administration
to unlock it). Do the same if another message warning box appears (i.e. click
OK again to clear it). You should be in the “Graphic Method Editor - ” window and see a chromatogram.
7. Choose menu item Calibration > Show Windows and retention window bars
(looks like “H” style error bars) will appear. Each retention time window bar
should be located above the chromatographic baseline and contain an
identified peak within its bounds. If there are any bars at the bottom of the
chromatogram located below the baseline, choose menu item Calibration >
Edit Components. Click on the first arsenic species peak that falls outside its
retention time window to select it. In the group of data fields located on the
right side of the window, click on the “Name” dropdown arrow (located on the
right side of the data entry field) and choose the appropriate species by name.
Next, click on the “ISTD” field’s dropdown arrow and choose “IS”. Be sure the

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“ISTD” checkbox is unchecked unless you are editing the “IS” peak; in this
case put a checkmark in the “ISTD” checkbox. It is usually not necessary to
alter the retention time window’s “Absolute” and “Relative” window
parameters, but you may do so if experience dictates that a change will be
beneficial. Click the Next or Prev button. Repeat these steps for each arsenic
species peak that was not properly identified because it was outside its
retention time window. Since TMAO is not present in any of the “S”
calibrators, choose File > New Data and open one of the “T” series calibrators
that contain TMAO (e.g., T4) and confirm its identity and retention time
window using the same process as was used for S4. When the editing of
peak retention time windows is completed, click on the menu bar item
“Return”. Choose File > Save followed by File > Exit.
8. Launch Microsoft® Excel and choose menu item HPLC > Create TC
Sequence File (the Excel macro “Extract TC Data.xls” must be installed in
Excel’s Startup folder). In the open file dialog box, navigate to the current
working HPLC data folder. Click on a RAW file then click the “Open” button.
Wait about 30 seconds until a “Done” message box appears. Excel will create
two sequence files, one containing just the calibrators (name ending with
“calib.seq”) and the other file containing all samples and calibrators (named
As.seq). You may leave Excel open.
Skip the following steps (a) through (c) unless, for some reason, the Excel
menu item HPLC > Create TC Sequence File cannot be run:
(a) In the TotalChrom™ Navigator window, choose the menu item Build >
Sequence and a dialog box called “Startup” will appear. Click on the
radio button labeled “Load sequence stored on disk” then click the OK
button. Navigate to the folder containing the run data and click on the
sequence file (ends with “.seq”) corresponding to the run (typically
named in the As_yymmdd.seq format). Click “Open”. A spreadsheet
styled sequence table will present itself in a window called “Sequence
Information – Channel A”. There will be a minimized window for channel
B data. Ignore this window. Look for the “Method” column and click on
the first cell in row 1 in this column. Right click the mouse and a
contextual menu will appear, choose ”Browse”. I n the resulting FileSelect dialog box, navigate and choose the method file (ending in “.mth”)
created earlier. Click ”Select”. The path and name of the new method
file will replace the default information in this cell. Right click this cell
again and choose Fill Down. The new file name information will fill down
to every cell in the “Method” column. Look for the “Study Name” column
and click on the first cell in row 1 in this column. Note that this cell
contains redundant information that is already in the Name column.
Press the delete key to clear this cell. Right click the mouse and a
contextual menu will appear, choose “Fill Down”. Right click this cell
again and choose Fill Down. This will clear every cell in the “Study
Name” column. Choose menu item File > Save. Do not close this
window yet.
(b) Position the mouse cursor over the first row number (in the Row column
on the far left side of the window) that is NOT a calibrator. The cursor
should be in the form of a fat plus sign. If it looks like small vertical
double-ended arrow, move the mouse slightly up or down until it changes
to a fat plus sign. Press and hold down the left mouse button and drag

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down across all the row numbers that are not calibrators. Check that you
have not accidentally included calibrators, otherwise, deselect all the
rows and try selecting again. Once you are sure that none of the
calibrators are selected, choose menu item Edit > Delete. Repeat this
process until only rows corresponding to calibrators are present in the
sequence table. Click the cell in the first row in the “Type” column and a
dropdown menu should appear. Choose “Cal:Replace”. Right-click on
the same cell and choose Fill Down. At this time, “Cal:Replace” should
appear in every cell in the ‘Type” column.
(c) Inspect the Sequence Editor window for Excel-styled workbook tabs at
the bottom. Locate the tab labeled “Calibration” (if you do not see it on
first look, click on the small right arrow just left of the first tab, this will
cause the tabs to “scroll” left and reveal additional tabs). Click on the top
cell in the “Cal Level” column. A dropdown menu will reveal “S0”, “S1”,
“S2” through to “T4” menu choices. Moving down the column, for each
calibrator you will need to assign its level by choosing correct menu item
from the dropdown menu. When all calibrators are assigned their
appropriate level, click on the cell in the “Calib Rpt” column
corresponding to “S4” calibrator. A pop-down menu should appear;
choose “Short”. Likewise, change the “Calib Rpt” for “T4” from “None” to
“Short”. Choose menu item File > Save As. A window showing a
Description field appears, just click OK and Save As dialog box will
appear. Name the new sequence file the same name as the original
sequence file except add the word “calib” to the end of the file name (be
sure to separate the words by a space). Click the Save button. Then
close the Sequence Editor window by choosing File > Exit from the menu
bar.
9. In the TotalChrom™ Navigator window, choose the menu item Sequence.
Open the calibrator sequence file created in the previous step (the file will be
named the corresponding date Asyymmdd_calib.seq). A sequence table will
open. By looking in the “name” field, ensure that this table only displays
information regarding calibrators from the run. (The names for calibrators
should match those in the Elan sample table created prior to the run, e.g.
002_S0.) If blanks, quality control material, samples, or any other names are
present in any row of the “name” field, delete the entire corresponding row in
which they are present. Once only calibrators are present in this table, ensure
that Cal:Replace is selected in the “type” field for all rows. Additionally,
ensure that the “Cal Level” field is complete. Using the drop-down menu, Cal
level fields for each row should match each level represented in the “name”
field. Lastly, ensure that the correct method is shown in the “method” field for
all rows. Save this sequence file (File > Save) and close.
10. In the TotalChrom™ Navigator window, again choose menu item Sequence.
This time, open the sequence file matching the date of the run that does not
include “calib” in the title. (It will look like Asyymmdd.seq.) A sequence table
containing all of the items represented in the run’s Elan sample file will
appear. In a manner similar to that in step 9, various field changes will have
to be made. This step is important for proper database importing. In the
“Type” field, use the drop-down menu to select the proper representation for
each item in the entire list. In this field, Calibrators should be marked
“Cal:Replace,” blanks should be marked “Blank,” quality control materials and
other control materials should be marked “Ctrl Sample,” and all other items,

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including those assessed for quantitative measure and equilibrators, should
be marked “Sample,” unless another title in the drop-down menu is
appropriate. Next, ensure that the “Cal level” field is populated for calibrator
sample rows only. If this field is not populated, select calibrator levels from a
drop down menu by clicking in the field. Make sure these selected calibrator
levels correspond to names in the “Type” field for each row. (For instance, a
name of 002_S0 in row 2 should correspond to a Cal level of “S0” in row 2.)
Next, ensure that the proper method is listed in the “Method” field of each row.
(This method should be the same that was “saved as” in step 5 of this
section). Lastly, at the bottom of the sequence table, click on the “calibration”
tab. A new sequence table will display; ensure that each row in the field “Cal
levels” is accurate. Save this sequence file (File > Save) and close.
11. In the TotalChrom™ Navigator window, choose the menu item Reprocess >
Batch. A new window appears entitled “Batch Reprocessing”. Choose menu
item File > Sequence and the “From Sequence” window appears. Locate the
top field labeled “Sequence file” and look for a button with an open folder icon
immediately to the right of the field. Click this button and navigate, if
necessary, to the folder containing the run’s sequence files. Click on the
sequence file whose name ends with “calib.seq” and click the Open button.
You will be returned to the previous window. Set each parameter in this
window to the values shown in TABLE 10-8.
TABLE 10-8: TOTALCHROM™ NAVIGATOR – REPROCESS BATCH
Parameter Name

Starting Row
Ending Row
Channel A
Channel B
Start Analysis
End Analysis
Batch Execution
Batch Printer
Batch Plotter
Enable Optional Reports in Method

Parameter Setting

:
:
:
:
:
:
:
:
:

Checked
Not Checked
Peak Detection
Calibration
Interactive
None
None

Not Checked
Grayed Out
Checked
Update existing raw file header with new sequence

Use Method is Result File

Overwrite Existing Result Files
Raw File Treatment

:
:

12. Click “OK”.
13. Choose menu item Reprocess > Start. The middle panel will contain a list of
raw file waiting to be processed. Reprocessing of the chromatographic raw
data will commence. The bottom panel in the window will update with each
file’s name as it is processed. When processing is done, this panel will be
clear of files. Close this window.

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14. In the TotalChrom™ Navigator window, choose the menu item Build > Method
and open the method file for this run. If you get an error message telling you
that you can only open this in read-only mode, then unlock the files by doing
these steps:
(a) Click “No” to cancel the error message. Select File > Exit to close the
Method Editor.
(b) In TotalChrom Navigator, select Admin > CAM Administration.
(c) In the CAM Admin Tool window, click in the file explorer-like window on
top of the “+” sign in front of “TotalChrom Servers” to expand it. Click on
the server name. A list of files will reveal themselves in the right-hand
window panel.
(d) Click on the first file, hold down the shift key and click on the last file in
the list. This will highlight all the files in the list. Press the keyboard’s
“delete” key. This does not delete the actual files but only unlocks them.
(e) Select File > Exit in the CAM Admin Tool window to close it.
(f) Return to Method Editor window by selecting Build > Method in the
TotalChrom™ Navigator window. Begin this step again.
15. Return to the menu bar and choose Window > Component List. Position the
mouse on the any of the four corners or edges of the Component List window
until the mouse cursor turns to a double-headed arrow. Expand the window
until it fills its parent window.
16. The Component List window is divided into three panels. The left panel lists
each component by peak number, retention time and component name.
Clicking on any of the components will reveal that component’s calibration
data and calibration curve in the middle and right panels, respectively.
Consecutively click on each component one at a time and individually inspect
each component’s calibration curve. It is important to note whether the
calibration curve meets requirements for linearity, slope and intercept.
(a) Watch for calibration points that obviously fail to display their expected
Response Ratio (i.e. fall away from the regression line compared to their
neighboring points). Typically, a point with a Response Ratio equal to
zero indicates that a component peak was missed during peak
identification phase of calibration processing. Likewise, a point that falls
far from the calibration curve might be because that component’s peak
was misidentified (perhaps confused for another component). Inspection
of nearby eluting component’s calibration curve might reveal an
oppositely misaligned point for the same calibration level. Return to
Graphic Editor and alter the retention time windows as necessary, so all
peaks will be correctly identified upon reprocessing.
(b) While the regression line does not have to intersect every point, be
especially mindful of “R-squared” value at the top left of the right panel.
R-squared must exceed 0.990 (“two nines”) for each component. Rsquared values >0.999 is common for this procedure.

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(c) Corrective action will need to be taken in any case of failure in the above
rules. Possible steps include:
(i) Checking chromatograms to see if an autosampler injection was
missed. If so, all samples will have to be re-analyzed.
(ii) Inspecting previous runs in database for deviating trends among
calibration curves from separate runs.
(iii) Inspecting chromatograms from previous runs to assess
chromatographic shifts in retention time, peak height, or peak
broadness. These shifts could be a result of errors in buffer
preparation, contaminated buffer solutions, an alteration of pH of
buffer solutions, or a poor column.
(iv) Deleting one extreme outlier point from the calibration curve if, in
doing so, adequate linearity is achieved. This step should not be
common practice (for no more than 2 runs in a row should an analyst
have to perform this corrective measure). Analysts must include
record of this action in a run summary email to supervisor(s).
17. If the calibration curves pass inspection, close the Method Editor.
18. In the TotalChrom™ Navigator window, choose the menu item Reprocess >
Batch to reopen the “Batch Reprocessing” window. Choose File > Sequence
and click the button with the open folder icon located right of the field labeled
“Sequence file”. Navigate, if necessary, to the folder containing the run’s
sequence files and click on the other sequence file whose name does not
contain “calib”. Click “Open”. Upon return to the previous window, set End
Analysis to “Quantitiation” and Batch Printer to “None”. All other parameters
should remain unchanged.
19. Click “OK” to close the front window. Next, click the green “start arrow”
button. Reprocessing of the chromatographic raw data will commence.
20. In the TotalChrom™ Navigator window, choose the menu item Reprocess >
Results. A new window should open called “Reprocess Results”. If you get
an error message telling you that you can only open this in read-only mode,
then unlock the files (follow the procedure described in step 4 of this section).
Select from the menu File > Open. In the open file dialog box, click on the
“Files of type:” dropdown menu and select “IDX files (*.idx)”. Navigate to the
folder containing this run’s data and click on the newest file (in the format of
“As--”). Click “Open”. A
chromatogram will be presented for the first sample in the sequence in the
Reprocess Results window. Carefully inspect the chromatogram one peak at
a time for correct peak identification and accurate baseline. If you are
satisfied that there are no integration problems, proceed to the next sample’s
chromatogram by selecting File > Next File from the menu bar. Examine all
chromatograms in this manner and make corrections in peak identity and
integration as necessary. Make notes concerning issues encountered with
individual chromatograms and changes that were made. If a chromatogram is
changed or edited in any way, be sure to select File > Save to save your
changes. See the chapter entitled “Developing Processing Parameters in the
Method” in the PerkinElmer TotalChrom™ Workstation Users Guide for a
detailed explanation on how to use integration events to optimize the
integration of a chromatogram. After review of each and every
chromatogram, select File > Exit from the Reprocess Results menu bar.

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21. Repeat step 18 except set both Start and End Analysis to “Report
Generation”. Set Batch Printer to “Find Print Factory Pro”. A new window will
open entitled “pdFactory Pro: <##> Jobs (## pages, ### Kb)”. When
reprocessing has completed, click the Save button on the pdFactory Pro
window. In the Save As dialog box that appears, navigate to the run’s data
folder and create a new pdf file named “As report”. Be sure to
include a space between “report” and the first word of the new file. Click the
Save” button. This pdf file is to be kept and backed up, for archival purposes,
in the same folder with all the other chromatographic data files for this run.
Click the “Close” button to close pdFactory Pro window.
22. Open Microsoft Excel and choose HPLC > Extract TC Data. In the dialog box
which follows, choose the sequence file created in step 10 of this section and
click Open. Immediately a macro will run that will transform the data into a
format that is easily exported into the database. Just before the macro
finishes, a Save As dialog box will open giving you the opportunity to save the
file as an Excel workbook. Give the file a name as follows: “As
results”. Note: For runs containing multiple groups of samples (each group
being bracketed by its own quality control material), separate filenames will be
necessary for each group. The newly-created “As results” Excel
file should be broken into multiple smaller files, labeled as “As
results Run 1”, “As results Run 2”, and so forth, where Run 1
corresponds to the first unique group in the spreadsheet, Run 2 corresponds
to the second unique group in the spreadsheet.
Each multi-tabbed Excel workbook contains a worksheet suitable for data
exportation to the MS SQL Server 7™ database. Clicking on additional tabs
will show worksheets for (a) summary table for easy visual review of the
data, (b) quality control results, (c) calibration data with regression statistics
and plotted calibration curves for each arsenic species, (d) instrument
stability chart showing degree of consistency of internal standard peak areas
plotted as a function of injection #, and (e) raw data (two tabs).
23. The data processing portion on the instrument controller computer is now
complete. At this point you may close Microsoft Excel® and TotalChrom™
Navigator.

11. RECORDING OF SAMPLE AND QC DATA
a. Transferring the Data to the Central Database
1. Transfer the “As results”.xls file (or the files representing each run)
via encrypted USB drive or other data media to the appropriate subdirectory
on the network drive where exported data are stored. (Note that directories
are named according to instrument\year\month\ and study name or ID, for
example,
“Q:\Nutritional\Instruments\ELAN\ELAN_DRC2H\2008\06\As080602”.)
2. From a computer that has access to the Microsoft Access™ or MS SQL
Server 7™ database used for tracking data start the program. A “GoTo2 :
Form” window should automatically open. If it does not, you may have to
open it manually.

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3. Click the “Add Sample Results to Database” button. New buttons will appear.
Click the “Import Instrument Data File” button. For “Instrument”, choose
“ELAN-DRC2D” (or the appropriate instrument). For “Assay”, choose “As
Speciation 2”. Choose the correct analyst from the drop-down list and the
appropriate study. It is not necessary to fill-in the “IS Lot Number” Field. Click
“Import”. Select the location of the data file on the network drive and press
the “Open” button.
4. In the “Imported Results” table, pressing the “Find X’s” button will show only
those samples whose sample ID is not recognized as a valid QC pool ID or
sample ID for this study. (Sample IDs are set up when the study is logged
into the database). If necessary, corrections to sample IDs and dilution
factors can be made in this table (e.g., correction of transcription errors and
adjustment for level of dilution). If samples were diluted for analysis, both the
sample ID and the dilution factor need to be edited in this table before the
values are transferred to the database. First, change the dilution factor to
reflect the way that the sample was analyzed then edit the sample ID to
remove any comments about the level of dilution at which the sample was
analyzed. (The replace command is useful here.)
5. When corrections to sample IDs are made, press the “Recheck” button to
evaluate the sample IDs. Any sample or analyte row marked “Not
Recognized” will not be transferred to the database when the “Transfer”
button is pressed.
6. Press the “Transfer” button to import data into the database.

b. QC Data
Once data is transferred to the Microsoft Access™ (or MS SQL Server 7™) database, quality
control (QC) samples must be assessed for pass or failure through the generation of QC reports.
The database allows for the printing of several types of QC reports. If necessary, keep a copy of
the report with the analysis printouts from the run (if the data needs to be printed). The QC
reports can be stored electronically.

12. FINAL REVIEW OF THE DATA
a. Analysis Printouts and Analyst Run Report
Per the guidelines of each study, bind the analysis printouts with a printout of the
calibration curve and curve statistics and place them in the study folder(s). For some
studies, this step is not necessary.

b. Plotting QC Results
When the Microsoft Access™ or MS SQL Server 7™ database is used, QC plots are
updated automatically when the data are imported into the database. Monitor these
plots regularly for any trends in the bench QC results. If trends are observed, contact
the laboratory supervisor.

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c. Supervisor Review
The Microsoft Access™ or MS SQL Server 7™ database allows the supervisor to
review the QC and sample results directly in the database. After the supervisor
reviews the data, he or she may mark results as “Ready to Report.”

13. REPLACEMENT AND PERIODIC MAINTENANCE OF KEY
COMPONENTS
a. ICP-MS Maintenance
Part numbers listed below are PerkinElmer part numbers from their 2006/2007 Consumables
Catalog. Equivalent high quality parts from other suppliers may be used as noted.
1. Peristaltic pump tubing for sample (0.03 inch i.d., Item # 09908587), rinse
station (can use either same tube type as for sample or 0.045-inch i.d., Item
#N0680375) and for waste (0.125-inch i.d., Item #N8122012): Keep at least 6
packages of 12 on hand of the sample tubing, 6 for rinse station and 2
packages of 12 on hand of the waste tubing. Other suppliers may offer the
same size/type of peristaltic tubing.
2. Autosampler probe assembly (Item # B3000161). Keep a spare on-hand.
3. Nebulizer capillary tubing (0.023-inch i.d., Item #09908265 or any source of
polyethylene tubing, 0.6 mm i.d. x 0.97 mm o.d.). Use to connect the
nebulizer and the peristaltic pump tubing. Keep one pack (10 feet) on hand.
4. Injector Support for ELAN DRC (Item # WE023951). Keep one spare on
hand.
5. Ball Joint Cassette Torch Injector Support Adapter (Item # W1012406).
6. Cassette Torch Mount for ELAN DRC II (Item # W1020672).
7. Torch O-Ring Kit (packages of four, Item # N8120100). Keep four spare
packages on hand.
8. Quartz torch. At least two spare torches should be on hand (Item #
N8122006).
9. Quartz Sample Injector, 2.0mm Ball Joint (Item # WE023948). At least two
spare injectors should be on hand.
10. RF coil (Item # WE021816). One spare should be on hand.
11. Platinum Skimmer (Item # WE027803 or equivalent) and platinum sampler
cones (Item # WE027802 or equivalent). Keep at least two spares of each on
hand.
12. Skimmer and sampler cone O-rings (Item # N8120512 and # N8120511, or
equivalent, respectively). Keep at least 10 spares of each on hand.
13. Series II replacement Ion lens (Item # WE018034). Keep two spares on hand.
14. Pump oil for the roughing pump (Item # N8122004 or equivalent). Keep four
bottles on hand. If an instrument is equipped with a Fomblin oil-based pump,

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only one bottle of Fomblin oil (Ausimont or equivalent) is necessary to keep
on-hand.
15. Polyscience chiller coolant (PE Sciex Coolant, Item # WE016558A): Two 1-L
bottles should be kept on hand.
16. If possible, have a backup Polyscience chiller (or equivalent). See a
PerkinElmer sales representative for part numbers.

14. LIMIT OF DETECTION AND LINEAR RANGE TESTED
The limits of detection (LOD) for arsenic species in urine specimens are based on data taken
from a minimum of 60 analytical runs. At least four levels are used in each run with one level
being below the LOD. The matrix blank can be used to satisfy the criterion of having a level
below the LOD. Using the data from at least 60 runs, regression can be used to validly predict
the standard deviation at the LOD concentration. The LOD will be three times this calculated
standard deviation, and this will represent the method detection limit. Report results below the
detection limit as “< LOD” (where “LOD” is the calculated lowest detection limit). The LOD
calculation is reevaluated once every two years.

TABLE 14-1: LIMITS OF DETECTION (LOD) AND LINEAR RANGE TESTED (LRT) FOR AS
SPECIES
Species
Chemical Name

Arsenobetaine
Arsenocholine

Abbreviated
Name

Limit of
Detection, g/L

Highest Concentration for

1.19

1000

Linear Range Tested, µg/L

0.28

1000

Trimethylarsine
oxide
Monomethylarsonic
acid
Dimethylarsinic acid

TMAO

0.25

1000

MMA

0.89

1000

DMA

1.8

1000

Arsenous (III) acid

As(III)

0.48

1000

Arsenic (V) acid

As(V)

0.87

1000

15. REPORTABLE RANGE OF RESULTS
Urine arsenic results are reportable in the range of greater than the LOD, where LOD is the
calculated limit of detection. When a sample result for any analyte is greater than the highest
calibrator for the same analyte within the run, the result needs to be confirmed. If a sample’s
result for any analyte is greater than 110% of the Linear Range Tested (“LRT”) concentration (see
TABLE 14-1), then the sample must be diluted with water before a repeat analysis can be
performed. Otherwise, the confirmation may be done without dilution in a run that includes an
additional standard or external reference material (“Extended Range Check”) having a known
analyte concentration equal to or greater than that measured in the sample, up to the LRT
concentration. The Extended Range Check shall not be included in the calculation of the
calibration curve; instead it will be analyzed like an unknown sample. Its result will serve to check

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the linearity of the regular calibration curve beyond the maximum calibration point. For the check
to qualify, the measured concentration of the Extended Range Check result must be within ±10%
of its nominal value. I f its value is within this target, the sample’s original result may be reported
for the analyte. If the Extended Range Check does not fall within the target specified, the sample
must be diluted to bring its analyte concentration within the method’s regular calibration range.
Results Greater Than Range of Linearity Tested: Perform an extra dilution on any urine sample
whose concentration is greater than those listed in Table 14-1 (the highest concentration for
linear range tested).

16. SPECIAL PROCEDURE NOTES – CDC MODIFICATIONS
None applicable for this method.

17. QUALITY CONTROL PROCEDURES
The Inorganic and Radiation Analytical Toxicology Branch uses the method described in this
protocol for environmental and occupational health screening studies.
This analytical method uses two types of Quality Control (QC) systems: With one type of QC
system, the analyst inserts bench QC specimens two times in each analytical run (a set of
consecutive assays performed without interruption) so that judgments may be made on the day of
analysis. With the other type of QC system, “blind” QC samples are placed in vials, labeled, and
processed so that they are indistinguishable from the subject samples (as many as possible).
The supervisor decodes and reviews the results of the blind specimens. With both systems,
taking these samples through the complete analytical process assesses all levels of the analyte
concentrations. The data from these materials are then used to estimate methodological
imprecision and to assess the magnitude of any time-associated trends. The bench QC pools
used in this method comprise two levels of concentration spanning the “low-normal” and “highnormal” ranges for each arsenic species. Both of these pools are analyzed after the calibration
standards are analyzed but before any patient samples are analyzed. These bench QCs should
be analyzed again at the end of the run. If a second run of samples are analyzed using the same
calibration curve as the first run, the QC results obtained from the second run’s own bench QC
samples need to be analyzed and treated independent of the first run.

a. Establish QC limits for each QC pool.
A run to assess the homogeneity of the pools is performed after the pools are aliquotted into
individual vials. Vials are randomly chosen and randomly analyzed, and the first and last vials
dispensed are always included in the homogeneity study. Unlike the characterization of the QC,
the homogeneity study can be completed in a single run. Once analysis is complete, the data is
evaluated in terms of QC recovery to determine whether or not trends exist in QC during the
dispensing of the pool. If the pool does not vary from beginning to end or problem vials can be
identified and eliminated, the characterization of the QC is the next step. If problems do exist, the
source(s) of the problem has to be identified and the pool has to be re-made and dispensed
again.

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To complete the characterization that will allow you to assess limits for each pool, analyze a
minimum of twenty samples of each pool (low and high) on 20 different days, preferably among
all of the instruments that will be used to analyze this method. During the 20 characterization
runs, previously characterized QCs or pools with target values assigned by outside laboratories
are also analyzed to evaluate each run’s QC. Once analysis is complete, calculate the mean and
standard deviation for each pool from the concentration results. These values will be used to
establish the limits for each pool.

b. Precision and Accuracy
QC Results Evaluation. After completing a run, consult the QC limits to determine whether the
run is “in control” for each of the seven analytes. The QC rules apply to the average of the
beginning and ending analyses of each of the bench QC pools. The QC rules are as follows:
1. If both the low-and the high-QC results are within the 2s limits, accept the run.
2. If one of two QC results is outside the 2s limits, apply the rules below and reject the
run if any condition is met.


13s – Average of both low QCs OR average of both high QCs is outside of a
3s limit.



22s – Average of both low QCs AND average of both high QCs is outside of
2s limit on the same side of the mean.



R4s sequential – Average of both low QCs AND average of both high QCs is
outside of 2s limit on opposite sides of the mean.



10x sequential – The previous nine average QCs results (for the previous
nine runs) were on the same side of the mean for either the low OR high QC.

If the run is declared “out of control,” the analysis results for all patient samples analyzed during
that run are invalid for reporting for the affected analytes.

c. Remedial Action If Calibration or QC Systems Fail to Meet Acceptable
Criteria
If an analyte fails to pass QC based upon the QC report, the following steps should be taken, if
possible:


Check the chromatograms for each blank, calibrator, QC, and sample for proper peak
integration and identification. Check that the internal standard peak was properly integrated
and identified. Change integration parameters or manually reintegrate peaks, if necessary,
and reprocess the run in TotalChom™.



Check the ICP-DRC-MS stability during the run by examining the degree of variability and
drift in internal standard raw peak areas over the course of the run. Irreproducibility that
exceeds 15% and drift >20% or sudden large changes in internal standard peak area likely
indicates that there was a problem in plasma stability.



Setup a new run for the reanalysis of the patient samples affected by the previous failed run.
Be sure to use freshly thawed calibrators and QC material.

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

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If these three steps do not result in correction of the out-of-control values for QC materials,
consult the supervisor for other appropriate corrective actions. No analytical results should
be reported for runs that are not in statistical control.

18. LIMITATIONS OF METHOD; INTERFERING SUBSTANCES AND
CONDITIONS
The argon chloride (ArCl) interferences on arsenic (75As) are eliminated by the operation of the
DRC™ under the parameters noted in the sections above during the speciated arsenic analysis.

19. REFERENCE RANGES
The reference range for each arsenic species (see TABLE 19-1) is based on literature reports
and from periodic review of accumulated data collected during the analysis of urine samples
representing a normal, healthy population believed to be free of unusual exposure to arsenic.
Where data is absent or scant, references ranges are based on the scientific literature, if
available.
TABLE 19-1: REFERENCE RANGES FOR ARSENIC SPECIES
Species Chemical Name

Reference Range1, µg/L

Arsenobetaine

0 Then
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tcLCDservicePath = strPenPath & "LCD.exe"
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progressIndicator = 0
numMissingFiles = 0
firstLowQCSample = True
firstHighQCSample = True

itDoesExist = False
peakRatio_blank = 0
peakRatio_zero_calib = 0
peakRatio_minus_bk = 0
internalStdName = "IS" 'default name for internal standard
concIS = 1
'default concentration of internal standard in ppb

massName = OGetSetting("VBA", "Sub_HPLCtransformVariables", "MassName_string", "As", True)
zeroLevelName = OGetSetting("VBA", "Sub_HPLCtransformVariables", "zeroCalibName_string",
"S0", True)
useIntercept = OGetSetting("VBA", "Sub_HPLCtransformVariables", "UseIntercept_boolean", True,
True)
lowBenchQCName = OGetSetting("VBA", "Sub_HPLCtransformVariables", "LowBenchQCName_string",
"LU-03102", True)
highBenchQCName = OGetSetting("VBA", "Sub_HPLCtransformVariables", "HighBenchQCName_string",
"HU-03104", True)
nameOfBlk = OGetSetting("VBA", "Sub_HPLCtransformVariables", "BlankName_string", "Bk", True)
columnDescription = OGetSetting("VBA", "Sub_HPLCtransformVariables",
"columnDescription_string", "not entered", True)
calibPrepDate = OGetSetting("VBA", "Sub_HPLCtransformVariables", "calibPrepDate_string", "",
True)
firstLevelName = Left(zeroLevelName, Len(zeroLevelName) - 1) & "1"
todaysDate = Date
currentTime = Time
If useIntercept Then
savedNameSuffix = " Results"
Else
savedNameSuffix = " Results (calib intercept not used)"
End If
fileSpec = Application.GetOpenFilename(FileFilter:="Seq Files (*.seq), *.seq", Title:="Choose
a Sequence File")
If fileSpec = False Then
Exit Sub
'assume user clicked the Cancel button
End If

Urine arsenic species HPLCICPDRCMS (Formerly Arsenic species in Urine)
DLS Method Code: 3000.1(Formerly 016A/01-OD)

IRAT-DLS
Page 80 of 107

thisFilename = Right(fileSpec, Len(fileSpec) - InStrRev(fileSpec, "\", -1, vbTextCompare))
workBookName = Left(thisFilename, Len(thisFilename) - 4)
originalStatusBar = Application.DisplayStatusBar
Application.DisplayStatusBar = True
On Error Resume Next
' Defer error handling
iRet = TcLoggedOn()
If iRet = 0 Then
Application.StatusBar = "Starting PEN LCD Service, please wait..."
Shell "NET START " & Chr(34) & PEN_Service_Name & Chr(34)
'C:\PenExe\TcWS\Ver6.2.0\Bin\LCD.exe
Pause5seconds
commandLine = tcNavProgramPath
Application.StatusBar = "Launching TotalChrom, please wait..."
RetVal = Shell(commandLine, vbMinimizedNoFocus)
Pause5seconds
If RetVal = 0 Then
MsgBox "TotalChrom could not be launched.", vbOKOnly + vbExclamation, "Macro Aborted"
Application.StatusBar = ""
Application.DisplayStatusBar = originalStatusBar
Application.ScreenUpdating = True
Exit Sub
End If
End If
Set
Set
Set
Set
Set
Set
Set
Set
Set
Set
Set
Set
Set
Set

dLevel = CreateObject("Scripting.Dictionary")
dLevel2 = CreateObject("Scripting.Dictionary")
dCompName = CreateObject("Scripting.Dictionary")
dCompName_numberOfLevels = CreateObject("Scripting.Dictionary")
dCompName_i = CreateObject("Scripting.Dictionary")
dAmount = CreateObject("Scripting.Dictionary")
dSeqIndex_resultFile = CreateObject("Scripting.Dictionary")
dSeqIndex_seqSampleName = CreateObject("Scripting.Dictionary")
dSeqIndex_rawFile = CreateObject("Scripting.Dictionary")
dSeqIndex_levelName = CreateObject("Scripting.Dictionary")
dSeqIndex_seqSampleType = CreateObject("Scripting.Dictionary")
dLevelName_iSeqCycleIndex = CreateObject("Scripting.Dictionary")
dPeakNameZeroLevel_peakRatio = CreateObject("Scripting.Dictionary")
dPeakNameBlank_peakRatio = CreateObject("Scripting.Dictionary")

Application.Workbooks.Add
Application.DisplayAlerts = False
For i = 1 To Workbooks.Count
Sheets(i).Delete
Next i
Application.DisplayAlerts = True
If Sheets(workBookName).Name = Empty Then
ActiveSheet.Name = Right(workBookName, 30)
Else
ActiveSheet.Name = Right(workBookName & " copy", 30)
End If
Application.StatusBar = "Extracting TotalChrom data, please wait... "
Application.ScreenUpdating = False
resultsSheetName = ActiveSheet.Name
Sheets.Add After:=Worksheets(Worksheets.Count)
stabilitySheetName = "IS Stability"
ActiveSheet.Name = stabilitySheetName
Range("A1").Select
Set stabilityShtRange = Selection
With Selection
.Value = "IS Area"
.HorizontalAlignment = xlRight
.Interior.ColorIndex = 15

Urine arsenic species HPLCICPDRCMS (Formerly Arsenic species in Urine)
DLS Method Code: 3000.1(Formerly 016A/01-OD)
.Interior.Pattern = xlSolid
End With
Sheets.Add After:=Worksheets(Worksheets.Count)
ActiveSheet.Name = Right(resultsSheetName & " Raw", 30)
rawSheetName = ActiveSheet.Name
Range("A1").Select
With Selection
.Value = "RT (min)"
.HorizontalAlignment = xlRight
.Interior.ColorIndex = 15
.Interior.Pattern = xlSolid
End With
Range("B1").Select
Sheets(resultsSheetName).Select
With ActiveSheet
.Range("A1").FormulaR1C1 = "Run ID"
.Range("B1").FormulaR1C1 = "Seq No."
.Range("C1").FormulaR1C1 = "Sample ID"
.Range("D1").FormulaR1C1 = "Type"
.Range("E1").FormulaR1C1 = "Study ID"
.Range("F1").FormulaR1C1 = "Result Date-Time"
.Range("G1").FormulaR1C1 = "Result File"
.Range("H1").FormulaR1C1 = "Method File"
.Range("I1").FormulaR1C1 = "Dilution"
.Range("J1").FormulaR1C1 = "Calib Name"
.Range("K1").FormulaR1C1 = "Calib Conc"
.Range("L1").FormulaR1C1 = "Analyte ID"
.Range("M1").FormulaR1C1 = "Ret Time"
.Range("N1").FormulaR1C1 = "Peak Ht"
.Range("O1").FormulaR1C1 = "Peak Area"
.Range("P1").FormulaR1C1 = "IS Peak Area"
.Range("Q1").FormulaR1C1 = "Ratio"
.Range("R1").FormulaR1C1 = "Ratio Blk"
.Range("S1").FormulaR1C1 = "Ratio minus Blk"
.Range("T1").FormulaR1C1 = "Ratio S0"
.Range("U1").FormulaR1C1 = "Calib Intercept"
.Range("V1").FormulaR1C1 = "Calib Slope"
.Range("W1").FormulaR1C1 = "Calib Correl"
.Range("X1").FormulaR1C1 = "Calib Number"
.Range("Y1").FormulaR1C1 = "Raw Amount"
.Range("Z1").FormulaR1C1 = "Adj Amount"
.Range("AA1").FormulaR1C1 = "Column Desc"
.Range("AB1").FormulaR1C1 = "Calib Prep Note"
.Range("AC1").FormulaR1C1 = "Sample Note"
End With
With Range("A1:AC1")
.Interior.ColorIndex = 15
.Interior.Pattern = xlSolid
End With
Range("D2").Select
ActiveWindow.FreezePanes = True
' Create a second sheet for Chromera-style export
Sheets.Add After:=Worksheets(Worksheets.Count)
chromeraSheetName = Right(resultsSheetName & " Chromera", 30)
ActiveSheet.Name = chromeraSheetName
Range("A5:J5").Select
Set chromeraDataShtRange = Selection
With ActiveSheet
.Range("A5").FormulaR1C1 = "Sample ID"
.Range("B5").FormulaR1C1 = "Replicate"

IRAT-DLS
Page 81 of 107

Urine arsenic species HPLCICPDRCMS (Formerly Arsenic species in Urine)
DLS Method Code: 3000.1(Formerly 016A/01-OD)
.Range("C5").FormulaR1C1
.Range("D5").FormulaR1C1
.Range("E5").FormulaR1C1
.Range("F5").FormulaR1C1
.Range("G5").FormulaR1C1
.Range("H5").FormulaR1C1
.Range("I5").FormulaR1C1
.Range("J5").FormulaR1C1
End With

=
=
=
=
=
=
=
=

IRAT-DLS
Page 82 of 107

"Analyte"
"Mass"
"Species"
"Retention Time"
"Peak Area"
"Peak Height"
"Concentration"
"Units"

With Selection
.Interior.ColorIndex = 15
.Interior.Pattern = xlSolid
End With
Range("A6").Select
' Select starting activecell on Sheet(resultsSheetName)
Sheets(resultsSheetName).Select
Range("A2").Select
Call TcInit
TcAccess.dll

'Initializes program with

c_SEQ = TcOpenConv("SEQ")
'c_SEQ is the first
conversation handle. Opens conversation with the Sequence topic in TcAccess.dll
If c_SEQ = 0 Then
'If c_SEQ = 0 then
conversation initiation and/or opening with SEQ Topic was not successful
MsgBox (TcErrorMsg)
'Display error that occurred,
End
'and end the program
End If
iRet
iRet
iRet
iRet
iRet
iRet
iRet
iRet

=
=
=
=
=
=
=
=

TcSet(c_SEQ,
TcGet(c_SEQ,
TcGet(c_SEQ,
TcGet(c_SEQ,
TcGet(c_SEQ,
TcGet(c_SEQ,
TcGet(c_SEQ,
TcGet(c_SEQ,

"FILE_NAME", fileSpec)
"SEQ_NUM_CYCLES", SeqNumCycles)
"FH_CDATE", seqCreationDate)
"FH_CTIME", seqCreationTime)
"FH_EDATE", seqEditDate)
"FH_ETIME", seqEditTime)
"FH_FILE_AUTHOR", seqFileAuthor)
"FH_ETIME", seqEditTime)

Sheets(chromeraSheetName).Select
Range("A1").FormulaR1C1 = "Batch Name: " & workBookName
Range("A2").FormulaR1C1 = "Description: Data Export from TotalChrom"
Range("A3").FormulaR1C1 = "Date Analyzed: " & seqCreationDate & " " & seqCreationTime
Range("A4").FormulaR1C1 = "Date Exported: " & todaysDate & " " & currentTime
iSeqNumCycles = CInt(SeqNumCycles)
For iSeqCycleIndex = 0 To iSeqNumCycles - 1
iRet = TcSet(c_SEQ, "SEQ_CYCLE_INDEX", iSeqCycleIndex)
iRet = TcGet(c_SEQ, "SD_RESULT_FILE", resultFile)
dSeqIndex_resultFile.Add iSeqCycleIndex, resultFile
iRet = TcGet(c_SEQ, "SD_SAMP_NAME", seqSampleName)
dSeqIndex_seqSampleName.Add iSeqCycleIndex, seqSampleName
iRet = TcGet(c_SEQ, "SD_RAW_FILE", rawFile)
dSeqIndex_rawFile.Add iSeqCycleIndex, rawFile
iRet = TcGet(c_SEQ, "SD_TYPE", seqSampleType)
dSeqIndex_seqSampleType.Add iSeqCycleIndex, seqSampleType
If Left(seqSampleType, 4) = "Cal:" Then
iRet = TcGet(c_SEQ, "SD_EXPT_NAME", seqLevelName)
Else
seqLevelName = ""
End If
dSeqIndex_levelName.Add iSeqCycleIndex, seqLevelName
dLevelName_iSeqCycleIndex.Add seqLevelName, iSeqCycleIndex
Next

Urine arsenic species HPLCICPDRCMS (Formerly Arsenic species in Urine)
DLS Method Code: 3000.1(Formerly 016A/01-OD)

IRAT-DLS
Page 83 of 107

calib_S1_exists = dLevelName_iSeqCycleIndex.Exists(firstLevelName)
If Not calib_S1_exists Then
rsp = MsgBox("Cannot find the first calibrator " & Chr(34) & firstLevelName & Chr(34) & "
in sequence file " & Chr(34) & thisFilename & Chr(34) & "." _
& vbCrLf & "Press Okay to continue without creating the calibration sheets. " &
vbCrLf & "Press Cancel to abort this macro.", vbOKCancel + vbInformation, "MESSAGE")
If rsp = vbCancel Then
ActiveWorkbook.Close SaveChanges:=False
Application.StatusBar = False
Application.DisplayStatusBar = originalStatusBar
Exit Sub
End If
End If
iRet = TcSet(c_SEQ, "SEQ_CYCLE_INDEX", 1)
iRet = TcGet(c_SEQ, "SD_PROCESS_FILE", filespec_mth)
iRet = TcCloseConv(c_SEQ)
c_MTH = TcOpenConv("MTH")
iRet = TcSet(c_MTH, "FILE_NAME", filespec_mth)
iRet = TcGet(c_MTH, "FH_FILE_DES", mthMethodDescription)
iRet = TcGet(c_MTH, "SMP_NUM_COMPONENTS", mthCompStr)
iRet = TcGet(c_MTH, "SMP_RSLT_UNITS", mthConcUnits)
mthCompNum = CInt(mthCompStr)
For i = 0 To mthCompNum - 1
iRet = TcSet(c_MTH, "SMP_COMP_INDEX", i)
iRet = TcGet(c_MTH, "CP_COMP_NAME", mthCompName)
iRet = TcGet(c_MTH, "CP_IS_ISTD", internalStdWasFound)
bIntStdWasFound = CBool(internalStdWasFound)
If bIntStdWasFound Then
internalStdName = mthCompName 'replace default name of internal std with actual name
jComponentIndex = i
Exit For
End If
Next i
iRet =
iRet =
iRet =
iRet =
concIS

TcSet(c_MTH, "SMP_COMP_INDEX", jComponentIndex)
TcSet(c_MTH, "SMP_LEV_INDEX", 1)
TcGet(c_MTH, "LEV_NAME", mthLevelName)
TcGet(c_MTH, "LEV_AMOUNT", mthLevelConc)
= mthLevelConc

iComponentIndex = 0
For i = 0 To mthCompNum - 1
iRet = TcSet(c_MTH, "SMP_COMP_INDEX", i)
iRet = TcGet(c_MTH, "CP_COMP_NAME", mthCompName)
If mthCompName <> internalStdName Then
iRet = TcGet(c_MTH, "CP_NUM_LEVELS", mthLevelCount)
dCompName(iComponentIndex) = mthCompName
dCompName_numberOfLevels(mthCompName) = mthLevelCount
For j = 0 To mthLevelCount - 1
iRet = TcSet(c_MTH, "SMP_LEV_INDEX", j)
iRet = TcGet(c_MTH, "LEV_NAME", mthLevelName)
iRet = TcGet(c_MTH, "LEV_AMOUNT", mthLevelConc)
dLevel2(i) = mthLevelName
dCompName_i(mthCompName & "|" & j) = mthLevelName
dLevel(mthCompName & "|" & mthLevelName) = mthLevelConc
Next j
iComponentIndex = iComponentIndex + 1
End If
Next i
iRet = TcCloseConv(c_MTH)

Urine arsenic species HPLCICPDRCMS (Formerly Arsenic species in Urine)
DLS Method Code: 3000.1(Formerly 016A/01-OD)

IRAT-DLS
Page 84 of 107

c_RAW = TcOpenConv("RAW")
If c_RAW = 0 Then
MsgBox (TcErroMsg)
End
End If
c_RST = TcOpenConv("RST")
'c_RST is the second conversation handle. Opens
conversation with the Result File topic in TcAccess.dll.
If c_RST = 0 Then
'If c_RST = 0 then conversation initiation and/or opening
with SEQ Topic was not successful.
MsgBox (TcErrorMsg)
'Display error that occurred and end the program.
End
End If
For iSeqCycleIndex = 0 To iSeqNumCycles - 1
resultFile = dSeqIndex_resultFile.Item(iSeqCycleIndex)
seqSampleName = dSeqIndex_seqSampleName.Item(iSeqCycleIndex)
seqSampleType = dSeqIndex_seqSampleType.Item(iSeqCycleIndex)
rawFile = dSeqIndex_rawFile.Item(iSeqCycleIndex)
seqLevelName = dSeqIndex_levelName.Item(iSeqCycleIndex)
iRet = TcSet(c_RST, "FILE_NAME", resultFile)
If iRet = -1 Then
ShowWarningMsg = True
numMissingFiles = numMissingFiles + 1
With ActiveCell
.FormulaR1C1 = "FILE NOT FOUND"
.Offset(0, 1).FormulaR1C1 = resultFile
.Offset(1, 0).Select
End With
Else
iRet = TcGet(c_RST, "SD_SAMP_NAME", rstSampleName)
iRet = TcGet(c_RST, "SD_SAMPLE_FILE", rstMethodName)
iRet = TcGet(c_RST, "AD_DATE_STARTED", rstCreationDate)
iRet = TcGet(c_RST, "AD_TIME_STARTED", rstCreationTime)
iRet = TcGet(c_RST, "SD_STUDY_NAME", rstStudyName)
iRet = TcGet(c_RST, "RST_NUM_PEAKS", rstPeakCount)
iRet = TcGet(c_RST, "SD_CYCLE_TEXT", rstSampleNotes)
iRet = TcGet(c_RST, "SMP_NUM_COMPONENTS", rstNumComponents)
iRet = TcGet(c_RST, "AD_NUM_POINTS", nPoints)
iRet = TcGet(c_RST, "SD_DIL_FACTOR", rstSampleDilFactor)
iRet = TcGet(c_RST, "SD_ACTUAL_IS_AMT", rstIntStdConc)
iRet = TcSet(c_RST, "RST_FIND_PEAK_NAME", internalStdName)
If iRet = 0 Then
iRet = TcGet(c_RST, "PK_AREA", IS_peakArea)
ElseIf Not bIntStdWasFound Then
IS_peakArea = 1
End If
stabilityShtRange.Offset(iSeqCycleIndex + 1, 0).Value = IS_peakArea
' check for existence of QC samples, if they exist then rename macro's QC variables
If firstLowQCSample Or firstHighQCSample Then
If seqSampleType = "Ctrl Sample" Then
If LCase(Left(seqSampleName, 3)) Like "[h,l][b,u][_,-]" Then
If UCase(Left(seqSampleName, 1)) = "L" Then
lowBenchQCName = seqSampleName
firstLowQCSample = False
' set low QC sample check to false
End If
If UCase(Left(seqSampleName, 1)) = "H" Then
highBenchQCName = seqSampleName
firstHighQCSample = False ' set high QC sample check to false
End If
End If
End If
End If

Urine arsenic species HPLCICPDRCMS (Formerly Arsenic species in Urine)
DLS Method Code: 3000.1(Formerly 016A/01-OD)

IRAT-DLS
Page 85 of 107

For iComponentIndex = 0 To (rstNumComponents - 1)
strComponent = CStr(iComponentIndex)
iRet = TcSet(c_RST, "SMP_COMP_INDEX", strComponent)
iRet = TcGet(c_RST, "CP_COMP_NAME", peakName)
iRet2 = TcSet(c_RST, "RST_FIND_PEAK_NAME", peakName)
If iRet2
iRet
iRet
iRet
iRet
iRet

=
=
=
=
=
=

0 Then
TcGet(c_RST,
TcGet(c_RST,
TcGet(c_RST,
TcGet(c_RST,
TcGet(c_RST,

"PK_RET_TIME", peakRT)
"PK_HEIGHT", peakHt)
"PK_AREA", peakArea)
"PK_RAW_AMOUNT", componentRawResult)
"PK_RESULT", componentResult)

peakRatio = Val(peakArea) / Val(IS_peakArea)
If seqSampleType = nameOfBlk Then
dPeakNameBlank_peakRatio(peakName) = peakRatio
End If
If dPeakNameBlank_peakRatio.Exists(peakName) Then
peakRatio_blank = dPeakNameBlank_peakRatio.Item(peakName)
Else
peakRatio_blank = 0
End If
'

peakRatio_minus_bk = Val(peakRatio) - Val(peakRatio_blank)
If seqLevelName = zeroLevelName Then
dPeakNameZeroLevel_peakRatio(peakName) = peakRatio
End If
comp_level_Name = peakName & "|" & seqLevelName
dAmount.Add comp_level_Name, peakRatio

equation1 = "Dilution * " & peakName & "_ConcIS * (Ratio_minus_blks Intercept + Ratio_S0)/Slope"
equation2 = "Dilution * " & peakName & "_ConcIS * Ratio_minus_blks/Slope"
If useIntercept = True Then
finalResultFormula = "=If(" & equation1 & " <0, 0, " & equation1 & ")"
Else
finalResultFormula = "=If(" & equation2 & " <0, 0, " & equation2 & ")"
End If
calibCurveCoeff_array = Split(calibCurveCoeffients, Chr(9), -1,
vbBinaryCompare)
calibCurveCoeff_0
calibCurveCoeff_1
calibCurveCoeff_5
peakRT = peakRT /

= Trim(calibCurveCoeff_array(0))
= Trim(calibCurveCoeff_array(1))
= Trim(calibCurveCoeff_array(5))
60

Else

peakRT = "-"
peakHt = 0
peakArea = 0
peakRatio = 0
peakRatio_blank = 0
peakRatio_minus_bk = 0
componentRawResult = 0
finalResultFormula = 0
End If
comp_level_Name = peakName & "|" & seqLevelName
mthLevelConc = dLevel.Item(comp_level_Name)
If dPeakNameZeroLevel_peakRatio.Exists(peakName) Then
peakRatio_zero_calib = dPeakNameZeroLevel_peakRatio.Item(peakName)
Else

Urine arsenic species HPLCICPDRCMS (Formerly Arsenic species in Urine)
DLS Method Code: 3000.1(Formerly 016A/01-OD)

IRAT-DLS
Page 86 of 107

peakRatio_zero_calib = 0
End If
Sheets(resultsSheetName).Select
With ActiveCell
.FormulaR1C1 = resultsSheetName
.Offset(0, 1).FormulaR1C1 = iSeqCycleIndex + 1
.Offset(0, 2).FormulaR1C1 = seqSampleName
.Offset(0, 3).FormulaR1C1 = seqSampleType
.Offset(0, 4).FormulaR1C1 = rstStudyName
.Offset(0, 5).FormulaR1C1 = rstCreationDate & " " & rstCreationTime
.Offset(0, 6).FormulaR1C1 = resultFile
.Offset(0, 7).FormulaR1C1 = rstMethodName
.Offset(0, 8).FormulaR1C1 = rstSampleDilFactor
.Offset(0, 9).FormulaR1C1 = seqLevelName
.Offset(0, 10).FormulaR1C1 = mthLevelConc
.Offset(0, 11).FormulaR1C1 = peakName
.Offset(0, 12).FormulaR1C1 = Format(peakRT, "0.00")
.Offset(0, 13).FormulaR1C1 = Format(peakHt, "0.000")
.Offset(0, 14).FormulaR1C1 = Format(peakArea, "0.000")
.Offset(0, 15).FormulaR1C1 = Format(IS_peakArea, "0.000")
.Offset(0, 16).FormulaR1C1 = peakRatio
.Offset(0, 17).FormulaR1C1 = IIf(peakRatio_blank <> "", peakRatio_blank, 0)
.Offset(0, 18).FormulaR1C1 = "= Ratio - Ratio_Blks"
.Offset(0, 19).FormulaR1C1 = IIf(peakName = internalStdName, 0,
peakRatio_zero_calib)
If peakName <> internalStdName Then
.Offset(0, 20).FormulaR1C1 = "=" & peakName & "_b" ' calibCurveCoeff_0
.Offset(0, 21).FormulaR1C1 = "=" & peakName & "_m" ' calibCurveCoeff_1
.Offset(0, 22).FormulaR1C1 = "=" & peakName & "_r2" ' calibCurveCoeff_5
End If
.Offset(0, 23).FormulaR1C1 = "=COUNT(INDIRECT(Analyte_ID & " & Chr(34) &
"_conc" & Chr(34) & "))"
'Number of calibrators used
.Offset(0, 24).FormulaR1C1 = componentRawResult
If peakName = internalStdName Then
finalResultFormula = 0
End If
.Offset(0, 25).FormulaR1C1 = finalResultFormula
finalResultName = seqSampleName & "_" & iSeqCycleIndex & "_" & peakName
.Offset(0, 25).Name = finalResultName
.Offset(0, 26).FormulaR1C1 = columnDescription
.Offset(0, 27).FormulaR1C1 = calibPrepDate
.Offset(0, 28).FormulaR1C1 = rstSampleNotes
.Offset(1, 0).Select
End With
' now write data to chromeraDataShtName
Sheets(chromeraSheetName).Select
With ActiveCell
.FormulaR1C1 = seqSampleName
.Offset(0, 1).FormulaR1C1 = iSeqCycleIndex + 1
.Offset(0, 2).FormulaR1C1 = massName
.Offset(0, 3).FormulaR1C1 = ""
.Offset(0, 4).FormulaR1C1 = peakName
.Offset(0, 5).FormulaR1C1 = Format(peakRT, "0.00")
.Offset(0, 6).FormulaR1C1 = Format(peakArea, "0.000")
.Offset(0, 7).FormulaR1C1 = Format(peakHt, "0.000")
If peakName = internalStdName Then
finalResultFormula = 0
End If
.Offset(0, 8).FormulaR1C1 = "=" & finalResultName
.Offset(0, 9).FormulaR1C1 = mthConcUnits
.Offset(1, 0).Select
End With
' finished writing data to chromeraDataShtName

Urine arsenic species HPLCICPDRCMS (Formerly Arsenic species in Urine)
DLS Method Code: 3000.1(Formerly 016A/01-OD)

IRAT-DLS
Page 87 of 107

peakIndex = Empty
peakName = Empty
peakHt = Empty
peakArea = Empty
componentResult = Empty
finalResultFormula = Empty
'
peakRatio_minus_bk = Empty
progressIndicator = 100 * iSeqCycleIndex / iSeqNumCycles
Application.StatusBar = "Extracting TotalChrom data, please wait... " &
progressIndicator & "% done."
Next
Sheets(rawSheetName).Select
ActiveCell.Value = seqSampleName
Selection.HorizontalAlignment = xlRight
With Selection.Interior
.ColorIndex = 15
.Pattern = xlSolid
End With
iRet = TcSet(c_RAW, "FILE_NAME", rawFile)
If iRet = -1 Then
ActiveCell.Offset(1, 0).Value = "MISSING RAW DATA FILE"
Else
iRet = TcSet(c_RAW, "RAW_POINT_INDEX", 1)
iRet = TcSet(c_RAW, "RAW_POINT_COUNT", nPoints)
iRet = TcGet(c_RAW, "RAW_DATA_POINTS", rawDataPoints)
iRet = TcGet(c_RST, "IN_SAMP_RATE", sampleRate)
iRet = TcGet(c_RST, "IN_TOTAL_RUN_TIME", runTime)
rawPointsArray = Split(rawDataPoints, Chr(10), -1, vbBinaryCompare)
nPoints = nPoints - 1
For i = 1 To nPoints
If iSeqCycleIndex = 0 Then
ActiveCell.Offset(i, -1).Value = CSng(i / sampleRate) / 60
End If
ActiveCell.Offset(i, 0).Value = Val(rawPointsArray(i - 1))
Next i
End If
ActiveCell.Offset(0, 1).Select
End If
Next
iRet = TcCloseConv(c_RAW)
iRet = TcCloseConv(c_RST)
' Go to Sheets(resultsSheetName) to name ranges
Sheets(resultsSheetName).Select
Range("C1").Select
Range(Selection, Selection.End(xlDown)).Select
nRows = Selection.Rows.Count
Range(ActiveCell, ActiveCell.Offset(nRows - 1, 0)).Select
Selection.Name = "Sample_Name"
Range("D1").Select
Range(ActiveCell, ActiveCell.Offset(nRows - 1, 0)).Select
Selection.Name = "Sample_Type"
Range("I1").Select
Range(ActiveCell, ActiveCell.Offset(nRows - 1, 0)).Select
Selection.Name = "Dilution"
Range("L1").Select
Range(ActiveCell, ActiveCell.Offset(nRows - 1, 0)).Select

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Selection.Name = "Analyte_ID"
Range("O1").Select
Range(ActiveCell, ActiveCell.Offset(nRows - 1, 0)).Select
Selection.Name = "Peak_Area"
Range("P1").Select
Range(ActiveCell, ActiveCell.Offset(nRows - 1, 0)).Select
Selection.Name = "IS_Peak_Area"
Range("Q1").Select
Range(ActiveCell, ActiveCell.Offset(nRows - 1, 0)).Select
Selection.Name = "Ratio"
Range("R1").Select
Range(ActiveCell, ActiveCell.Offset(nRows - 1, 0)).Select
Selection.Name = "Ratio_Blks"
Range("S1").Select
Range(ActiveCell, ActiveCell.Offset(nRows - 1, 0)).Select
Selection.Name = "Ratio_minus_blks"
Range("T1").Select
Range(ActiveCell, ActiveCell.Offset(nRows - 1, 0)).Select
Selection.Name = "Ratio_S0"
Range("U1").Select
Range(ActiveCell, ActiveCell.Offset(nRows - 1, 0)).Select
Selection.Name = "Intercept"
Range("V1").Select
Range(ActiveCell, ActiveCell.Offset(nRows - 1, 0)).Select
Selection.Name = "Slope"
Range("Z1").Select
Range(ActiveCell, ActiveCell.Offset(nRows - 1, 0)).Select
Selection.Name = "Adj_Amount"
ActiveCell.CurrentRegion.Select
Selection.Name = "TC_data"
Selection.AutoFilter
Range("A:A").HorizontalAlignment = xlCenter
Range("A1").Select
' Create Calibration sheets
If calib_S1_exists Then
mthCompNum = dCompName.Count
For iComponentIndex = mthCompNum - 1 To 0 Step -1
peakName = dCompName.Item(iComponentIndex)
Sheets.Add After:=Sheets(1)
Sheets(2).Name = peakName
Range("A1").Formula = "CALIBRATION TABLE - Peak Area Ratios for " & peakName
With Selection.Font
.Name = "Arial"
.FontStyle = "Bold"
.Size = 10
End With
Range("A2").Formula = "Calib Level"
Range("B2").Formula = "Conc"
Range("C2").Formula = "Ratio Conc"
Range("D2").Formula = "Ratio Peak Areas"
Range("E2").Formula = "Deviation %"
Range("B2:E2").HorizontalAlignment = xlRight
Range("A3").Select
mthLevelCount = dCompName_numberOfLevels.Item(peakName)

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For i = 0 To mthLevelCount - 1
peakName_index = peakName & "|" & i
mthLevelName = dCompName_i.Item(peakName_index)
comp_level_Name = peakName & "|" & mthLevelName
mthLevelConc = dLevel.Item(comp_level_Name)
peakRatio = dAmount.Item(comp_level_Name)
ActiveCell.Offset(i, 0).Value = mthLevelName
ActiveCell.Offset(i, 1).Value = mthLevelConc
ActiveCell.Offset(i, 2).FormulaR1C1 = "= RC[-1]/" & peakName & "_ConcIS"
ActiveCell.Offset(i, 3).FormulaR1C1 = IIf(peakRatio = Empty, 0, peakRatio)
If i > 0 Then
ActiveCell.Offset(i, 4).FormulaR1C1 = "= (" & peakName & "_ConcIS*(RC[-1] - "
& peakName & "_b)/" & peakName & "_m)/RC[-3] - 1"
End If
Next i
Range("B3").Select
Range(Selection, Selection.End(xlDown)).Select
Selection.Name = peakName & "_conc"
Selection.NumberFormat = "0.00;-0.00;0.00"
Selection.Offset(0, 1).Select
Selection.Name = peakName & "_ratioConc"
Selection.NumberFormat = "0.0000;-0.0000;0.0000"
Selection.Offset(0, 1).Select
Selection.Name = peakName & "_ratioArea"
Selection.NumberFormat = "0.00000;-0.00000;0.00000"
Selection.Offset(0, 1).Select
Selection.Name = peakName & "_deviation"
Selection.NumberFormat = "0.0%"
Range("B20").Select
Range(Selection, Selection.Offset(4, 1)).Select
Selection.FormulaArray = "=LINEST(" & peakName & "!" & peakName & "_ratioArea" _
& "," & peakName & "_ratioConc, TRUE, TRUE)"
ActiveCell.Name = peakName & "_m"
ActiveCell.Offset(0, 1).Name = peakName & "_b"
ActiveCell.Offset(2, 0).Name = peakName & "_r2"
ActiveCell.Offset(6, 1).FormulaR1C1 = "=(R[-6]C - R[-23]C[1])/R[-6]C[-1]"
ActiveCell.Offset(6, 1).NumberFormat = "0.000%"
ActiveCell.Offset(7, 0).Value = "IS Conc -->"
ActiveCell.Offset(7, 1).FormulaR1C1 = concIS
ActiveCell.Offset(7, 1).NumberFormat = "0.0000"
ActiveCell.Offset(7, 1).Name = peakName & "_ConcIS"
Range("A19").Formula = peakName & " Regression Statistics"
Range("A20").Formula = "Slope, Intercept"
Range("A21").Formula = "SE-m, SE-b"
Range("A22").Formula = "r2, SE-y"
Range("A23").Formula = "F, df"
Range("A24").Formula = "SS-reg, SS-resid"
Columns("A:A").ColumnWidth = 15
Columns("B:B").ColumnWidth = 10
Columns("C:C").ColumnWidth = 10
Columns("D:D").ColumnWidth = 15
Columns("E:E").ColumnWidth = 10
Columns("F:G").ColumnWidth = 1
AddChart peakName & "_ratioConc", peakName & "_ratioArea", peakName, True
Range("A1").Select
Next iComponentIndex
End If
' Done with creating Calibration sheets

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progressIndicator = 100
Application.StatusBar = "Extracting TotalChrom data, please wait... " & progressIndicator &
"% done."
' Clear
Set
Set
Set
Set
Set
Set
Set
Set
Set
Set
Set
Set
Set
Set

dictionary objects
dLevel = Empty
dLevel2 = Empty
dCompName = Empty
dCompName_numberOfLevels = Empty
dCompName_i = Empty
dAmount = Empty
dSeqIndex_resultFile = Empty
dSeqIndex_seqSampleName = Empty
dSeqIndex_seqSampleType = Empty
dSeqIndex_rawFile = Empty
dSeqIndex_levelName = Empty
dLevelName_iSeqCycleIndex = Empty
dPeakNameZeroLevel_peakRatio = Empty
dPeakNameBlank_peakRatio = Empty

' Create the IS Stability sheet
If Not bIntStdWasFound Then
Application.DisplayAlerts = False
Sheets(stabilitySheetName).Delete
Application.DisplayAlerts = True
Else
Sheets(stabilitySheetName).Select
Range(Selection.Offset(1, 0), Selection.End(xlDown)).Select
Selection.Name = "IS_area"
Charts.Add
ActiveChart.ChartType = xlLineMarkers
ActiveChart.SetSourceData Source:=Sheets(stabilitySheetName).Range("IS_area"),
PlotBy:=xlColumns
ActiveChart.Location Where:=xlLocationAsObject, Name:="IS Stability"
With ActiveChart
.HasTitle = True
.ChartTitle.Characters.Text = "IS Peak Stability"
.Axes(xlCategory, xlPrimary).HasTitle = True
.Axes(xlCategory, xlPrimary).AxisTitle.Characters.Text = "Injection Number"
.Axes(xlValue, xlPrimary).HasTitle = True
.Axes(xlValue, xlPrimary).AxisTitle.Characters.Text = "Raw Peak Area"
End With
ActiveChart.Axes(xlValue).Select
With ActiveChart.Axes(xlValue)
.MinimumScale = 0
.MaximumScaleIsAuto = True
.MinorUnitIsAuto = True
.MajorUnitIsAuto = True
.Crosses = xlAutomatic
.ReversePlotOrder = False
.ScaleType = xlLinear
.DisplayUnit = xlNone
.TickLabels.NumberFormat = "0.0;-0.0;0"
.TickLabels.NumberFormat = "0;-0;0"
.TickLabels.AutoScaleFont = True
End With
ActiveChart.Axes(xlValue).Select
With Selection.TickLabels.Font
.Name = "Arial"
.Size = 8
.Strikethrough = False
.Superscript = False
.Subscript = False
.OutlineFont = False
.Shadow = False

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.Underline = xlUnderlineStyleNone
.ColorIndex = xlAutomatic
.Background = xlAutomatic
End With
ActiveChart.Axes(xlCategory).Select
Selection.TickLabels.AutoScaleFont = True
With Selection.TickLabels.Font
.Name = "Arial"
.Size = 8
.Strikethrough = False
.Superscript = False
.Subscript = False
.OutlineFont = False
.Shadow = False
.Underline = xlUnderlineStyleNone
.ColorIndex = xlAutomatic
.Background = xlAutomatic
End With
With Selection.TickLabels
.Alignment = xlCenter
.Offset = 100
.Orientation = xlHorizontal
End With
ActiveChart.SeriesCollection(1).Select
ActiveChart.SeriesCollection(1).Name = "IS"
With ActiveSheet
.ChartObjects(1).Height = 400
.ChartObjects(1).Width = 500
.ChartObjects(1).Left = .Columns("A").Left
.ChartObjects(1).Top = .Rows("1").Top
.ChartObjects(1).Border.LineStyle = xlLineStyleNone
End With
ActiveChart.Deselect
ActiveWindow.Zoom = 100
End If
' Done with creating the IS Stability sheet
' Create the pivot table
Sheets(1).Select
ActiveWorkbook.PivotCaches.Add(SourceType:=xlDatabase,
SourceData:="TC_data").CreatePivotTable TableDestination:="", TableName:="PivotTable_TCData",
DefaultVersion:=xlPivotTableVersion10
ActiveSheet.PivotTableWizard TableDestination:=ActiveSheet.Cells(3, 2)
ActiveSheet.Cells(3, 2).Select
With ActiveSheet.PivotTables("PivotTable_TCData")
.ColumnGrand = False
.HasAutoFormat = False
.DisplayErrorString = True
.ErrorString = "!error"
.RowGrand = False
End With
ActiveSheet.PivotTables("PivotTable_TCData").PivotCache.RefreshOnFileOpen = True
ActiveSheet.PivotTables("PivotTable_TCData").AddFields RowFields:=Array("Sample ID" _
, "Seq No."), ColumnFields:="Analyte ID"
With ActiveSheet.PivotTables("PivotTable_TCData").PivotFields("Adj Amount")
.Orientation = xlDataField
.Caption = "Amount"
.Function = xlAverage
End With
ActiveSheet.PivotTables("PivotTable_TCData").PivotFields("Analyte ID").Subtotals = Array _
(False, False, False, False, False, False, False, False, False, False, False, False)
ActiveSheet.PivotTables("PivotTable_TCData").PivotFields("Seq No.").Subtotals = Array _
(False, False, False, False, False, False, False, False, False, False, False, False)
ActiveSheet.PivotTables("PivotTable_TCData").PivotFields("Sample ID").Subtotals = Array _
(False, False, False, True, False, False, False, False, False, False, False, False)

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With ActiveSheet.PivotTables("PivotTable_TCData").PivotFields("Amount")
.NumberFormat = "0.000"
End With
Range("B4").Select
With ActiveSheet.PivotTables("PivotTable_TCData").PivotFields("Seq No.")
.Orientation = xlRowField
.Position = 2
End With
Range("A4").Select
With ActiveSheet.PivotTables("PivotTable_TCData").PivotFields("Seq No.")
.Orientation = xlRowField
.Position = 2
End With
With ActiveSheet.PivotTables("PivotTable_TCData").PivotFields("Sample ID")
For i = 2 To .PivotItems.Count
.PivotItems(i).Visible = False
Next i
End With
With ActiveSheet.PivotTables("PivotTable_TCData").PivotFields("Sample ID")
If .PivotItems(lowBenchQCName).Position > 0 Then
itDoesExist = True
End If
If itDoesExist Then
.PivotItems(lowBenchQCName).Visible = True
.PivotItems(highBenchQCName).Visible = True
.PivotItems(1).Visible = False
End If
End With
With ActiveSheet.PivotTables("PivotTable_TCData").PivotFields("Analyte ID")
.PivotItems("IS").Visible = False
End With
With ActiveSheet.PivotTables("PivotTable_TCData")
.PivotSelect "", xlDataAndLabel, True
.Format xlTable1
.Columns.AutoFit
.PivotFields("Sample ID").LayoutBlankLine = False
End With
ActiveWorkbook.ShowPivotTableFieldList = False
Application.CommandBars("PivotTable").Visible = False
' Add a button that runs the macro for refreshing the pivot table
Set btn = ActiveSheet.Buttons.Add(3, 3, 70, 16)
macroName = ActiveWorkbook.Name & "!RefreshPivotTable"
btn.OnAction = macroName
btn.Placement = xlFreeFloating
btn.PrintObject = False
btn.Characters.Text = "Refresh Data"
With btn.Characters(Start:=1, Length:=16).Font
.Name = "Arial"
.FontStyle = "Regular"
.Size = 10
.Strikethrough = False
.Superscript = False
.Subscript = False
.OutlineFont = False
.Shadow = False
.Underline = xlUnderlineStyleNone
.ColorIndex = xlAutomatic
End With
' Done with making a button

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Columns("B:Z").ColumnWidth = 12
Range("B4").Select
' Done with making the Pivot Table sheet
qcResultsShtName = "QC Results"
ActiveSheet.Name = qcResultsShtName
Sheets(qcResultsShtName).Move After:=Sheets(resultsSheetName)
' Create a new module in the workbook and insert code
ExportCode workBookName
' Make a chart showing a chromatogram
Sheets(rawSheetName).Select
sampleNamesAddress = Range(Cells(1, 1), Cells(1, iSeqNumCycles)).Address
plotAddress = Range(Cells(1, 1), Cells(nPoints + 1, 2)).Address
Charts.Add
ActiveChart.ChartType = xlXYScatterLinesNoMarkers
ActiveChart.SetSourceData Source:=Sheets(rawSheetName).Range(plotAddress), _
PlotBy:=xlColumns
ActiveChart.Location Where:=xlLocationAsObject, Name:=rawSheetName
With ActiveChart
.HasTitle = True
.ChartTitle.Characters.Text = resultsSheetName
.Axes(xlCategory, xlPrimary).HasTitle = True
.Axes(xlCategory, xlPrimary).AxisTitle.Characters.Text = _
"Retention Time (min)"
.Axes(xlValue, xlPrimary).HasTitle = True
.Axes(xlValue, xlPrimary).AxisTitle.Characters.Text = "Intensity (cps)"
End With
ActiveChart.Axes(xlValue).MajorGridlines.Select
Selection.Delete
ActiveSheet.ChartObjects(1).Select
On Error Resume Next
With ActiveSheet
.ChartObjects(1).Left = .Columns("C").Left
.ChartObjects(1).Top = .Rows("5").Top
.ChartObjects(1).Height = 500
.ChartObjects(1).Width = 700
.ChartObjects(1).Border.LineStyle = xlLineStyleNone
End With
With ActiveChart.Axes(xlValue)
.MinimumScale = 0
.MaximumScaleIsAuto = True
.MinorUnitIsAuto = True
.MajorUnitIsAuto = True
.Crosses = xlAutomatic
.ReversePlotOrder = False
.ScaleType = xlLinear
.DisplayUnit = xlNone
End With
ActiveChart.Axes(xlValue).Select
Selection.TickLabels.AutoScaleFont = False
With Selection.TickLabels.Font
.Name = "Arial"
.Size = 9
.Strikethrough = False
.Superscript = False
.Subscript = False
.OutlineFont = False
.Shadow = False
.Underline = xlUnderlineStyleNone
.ColorIndex = xlAutomatic
.Background = xlAutomatic
End With
ActiveChart.Axes(xlCategory).Select
Selection.TickLabels.AutoScaleFont = True

Urine arsenic species HPLCICPDRCMS (Formerly Arsenic species in Urine)
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With Selection.TickLabels.Font
.Name = "Arial"
.Size = 9
.Strikethrough = False
.Superscript = False
.Subscript = False
.OutlineFont = False
.Shadow = False
.Underline = xlUnderlineStyleNone
.ColorIndex = xlAutomatic
.Background = xlAutomatic
End With
ActiveChart.Axes(xlValue).AxisTitle.Select
Selection.AutoScaleFont = True
With Selection.Font
.Name = "Arial"
.Size = 10
.Strikethrough = False
.Superscript = False
.Subscript = False
.OutlineFont = False
.Shadow = False
.Underline = xlUnderlineStyleNone
.ColorIndex = xlAutomatic
.Background = xlAutomatic
End With
ActiveChart.Axes(xlCategory).AxisTitle.Select
Selection.AutoScaleFont = True
With Selection.Font
.Name = "Arial"
.Size = 10
.Strikethrough = False
.Superscript = False
.Subscript = False
.OutlineFont = False
.Shadow = False
.Underline = xlUnderlineStyleNone
.ColorIndex = xlAutomatic
.Background = xlAutomatic
End With
ActiveChart.Legend.Select
Selection.AutoScaleFont = True
With Selection.Border
.Weight = xlHairline
.LineStyle = xlAutomatic
End With
Selection.Shadow = False
With Selection.Interior
.ColorIndex = 15
.PatternColorIndex = 1
.Pattern = xlSolid
End With
ActiveChart.Legend.Select
Selection.AutoScaleFont = True
With Selection.Font
.Name = "Arial"
.Size = 9
.Strikethrough = False
.Superscript = False
.Subscript = False
.OutlineFont = False
.Shadow = False
.Underline = xlUnderlineStyleNone
.ColorIndex = xlAutomatic
.Background = xlAutomatic
End With
ActiveChart.ChartTitle.Select
With Selection.Characters.Font

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.Name = "Arial"
.FontStyle = "Bold"
.Size = 10
.Strikethrough = False
.Superscript = False
.Subscript = False
.OutlineFont = False
.Shadow = False
.Underline = xlUnderlineStyleNone
.ColorIndex = xlAutomatic
End With
If Not firstLowQCSample Then
Set c = Range(sampleNamesAddress).Find(lowBenchQCName, LookIn:=xlValues)
If Not c Is Nothing Then
thisAddress = c.Address
End If
Range(thisAddress, Range(thisAddress).End(xlDown)).Select
Selection.Copy
ActiveSheet.ChartObjects("Chart 1").Activate
ActiveChart.SeriesCollection.Paste Rowcol:=xlColumns, SeriesLabels:=True,
CategoryLabels:=False, Replace:=False, NewSeries:=True
Application.CutCopyMode = False
End If
If Not firstHighQCSample Then
Set c = Range(sampleNamesAddress).Find(highBenchQCName, LookIn:=xlValues)
If Not c Is Nothing Then
thisAddress = c.Address
End If
Range(thisAddress, Range(thisAddress).End(xlDown)).Select
Selection.Copy
ActiveSheet.ChartObjects("Chart 1").Activate
ActiveChart.SeriesCollection.Paste Rowcol:=xlColumns, SeriesLabels:=True,
CategoryLabels:=False, Replace:=False, NewSeries:=True
Application.CutCopyMode = False
End If
If (Not firstLowQCSample) Or (Not firstHighQCSample) Then
ActiveChart.SeriesCollection(1).Delete
End If
For Each s In ActiveChart.SeriesCollection
s.Smooth = True
Next s
ActiveChart.Deselect
' Done making a chart showing a chromatogram
' Add a button that runs the macro for adding a new series
Set btn = ActiveSheet.Buttons.Add(5, 25, 100, 25)
'
ActiveSheet.Shapes(ActiveSheet.Shapes.Count).Select
macroName = ActiveWorkbook.Name & "!Chart_InsertNewSeries"
btn.OnAction = macroName
btn.Placement = xlFreeFloating
btn.PrintObject = False
btn.Characters.Text = "Add Chromatogram"
With btn.Characters(Start:=1, Length:=16).Font
.Name = "Arial"
.FontStyle = "Regular"
.Size = 10
.Strikethrough = False
.Superscript = False
.Subscript = False
.OutlineFont = False

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.Shadow = False
.Underline = xlUnderlineStyleNone
.ColorIndex = xlAutomatic
End With
Range("A1").Select
' Done with making a button
' Move the Chromera data sheet to the front, unless the first calibrator was
' not found then delete the Chromera data sheet
If calib_S1_exists Then
Sheets(chromeraSheetName).Move After:=Sheets.Count
Else
Application.DisplayAlerts = False
Sheets(chromeraSheetName).Delete
Sheets(qcResultsShtName).Delete
Application.DisplayAlerts = True
Sheets(resultsSheetName).Select
Range("Adj_Amount").Select
For Each c In Selection
If IsError(c.Value) Then
c.Value = 0
End If
Next c
End If
' Go to the first sheet, hide columns, turn on screen updating and return the status bar to it's
original state
Sheets(resultsSheetName).Select
Range("R:X").EntireColumn.Hidden = True
Range("A1").Select
With ActiveWorkbook.BuiltinDocumentProperties
.Item("Title").Value = workBookName & savedNameSuffix
.Item("Subject").Value = "Results imported from TotalChrome " & strVersionNo
.Item("Author").Value = seqFileAuthor
.Item("Category").Value = "summary results"
.Item("Keywords").Value = ""
.Item("Comments").Value = "The TotalChrom sequence file used to export data " _
& "to this Excel workbook was created " & seqCreationDate & " " & seqCreationTime &
"." _
& vbCrLf & mthMethodDescription
.Item("Manager").Value = ""
.Item("Company").Value = ""
End With
Application.StatusBar = False
Application.DisplayStatusBar = originalStatusBar
Application.ScreenUpdating = True
If ShowWarningMsg = True Then
MsgBox "Data from " & CStr(numMissingFiles) & " TotalChrom files failed to be extracted.
Check for " & vbCrLf & _
"missing or renamed .RST files in the sequence file " & Chr(34) & thisFilename &
Chr(34) & vbCrLf & _
"and the source folder containing its data files.", _
vbOKOnly + vbExclamation, "Warning"
End If
' Bring up the SaveAs dialog box, if the PromptForSaveAsDialog switch is true
If True Then
currentDir = CurDir
filePathName = "C:\HPLC\Data\" & workBookName
dirExist = Dir(filePathName, vbDirectory)
' Retrieve the first entry.
If dirExist <> "" Then
ChDir filePathName
Else
ChDir "C:\"
End If

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nameSaveAsFile = Application.GetSaveAsFilename(InitialFileName:=workBookName & _
savedNameSuffix, FileFilter:="Excel normal workbook (*.xls),*.xls")
If nameSaveAsFile <> False Then
ActiveWorkbook.SaveAs fileName:=nameSaveAsFile, _
FileFormat:=xlWorkbookNormal, AddToMru:=True, _
ConflictResolution:=xlUserResolution
End If
ChDir currentDir
End If
End Sub
'
' breakHere = IIf(componentRawResult > 0, True, False)
' breakHere = IIf(iSeqCycleIndex = 18, True, False)
' thisFilename = Right(filespec, Len(filespec) - InStrRev(filespec, "\", -1, vbTextCompare))
' workBookName = Left(thisFilename, Len(thisFilename) - 4)
Sub AddChart(x_rangeToPlot, y_rangeToPlot, chartName, useIntercept)
' Subroutine for "TransformHPLCdata"
' Macro created 12/5/2002
' Written by Carl P. Verdon, Ph.D.
'
Charts.Add
ActiveChart.ChartType = xlXYScatter
ActiveChart.SetSourceData Source:=Sheets(chartName).Range(x_rangeToPlot, _
Range(x_rangeToPlot).Offset(0, 1)), PlotBy:=xlColumns
ActiveChart.Location Where:=xlLocationAsObject, Name:=chartName
ActiveSheet.ChartObjects(1).Select
On Error Resume Next
With ActiveSheet
.ChartObjects(1).Left = .Columns("H").Left
.ChartObjects(1).Top = .Rows("1").Top
.ChartObjects(1).Height = 400
.ChartObjects(1).Width = 400
.ChartObjects(1).Border.LineStyle = xlLineStyleNone
End With
With ActiveChart
.HasTitle = True
.ChartTitle.Characters.Text = chartName
.HasLegend = False
.DisplayBlanksAs = xlNotPlotted
.PlotVisibleOnly = True
.SizeWithWindow = False
.Axes(xlCategory, xlPrimary).HasTitle = True
.Axes(xlCategory, xlPrimary).AxisTitle.Characters.Text = _
"Concentration (Ratio [Calib]/[IS])"
.Axes(xlValue, xlPrimary).HasTitle = True
.Axes(xlValue, xlPrimary).AxisTitle.Characters.Text = _
"Ratio (Peak Area/IS Peak Area)"
End With
seriesFormulaText = "=SERIES('Calib'!RC,'Calib'!" + x_rangeToPlot + _
",'Calib'!" + y_rangeToPlot + ",1)"
ActiveChart.SeriesCollection(1).Formula = seriesFormulaText
ActiveChart.SeriesCollection(1).Select
With Selection.Border
.Weight = xlHairline
.LineStyle = xlNone
End With
With Selection
.MarkerBackgroundColorIndex = 27
.MarkerForegroundColorIndex = 1
.MarkerStyle = xlDiamond
.Smooth = False
.MarkerSize = 7
.Shadow = False
End With
If useIntercept Then
ActiveChart.SeriesCollection(1).Trendlines.Add(Type:=xlLinear, Forward:=0, _

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Backward:=0, DisplayEquation:=True, DisplayRSquared:=True, _
Name:="Regression").Select
Else
ActiveChart.SeriesCollection(1).Trendlines.Add(Type:=xlLinear, Forward:=10, _
Backward:=0, Intercept:=0, DisplayEquation:=True, DisplayRSquared:=True, _
Name:="Regression").Select
End If
ActiveChart.Axes(xlValue).Select
With ActiveChart.Axes(xlValue)
.MinimumScale = 0
.MaximumScaleIsAuto = True
.MinorUnitIsAuto = True
.MajorUnitIsAuto = True
.Crosses = xlAutomatic
.ReversePlotOrder = False
.ScaleType = xlLinear
.DisplayUnit = xlNone
.TickLabels.NumberFormat = "0.00;-0.00;0"
End With
ActiveChart.Axes(xlValue).MajorGridlines.Select
Selection.Delete

ActiveChart.SeriesCollection(1).Trendlines(1).Select
With Selection.Border
.ColorIndex = 57
.Weight = xlThin
.LineStyle = xlContinuous
End With
ActiveChart.ChartTitle.Select
Selection.Characters.Text = chartName
Selection.AutoScaleFont = False
With Selection.Characters(Start:=1, Length:=2).Font
With Selection.Characters.Font
.Name = "Arial"
.FontStyle = "Bold"
.Size = 12
.Strikethrough = False
.Superscript = False
.Subscript = False
.OutlineFont = False
.Shadow = False
.Underline = xlUnderlineStyleNone
.ColorIndex = xlAutomatic
End With
ActiveChart.SeriesCollection(1).Trendlines(1).DataLabel.Select
With Selection.Border
.ColorIndex = 1
.Weight = xlThin
.LineStyle = xlContinuous
End With
Selection.Shadow = True
With Selection.Interior
.ColorIndex = 2
.PatternColorIndex = 1
.Pattern = xlSolid
End With
Selection.AutoScaleFont = True
With Selection.Font
.Name = "Arial"
.FontStyle = "Bold"
.Size = 10
.Strikethrough = False
.Superscript = False
.Subscript = False
.OutlineFont = False
.Shadow = False

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.Underline = xlUnderlineStyleNone
.ColorIndex = xlAutomatic
.Background = xlAutomatic
End With
Selection.NumberFormat = "0.0000"
Selection.Left = 88
Selection.Top = 76
ActiveChart.Deselect
End Sub
Sub addCalibSheet(speciesName, useIntercept, numberOfCalibrators)
' Subroutine for "TransformHPLCdata"
' Macro created 12/5/2002
' Written by Carl P. Verdon, Ph.D.
'
Sheets("Calib").Copy After:=Sheets(Sheets.Count)
Sheets(Sheets.Count).Name = speciesName
Sheets(speciesName).Select
Range("C2").Select
Range(ActiveCell, Cells(ActiveCell.Row, lastCol - 1)).Select
ncols = Selection.Columns.Count
For i = 1 To ncols
If ActiveCell.Formula <> speciesName Then
ActiveCell.EntireColumn.Delete
Else
ActiveCell.Offset(0, 1).Select
End If
Next
speciesName_calibConc = speciesName + "_calibConc"
Range("B3").Select
Range(ActiveCell, Selection.End(xlDown)).Select
Selection.Name = speciesName_calibConc
Selection.Offset(0, 1).Name = speciesName
targetRange1 = numberOfCalibrators + 10
colNumber = 2
Range(Cells(targetRange1, colNumber), Cells(targetRange1 + 4, _
colNumber + 1)).Select
Selection.FormulaArray = "=LINEST(Calib!" + speciesName + _
"," + speciesName_calibConc + "," + CStr(useIntercept) + ",TRUE)"
ActiveSheet.Select
Cells(targetRange1 - 1, colNumber - 1).FormulaR1C1 = speciesName + _
" Regression Statistics"
Cells(targetRange1 + 0, colNumber - 1).FormulaR1C1 = "Slope, Intercept"
Cells(targetRange1 + 1, colNumber - 1).FormulaR1C1 = "SE-m, SE-b"
Cells(targetRange1 + 2, colNumber - 1).FormulaR1C1 = "r2, SE-y"
Cells(targetRange1 + 3, colNumber - 1).FormulaR1C1 = "F, df"
Cells(targetRange1 + 4, colNumber - 1).FormulaR1C1 = "SS-reg, SS-resid"
Cells(targetRange1, colNumber).Name = speciesName + "_slope"
Cells(targetRange1, colNumber + 1).Name = speciesName + "_intercept"
Range(Cells(targetRange1, colNumber), Cells(targetRange1 + 1, _
colNumber + 1)).Select
Selection.NumberFormat = "0.0000"
Cells(targetRange1 - 1, colNumber - 1).Select
With Selection.Font
.Name = "Arial"
.FontStyle = "Bold"
.Size = 10
End With
Cells(targetRange1 + 1, colNumber + 2).FormulaR1C1 = "=R[-1]C[-1]/R[-1]C[-2]"
Cells(targetRange1 + 1, colNumber + 2).NumberFormat = "0%;-0%;0%"
Columns("A:A").ColumnWidth = 14

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Range("A1").Select
AddChart speciesName_calibConc, speciesName, speciesName, useIntercept
End Sub
Sub AddMenuItems()
Dim MenuControls As Object, MenuItem As Object, menuExists As Boolean
menuExists = False
Set MenuControls = Application.CommandBars(1).Controls
For Each menuPopup In MenuControls
If menuPopup.Caption = "&HPLC" Then
menuExists = True
End If
Next
If Not menuExists Then
Set newMenuItem = CommandBars(1).Controls.Add(Type:=msoControlPopup, Before:=9)
With newMenuItem
.Caption = "&HPLC"
End With
With CommandBars(1).Controls("&HPLC")
.Controls.Add(Type:=msoControlButton, Before:=1).Caption = "&Create TC Sequence File"
.Controls("&Create TC Sequence File").OnAction = "MakeSequenceList"
.Controls.Add(Type:=msoControlButton, Before:=2).Caption = "Create &Summary.csv File"
.Controls("Create &Summary.csv File").OnAction = "LaunchSummaryExe"
.Controls.Add(Type:=msoControlButton, Before:=3).Caption = "Transform &HPLC Data"
.Controls("Transform &HPLC Data").OnAction = "TransformHPLCdata"
.Controls.Add(Type:=msoControlButton, Before:=4).Caption = "&Extract TC Data"
.Controls("&Extract TC Data").OnAction = "ExtractTCData"
.Controls("&Extract TC Data").BeginGroup = True
.Controls.Add(Type:=msoControlButton, Before:=5).Caption = "&Get Daily Performances"
.Controls("&Get Daily Performances").OnAction = "getDailyPerformIntensities"
.Controls.Add(Type:=msoControlButton, Before:=6).Caption = "&Import XY Data"
.Controls("&Import XY Data").OnAction = "Import_XY_data"
.Controls.Add(Type:=msoControlButton, Before:=7).Caption = "Set &Parameters"
.Controls("Set &Parameters").BeginGroup = True
.Controls("Set &Parameters").OnAction = "SetRegisters"
End With
End If
End Sub
Sub DeleteMenuItems()
Dim MenuControls As Object, MenuItem As Object, menuExists As Boolean
menuExists = False
Set MenuControls = Application.CommandBars(1).Controls
For Each menuPopup In MenuControls
If menuPopup.Caption = "&HPLC" Then
menuExists = True
End If
Next
If menuExists Then
CommandBars(1).Controls("&HPLC").Delete
End If
End Sub
Public Sub Auto_Open()
Application.Workbooks.Add
AddMenuItems

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End Sub
Public Sub Auto_Close()
DeleteMenuItems
End Sub
Public Sub SetRegisters()
'
' concIS As Variant
' injNumberPattern As String
' howManyBlanks As Variant
' altFormulaCommentCode As String
'
pathToMethodParametersFile = OGetSetting("VBA", "Sub_HPLCtransformVariables",
"pathToMethodParametersFile_str", "C:\HPLC\Macro\Config.txt", True)
useIntercept = OGetSetting("VBA", "Sub_HPLCtransformVariables", "UseIntercept_boolean",
False, True)
howManyBlanks = OGetSetting("VBA", "Sub_HPLCtransformVariables", "HowManyBlanks_integer", 3,
True)
blankName = OGetSetting("VBA", "Sub_HPLCtransformVariables", "BlankName_string", "Bk", True)
lowBenchQCName = OGetSetting("VBA", "Sub_HPLCtransformVariables", "LowBenchQCName_string",
"LU-03102", True)
highBenchQCName = OGetSetting("VBA", "Sub_HPLCtransformVariables", "HighBenchQCName_string",
"HU-03104", True)
massName = OGetSetting("VBA", "Sub_HPLCtransformVariables", "MassName_string", "As", True)
dilFactorDelimiter = OGetSetting("VBA", "Sub_HPLCtransformVariables",
"dilFactorDelimiter_string", "^", True)
columnDescription = OGetSetting("VBA", "Sub_HPLCtransformVariables",
"columnDescription_string", "not entered", True)
zeroCalibName = OGetSetting("VBA", "Sub_HPLCtransformVariables", "zeroCalibName_string",
"S0", True)
calibPrepDate = OGetSetting("VBA", "Sub_HPLCtransformVariables", "calibPrepDate_string", "",
True)
listBoxEntries = Array(1, 2, 3, 4, 5, 6, 7)
With SetVariablesForm1
.TextBox1.Value = pathToMethodParametersFile
.TextBox2.Value = blankName
.TextBox3.Value = lowBenchQCName
.TextBox4.Value = highBenchQCName
.TextBox5.Value = massName
.TextBox6.Value = columnDescription
.TextBox7.Value = zeroCalibName
.TextBox8.Value = calibPrepDate
.OptionButton3.Value = useIntercept
.OptionButton4.Value = Not useIntercept
.ComboBox1.List() = listBoxEntries
.ComboBox1.Text = howManyBlanks
End With

SetVariablesForm1.CommandButtonDone.Default = True
CommandButtonDoneClicked = False
SetVariablesForm1.Show
If CommandButtonDoneClicked Then
CommandButtonDoneClicked = True
SetVariablesForm1.Hide
End If
If CommandButtonDoneCancel Then
CommandButtonDoneClicked = False
SetVariablesForm1.Hide
End If
If CommandButtonDoneClicked Then

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With SetVariablesForm1
pathToMethodParametersFile = .TextBox1.Value
blankName = .TextBox2.Value
lowBenchQCName = .TextBox3.Value
highBenchQCName = .TextBox4.Value
massName = .TextBox5.Value
columnDescription = .TextBox6.Value
zeroCalibName = .TextBox7.Value
calibPrepDate = .TextBox8.Value
howManyBlanks = CInt(.ComboBox1.Text)
If .OptionButton3.Value = True Then
useIntercept = True
End If
If .OptionButton4.Value = True Then
useIntercept = False
End If
End With
Else
Exit Sub
End If
OSaveSetting "VBA", "Sub_HPLCtransformVariables",
pathToMethodParametersFile, True
OSaveSetting "VBA", "Sub_HPLCtransformVariables",
OSaveSetting "VBA", "Sub_HPLCtransformVariables",
OSaveSetting "VBA", "Sub_HPLCtransformVariables",
True
OSaveSetting "VBA", "Sub_HPLCtransformVariables",
True
OSaveSetting "VBA", "Sub_HPLCtransformVariables",
True
OSaveSetting "VBA", "Sub_HPLCtransformVariables",
OSaveSetting "VBA", "Sub_HPLCtransformVariables",
dilFactorDelimiter, True
OSaveSetting "VBA", "Sub_HPLCtransformVariables",
columnDescription, True
OSaveSetting "VBA", "Sub_HPLCtransformVariables",
OSaveSetting "VBA", "Sub_HPLCtransformVariables",

"pathToMethodParametersFile_str",
"UseIntercept_boolean", useIntercept, True
"BlankName_string", blankName, True
"HowManyBlanks_integer", howManyBlanks,
"LowBenchQCName_string", lowBenchQCName,
"HighBenchQCName_string", highBenchQCName,
"MassName_string", massName, True
"dilFactorDelimiter_string",
"columnDescription_string",
"zeroCalibName_string", zeroCalibName, True
"calibPrepDate_string", calibPrepDate, True

'
prompt = "Set the search criteria for the macro Delete Rows by Criteria. " & _
'
"Wild card values # (any single number), ? (any single character) and * (any number
of characters/numbers) are allowed."
'
criteria = Application.InputBox(prompt:=prompt, Type:=2, Default:=criteria)
End Sub
Public Function OGetSetting(ByVal AppName As String, ByVal Section As String, ByVal KeyName As
String, ByVal Default As Variant, Optional ByVal System As Boolean = False) As String
Set wshShell = CreateObject("WScript.Shell")
On Error GoTo errBadRead
OGetSetting = wshShell.RegRead(IIf(System, "HKLM", "HKCU") & "\Software\Microsoft\" & AppName
& "\" & Section & "\" & KeyName)
Set wshShell = Nothing
Exit Function
errBadRead:
OGetSetting = Default
Set wshShell = Nothing
End Function
Public Function OSaveSetting(ByVal AppName As String, ByVal Section As String, ByVal KeyName As
String, ByVal Value As Variant, Optional ByVal System As Boolean = False)
Set wshShell = CreateObject("WScript.Shell")
On Error GoTo errBadWrite
Call wshShell.RegWrite(IIf(System, "HKLM", "HKCU") & "\Software\Microsoft\" & AppName & "\" &
Section & "\" & KeyName, Value)

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Set wshShell = Nothing
Exit Function
errBadWrite:
Set wshShell = Nothing
End Function
Public Function ODeleteSetting(ByVal AppName As String, ByVal Section As String, ByVal KeyName As
String, Optional ByVal System As Boolean = False)
Set wshShell = CreateObject("WScript.Shell")
On Error GoTo errBadDelete
Call wshShell.RegDelete(IIf(System, "HKLM", "HKCU") & "\Software\Microsoft\" & AppName & "\"
& Section & "\" & KeyName)
Set wshShell = Nothing
Exit Function
errBadDelete:
Set wshShell = Nothing
End Function
Public Function Pause5seconds()
Dim pauseSeconds As Long
pauseSeconds = 5
newHour = Hour(Now())
newMinute = Minute(Now())
newSecond = Second(Now()) + pauseSeconds
waitTime = TimeSerial(newHour, newMinute, newSecond)
Application.Wait waitTime
End Function
Public Function ExportCode(ByVal wkbkName As String)
' Macro created by Carl Verdon, 2/24/2005
' Updated by C. Verdon 8/9/2006
'

Dimension variables
Dim strCode1 As String
Dim strCode2 As String
Dim strProjName As String
Dim strChartName As String
Dim modObj As Object
Dim modObjItem As Object

Inialize variables
strProjName = "VBAProject"
strChartName = "Chart 1"
strCode1 = _
"Sub Chart_InsertNewSeries()" & vbCrLf & _
"
" & vbCrLf & _
"
Dim c as Range" & vbCrLf & _
"
Dim s as Series" & vbCrLf & _
"
" & vbCrLf & _
"
Application.ScreenUpdating = False" & vbCrLf & _
"
Range(Cells(1, ActiveCell.Column), Cells(1, ActiveCell.Column + Selection.Columns.Count
- 1)).Select" & vbCrLf & _
"
Set c = Selection" & vbCrLf & _
"
If (ActiveCell.FormulaR1C1 <> Empty) And (ActiveCell.Column <> 1) Then" & vbCrLf & _
"
Range(Selection, Selection.End(xlDown)).Select" & vbCrLf & _
"
Selection.Copy" & vbCrLf & _
"
ActiveSheet.ChartObjects(" & Chr(34) & strChartName & Chr(34) & ").Activate" &
vbCrLf & _
"
ActiveChart.SeriesCollection.Paste Rowcol:=xlColumns, SeriesLabels:=True,
CategoryLabels:=False, Replace:=False, NewSeries:=True" & vbCrLf & _
"
Application.CutCopyMode = False" & vbCrLf & _
"
For each s in ActiveChart.SeriesCollection" & vbCrLf & _
"
s.Smooth = True" & vbCrLf & _
"
Next s" & vbCrLf & _
"
ActiveChart.Deselect" & vbCrLf & _
"
c.Select" & vbCrLf & _
"
Else" & vbCrLf & _
"
MsgBox" & Chr(34) & "This column does not contain signal data. Select a column

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header for a column that contains signal data." & Chr(34) & vbCrLf & _
"
End If" & vbCrLf & _
"
" & vbCrLf & _
"End Sub"
strCode2 = _
"Sub RefreshPivotTable()" & vbCrLf & _
"
" & vbCrLf & _
"
Application.ScreenUpdating = False" & vbCrLf & _
"
ActiveSheet.PivotTables(" & Chr(34) & "PivotTable_TCData" & Chr(34) &
").PivotCache.Refresh" & vbCrLf & _
"
Columns(" & Chr(34) & "B:Z" & Chr(34) & ").ColumnWidth = 12" & vbCrLf & _
"
" & vbCrLf & _
"End Sub"
'

Main
Set modObj = Application.VBE.VBProjects(strProjName).Collection
Set modObjItem = modObj.Item(modObj.Count)
modObjItem.VBComponents.Add (vbext_ct_StdModule)
modObjItem.VBComponents.Item("Module1").CodeModule.AddFromString (strCode1)
modObjItem.VBComponents.Item("Module1").CodeModule.AddFromString (strCode2)

End Function
' Macro created 4/18/2006 by Carl Verdon
Public Function MakePivotTable( _
ByVal mySourceData As Range, _
ByVal myTableName As String, _
ByVal myRowField As String, _
ByVal myColField As String, _
ByVal myDataField As String, _
Optional ByVal startRow As Integer, _
Optional ByVal startCol As Integer _
)
Application.ScreenUpdating = False
If startRow = Empty Then startRow = 3
If startCol = Empty Then startCol = 1
Set sourceDataArea = mySourceData
ActiveWorkbook.PivotCaches.Add(SourceType:=xlDatabase, _
SourceData:=sourceDataArea).CreatePivotTable _
TableDestination:="", _
TableName:=myTableName, _
DefaultVersion:=xlPivotTableVersion10
ActiveSheet.PivotTableWizard TableDestination:=ActiveSheet.Cells(startRow, startCol)
ActiveSheet.Cells(startRow, startCol).Select
With ActiveSheet.PivotTables(myTableName)
.ColumnGrand = False
.HasAutoFormat = False
.DisplayErrorString = True
.ErrorString = "?Data"
.NullString = "-"
.PreserveFormatting = False
.RowGrand = False
End With
ActiveSheet.PivotTables(myTableName).AddFields RowFields:=myRowField,
ColumnFields:=myColField
ActiveSheet.PivotTables(myTableName).HasAutoFormat = True
With ActiveSheet.PivotTables(myTableName).PivotFields(myDataField)
.Orientation = xlDataField
.Caption = "Ave Data"
.Function = xlAverage

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End With
With ActiveSheet.PivotTables(myTableName).PivotFields("Ave Data")
.NumberFormat = "0.0"
End With
With ActiveSheet.PivotTables(myTableName).PivotFields(myColField)
.PivotItems("(blank)").Visible = False
End With
With ActiveSheet.PivotTables(myTableName).PivotFields(myRowField)
.PivotItems("(blank)").Visible = False
End With
ActiveWorkbook.ShowPivotTableFieldList = False
Application.CommandBars("PivotTable").Visible = False
ActiveSheet.Cells(startRow, startCol).Select
ActiveSheet.Name = "Calib"
'
'
'

Sub RefreshPivotTable1()
ActiveSheet.PivotTables("PivotTable1").PivotCache.Refresh
End Sub

End Function

End of Program Listing

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b. Sample Chromatographic Report
Example chromatogram of a bench QC sample below:

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c. Sample HPLC Batch Run “Results” File in Microsoft Excel®
A screen shot of a "results” file in Microsoft Excel® after transformation by the Excel Macro
“Extract TC Data”.

Division of Laboratory Sciences
Laboratory Protocol

Analyte:
Matrix:
Method:
Method Code:
Branch:

Iodine & Mercury
Urine
Inductively Coupled Plasma Dynamic Reaction Cell Mass
Spectrometry (ICP-DRC-MS)
3002.1
Inorganic Radiation Analytical Toxicology

Prepared By:
Supervisor:

Ge Xiao, PhD
author’s name

Kathleen L. Caldwell, PhD
Supervisor’s name

Branch Chief:

Robert L. Jones, PhD

Adopted:

01 September 2005

Updated:

31 May 2010

Branch Chief

signature

date

signature

date

signature and date

date

Director's Signature Block:
Reviewed:
signature

date

signature

date

signature

date

signature

date

signature

Modifications/Changes: see Procedure Change Log

date

Procedure Change Log
Procedure: Iodine & Mercury DLS Method Code: 3002.1

Date
09/19/2011

Changes Made
Use of magnetic stirrer with diluent during preparation
documented.

By
DH

Rev’d
By
(Initials)
JJ

Date
Rev’d
09/20/2011

Laboratory Procedure Manual
Analyte:

Iodine & Mercury

Matrix:

Urine
Inductively Coupled Plasma Dynamic
Reaction Cell Mass Spectrometry
(ICP-DRC-MS)

Method:

Method No:

3002.1

Revised:
as performed by:
Inorganic Radionuclides and Toxicology
Division of Laboratory Sciences
National Center for Environmental Health
contact:

Dr. Robert L. Jones
Phone: 770-488-7991
Fax:
770-488-4097
Email: [email protected]
James L. Pirkle, M.D., Ph.D.
Director, Division of Laboratory Sciences

Important Information for Users

CDC periodically refines these laboratory methods. It is the responsibility of the user to contact
the person listed on the title page of each write-up before using the analytical method to find out
whether any changes have been made and what revisions, if any, have been incorporated.

Iodine and Mercury in Urine

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Iodine and Mercury in Urine

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DLS Method Code: 3002.1

TABLE OF CONTENTS
1.

CLINICAL RELEVANCE AND TEST PRINCIPLE ............................................................... 1
a.

Clinical Relevance

Test Principle

SAFETY PRECAUTIONS....................................................................................................... 2

DATA SYSTEM MANAGEMENT ........................................................................................... 3
a.

Data Entry and Transfer

Routine Computer Hard Drive Maintenance

Data Backup

(1) Schedule of Data Backups
(2) Backup Procedures

3
3

Documentation of System Maintenance

4.
COLLECTING, STORING, AND HANDLING SPECIMENS; CRITERIA FOR
REJECTING SPECIMENS ................................................................................................................... 5
a.

Specimen Type

Specimen Collection, Handling and Storage

Criteria for an Unacceptable Specimen

PROCEDURES FOR MICROSCOPIC EXAMINATIONS.................................................... 6

CHEMICALS, STANDARDS, AND QUALITY CONTROL MATERIAL ............................. 6
a.

Chemicals

Reagent Preparation

STANDARDS PREPARATION .............................................................................................. 9

INSTRUMENT & SOFTWARE FOR THE ICP-DRC- MS .................................................. 12

10.

a. ICP-DRC-MS System

Other equipment

Supplies

CALIBRATION AND CALIBRATION-VERIFICATION PROCEDURES .......................... 14
a. Calibration Curve

b. Calibration Verification

OPERATING PROCEDURES; CALCULATIONS; INTERPRETATION OF RESULTS . 15

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DLS Method Code: 3002.1

11.

a. Preliminaries

b. Sample Preparation

c. Instrument Setup and Configuration

d. Recording of Data

FINAL REVIEW OF THE DATA ........................................................................................... 23
a.

AbnormalPatient Results

Evaluating Bench QC of a run

12.

REPLACEMENT AND PERIODIC MAINTENANCE OF KEY COMPONENTS ............. 24

13.

LIMIT OF DETECTION ......................................................................................................... 25

14.

REPORTABLE RANGE OF RESULTS............................................................................... 25

15.

SPECIAL PROCEDURE NOTES – CDC MODIFICATIONS ............................................ 26

16.

QUALITY CONTROL PROCEDURES ................................................................................ 26

17.

Establish QC limits for each QC pool.

Evaluating the QC of a run

Remedial action if Calibration or QC systems fail acceptable criteria

REFERENCE RANGES........................................................................................................ 28

18. ACTION-LEVEL RESULTS ........................................................................................................ 28
19. SPECIMEN STORAGE AND HANDLING DURING TESTING .............................................. 28
20. ALTERNATE METHODS FOR PERFORMING TEST AND STORING SPECIMENS IF
TEST SYSTEM FAILS......................................................................................................................... 28
21. TEST - RESULT REPORTING SYSTEM; PROTOCOL FOR REPORTING CRITICAL
CALLS (IF APPLICABLE) .................................................................................................................. 29
22. TRANSFER OR REFERRAL OF SPECIMENS; PROCEDURES FOR SPECIMEN
ACCOUNTABILITY AND TRACKING ............................................................................................... 29
23.

REFERENCES ...................................................................................................................... 29

24.

APPENDIX A – RUGGEDNESS TESTING……………...................................................30

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Iodine and Mercury in Urine
DLS Method Code: 3002.1

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CLINICAL RELEVANCE AND TEST PRINCIPLE

a. Clinical Relevance
Iodine (I), an essential element for thyroid function, is necessary for normal growth,
development, and functioning of the brain and body. Iodine-deficiency disorders (IDDs) are
well-documented global health problems affecting more than a billion people worldwide.
Consequences of IDD include goiter, cretinism, intellectual impairment, brain damage, mental
retardation, stillbirth, spontaneous abortions, miscarriages, congenital deformities, and
increased perinatal mortality. Progress toward eliminating IDDs has been substantial; an
estimated 70% of the world’s edible salt currently is iodized. Most excess iodine is excreted,
and most people can tolerate fairly large amounts without experiencing problems. People
with a tendency towards autoimmune thyroid disease are less tolerant of excess iodine. If a
person has previously been iodine deficient, that person may be at risk for iodine-induced
hyperthyroidism. Excessive iodine intake by a mother can pose a reproductive risk. Since
urinary iodine values directly reflect dietary iodine intake, urinary iodine analysis is the
recommended and most common method for biochemically assessing the iodine status of a
population (1). On the other hand, Mercury (Hg) is a toxic non-essential element that can
affect various organ systems within the body but especially the central nervous system. The
main sources of mercury intake in humans are fish, dental amalgams, and occupational
exposure. The main organs affected by mercury are the brain and the kidneys (2). Psychic
and emotional disturbances are the initial signs of chronic intoxication by elemental mercury
vapors or salts. Parasthesia, neuralgias, renal disease, digestive disturbances, and ocular
lesions may develop (3). Massive exposure over a longer period of time results in violent
muscular spasms, hallucinations, delirium, and death (4). The determination of total Hg in
blood and urine are both used to assess the internal exposure. Since urine can be collected
non-invasively, it is more commonly used to assess exposure to mercury, particularly in
occupational health settings where biomonitoring of random spot urine samples is routinely
practiced. This method is used to achieve rapid and accurate quantification of iodine (I) and /
or mercury (Hg) in urine. The method can be used to analyze urine for both elements at the
same time, or just one of the elements.

b. Test Principle
Urine iodine and mercury concentrations are determined by ICP-DRC-MS (Inductively
Coupled Plasma Dynamic Reaction Cell Mass Spectroscopy). This multielement analytical
technique is based on quadrupole ICP-MS technology (5) and includes DRC™ technology (6,
7). Coupling radio frequency power into a flowing argon stream seeded with electrons creates
the plasma, the heat source, which is ionized gas suspended in a magnetic field.
Predominant species in the plasma are positive argon ions and electrons. Diluted urine
samples are converted into an aerosol using a nebulizer inserted within the spray chamber. A
portion of the aerosol is transported through the spray chamber and then through the central
channel of the plasma, where it is exposed to temperatures of 6000-8000 K. This thermal
energy atomizes and ionizes the sample. The ions and the argon enter the mass
spectrometer through an interface that separates the ICP, which is operating at atmospheric
pressure (approximately 760 torr), from the mass spectrometer, which is operating at

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-5

approximately 10 torr. The mass spectrometer permits detection of ions at each mass-tocharge ratio in rapid sequence, which allows the determination of individual isotopes of an
element. Once inside the mass spectrometer, the ions pass through the ion optics, then
through DRC™, and finally through the mass-analyzing quadrupole before being detected as
they strike the surface of the detector. The ion optics uses an electrical field to focus the ion
beam into the DRC™. The DRC™ component is pressurized with an appropriate reaction
gas and contains a quadrupole. Electrical signals resulting from the detection of the ions are
processed into digital information that is used to indicate the intensity of the ions and
subsequently the concentration of the element. Traditionally ICP-MS has been a trace
analysis technique and the typical measurement ranges from < 1 µg/L to around 100 µg/L.
DRC technology can be used to provide additional control of ICP-MS sensitivity. In this
method, adjustments of the reaction cell parameters dampen the sensitivity of iodine (isotope
mass 127) to extend the useful concentration measurement range to higher concentrations.
The reaction cell parameters in this method are also used to increase the sensitivity of
mercury (isotope mass 202) by a process known as collisional focusing. Both of these
processes are accomplished by filling the Dynamic Reaction Cell™ (DRC) with 100% argon.
Urine samples are diluted 1+1+ 8 (sample+ water + diluent) with water and diluent containing
tellurium and bismuth for internal standardization.

SAFETY PRECAUTIONS

Precautionary information that is important to protecting personnel and safeguarding
equipment will be presented inside a box, such as this one, throughout the procedure
where appropriate.
Follow universal precautions. Wear gloves, a lab coat, and safety glasses while handling
human blood, plasma, serum, urine or other bodily fluid or tissue. Place disposable plastic,
glass, and paper (e.g., pipette tips, autosampler tubes and gloves) that come in contact with
human biological fluids, such as urine, in a biohazard autoclave bag. Keep these bags in
appropriate containers until they are sealed and autoclaved. When work is finished, wipe
down all work surfaces where human biological fluid was handled with a 10% (v/v) sodium
hypochlorite solution or equivalent. The use of the foot pedal on the Micromedic Digiflex™ is
recommended because it reduces analyst contact with work surfaces that have been in
contact with human biological fluid and also keeps the hands free to hold specimen cups and
autosampler tubes. Dispose of all biological samples and diluted specimens in a biohazard
autoclave bag at the end of the analysis according to CDC/DLS guidelines for disposal of
hazardous waste.
PerkinElmer provides safety information that should be read before operating the instrument.
This information is found in the PerkinElmer ELAN ICP-DRC-MS System Safety Manual.
Possible hazards include ultraviolet radiation, high voltages, radio-frequency radiation, and
high temperatures.

Iodine and Mercury in Urine

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Caution!
Exercise caution when handling and dispensing concentrated nitric acid and base
Tetramethylammonium hydroxide (TMAH). Always remember to add acid to water. Nitric
acid and TMAH are caustic chemicals that are capable of severe eye and skin damage.
Wear powder-free gloves, a lab coat, and safety glasses. If nitric acid or TMAH comes in
contact with any part of the body, quickly wash the exposed area with copious quantities
of water for at least 15 minutes.

DATA SYSTEM MANAGEMENT

To maintain the integrity of specimen and analytical data generated by this method, eliminate
hand entry of specimen identifiers or analytical results whenever possible, proofread all
transcribed data, and regularly defragment and back up the ICP-MS computer’s hard drive.

a. Data Entry and Transfer
Whenever possible, use bar code scanners to enter sample identifiers into the ICP-DRCMS computer software to avoid errors associated with the keyboard-entry process and to
speed up sample processing. When bar code scanners cannot be used, proofread
transcribed data after entry. Handle or transfer data electronically when reporting or
moving data to other computerized data-handling software. In the Inorganic Radiation
and Analytical Toxicology Branch, sample analysis results generated by this method are
stored for long periods in Microsoft Access™ or MS SQL Server database software. The
results should include at least the analysis date, analytical run number, quality-control
(QC) results for the run, results of specimen analysis by specimen identification (ID), and
method identifier.

b. Routine Computer Hard Drive Maintenance
Defragment the computer hard drive regularly by using software such as Microsoft
®
Windows Disk Defragmenter (located in Start > Programs > Accessories > System
Tools) or an equivalent backup program to maximize computer performance and
maintain data integrity for files on the hard drive. An entry will automatically be made in
the Windows™ system event log when this process is done and will provide
documentation of this step.

c. Data Backup
(1) Schedule of Data Backups
Weekly. Full data backups onto one or more recordable compact discs (CD-R) or
digital video discs (DVD).
Daily. Full data backups onto an external hard drive.

(2) Backup Procedures
Whenever making a backup (daily or weekly) include the directories and
subdirectories: C:\elandata (include all subdirectories)

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Before making weekly backups, saving a copy of the Windows™ event log in the
active “elandata” directory will ensure archiving of all recent software system events
(including communications between ICP-DRC-MS and ELAN® software, as well as
times of hard drive defragmentation, and other Windows™ system events).
(a) External Hard Disk Backups
Connect the ELAN data system computer to an external hard disk with sufficient
storage capacity to store several copies of the backup files (≥ 18 gigabytes).
®
Configure Microsoft Windows Backup™ (located in Start > Programs >
Accessories > System Tools) program to do a daily backup of the ELAN data
system computer’s data directories (see Backup Procedures)
(b) Compact Disc Backups
Use CD-R disks only (recordable compact disks), not CD-RW disks (rewritable
compact disks). Record the CD-R so that after creation the recordable compact
disk cannot be written to again (to prevent any accidental over-writing of stored
data). Use Adaptec “Easy CD Creator”™ or equivalent software to backup.
(c) Removing Data from the ICP-DRC-MS Computer Hard Drive
When the active “elandata” directory on the ICP-DRC-MS computer hard drive
becomes too large to fit onto a single recordable compact disk, remove the oldest
data on the hard drive so that a regular backup can be done onto a single CD-R.
Usually, this procedure can be done annually.
•

•

•
•

Back up the oldest data on the hard drive in duplicate onto two CD-R disks.
Manually select each dataset folder (subdirectories under
“C:\elandata\dataset” and other relevant files (i.e., optimization, tuning, and
sample files) that are to be included on these backups.
Verify that backup CD-R disks operate correctly before deleting any data
from the hard drive. To verify the operation of a CD-R disk, open any file on
the disk by using the appropriate computer software (ICP-DRC-MS
software).
After verifying that all backups are operational, delete the original data from
the hard drive.
Keep one copy of the CD-R disk in a building other than the laboratory in
case of fire (currently, the storage room is room 1011 in building 103). Keep
the other near the ICP-MS laboratory.

(d) Backup of Sensitive Data
Make a backup for sensitive data on duplicate, recordable compact disk. Store
the two CD-R disks in two different buildings.

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d. Documentation of System Maintenance
(1) Computer Maintenance:
Record any maintenance of computer hardware and ICP-DRC-MS software in the
instrument logbook. Place other electronic records relating to integrity of the data and
hard drive in the Windows™ event log. Back up the event log on a regular basis by
saving a copy in the active “elandata” directory. The event log will then be backed up
along with the ELAN data when backup CD-R disks and tapes are made.

(2) Instrument Maintenance:
Document system maintenance in hard copies of data records (i.e., daily maintenance
checklists, PerkinElmer service records, and instrument log book) as well as in electronic
records relating to instrument optimization (default.dac), tuning (default.tun).

COLLECTING, STORING, AND HANDLING SPECIMENS; CRITERIA
FOR REJECTING SPECIMENS

a. Specimen Type: Specimen type is urine. No special instructions for fasting or special diets
are required of patient or study subjects.
b. Specimen Collection, Handling and Storage
(1) No special instructions for fasting, special diets are required.

(2) The specimen type is urine with preservative (for mercury). The preservative is a solution of
approximately 2 M sulfamic acid and. It is added for the purpose of preventing loss of
mercury from the urine before analysis. Urine should be mixed with the preservative
as soon as possible after initial collection in the proportion of 10 µL of preservative
solution per 1 mL of urine (example: To a tube containing 50 µL of preservative, up to
5 mL of urine can be added for urine mercury analysis). Mix the urine well after
addition of the preservative. See Section 6.b.2 for details on preparation of the
preservative solution.
(3) Optimal amount of specimen is 1.8 mL; minimum amount in a cryo-vial is about 0.75
mL. 500 µL needed for an analysis.
(4) Acceptable containers for allotment of urine for this method include 15 mL PP
centrifuge tubes (e.g., Becton, Dickinson and Company model number 352097). Use
sterile collectors for specimen acquisition.
(5) Screen specimen collection cups, containers and sample tubes for iodine and mercury
contamination before use.
(6) Specimen stability has been demonstrated for 1 year at ≤ -20°C.
(7) Specimen characteristics that may compromise test results are indicated above and
include high storage temperature or no preservative.

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(8) Specimen handling conditions are outlined in the division protocol for urine collection
and handling. Copies are available in the branch, laboratory and special activities
specimen-handling offices. The protocol addresses collection, transport, and specialequipment requirements. In general, transport and store urine specimens at ≤ -20°C.
Upon receipt, freeze the specimens at ≤ -20°C until time for analysis. The analyst puts
the remaining samples in the freezer after analytical aliquots are done and refreezes
them at ≤ -20°C. Samples that are thawed and refrozen several times will not be
compromised.
c. Criteria for an Unacceptable Specimen
The criteria for an unacceptable specimen are either a low volume (< 0.75 mL), suspected
contamination due to improper collection procedures or collection devices, or failure to
add the proper preservative to urine to prevent the loss of mercury. The volume of urine
used in a single analysis is 0.5 mL, but sample volumes <0.75 mL may not allow for
proper pipetting. Requested volume is >1.8mL to allow for repeat / confirmation analysis
if necessary. Specimen contact with dust or dirt may compromise test results. In all
cases, request a second urine specimen.

PROCEDURES FOR MICROSCOPIC EXAMINATIONS
Not applicable for this procedure.

CHEMICALS, STANDARDS, AND QUALITY CONTROL MATERIAL

a. Chemicals
(d) Water, high purity (≥18 MΩ⋅cm resistivity using a NANOpure Diamond Ultrapure
Water System or equivalent).
(e) TritonX-100™ (Aldrich Chemical Co., Milwaukee, WI, or any source whose product is
low in trace-metal contamination).
(f) Sulfamic Acid (Columbus Chemical Industries, Columbus, WI or equivalent).
(g) Concentrated (16M or ~70%) nitric acid (Environmental Grade from GFS Chemicals
Inc., Columbus, OH or equivalent). If other stock concentrations are used, volumes
must be adjusted accordingly.
(h) Concentrated (12M or ~37%) hydrochloric acid (Superior Reagent HCl from GFS
Chemicals Inc., Columbus, OH or equivalent). If other stock concentrations are used,
volumes must be adjusted accordingly.
(i) Ethyl Alcohol (Ethanol) (C2H5OH), ACS/USP Absolute, Anhydrous, 200 proof
(Aaper-Pharmco Products, Inc., Shelbyville, KY or equivalent low in trace-metal
contamination).
(j) Mercury (Hg) Stock Standard 1,000 mg Hg / L in 3-10% HNO3 or HCl and 10,000 mg
Hg / L in 3-10% HNO3 or HCl (Inorganic Ventures, Lakewood, NJ or equivalent NIST
traceable vendor / product).

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(k) Iodide (I-) Stock Standard: 1,000 mg I / L in H2O+stabilizer (Inorganic Ventures,
Lakewood, NJ or equivalent NIST traceable stock standard).
(l) Tellurium (Te) Stock Standard: 1,000 mg Te / L in 2-10% HNO3 (Inorganic Ventures,
Lakewood, NJ or equivalent NIST traceable stock solution).
(m) Gold (Au) Stock Standard: 1,000 mg Au / L in 2-10% HCl (Inorganic Ventures,
Lakewood, NJ or equivalent NIST traceable stock standard).
(n) Ethylenediaminetetraacetic Acid (EDTA, Sigma-Aldrich Chemicals, St.Louis, MO or
equivalent source).
(o) Tetramethylammonium hydroxide (TMAH), 25% w/w, or equivalent (AlfaAesar, 30
Bond St., Ward Hill, MA 01835).
(p) Sodium Hypochlorite (Bleach) or equivalent for preparation of 10% bleach solution
used for biological decontamination (i.e. ACTIVATE “Fresh Mix Bleach in a Bottle”,
an approved equivalent product that mixes the 10% bleach solution with each spray).
(q) Liquid argon (supplied by Speciality Gases or other contract agency) equipped with
approved gas regulator (Matheson Gas Products, Secaucus, NJ – or equivalent).

b. Reagent Preparation
(1) Triton X-100 intermediate solution (1% v/v Triton X-100 in water)
For ease of the regular preparation of other solutions (diluent and urine preservative),
first prepare a 1% Triton X-100™ stock solution. Add 20 mL of Triton X-100™ to a
pre-acid-washed 2 L, narrow-mouth container that is partially filled with ≥18 M Ω⋅cm
water. Fill to 2 L with ≥18 MΩ⋅cm water. Add an acid-washed, Teflon™ coated
stirring bar, and stir on a magnetic stirrer until the Triton X-100™ has completely
dissolved into solution (several hours) or mix well and allow to stand overnight for
complete dissolution.
(2) Preservative for collected urine or intermediate standards
(a) Preservative for collected urine samples
(200 g/L sulfamic acid, 0.01% Triton X-100™).
Partially fill a pre-screened or pre-acid-washed 50mL polypropylene centrifuge
-1
tube with ≥18 Mohm cm water. Add 10 g of sulfamic acid and 0.5mL of 1%
TritonX-100 intermediate solution. Fill to the 50mL mark with ≥18 Mohm cm-1
water. Dissolve the sulfamic acid by mixing well (use of a vortexer, or warm
water bath is helpful in this process). Store at room temperature. Expiration is
one year from preparation.
(b) Preservative for intermediate standards
(200 g/L sulfamic acid)
Partially fill a pre-screened or pre-acid-washed 50mL polypropylene centrifuge
-1
tube with ≥18 Mohm cm water. Add 10 g of sulfamic acid. Fill to the 50mL
mark with ≥18 Mohm cm-1 water. Dissolve the sulfamic acid by mixing well (use
of a vortexer, or warm water bath is helpful in this process). Store at room
temperature. Expiration is one year from preparation.
(3) Diluent.
(a) Internal Standard Intermediate Solution. (100 mg/L Te in ≥18 MΩ⋅cm water)
To facilitate the “as needed” preparation of the diluent, preparation of a supply of
internal standard intermediate solution is recommended. Partially fill a pre-

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screened or pre-acid-washed 50mL polypropylene centrifuge tube with ≥18
Mohm cm-1 water. Add 5mL Te stock (1000mg/L) then fill to the 50 mL mark
with ≥18 Mohm cm-1 water. Mix well and store at room temperature. To prepare
different volumes adjust the solution constituents proportionally.
(b) Diluent (1% (v/v) TMAH, 0.02% Triton X-100™, 25 µg/L Te, 5% (v/v) C2H5OH,
500 µg/L Au, 0.5 g/L EDTA ).
Acid-rinse a narrow-mouth 2 L container (Teflon™ preferred), and partially fill
with ≥18 MΩ⋅cm water. Add the following, mixing in between each addition: 20
mL of 25% (v/v) TMAH, 40 mL of 1% Triton X-100™, 100 mL ethanol, 1 mL of
1,000 mg/L Au, 1g EDTA, and 0.5mL of internal standard intermediate solution
(100 mg/L Te). Dilute to 2 L with ≥18 MΩ⋅cm water. Store at room temperature
and prepare as needed. To prepare larger volumes of diluent, add proportionally
larger volumes of the solution constituents. Use this diluent to prepare all
standards and samples during the sample preparation / dilution process, which
should occur just before analysis. It is important to make all calibrators, blanks,
QC, and samples in a run from the same diluent solution so that the
concentration of the internal standard is consistent. Diluent homogeneity (e.g.
internal standard concentration) is enhanced by stirring the diluent on a stir plate
at lowest vortex speed throughout calibrator and sample preparation.
(4) ICP-DRC-MS Rinse Solution ( 1% (v/v) TMAH, 0.02% Triton X-100™, 5% (v/v)
C2H5OH and 500 µg/L Au).
To prepare, acid-rinse a 4 L narrow-mouth Teflon™ container and partially fill with ≥18
MΩ⋅cm water. Add 40 mL of 25% (v/v) TMAH and 80 mL of 1% Triton X-100™ (see
section 6.b.1 for preparation procedure), 200 mL C2H5OH and 2 mL of 1,000 mg/L Au.
Dilute to 4 L with ≥18 MΩ⋅cm water. Store at room temperature and prepare as
needed. To prepare larger volumes of rinse solution, add proportionally larger volumes
of the solution constituents. Pump this solution into the sample introduction system
between samples to prevent carry over of the analytes of interest from one sample
measurement to the next.
(5) Base Urine Preparation
The base urine used in this method is a pool of urine collected from anonymous
donors. Collect urine in containers screened for iodine and mercury content. After
receiving donations, analyze the urine to determine iodine and mercury
concentrations. The final base urine pool should be ≤ 70 µg/L iodine and ≤ 0.1 µg/L
mercury. Donated urine specimens with acceptable concentrations of iodine and
mercury, are pooled and then dispensed into smaller-volume tubes (i.e., 50 mL
polypropylene tubes) for daily use. For short-term storage (a few days), store at
approximately 2-4°C. For long-term storage, store at ≤ -20°C. A 2 L base urine pool
should be enough for ~350 analysis runs (~ 14,000 samples at 40 samples per run).
Combine this base urine with intermediate working standards prior to analysis each
day to prepare a matrix-matched calibration curve for each run.

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STANDARDS PREPARATION

CAUTION!
Mercury compounds are toxic! Take extra care to avoid accidental ingestion or inhalation of these
materials. Wear appropriate personal protective gear. Above all, wear a laboratory coat
and latex or nitrile gloves. Clean up any spill that might occur according to applicable
hazardous material spill procedures.
Materials:
Flasks: one 500 mL (plastic or glass) for 10% v/v HCl
one 50mL (glass) for Hg Intermediate Stock Standard B
four 100 mL (glass) for other Intermediate Stock Standards
(Hg-A, IOD-A, IOD-B, Hg-C)
five 100mL (glass) for Intermediate Working Standards 1-5.
Three 100mL (glass) for range of linearity (calibration verification) solutions
Pipette volumes: 80-1000 µL
Glass Bottles (~0.5oz) for final storage of standards solutions (approximately 30).
Stock solutions: iodine (1000 mg/L) & mercury (1000 mg/L).
Hydrochloric acid: Concentrated (12M or 37%) HCl
a.
(1)

(2)
(3)

Materials preparation
Clean the flasks:
(a) Acid-wash flasks: Rinse each with 5% (v/v) nitric acid solution followed by
rigorous rinsing with ≥18 MΩ⋅cm water. Repeat this process several times
depending on prior use of the containers. After adequate acid washing, flasks
should be clean of residual Hg and I from previous usage.
(b) Monitoring: This step is not usually necessary, but if performed, compare
analysis results of water poured out of these flasks with results for water taken
directly from the same water purification system to decide whether or not
additional cleaning of the containers is needed. Analysis of the water can be
done directly without dilution or calibration using the normal ICP-DRC-MS
method file. Acceptable counts for clean vessels should be negligibly different
from water blank counts. Typical measured intensity observed for Iodine in water
is less than 100cps and Mercury <30cps (counts vary between instruments due
to sensitivity differences). If background counts are too high, repeat step 7.a.1.a.
Sulfamic Acid Preservative: Prepare 200 g/L sulfamic acid preservative as per
Section 6.b.2.
Prepare 10% v/v HCl: Add 25mL of concentrated HCl (12 M or 37%) to
approximately 400mL water in an acid-washed 500 mL volumetric flask (see Section
7. Materials: Flasks). Dilute to the mark with ≥18 MΩ⋅cm water. Mix well. Store at
room temperature. Expiration date is 1 year from preparation.

b. Intermediate Stock Standards Preparation
(1) Mercury Intermediate Stock Standards Preparation:
(a) Hg Intermediate Stock Calibrator Solution A
(‘Hg-A’, 1 mg/L mercury in 10% v/v HCl).
Partially fill an acid-cleaned 100mL glass volumetric flask with 10% v/v HCl. Add 100
µL of 1000mg/L Hg stock standard. Dilute to the 100 mL mark with 10% v/v HCl.

Iodine and Mercury in Urine
DLS Method Code: 3002.1

(2)

(3)

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Mix well before use or storage. Store an aliquot of this solution in a properly labeled
glass bottle at refrigerated temperatures (~2-4ºC).
Hg Intermediate Stock Calibrator Solution B
(‘Hg-B’, 0.1 mg/L mercury in 10% v/v HCl).
Partially fill an acid-cleaned 50mL glass volumetric flask with 10% v/v HCl. Mix the
Hg Intermediate Stock Solution A (‘Hg-A’) well, then pipette 5mL of it into the partially
filled, 50mL flask. Dilute to the 50 mL mark with 10% v/v HCl. Mix well before use or
storage. Store an aliquot of this solution in a properly labeled glass bottle at
refrigerated temperatures (~2-4ºC).
Hg Intermediate Stock Calibration Verification Solution
(‘Hg-C’, 100 mg/L mercury in 10% v/v HCl).
Partially fill an acid-cleaned 100mL glass volumetric flask with 10% v/v HCl. Add
1000 µL of 10,000 mg/L Hg stock standard. Dilute to the 100 mL mark with 10% v/v
HCl. Mix well before use or storage. Store an aliquot of this solution in a properly
labeled glass bottle at refrigerated temperatures (~2-4ºC).

c. Iodine Intermediate Stock Standards Preparation:
(1)

(2)

Iodine Intermediate Stock Calibrator and Calibration Verification Solution A
(‘IOD-A’, 100 mg/L iodine in water)
Partially fill an acid-rinsed 100mL glass volumetric flask with ≥18 MΩ⋅cm water. Add
10 mL of 1000mg/L I stock standard. Dilute to the 100 mL mark with ≥18 MΩ⋅cm
water. Mix well before use or storage. Store an aliquot of this solution in a properly
labeled glass bottle at refrigerated temperatures (~2-4ºC).
Iodine Intermediate Stock Calibrator Solution B
(‘IOD-B’, 10 mg/L iodine in water)
Partially fill an acid-rinsed 100mL glass volumetric flask with ≥18 MΩ⋅cm water. Mix
the I Intermediate Stock Solution A (‘IOD-A’) well, then pipette 10mL of it into the
partially filled, 100mL flask. Dilute to the 100 mL mark with ≥18 MΩ⋅cm water. Mix
well before use or storage. Store an aliquot of this solution in a properly labeled
glass bottle at refrigerated temperatures (~2-4ºC).

d. Intermediate Working Standards Preparation
(1)

Partially fill five 100 mL glass volumetric flasks to a few centimeters below the
meniscus with ≥18 MΩ⋅cm water. Add 1mL of the 200 g/L sulfamic acid preservative
solution to each flask and mix well. Pipette the appropriate volume (see the table
below) of each intermediate stock solution into the five flasks. Mix these well, then
dilute each to a final volume of 100mL with ≥18 MΩ⋅cm water. Different volumes can
be prepared by spiking with proportionally smaller or larger additions of components.

Intermediate Working Standards Preparation volumes (µL).
Working
Hg Interm. Stock
Hg Interm. Stock
I Interm. Stock
Standard
STD B (‘Hg-B’)
STD A (‘Hg-A’)
STD B (‘IOD-B’)
Number
(0.1 mg/L)
(1 mg/L)
(10 mg/L)
80
80
STD 1
240
240
STD 2
800
800
STD 3
240
STD 4
800
STD 5
(2)

I Interm. Stock
STD A (‘IOD-A’)
(100 mg/L)

240
800

The final concentrations of iodine and mercury in each of the intermediate working
standards can be calculated by the formula below (see table below for final

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concentrations). The values entered into the ICP-DRC-MS software should be the
concentrations of the intermediate working standard.
Int. Work. Std. Conc. (µg/L) = Int. Stock Std. Conc. (µg/L) x Int. Stock Std Spike (L)
0.100 L

STD 1
STD 2
STD 3
STD 4
STD 5
(3)

Intermediate Working Standards Concentrations (ug/L)
Mercury
Iodine
0.08
8
0.24
24
0.8
80
2.4
240
8.0
800

Mix standards well and allow to equilibrate. If time allows, test one aliquot of the
calibrators before aliquoting into labeled glass bottles for storage with bench QC and
reference materials (as available) before using it for patient sample analysis. Store at
refrigerator temperatures (~2-4ºC). Expiration date is 6 month from preparation.

e. Working Standards (Calibrators)
The working calibrators are dilutions of the five intermediate working standards into a urine
matrix (base urine) for the purpose of a matrix-matched external calibration of an analytical
run (i.e. the run calibrators). Prepare the working calibration standards along with patient
samples and QC using the same diluent solution. Diluent homogeneity (e.g. internal standard
concentration) is enhanced by stirring the diluent on a stir plate at lowest vortex speed
throughout calibrator and sample preparation. Use the same base urine for all calibrators
and urine blanks to be used within the run. To prepare the working calibration standards,
transfer 500 µL of the appropriate aqueous intermediate working standard, 500 µL of base
urine, and 4,000 µL of diluent to a 15 mL polypropylene centrifuge tube by using the
Micromedic Digiflex™. Cap the tube and mix well before analysis. Section 20 describes
procedures for situations where prepared dilutions cannot be analyzed within the same
workday as preparation. .
f.

Range of Linearity (RLT) / Calibration Verification Intermediate Working Standards
Partially fill three 100 mL glass volumetric flasks to a few centimeters below the meniscus
with ≥18 MΩ⋅cm water. Add 1mL of the 200 g/L sulfamic acid preservative solution to each
flask and mix well. Pipette the appropriate volume (see the table below) of each intermediate
stock solution into the three flasks. Mix these well, then dilute each to a final volume of
100mL with ≥18 MΩ⋅cm water. Mix standards well and allow to equilibrate. If time allows,
test one aliquot of the calibrators before aliquoting into labeled glass bottles for storage. Test
the solutions to verify the concentrations with bench QC and reference materials (as
available) before using for patient sample analysis. Store at refrigerator temperatures (~24ºC). Expiration date is 6 month from preparation.
Range of Linearity (RLT) / Calibration Verification (CV) Intermediate Working Standards
Preparation
RLT / CV
Spike Vol.
Spike Vol.
working
Hg Intermediate
I Intermediate
Hg Conc.
Iodine Conc.
standard
Stock Std C
Stock Std A
(ug/L)
(ug/L)
number
(‘Hg-C’, 100 mg/L)
(‘IOD-A’, 100 mg/L)
RLT / CV 1
100 ug/L
1,200 ug/L
100 µL
1,200 µL
RLT / CV 2
200 ug/L
2,000 ug/L
200 µL
2,000 µL

Iodine and Mercury in Urine

ITN-DLS

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RLT / CV 3

Page 12 of 34
250 µL

250 ug/L

3,000 µL

3,000 ug/L

Different volumes can be prepared by spiking with proportionally smaller or larger additions
of components. Different concentrations of RLT / CV solutions can be prepared by making
dilutions of these preparations using water immediately prior to their preparation at the
Digiflex (i.e. a 50 ug/L Hg solution can be prepared by initially diluting the RLT / CV
intermediate stock solution 2x using water prior to preparation of the working RLT / CV
standard) or by spiking proportionally different volumes of the Hg or I intermediate stock
standards when making the RLT / CV intermediate working standards.
g.

Quality Control Material
(1)

Bench QC Materials
Analyze low and high bench QC material in each run to determine the validity of the
concentration measurements being made. Quality control (QC) materials are made by
spiking human urine collected from anonymous donors (see section 6.b.5) with single
element iodide and mercury standards. Prepare these pools periodically, as supply
dictates, by spiking base urine to desired concentrations. Prepare new pools far
enough in advance so that both old and new pools can be analyzed together for a
period of time (preferably at least 20 runs) before switching to the new QC materials.
The two urine QC pools made for iodine and mercury assay are designated as:
QC level

QC Designation ID

low pool

LU-yy###

high pool

HU-yy###

Where substitutions are: yy = the last two digits of production year, and ### = assigned
pool identification number.
QC material that is to be used for bench quality control run judge purposes will need to
be “characterized” as described in the section Establish QC limits for each QC pool.
(2)

Reference Materials
Analyze reference materials on a regular basis to evaluate / verify method
performance. When available, use standard reference materials (NIST). Freeze
dried certified reference materials (i.e. NIST SRM 2670) can be aliquoted into smaller
volumes after reconstitution and stored at ≤ -20°C for use when needed.

INSTRUMENT & SOFTWARE FOR THE ICP-DRC- MS

a. ICP-DRC-MS System
(1)

Inductively Coupled-Plasma Dynamic-Reaction Cell Mass Spectrometer ELAN DRC
Plus or DRC II (PerkinElmer Instruments, Headquarters Office, 710 Bridgeport Ave.,
Shelton, CT 06484-4794). Parameters of x-y alignment, mass calibration, autolens
voltages, and nebulizer gas flow rates are optimized regularly. Other DRC™
parameters are optimized for each specific instrument.

Iodine and Mercury in Urine
DLS Method Code: 3002.1

ITN-DLS
Page 13 of 34

ELAN ICP-DRC-MS Method Parameters
Parameter
Setting
RF Power
1.45 KW
Argon nebulizer gas flow is the same
Approx 0.9-1 LPM
as the DRC mode nebulizer gas flow
Detector mode
Pulse
Measurement units
Cps
Autolens
On
Blank subtraction
After internal standard
Curve type
Simple Linear
Sample units
µg/L
Sweeps/reading
90
Readings/replicate
1
Replicates
3
Dwell time
30 ms for I and Te and 100 ms for Hg
Cell gas
Argon
Cell gas flow rate
~0.3 - 0.5 mL/min (optimize as needed)
~0.75 for iodine and ~0.35 for Hg
RPQ
(optimize as needed)
Equations (on Te)
0.154312 * Xe 129 – 0.009437 * Ba 137
(2)

ELAN instrument control and data handling software, version 3.0 with service pack 2
or equivalent (PerkinElmer Instruments, Shelton CT).

(3)

Cyclonic spray chamber (PerkinElmer Instruments, Shelton CT), or equivalent.

(4)

Concentric glass nebulizer, (P/N SB-50-A2, J. E. Meinhard Associates, CA) or
equivalent.

b. Other equipment
(1)

Water purification system (NANOpure Diamond Ultrapure Water System, Barnstead
International, Bedford, MA or equivalent) for providing ultrapure water with a
resistivity ≥18 MΩ⋅cm.

(2)

Analytical balance for routine weighing of material to the nearest tenth of a gram and
with a loading capacity of at least 200 g.

(3)

Micromedic Digiflex™ automatic pipette (or equivalent) to facilitate sample dilution /
preparation equipped with 10.0 mL dispensing syringe, 2.0 mL sampling syringe,
0.75 mm tip, and foot pedal (LABREPO, Inc., 101 Witmer Rd., Suite 700, Horsham,
PA 19044).

c. Supplies
(1)

5-100 µL pipette tips, 960 tips per case (Eppendorf® catalogue # 2235137-1,
distributed by Eppendorf North America, Westbury NY), or equivalent.

(2)

20-300 µL pipette tips, 960 tips per case (Eppendorf® catalogue # 2235144-3,
distributed by Eppendorf North America, Westbury NY), or equivalent.

(3)

1,000 µL pipette tips, 960 tips per case (Eppendorf® catalogue # 2249044-3,
distributed by Eppendorf North America, Westbury NY), or equivalent.

Iodine and Mercury in Urine
DLS Method Code: 3002.1

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Page 14 of 34

(4)

5 mL pipette tips, 500 tips per case (Eppendorf® catalogue # 2235081-1, distributed
by Eppendorf North America, Westbury NY), or equivalent.

(5)

Acid-cleaned volumetric flasks, 100 mL (qty 12) and 50 mL (qty 1) for standards
preparation (glass preferred) and 500 mL (qty 1) for 10% HCl preparation (glass or
plastic acceptable). To acid-wash flasks, rinse with 5% (v/v) reagent-grade nitric acid,
followed by rigorous rinsing with ≥18 MΩ⋅cm water. Repeat this process several
times depending on prior use of the containers.

(6)

2 Acid-cleaned 2 liter PE bottles. To acid-wash containers, rinse with 5% (v/v)
reagent-grade nitric acid, followed by rigorous rinsing with ≥18 MΩ⋅cm water. Repeat
this process several times depending on prior use of the containers.

(7)

2 acid-cleaned 4 liter PE bottles. To acid-wash containers, rinse with 5% (v/v)
reagent-grade nitric acid, followed by rigorous rinsing with ≥18 MΩ⋅cm water. Repeat
this process several times depending on prior use of the containers.

(8)

Glass vials (10-30mL) for storage of calibration standards after preparation. (i.e.
clear Qorpak vials, All-Pak, Bridgeville, PA or equivalent).

(9)

Kay-Dry™ paper towels and Kim-Wipe™ tissues (Kimberly-Clark Corp., Roswell GA,
or equivalent vendor).

(10)

Teflon™-coated magnetic stirs bars (2) (Catalog Number 58948-974 or equivalent),
VWR Scientific Products, Buffalo Grove, IL.

(11)

Cotton swabs (Hardwood Products Co. ME, or equivalent vendor).

(12)

15 mL (# 352097) and 50 mL (#352098) polypropylene centrifuge tubes or
equivalent: (Becton Dickinson Labware, 1 Becton Drive, Franklin Lakes, New Jersey
07417 or equivalent).

(13)

Nitrile or Latex, powder-free examination gloves (N-Dex®, Best Manufacturing Co.,
Menlo, GA, or equivalent vendor).

CALIBRATION AND CALIBRATION-VERIFICATION PROCEDURES

a. Calibration Curve
Generate a simple linear calibration curve for iodine & mercury by using a series of five
external calibrators whose concentrations are defined in the calibration page of the
quantitative analysis method software. The calibration curve plots the ratio of the
observed intensities for iodine & mercury and the internal standards versus the
concentration of the calibrators. Compare the ratio of the observed intensities for iodine &
mercury and the internal standards in the patient sample to those obtained from the
calibrators to determine the concentration of iodine & mercury in the sample.

b. Calibration Verification
CLIA requires the verification of accuracy of instrument response to analyte
concentration be completed at least every 6 months. Each time this method is
performed, the run contains calibration curve which meets this requirement for
concentrations up to that of the highest calibrator. To verify accuracy of instrument

Iodine and Mercury in Urine
DLS Method Code: 3002.1

ITN-DLS
Page 15 of 34

response at concentrations higher than the highest calibrator take the following
steps.
(1) Bi-annual tests as defined in the DLS Policy and Procedures manual: Analyze
the Range of Linearity (RLT) / Calibration Verification (CV) working standard #3
at least every 6 months. If the observed concentrations are not within 10% of
the target value, the lab supervisor should be notified and the issue should be
investigated. Verify that normal background measured intensities have been reachieved on the ICP-MS following analysis of elevated standards for calibration
verification prior to performing further analysis.
(2) As-needed confirmations (per supervisor discretion): When a sample
concentration is greater than 110% of the highest calibrator in the run, include
an RLT / CV working standard, standard reference material, or certified
reference material with equivalent (within 10%) or greater concentration than the
sample. Section 7.e. describes the preparation of three concentration levels of
an RLT / CV working standard. It is the analyst’s discretion which concentration
is prepared and used so long as it is within 10% of the concentration being
verified or higher. In order to avoid needless contamination of the ICP-MS
sample introduction system with high concentrations of analytes, use the lowest
appropriate analyte concentration to meet the need.
Any reference material sample from a historical proficiency testing program
challenge can be substituted for this verification purpose IF
(a)
(b)
(c)
(d)
(e)

The target value has been assigned by an external source (i.e. NIST, or
the proficiency testing program).
The concentration of the external reference material is within 10% or is
higher than the concentration of the material you need it to confirm.
There is confidence that there is no contamination of previously used
external reference material.
A note to file is made that this was done.
If the observed concentrations are not within 10% of the target value the
lab supervisor should be notified and the issue should be investigated.

10. OPERATING PROCEDURES; CALCULATIONS; INTERPRETATION
OF RESULTS
a. Preliminaries
(1) For information about the reportable range of results and how to handle results
outside this range, refer to the Reportable Range of Results section of this
document (section 16).
(2)
Allow frozen urine specimens, QC specimens, and base urine to reach ambient
temperature. Vortex the sample well so that no particulates remain on the bottom
of the tube before taking an aliquot for analysis.
b. Sample Preparation

Iodine and Mercury in Urine

ITN-DLS

DLS Method Code: 3002.1

Page 16 of 34

(1) Thaw the frozen urine specimens; allow them to reach ambient temperature (about
20°C).
(2) Set up a series of 15 mL polypropylene centrifuge tubes corresponding to the
number of blanks, standards, QCs, and patient samples to be analyzed.
(3) Prepare the following solutions into the 15 mL polypropylene centrifuge tubes by
using the Micromedic Digiflex™.
Preparation of Samples for Analysis (All Volumes in µL)
ID

Water

Intermediate
Working Std.

Base
Urine

Urine
Sample
or QC
-

Diluent

Urine blank
500
500
4,000
Calibration
500
500
4,000
standards
Aqueous blank
1,000
4,000
Urine sample or
500
500
4,000
QC
2x dilution of
750
250
4,000
Urine sample*
Note: These volumes are used because the total volume each sample this method
consumed for is around 3,000 µL
* Volumes listed here are an example of how to combine the correct proportions of
water, urine, and diluent in making a 2x dilution. Other volumes of the same
proportions can also be used. Other dilutions can be prepared as needed by
adjusting the proportion of urine to the total volume of diluted sample. Use pipettes
at greater than 10% volume capacity for best accuracy.
(a) Prepare an aqueous blank that consists of 1,000 µL of ≥18 MΩ⋅cm water and
4,000 µL diluent. Use the aqueous blank for the QC pools and patient samples.
(b) Prepare five urine blanks that consist of 500 µL of base urine (same material
used for preparation of the urine calibration standards), 500 µL of ≥18 MΩ⋅cm
water, and 4,000 µL of diluent. Run one of these as the blank for the calibration
standards. Run two urine blanks after standard 5 (as sample IDs UrBlkChk1
and UrBlkChk2, respectively). Analyze two urine blanks before the calibration
blank to condition the system.
(c) Prepare the working calibration standards as described in section 7.d.
(d) Prepare dilutions (10x) of the QC and patient urine samples using manual or
automated pipettors: 500 µL of ≥18 MΩ⋅cm water, 4,000 µL of the diluent and
500 µL of the patient or QC urine sample.
(e) Cap all of the blanks, standards, and samples and mix them well.
(f) It may be necessary to operate the instrument with cell gas flowing at the
method flow rate for at least 30-45 minutes before the run begins. This may be
done by analyzing the rinse solution for 8-10 sample cycles prior to anlayiss of
the first conditioning blank. This is to allow the conditions within the reaction
cell to equilibrate before the run begins. Note: The cell gas will automatically
turn off after 1 hour of no ICP-MS DRC mode analysis.
(g) Uncap and place the dilution preparation of the blanks, standards, QC, and
®
patient samples in the autosampler of the ELAN ICP-DRC-MS immediately
prior to start of the analysis run.
(h) Section 20 describes procedures for situations where prepared dilutions cannot
be analyzed within the same workday as preparation.

Iodine and Mercury in Urine
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ITN-DLS
Page 17 of 34

c. Instrument Setup and Configuration
(1)
(2)
(3)

(4)
(5)

(6)

(7)
(8)

Turn on the computer, printer, peristaltic pump, and autosampler. Log into the
computer operating system.
®
Start the ELAN ICP-DRC-MS software from Windows™ and note whether all
graphical indicators of instrument readiness are green. If not, take the
appropriate actions described in the instrument’s software and hardware manual.
Perform necessary daily maintenance checks as described in Chapter 5 of the
®
ELAN ICP-DRC-MS Hardware Guide (e.g., argon supply, interface components,
cleanliness, positioning and interface pump oil condition). Note the base vacuum
pressure in the INSTRUMENT window of the software (Before igniting the
-7
-6
plasma, the vacuum is typically between 8 x 10 and 1.8 x 10 torr). Record
any maintenance procedures along with the base vacuum pressure in the Daily
Maintenance Checklist (See example of daily checklist in the Appendix).
Set up the peristaltic pump tubing for the autosampler, rinse station, and spraychamber waste line. Position the tubing and close the pump clamps.
Start the peristaltic pump by pressing the appropriate arrow in the DEVICES
window (Make sure that the rotational direction is correct for the way the tubing is
set up in the peristaltic pump). Fill the rinse station reservoir quickly by pressing
the “Fast” button in the DEVICES window. After the rinse station is filled with the
rinse solution, type in “20” in the rpm field of the DEVICES window to set the
pump speed. If the spray chamber rinse line is not draining the spray chamber
correctly or the rinse solution is not flowing properly to the rinse station, adjust
the tension screws on the peristaltic pump.
Read this step through entirely before proceeding. It is important to get the
tension on the autosampler tubing correct, or it will adversely affect the precision
of the ICP-DRC-MS measurements. Through the METHOD/SAMPLING window
in the software, press the “Probe” button, then the “Go to Rinse” button to lower
the autosampler probe into the rinse solution. Watch as the solution is taken up
through the autosampler probe tubing. When the leading edge of the solution is
visible, press “Stop” in the DEVICES window. The leading edge of solution in the
autosampler tubing line should stop moving. If it does not stop, tighten the
tension screw for this line on the back of the peristaltic pump. Loosen the
peristaltic pump tubing screw for the autosampler tubing until the leading edge of
solution in the autosampler tubing begins to move again, then tighten the screw
just enough to make the solution edge stop. Tighten the screw another eighth to
a quarter of a turn. Next, start the peristaltic pump by pressing the appropriate
arrow in the DEVICES window (make sure that the rotational direction is correct
for the way the tubing is set up in the peristaltic pump).
In the INSTRUMENT window of the software, press the “Start” button to ignite
the plasma. After the plasma ignites, restart the peristaltic pump.
Allow approximately 30 to 45 minutes warm-up time for the ICP-DRC-MS (with
plasma running). After this warm-up time, complete the appropriate daily
®
optimization procedures as described in Chapter 3 of the ELAN DRCII Software
Guide. Include beryllium (m/z 9) in the mass calibration, autolens optimization,
and daily performance check by using a 1-10 µg/L multielement solution. Fill in
the Daily Maintenance Checklist according to the completed optimization
procedures. Save new tuning (mass-calibration) parameters to the file
“default.tun.” Periodically, save these parameters also in a separate file
containing the analysis date “default_YYMMDD.tun”. Save new optimization
parameters (i.e., detector voltages, autolens values and nebulizer gas flow rate)
to the file “default.dac”. Periodically, save these parameters also in a separate
file containing the analysis date “default_YYMMDD.dac” (where YY=year,
MM=month and DD=day).

Iodine and Mercury in Urine
DLS Method Code: 3002.1
(9)

ITN-DLS
Page 18 of 34

To set up the run in the software, click on “Open Workspace” from the “File”
menu. Select the workspace file “CDC_Urine_ I_Hg.wrk.”. Select “Review Files”
from the “File” menu. From this window, you will be able to set up the correct files
and directories for data for your analysis. Select the method, report template,
tuning, and optimization files later. There is no need to select a calibration or
polyatomic file (If this workspace has not been created on the instrument
computer being used, follow the directions in the ELAN ICP-DRC-MS software
manual to set it up using the parameters described in this write-up).
•
Data set: If this is the first run of the day, create a new data set by using
the date as the name (Use the format 20050801 for August 1, 2005). If a
run has already been performed today, select the data set for today’s
date.
•
Sample: If an analysis has been performed that is similar to the one you
are going to do, select the sample file corresponding to it. Edit it later for
the present analysis.
(10) In the SAMPLES/BATCH window, update the table to reflect the current sample
set (e.g., autosampler locations, sample identification (ID), analysis methods and
peristaltic pump speeds). Two method files (CDC_UIHg_methITU007B_urblk.mth
and CDC_UIHg_methITU007B_aqblk.mth) will be used. These two methods
differ only in the autosampler locations of the blank and calibration solutions. Use
the “UR” method file to run the base urine blank and the calibration standards at
the very beginning of the run. Because of the autosampler positions defined in
the method file (these are editable), the urine blank must go in autosampler
location 11 and the urine calibration standards 1-5 must go in autosampler
locations 12-16, respectively. Use the “AQ” method file to run the aqueous blank
before the first sample. Because of the autosampler positions defined in the
method file (these are editable), the aqueous blank must go in autosampler
location 19. Except for defining the blank and calibration standards’ autosampler
locations, it does not matter which of these files is used when analyzing a sample
since all other analysis parameters are identical in the method files. A typical
SAMPLE/BATCH window for this method will look like Table below. (Note: All
other autosampler positions besides those specified above are arbitrary.)

Iodine and Mercury in Urine
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ITN-DLS
Page 19 of 34

Typical Sample File Setup for a Urine Iodine & Mercury Analysis Run
A/S
Measurements
Sample ID
Method File*
Location
Action
37
DRC Rinse Delay
Run sample
CDC_UIHg_methITU007B_urblk.mth
38
DRC Rinse Delay
Run sample
CDC_UIHg_methITU007B_urblk.mth
Conditioning
39
Run sample
CDC_UIHg_methITU007B_urblk.mth
Urine Blank
Conditioning
Run sample
CDC_UIHg_methITU007B_urblk.mth
40
Urine Blank
Run blank,
standards, and
100
UrBlkChk1
CDC_UIHg_methITU007B_urblk.mth
sample
101
UrBlkChk2
Run sample
CDC_UIHg_methITU007B_urblk.mth
Run blank and
20
AqBlkChk
CDC_UIHg_methITU007B_aqblk.mth
sample
23
Low-bench QC
Run sample
CDC_UIHg_methITU007B_aqblk.mth
24
High-bench QC
Run sample
CDC_UIHg_methITU007B_aqblk.mth
35
Sample 1
Run sample
CDC_UIHg_methITU007B_aqblk.mth
36
Sample 2
Run sample
CDC_UIHg_methITU007B_aqblk.mth
37
Sample 3
Run sample
CDC_UIHg_methITU007B_aqblk.mth
Etc.
……
74
Sample 40
Run sample
CDC_UIHg_methITU007B_aqblk.mth
25
Low-bench QC
Run sample
CDC_UIHg_methITU007B_aqblk.mth
26
High-bench QC
Run sample
CDC_UIHg_methITU007B_aqblk.mth
Modification of the method file is allowed for situations such as
1. running only Hg or only I (and the internal standard)
2. analyzing calibrators at different autosampler locations (when you want to
perform multiple runs with different sets of calibrators).
In such cases, any new file created should be renamed to have the original
filenames (above) at the beginning. Examples include
CDC_UIHg_methITU007B_urblk_Hg.mth (when only analyzing for Hg) or
CDC_UIHg_methITU007B_urblk_calset2.mth (when performing a second run with
a different set of calibrators).
The autosampler positions of QCs and patient samples do not have to be those
shown above, but the order in which these are run (DRC mode delay time of
approximately 1 hour with rinse solution aspirating, followed by 2 UrBlkChk
conditioners, a urine blank for the calibrators, calibration standards 1-5, urine blank
checks 1 and 2, low-bench QC, high-bench QC, 40-80 patient samples including 1
blind QC sample, low-bench QC, and high-bench QC) should be as shown in Table
above.
The settings in Table below should be used for uptake and rinse times for all
samples, QC’s, and standards. (These values are already stored in the method
files for the blanks and standards.)
Sample File Timing Parameters for a Urine Iodine & Mercury Analysis Run
Pump Speed*
Duration
Sample flush
-20 rpm
45 s
Read delay and analysis
-20 rpm
50 s
Wash
-48 rpm
100 s

Iodine and Mercury in Urine
DLS Method Code: 3002.1

ITN-DLS
Page 20 of 34

The QC tab/sample tab in the method should be setup so that a 200s rinse
time will occur after any sample whose Hg concentration exceeds 30 µg/L or
whose I concentration exceeds 2000 µg/L.
* Note: Negative values for pump speed indicate direction of pump rotation.
Make sure that pump tubing is set up appropriately to match the direction of
pump rotation.

(10)

(11)

(12)

If using the lab database for long-term recording and handling of data (Section
®
10.d.(2)(d)), do not use the Elan software to automatically correct for sample
dilutions. When dilutions of any sample are run, the sample ID should be edited to
reflect the level of dilution being performed (A two-fold dilution of “Sample 1” could
be recorded in the sample ID as “Sample 1 (2X)”. The exact wording is not critical).
Edit this sample ID during the data-import process to the database so that it is
recognized as the appropriate sample. (See Section 10.d.(2)(d)).
Before beginning the analysis run, start the flow of the reaction-cell gas (argon) and
allow the cell conditions to equilibrate. Make sure that the reaction-cell gas
pressure to the instrument is approximately 7 psi on the cell-gas cylinder regulator.
In the “Manual Adjust” page of the “Optimization” window, enter a value of “0” in the
appropriate cell-gas field (cell-gas A for this method). Then enter a very low, nonzero cell gas flow rate in the same field (i.e. 0.01mL/min). A clicking should be
heard from the ICP-DRC-MS cell-gas solenoid as the flow turns on. Flush the cell
gas for 60 seconds by lifting the flush level at the front of the instrument (The flush
step may not be necessary if this same gas cell was used recently and no gas
tubing has since been disconnected). After the cell gas flush, enter the methodappropriate cell gas flow rate in the same field. Monitor the flow in the ELAN
software, on the Instrument window (diagnostics tab). It is usually necessary to
operate the instrument with cell gas flowing at the method flow rate for 30 minutes
to 1 hour before the run begins. This is to allow the conditions within the reaction
cell to equilibrate before the run begins. Necessity of this equilibration time can be
determined by monitoring stability of the observed iodine and mercury
concentration of a standard analyzed multiple times within a run. Note: The cell
gas will automatically turn off after 1 hour of no DRC-mode analysis.
After the parameters in the SAMPLE/BATCH window are edited for the run, place
the solutions in the autosampler tray according to the setup of the
SAMPLE/BATCH window and method files. Highlight (click and drag with the
mouse) the table rows of the samples that are to be included in the run, then click
on “Analyze Batch.” If sample analysis is to finish the run unattended after normal
laboratory hours, the AutoStop (Instrument window- AutoStop Tab) may be
enabled to shut off the plasma when the analysis is completed. If the AutoStop is
not enabled, the DRC gas will shut off approximately 1 hour after the last sample
analysis in DRC mode, but the plasma will remain lit. If the plasma is to remain
operating unattended, ensure that adequate rinse solution is available for the time
that the instrument will be unattended.
Instrument Shut Down
(a) Rinse the sample introduction system with water, then with no liquid (dry).
(b) Stop the peristaltic pump.
(d) Shut off ICP-DRC-MS plasma.
(e) Loosen tensioning bars and tubing.
(f) At the controller computer, visit the ELAN Instrument Control Session

Iodine and Mercury in Urine

ITN-DLS

DLS Method Code: 3002.1

Page 21 of 34

application and open the “Dataset” window. Confirm that all samples ran
successfully and that the corresponding data for each sample is listed in this
window.
d. Recording of Data
(1) QC Data
Store the results of the QC samples analyzed in each run in the Microsoft
Access™ (or MS SQL Server) database when all other data for the run is imported
®
from the ELAN software See Section 10.d.(2)(d) for a description of how to import
data into the Microsoft Access™ database).
(2) Analytical Results
(a) Analysis Printouts and Analyst Run Report
Bind the analysis printouts with a printout of the calibration curve and curve
statistics as the top page and place them in the study folder(s). Write the
following information on the cover sheet of the analysis printouts: Run date, run
number, study ID, and analyst ID (the Run ID from the database is also
helpful). Store the results of the patient samples analyzed in each run in the
Microsoft Access™ (or MS SQL Server) database when all other data for the
®
run is imported from the ELAN software. See Section10.d.(2)(b) for a
description of how to import data into the laboratory database. If the database
allows for the printing of a run summary report that indicates whether any
particular patient-sample results are outside of the normal concentration
reference range or whether any measurement failed precision limits, it may be
helpful to print it out after each analysis. These reports can be helpful to keep
in a notebook for future reference (See Section 10.d.(2)(b) for a description of
how to import data into the database to print out a customized sample report).
(b) Using the Microsoft Access™ Database
After an analysis run, export the results to a .TXT file and then import into the
Microsoft Access™ or MS SQL Server database that handles data for the
laboratory branch.
i.

Data Export Process (from ELAN software to .TXT or .CSV file)
®
In the ELAN ICP-DRC-MS software, select “Review Files” from the “File”
menu. From this window, you must open the files and directories that were
used when collecting the data of the run that you wish to export (If the
analysis has just ended, all of these files and directories will still be open).
®
NOTE: A second copy of the ELAN software can be run as an
Edit/Reprocess copy without affecting an ongoing analysis by the first copy
of the software running in Windows. After you open the relevant files, go to
the “Report” page in the METHOD window. Deselect the box that prints a
paper copy of data and select the box that sends data to a file. Select the
“Report Options Template” named “CDC_Database_output.rop” and type in
a report filename using a format such as “20050801a_group55.txt” (or
20050801a_group55.csv) to designate data from analysis of group 55 from
August 1, 2005, run #1. Under “Report Format”, choose the “Use
Separator” option, and under the “File Write” section choose “Append.”
®
Finally, reprocess the data of interest (See PerkinElmer ELAN II Software
Manual). Make sure you apply the correct blank to the correct samples and
QCs (use the urine blank for all of the calibration standards, UrBlkChk1,

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and UrBlkChk2. Use the aqueous blank for all analyses of patient samples
and QC samples).
ii.

Data Import Process (from .TXT or .CSV file to Microsoft Access™
database).
Transfer the .TXT or .CSV file to the appropriate subdirectory on the
network drive where exported data are stored(Note that directories are
named according to instrument/year/month/ and study name or ID, such as
®
I:/Instruments/ELAN DRCIIG/2005/08/Study 2005-xx). From a computer
that has access to Microsoft Access™ or MS SQL Server database used
for tracking data, log in using your user ID#. After you log into the
database, open the select “Import Instrument File” from the “Front End
Set”. Enter the appropriate information to identify the run, assay, study,
instrument, and analyst and press the “Import” button. Select the location
of the data file on the network drive and press the “Open” button. In the
“Imported Results” table, pressing the “Find X’s” button will show only
those samples whose sample ID is not recognized as a valid QC pool ID or
sample ID for this study (Sample IDs are set up when the study is logged
into the database). Corrections to sample IDs and dilution factors can be
made in this table (e.g., correction of transcription errors and adjustment
for level of dilution). If samples were diluted for analysis (Section 10.c.(10)),
both the sample ID and the dilution factor need to be edited in this table
before the values are transferred to the database. First, change the dilution
factor to reflect the way that the sample was analyzed then edit the sample
ID to remove any comments about the level of dilution at which the sample
was analyzed (The replace command is useful here). When corrections to
sample IDs are made, press the “Recheck” button to evaluate the sample
IDs. Any sample or analyte row marked “Not Recognized” will not be
transferred to the database when the “Transfer” button is pressed. From
this point, the data should be labeled with the appropriate settings for QC
accept / reject, final value status, and comment.

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11. FINAL REVIEW OF THE DATA
a.

Abnormal Patient Results:
a.

Boundaries Requiring Confirmatory Measurement:

(a) Results Lower than the First Lower Boundary (1LB): Concentrations observed less
than the “first lower boundary” (defined in the laboratory database as the “1LB”) should be
confirmed by repeat analysis of a new sample preparation. The concentration assigned to
the 1LB for an element is determined by study protocol. The default 1LB for iodine is 10
ug/L (there is no lower confirmation boundary for mercury). Report the original result, as
long as the confirmation is within 10% of the original. Continue repeat analysis until a
concentration can be confirmed.
(b) Results Greater than the First Upper Boundary (1UB): Concentrations
observed greater than the “first upper boundary” (defined in the laboratory
database as the “1UB”) should be confirmed by repeat analysis of a new
sample preparation. The concentration assigned to the 1UB for an element is
determined by study protocol. The default concentrations are 800 ug/L for I
and 5 ug/L for Hg. Report the original result, as long as the confirmation is
within 10% of the original. Continue repeat analysis until a concentration can
be confirmed.
(c) Results Greater Than Highest Calibrator: When a sample result is greater
than the highest calibrator, the result should be confirmed in an analysis run
which includes a standard or external reference material with equivalent (within
10%) or greater concentration than the sample.
(d) Results Greater Than Range of Linearity Tested (RLT): Perform an extra
dilution on any urine sample whose concentration is greater than the range of
linearity tested / calibration verification sample analyzed along with it (Section
9.b). See table in section 10.b for details of preparing a sample with extra
dilution.
b. Inadequate Precision in Confirmation of a Measurement: If a sample is
reanalyzed to obtain a confirmation of an initially elevated result, the confirmation
should be within 10% of the original result.
c.

Inadequate Precision Within One Measurement: If the range of the three replicate
readings (maximum replicate concentration value - minimum replicate concentration
value) for a single sample analysis is greater than 30 ug/L for I or 1 ug/L for Hg (“>Lim
Rep Delta” in the database) and the range of the three replicate readings is greater
than 10% of the observed concentration, do not use the measurement for reporting.
Repeat the analysis of the sample.

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d. Analyst Reporting of Abnormal Patient Results: Concentrations observed for
iodine less than the second lower boundary” (defined in the laboratory database as the
2LB) or greater than the “second upper boundary” (defined in the laboratory database
as the “2UB”) should be reported to the QC reviewer as an either an “abnormally low
result” or an “elevated result”, respectively. The concentrations assigned to the 2LB
and the 2UB for an element is determined by study protocol. The default second
boundary concentrations for iodine are 10 ug/L (2LB) and 2000 ug/L (2UB). The
default boundaries for mercury are 5 ug/L (1UB) and 10 ug/L (2UB). There are no
lower boundaries for mercury. The analyst should report any patient results confirmed
to be greater than the second upper boundary to the QC reviewer as an “elevated
result”. There is no routine notification for elevated levels for the metals determined in
this method. The protocol for supervisors reporting elevated results to medical
personnel is defined according to the study protocol.

Evaluating Bench QC Results: See sections 16.b-c for how to apply division QC rules and take
corrective actions if necessary.
i.

Submitting Final Work for Review
Once results have been imported, reviewed, and set as final in the database by the
analyst,
a.

Submit an email to the QC reviewer informing them of the readiness of
the data for final review. The email should include
1. Instrument ID, run Date, run number, study ID, group ID.
2. Any bench QC failures (include reasons if known).
3. Any patient sample results less than the 2LB or greater than the 2UB
should be reported in the email as either an abnormally low
concentration (<2LB) or an “elevated result” (>2UB).
4. Anything out of the ordinary about this analytical work which could
have a bearing on the availability (i.e. insufficient sample to analyze),
accuracy, or precision of the results.

Include all items called for by the study folder cover sheet in the study
folder (i.e. printouts from the ICP-MS, bench QC evaluation) together in
the study folder before submitting the folder for review when analysis is
complete.

12. REPLACEMENT AND PERIODIC MAINTENANCE OF KEY
COMPONENTS
Part numbers listed below are PerkinElmer part numbers from their 2005 Consumables
Catalog. Equivalent parts may be substituted.
a. Autosampler probe assembly (part # B300-0161) or equivalent. Keep one spare on
hand.
b. Peristaltic pump tubing for sample (0.03 inch i.d., part #09908587), rinse station (can
use either same tube type as for sample or 0.045 inch i.d., part #N0680375) and for

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waste (0.125 inch i.d., part #N8122012): Keep at least 6 packages of 12 on hand of the
sample tubing, 6 for rinse station and 2 packages of 12 on hand of the waste tubing.
Other suppliers may offer the same size/type of peristaltic tubing.
c. Nebulizer capillary tubing (used to connect the nebulizer and the peristaltic pump
tubing, part #09908265 or any source of polyethylene tubing, 0.6 mm i.d. x 0.97 mm
o.d.). Keep one pack (10 feet) on hand.
d. Injector Support/Torch Base (part #N8120116). Keep one spare on hand.
e. Torch O-Ring Kit (packages of four, part #N8120100). Keep four spare packages on
hand.
f.

Quartz torch. At least two spare torches should be on hand (part #N8122006).

g. Quartz 2mm Bore Injector (part #WE023948).
h. RF coil Assembly, self aligning (part #WE021816). One spare should be on hand.
i.

Nickel Skimmer (part #WE021137) and sampler cones (part #WE021140). Keep at
least two spares of each on hand.

Skimmer and sampler cone O-rings (part #N8120512 and #N8120511, respectively).
Keep at least 10 spares of each on hand.

k. Series II replacement Ion lens (part #WE018034). Keep two spares on hand.
l.

Pump oil for the roughing pump (part #N8122004). Keep four bottles on hand.

m. Polyscience chiller coolant (PE Sciex Coolant, part #WE016558): Two 1 L bottles
should be kept on hand. If possible, have a backup autosampler and chiller. See a
PerkinElmer sales representative for part numbers.
n. Nebulizer, quartz concentric ~1mL/min liquid flow rate like part # 500-70QQDAC
(Precision Glass Blowing, Centennial, CO, www.precisionglassblowing.com). This
nebulizer is designed to use quick disconnects part # 500-QD (liquid) and # 500-AC
(argon).
o. Spray chamber, quartz concentric like PerkinElmer part # WE025221 (PerkinElmer,
Shelton, CT, www.perkinelmer.com). Available direct from manufacturer as part # 40020 (Precision Glass Blowing, Centennial, CO, www.precisionglassblowing.com) or from
various distributors.

13. LIMIT OF DETECTION
The limits of detection (LOD) for iodine and mercury in urine specimens is based on three times
the standard deviation of approximately 20 or more measurements of urine blanks or low
concentration urine samples, each analyzed in a separate run. This represents the method
detection limit. Report results below the detection limit as “< LOD” (where “LOD” is the calculated
lowest detection limit). The LOD calculation may be reevaluated annually.

14. REPORTABLE RANGE OF RESULTS
Urine Iodine & Mercury results are reportable in the range of greater than the LOD, where LOD is
the calculated lower detection limit.

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15. SPECIAL PROCEDURE NOTES – CDC MODIFICATIONS
None applicable for this operation.

16. QUALITY CONTROL PROCEDURES
The Inorganic and Radiation Analytical Toxicology Branch uses the method described in this
protocol for environmental and occupational health screening studies.
This analytical method uses two types of Quality Control (QC) systems: With one type of the
QC system, the analyst inserts bench QC specimens two times in each analytical run (a set of
consecutive assays performed without interruption) so that judgments may be made on the day
of analysis. With the other type of QC system, “blind” QC samples are placed in vials, labeled,
and processed so that they are indistinguishable from the subject samples (as much as
possible). If it is not possible to have the blind QC inserted into the sample group before
receipt into the lab, an additional low and high QC pool should be made available to the analyst
so that they can manually insert the material into the run. This type of “blind QC” should match
the matrix of the patient samples as much as possible and the acceptable concentration limits
(characterized limits) should be unknown by the analyst(s). The supervisor decodes and
reviews the results of the blind specimens. With both systems, taking these samples through
the complete analytical process assesses all levels of the analyte concentrations. The data
from these materials are then used to estimate methodological imprecision and to assess the
magnitude of any time-associated trends. The bench QC pools used in this method comprise
two levels of concentration spanning the “low-normal” and “high-normal” ranges. Both of these
pools are analyzed after the calibration standards are analyzed but before any patient samples
are analyzed so that judgments on the iodine and mercury calibration curves may be made
before analysis of patient samples. These bench QCs should be analyzed again at the end of
the run.

a. Establish QC limits for each QC pool.
Perform an analysis of the mean and standard deviation for each pool from the
concentration results observed in at least 20 characterization runs. During the 20
characterization runs, previously characterized QCs or pools with target values assigned by
outside laboratories to evaluate each run’s QC. In addition to providing QC limits, the
characterization runs also serve to establish homogeneity of the pools.

b. Evaluating the Quality Control of a Run. After completing a run, and importing the
results into the database, export the QC results to the SAS program where the run will be judged
to be in or out of control. The QC limits are based on the average and standard deviation of the
beginning and ending analyses of each of the bench QC pools, so it will not be possible to know if
the run is officially accepted or rejected until it is completed. The following is an explanation of the
division QC rules which will be applied by the SAS program.
(1)

If both QC run means (low & high bench QC) are within 2Sm limits and individual
results are within 2Si limits, then accept the run.

(2)

If 1 of the 2 QC run means is outside a 2Sm limit - reject run if:

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(a)

Extreme Outlier – Run mean is beyond the characterization mean +/- 4Sm

(b)

1 3S Rule - Run mean is outside a 3Sm limit

(c)

2 2S Rule - Both run means are outside the same 2Sm limit

(d)

10 X-bar Rule – Current and previous 9 run means are on same side of the
characterization mean

(e)

If one of the 4 QC individual results is outside a 2Si limit - reject run if:

(f)

R 4S Rule – Within-run ranges for all pools in the same run exceed 4Sw (i.e.,
95% range limit)

Note: Since runs have multiple results per pool for 2 pools, the R 4S rule is pplied within
runs only. Abbreviations:
Si = Standard deviation of individual results (the limits are not shown on the chart unless
run results are actually single measurements).
Sm = Standard deviation of the run means (the limits are shown on the chart).
Sw = Within-run standard deviation (the limits are not shown on the chart).

c. Remedial Action If Calibration or QC Systems Fail to Meet Acceptable
Criteria
(1)

If the division SAS program declares the run out of control” for any analyte, ONLY the
analytes which were “out of control” are invalid for reporting from the run. Set all run
results for those 1 or 2 analytes as “QC Rejected” in the database. Evaluate the reason
for QC failure and take corrective action. Below is a list of areas to evaluate.

(2)

Check the calibration curve(s) for linearity and for an intercept near zero. Calibration
points not falling closely to the regression line may indicate a calibrator which was
improperly prepared, analyzed, or needs to be made new. Be sure to use freshly
prepared calibrators and QC material. Typical correlation coefficients (r2) are > 0.999. If
possible, prepare new dilutions or preparations of calibrators which are outliers and
reanalyze with the run to replace the original calibrator analysis. An individual calibration
point may be removed from the curve if it is obviously an outlier. If the highest calibration
point is removed, the highest calibrator used in the analysis should be specified in the
laboratory database when the results are imported. If a certain calibrator is problematic
repeatedly, investigate the problem and take corrective action to prevent the problem
from continuing.

(3)

Check for high blanks which lead to over-subtraction from analysis results.

(4)

Check the ICP-DRC-MS stability during the run by examining the degree of variability
and drift in internal standard raw peak areas over the course of the run. Irreproducibility
that exceeds 15% and drift >20%, or sudden large changes in internal standard peak
area, likely indicates that there was a problem in plasma stability.

If these steps do point to appropriate corrective action, for the out-of-control values for QC
materials, consult the supervisor for other appropriate corrective actions. No analytical results
should be reported for runs that are not in statistical control.

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17. REFERENCE RANGES

Reference Ranges for Elements Measured (all units µg/L)

Element/
Isotope
Monitored
I-127*
*
Hg-202

Reference Ranges
th
th
(10 - 95 Percentile, µg/L)
(weighted, non-creatinine
corrected NHANES 2001 &
2002 results)
(9)

41- 803
(8)
< 4.63

* Te-130 used as internal standard

18. ACTION-LEVEL RESULTS
Due to the uncertainty of the health implications of elevated concentrations of these elements,
there is no routine notification for elevated levels of urine iodine or mercury. Action levels for
reporting to supervising physicians are determined on a study-by-study basis.

19. SPECIMEN STORAGE AND HANDLING DURING TESTING
Specimens may reach and maintain ambient temperature during analysis. Take stringent
precautions to avoid external contamination. After the samples are analyzed, return them to ≤ º
20 C freezer storage as soon as possible.

20. ALTERNATE METHODS FOR PERFORMING TEST AND STORING
SPECIMENS IF TEST SYSTEM FAILS
If prepared working calibrators, samples, and QC cannot be analyzed within the same workday of
the preparation, they may be capped and stored at refrigerator temperatures (~2-4°C) for up to 48
hours before analysis. Samples can be stored at room temperature during this time if they will
only be analyzed for iodine. (see Appendix A, Parameter Test #5)
º

If the analytical system fails, then store urine specimen at ≤ 4 C until the analytical system is
restored to functionality. If long-term interruption (longer than 4 weeks) is anticipated, then store
º
urine specimens at ≤ -20 C. If this method is not available, a Flow Injection Mercury System
method (FIMS) can be used as an alternative for urine mercury analysis and a
spectrophotometric analysis method can be used as an alternative for urine iodine analysis.

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21. TEST - RESULT REPORTING SYSTEM; PROTOCOL FOR
REPORTING CRITICAL CALLS (IF APPLICABLE)
Report test results as outlined in the DLS Policies and Procedures Manual. For critical calls, the
supervisor should notify the supervising physician or principal investigator as soon as possible.
The most expeditious means should be used (e.g., telephone or E-mail).

22. TRANSFER OR REFERRAL OF SPECIMENS; PROCEDURES FOR
SPECIMEN ACCOUNTABILITY AND TRACKING
Location, status, and final disposition of the specimens will be tracked at least by paper document
in the “Study Folder” (created before analysts receive the samples). Apart from this specimen
tracking form, this folder will also contain the paper print outs of results from analysis of the
specimens. Maintain records for a minimum of 3 years. Use only numerical identifiers for
samples within the laboratory (e.g., case ID numbers) in order to safeguard confidentiality. Only
the medical supervisor (MS) or project coordinator (PC) i.e. non CDC personnel should have
access to the personal identifiers.

23. REFERENCES
(1)

Hollowell JG, Staehling NW, Hannon WH, et al. 1998 iodine nutrition in the United
States. Trends and public health implications: iodine excretion data from National
Health and Nutrition Examination Surveys I and III (1971-1974 and 1988-1994). J
Clin Endocrinol Metab 1998; 83:3401-8.

(2)

Carson BL, Ellis HV III, McCann JL. Toxicology and biological monitoring of metals in
humans. Chelsea (MI): Lewis Publishers, Inc.; 1986: p.150-156.

(3)

Handbook of Toxicity of Inorganic Compounds, edited by Sieler, H.G., Sigel, H.,
Sigel, A, Marcel Dekker, INC., 1988: p. 419-436.

(4)

World Health Organization, Environmental health Criteria 118: Inorganic mercury,
Geneva,1991

(5)

Thomas R, Practical Guide to ICP-MS. New York: Marcel Dekker; 2004.

(6)

Tanner SD, Baranov VI., Theory, design and operation of a DRC™ for ICP-MS.
Atomic Spectroscopy 1999; 20(2): 45-52.

(7)

Tanner SD, Baranov VI, Bandura DR, Reaction cells and collision cells for ICP-MS: a
tutorial review. Spectrochimica Acta part B 57, 2002: 1361-1452.

(8)

Third National Report on Human Exposure to Environmental Chemicals (CDC, July,
2005). National Health and Nutrition Examination Survey, 2001-2002.

(9)

Caldwell K, Jones R, Hollowell J. Urinary Iodine Concentration: United States
NHANES 2001-2002. Thyroid 2005; 15(7): 687-693.

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24. APPENDIX
Appendix A. Ruggedness Testing Results.
Parameter Test #1: Evaluate the impact on analysis results if the set RF Power is increased to
1600W (instrument maximum) or decreased to 1150W (by 20%) for the analytical run. (Method
RF Power setting described in section 8.a.1).
Test Details:
1. Three different RF power settings were tested in separately prepared, consecutive runs on
the instrument without turning off the plasma. At least 15 minutes stabilization time was
allowed between each run after the RF power was changed. “Junk urine” samples (38) were
analyzed between the beginning and ending QC of each run. All other method parameters
were kept per method.
2. Run #1 (method default, 1450W).
3. Run #2 (Decreased RF power by 20% to 1150W).
4. Run #3 (Increased RF power to instrument maximum, 1600W).
Parameter Test 1 Results.
Test performed 3/17-18/10 by Dana Henahan.
QC
Pool ID

LU-03250_UIHG_c

RF Power Tested

I (ug/L)

Hg (ug/L)

Characterized Mean
Characterized 2SD Range

92.7
88.2 – 97.2

0.21
0.14 – 0.29

1150W

(Reduced)

93.2

0.15

1450W

(Per Method)

92.1

0.18

1600W

(Increased)

92.4

0.20

308
293 – 322

2.30
2.05 – 2.54

Characterized Mean
Characterized 2SD Range

HU-03251_UIHG_c

1150W

(Reduced)

294

2.15

1450W

(Per Method)

307

2.23

1600W

(Increased)

315

2.19

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Appendix A. Ruggedness Testing Results. (continued)
Parameter Test #2: Evaluate the impact on analysis results if the Cell Gas Flow Rate is
increased or decreased by 20% for the analytical run. (Method Cell Gas Flow Rate setting
described in section 8.a.1).
Test Details:
1. Three different Cell Gas Flow Rates were tested in separately prepared, consecutive runs on
the instrument without turning off the plasma. At least 15 minutes stabilization time was
allowed between each run after the cell gas flow rate was changed. “Junk urine” samples
(38) were analyzed between the beginning and ending QC of each run. All other method
parameters were kept per method.
2. Run #1 (method default = 0.3 mL/min)
3. Run #2 (decreased Cell Gas Flow Rate by 20% to 0.24 mL/min).
4. Run #3 (increased Cell Gas Flow Rate by 20% to to 0.36 mL/min).

Parameter Test 2 Results.
Test performed 3/23/10 by Dana Henahan.
QC
Pool ID

Cell Gas Flow Rate Tested

I (ug/L)

Hg (ug/L)

Characterized Mean
Characterized 2SD Range

92.7
88.2 – 97.2

0.21
0.14 – 0.29

(Reduced)

92.1

0.21

0.30 mL/min (Per Method)

93.4

0.19

0.36 mL/min

93.9

0.19

308
293 – 322

2.30
2.05 – 2.54

0.24 mL/min
LU-03250_UIHG_c

(Increased)

Characterized Mean
Characterized 2SD Range
0.24 mL/min
HU-03251_UIHG_c

(Reduced)

289

0.30 mL/min (Per Method)

293

2.34

0.36 mL/min

297

2.37

(Increased)

2.23

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Appendix A. Ruggedness Testing Results. (continued)
Parameter Test #3: Evaluate the impact on analysis results if the RPq is increased or decreased
by 20% for the analytical run. (Method RPQ setting described in section 8.a.1).
Test Details:
1. Three different RPQ settings were tested in separately prepared, consecutive runs on the
instrument without turning off the plasma. At least 15 minutes stabilization time was allowed
between each run after DRC RPQ was changed. “Junk urine” samples (38) were analyzed
between the beginning and ending QC of each run. All other method parameters were kept
per method.
2. Run #1 (method default DRC RPQ: IOD= 0.8 Te= 0.8 Hg= 0.4).
3. Run #2 (decreased DRC RPQ 20%: IOD= 0.6 Te= 0.6 Hg= 0.3).
4. Run #3 (increased DRC RPQ 20% : IOD= 0.9 Te= 0.9 Hg= 0.5).
(Note: 0.9 is highest RPQ instrument will allow).
Parameter Test 3 Results.
Test performed 3/24/10 by Dana Henahan.
QC
RPQ Tested
Pool ID
Characterized Mean
Characterized 2SD Range

LU-03250_UIHG_c

HU-03251_UIHG_c

I (ug/L)

Hg (ug/L)

92.7
88.2 – 97.2

0.21
0.14 – 0.29

DRC RPQ: (Reduced)
IOD= 0.6 Te= 0.6 Hg= 0.3

92.6

0.16

DRC RPQ: (Per Method)
IOD= 0.8 Te= 0.8 Hg= 0.4

91.0

0.20

DRC RPQ: (Increased)
IOD= 0.9 Te= 0.9 Hg= 0.5

93.2

0.19

Characterized Mean
Characterized 2SD Range

308
293 – 322

2.30
2.05 – 2.54

DRC RPQ: (Reduced)
IOD= 0.6 Te= 0.6 Hg= 0.3

313

2.30

DRC RPQ: (Per Method)
IOD= 0.8 Te= 0.8 Hg= 0.4

306

2.41

DRC RPQ: (Increased)
IOD= 0.9 Te= 0.9 Hg= 0.5

311

2.39

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Appendix A. Ruggedness Testing Results. (continued)
Parameter Test #4: Evaluate the impact on analysis results if the axial field voltage (AFV) is
increased or decreased by 20% for the analytical run. (Method AFV setting described in section
8.a.1).
Test Details:
1. Three different DRC AFV were tested in separately prepared, consecutive runs on the
instrument without turning off the plasma. At least 15 minutes stabilization time was allowed
between each run after the axial field voltage was changed. “Junk urine” samples (38) were
analyzed between the beginning and ending QC of each run. All other method parameters
were kept per method.
2. Run #1 (method default DRC AFV = 275)
3. Run #2 (decreased DRC AFV by 20% to 220).
4. Run #3 (increased DRC AFV by 20% to 330).
Parameter Test 4 Results.
Test performed 3/25/10 by Dana Henahan.
QC
Axial Field Voltage Tested
Pool ID
Characterized Mean
Characterized 2SD Range
LU-03250_UIHG_c

Hg (ug/L)

92.7
88.2 – 97.2

0.21
0.14 – 0.29

220

(Reduced)

92.1

0.19

275

(Per Method)

91.5

0.21

330

(Increased)

93.3

0.18

308
293 – 322

2.30
2.05 – 2.54

(Reduced)

303

2.12

275

(Per Method)

299

2.18

330

(Increased)

304

2.20

Characterized Mean
Characterized 2SD Range
HU-03251_UIHG_c

I (ug/L)

220

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Appendix A. Ruggedness Testing Results. (continued)
Parameter Test #5: Method descriptions and SOP assume preparation and analysis on same
day. Evaluate the impact on analysis results if the analytical run is prepared to analyze but
circumstances do not allow for analysis to occur until 24 or 48 hours later.
Test Details:
1. Three separate run sets (A, B, and C) including blanks, calibrators, and QC were prepared at
one sitting from the same starting materials. Set ‘A’ was analyzed immediately per the
assumption of the method. Set’s ‘B’ and ‘C’ were capped and stored for 24 and 48 hours,
respectively, before analysis. Room temperature (~20°C) and refrigerated (~2-4°C) were
both tested. All other method parameters were kept per method. Each analytical run was
made normal length by including 38 dummy urine samples between QC checks.
2. On day two, a fresh run set (“D”) was prepared and analyzed immediately for comparison to
results from set “B” (analyzed as run 2 of the day).
3. On day two, another fresh run set (“E”) was prepared and analyzed immediately for
comparison to results from set “C” (analyzed as run 2 of the day).
Parameter Test 5 Results. Test performed 4/13-15/10 by Dana Henahan.
QC
Time From Prep
I (ug/L)
Pool ID
To Analysis Tested
Characterized Mean
2SD Range
Storage
Temperature
LU-03250
_UIHG_c

HU-03251
_UIHG_c

92.7
88.2 – 97.2
Room
2-4 °C
Temp

Hg (ug/L)
0.21
0.14 – 0.29

Room
Temp

2-4 °C

Run Set A (Freshly Prepared)

92.3

93.5

0.19

Run Set B (After 24hr)

92.8

93.8

0.22

0.19

Run Set C (After 48hr)

93.6

93.0

0.22

0.21

Characterized Mean
2SD Range
Storage
Temperature

308
293 – 322

2.30
2.05 – 2.54

Room
Temp

2-4 °C

Room
Temp*

2-4 °C

Run Set A (Freshly Prepared)

306

309

2.31

2.23

Run Set B (After 24hr)

304

307

2.61*

2.37

Run Set C (After 48hr)

301

304

2.58*

2.32

* Note: Test results show that room temperature storage for 24-48hrs is NOT adequate for Hg
analysis. Only if samples are refrigerated can a 24-48hr delay be allowed for Hg analysis.

Division of Laboratory Sciences
Laboratory Protocol
Analytes:
Matrix:
Method:

Zinc, Copper and Selenium
Serum
Serum Multi-Element ICP-DRC-MS

Method Code:
Branch:

ICPDRCMS-3006.1
Inorganic Radiation Analytical Toxicology

Prepared By:

Ge Xiao PhD
author's name

Supervisor:

signature

date

signature

date

Kathleen L. Caldwell PhD
supervisor's name

Branch Chief:

Robert L. Jones PhD
Branch Chief

Adopted:

Signature and date

19 October 2006
date

Updated:

23 October 2006
date

Director's Signature Block:
Reviewed:
signature

date

signature

date

signature

date

signature

date

signature

date

Procedure Change Log
Procedure: Serum Multi-Element DLS Method Code: 3006.1

Date
11/25/2011

Changes Made
Use of concentrated nitric acid during preparation of
calibrators and calibration verification standards
documented.

By
GM

Rev’d
By
(Initials)

Date
Rev’d

Laboratory Procedure Manual
Analytes:

Zinc, Copper and Selenium

Matrix:

Serum

Method:

Serum Multi-Element ICP-DRC-MS

Method No: ICPDRCMS-3006.1
Revised:

As performed by:

Inorganic Radiation Analytical Toxicology
Division of Laboratory Sciences
National Center for Environmental Health

Contact:

Dr. Kathleen L. Caldwell
Phone: 770-488-7990
Fax:
770-488-4097
Email: [email protected]
Dr. James L. Pirkle, M.D., PhD
Director, Division of Laboratory Sciences

Important Information for Users
The Centers for Disease Control and Prevention (CDC) periodically refines these
laboratory methods. It is the responsibility of the user to contact the person
listed on the title page of each write-up before using the analytical method to find
out whether any changes have been made and what revisions, if any, have been
incorporated.

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 1 of 73

Table of Contents
Cross reference to DLS CLIA and Policy and Procedures . . . . . . . . . . .. . . . . . . . 5
Index of tables and figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1) Clinical Relevance & Summary of Test Principle
a. Clinical Relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
b. Test Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2) Limitations of Method; Interfering Substances and Conditions
a. Interferences Addressed by This Method
i.

Correction & Elimination of Interferences (64Ni, 36Ar14N2) on Zinc (64Zn). . . 9
1
2

Mathematical Correction for Nickel (64Ni) Interference:
Elimination 36Ar14N2 Interference

ii.

Elimination of Interferences (40Ar25Mg, 36Ar14N21H) on Copper (65Cu) . . . 9

iii.

Correction & Elimination of Interferences (78Kr, 38Ar40Ar, 38Ar40Ca) on
Selenium (78Se). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

b. Limitations of Method (Interferences Remaining in Method)
i. 48Ca16O1H Interference on Copper (65Cu). . . . . . . . . . . . . . . . . . . . . . . . . . . 9
ii. Time between dilution of serum materials and analysis . . . . . . . . . . . . . . . . 9
3) Procedures for Collecting, Storing, and Handling Specimens; Criteria for
Specimen Rejection
a. Procedures for Collecting, Storing, and Handling Specimens . . . . . . . . . . . . . 10
b. Criteria for Specimen Rejection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
c. Transfer or Referral of Specimens; Procedures for Specimen Accountability
and Tracking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . 11
4) Safety Precautions
a. General Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 11
b. Waste Disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .12
5) Instrument & Material Sources
a. Sources for ICP-MS Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
b. Sources for ICP-MS Parts & Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
c. Sources for ICP-MS Maintenance Equipment & Supplies . . . . . . . . . . . . . . . . 18

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 2 of 73

d. Sources for General Laboratory Equipment & Consumables . . . . . . . . . . . . . . 19
e. Sources for Chemicals, Gases, & Regulators . . . . . . . . . . . . . . . . . . . . . . . . . . 21
6) Preparation of Reagents and Materials
a. Diluent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
b. Base Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
c. ICP-DRC-MS Rinse Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
d. Standards and Calibrators
i. Multi-Element Intermediate Stock Standard . . . . . . . . . . . . . . . . . . . . . . . .

ii. Multi-Element Intermediate Working Calibration Standards . .. . . . . . . . . . . 26
iii. Working Multi-Element Calibrators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

iv. Multi-Element Intermediate Working Calibration Verification Standards …

v. Working Multi-Element Calibration Verification Standards…………………… 28
vi. Internal Quality Control Materials (“Bench” QC) . . . . . . . . . . . .. . . . . . . . . . 28
7) Analytical Instrumentation & Parameters
a. Instrumentation & Equipment Setup
i. ICP-DRC-MS
1. Modifications made to ICP-DRC-MS . . . . . . . . . . . . . . . . . . . . . . . . . .

2. Sample introduction system setup . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3. Configuration of tubing for liquid handling . . . . . . . . . . . . . . . . . . . . . .

4. Cones used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5. Gases & Regulators setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6. Chiller / Heat Exchanger. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ii. Computer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

iii. Autosampler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

b. Parameters for Instrument and Method (see Table 1, pp. 47-49) . . . . . . . . . . . 34
8) Method Procedures
a. Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
i. Types of Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ii. Calibration Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

b. Daily Analysis of Samples
i. Preparation of the Analytical Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . .

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 3 of 73

ii. Preparation of Samples for Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

iii. Specimen Storage and Handling during Testing . . . . . . . . . . . . . . . . . . . .

iv. Starting the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v. Monitoring the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vi. Records of Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii. Transfer of Results to the Laboratory Database . . . . . . . . . . . . . . . . . . . . .

viii. Analyst Evaluation of Run Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix. Submitting Final Work for Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

x. Overnight operation (or Any Use of Autostop) . . . . . . . . . . . . . . . . . . . . . . . 47
c. Equipment Maintenance
i. ICP-MS Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
ii. Data Backup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
9) Interpretation of the Results
a. Reportable Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
b. Reference Ranges (Normal Values) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
c. Action Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
10) Method Calculations
a. Method Limit of Detection (LOD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
b. Method Limit of Quantitation (LOQ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
c. QC Limits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
11) Alternate Methods for Performing Test and Storing Specimens If Test System
Fails . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Appendix A (Ruggedness Test Results) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Appendix B (Tables and Figures) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1
This page intentionally left blank

Page 4 of 73

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

1.
2.
3.
4.
5.

6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.

Page 5 of 73

Cross reference to DLS CLIA and Policy and Procedures policy
Summary of Test Principle and Clinical Relevance
1) a. b.
Safety Precautions
4) a.b.
Computerization; Data System Management
8) b.vi vii ix
Specimen Collection, Storage, and Handling Procedures; Criteria for Specimen
Rejection
3) a.b.
Procedures for Microscopic Examinations; Criteria for Rejection of Inadequately
Prepared Slides
- As no microscope used in this process there are no procedures for
microscopic examinations; and as no slides are prepared for this analysis
there is no criteria for rejection of inadequately prepared slides
Preparation of Reagents, Calibrators (Standards), Controls, and All Other
Materials; Equipment and Instrumentation
5) a. i ii iii b. 6) a. b. c. d. 7) a. b. 8) c i ii
Calibration and Calibration Verification Procedures
8) ii
Procedure Operating Instructions; Calculations; Interpretation of Results
8) b. i ii iv v x
Reportable Range of Results
9) a.
Quality Control (QC) Procedures
8) a. i
Remedial Action If Calibration or QC Systems Fail to Meet Acceptable Criteria
8) ii 1, ii 2, e
Limitations of Method; Interfering Substances and Conditions
2) a. b.
Reference Ranges (Normal Values)
9) b.
Critical Call Results ("Panic Values")
9) c.
Specimen Storage and Handling during Testing
8) b. iii
Alternate Methods for Performing Test or Storing Specimens If Test System Fails
11)
Test Result Reporting System; Protocol for Reporting Critical Calls (If Applicable)
9) c.
Transfer or Referral of Specimens; Procedures for Specimen Accountability and
Tracking
3) c.
References

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 6 of 73

List of Tables
Table 1. Instrument and Method Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56
Table 2. Suggested Maximum Analyte Concentrations for Base Serum . . . . . . . . . . 58
Table 3. Concentrations of Analytes in the Multi-Element Intermediate Stock
Standard from High Purity Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Table 4. Preparation of Multi-element Intermediate Working Standards . . . . . . . . . 59
Table 5. Acceptable Ways to Perform Two Consecutive Analytical Runs, Bracketing
with Bench Quality Control Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Table 6. A Typical SAMPLE/BATCH Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Table 7. Preparation of Multi-element Intermediate Working Standards . . . . . . . . . 61
Table 8. Range of Reporting and Calibration Verification Requirements . . . . . . . . . 61
Table 9. Boundary Concentrations and Replicate Range Maximums . . . . . . . . . . . . 62
Table 10. Normal Ranges for Serum Concentrations . . . . . . . . . . . . . . . . . . . . . . . .

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 7 of 73

List of Figures

Figure 1.

Configuration of Tubing and Devices for Liquid Handling . . . . . . . . . . . . . 63

Figure 2.

ELAN ICP-DRC-MS Method Screen Shots

Figure 3.

a. Timing Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

b. Processing Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c. Equations Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

d. Calibration Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

e. Sampling Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

f. Report Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ESI SC4 Autosampler Screen Shots
a. Main page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

b. Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
c. Communication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
d. 5x12 Rack Setup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
e. 50mL Tube Rack Setup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 8 of 73

1) Clinical Relevance & Summary of Test Principle
a. Clinical Relevance:
This method is used to achieve rapid and accurate quantification of three
elements of toxicological and nutritional interest including Zinc (Zn), Copper (Cu)
and Selenium (Se). The method may be used to screen serum when people are
suspected to be acutely exposed to these elements or to evaluate chronic
environmental or other non-occupational exposure.
b. Test Principle:
Inductively coupled plasma dynamic reaction cell mass spectrometry (ICP-DRCMS) is a multi-element analytical technique capable of trace level elemental
analysis [1-4]. This ICP-DRC-MS method is used to measure the entire panel of
3 elements, or any subgroup of these. Liquid samples are introduced into the
ICP through a nebulizer and spray chamber carried by a flowing argon stream.
By coupling radio-frequency power into flowing argon, plasma is created in which
the predominant species are positive argon ions and electrons and has a
temperature of 6,000-8,000 K. The sample passes through a region of the
plasma and the thermal energy atomizes the sample and then ionizes the atoms.
The ions, along with the argon, enter the mass spectrometer through an interface
that separates the ICP (at atmospheric pressure, ~760 torr) from the mass
spectrometer (operating at a pressure of 10-5 torr). The ions pass through a
focusing region, the dynamic reaction cell (DRC), the quadrupole mass filter, and
finally are counted in rapid sequence at the detector allowing individual isotopes
of an element to be determined. In this method, the instrument is operated in
‘DRC’ mode where the cell is pressurized with 99.99+% ammonia gas which
collides or reacts with the incoming ions to eliminate interfering ions and leave
the ion of interest to be detected. After leaving the DRC cell, the ions are
focused with ion optics into a quadrupole mass analyzer with a nominal mass
resolution of 0.7amu. The quadrupole is sequentially scanned to specific mass
to charge ratio of each analyte and intensity is detected with a pulse detector.
Electrical signals resulting from the detection of ions are processed into digital
information that is used to indicate first the intensity of the ions and then the
concentration of the element. This method was originally based on the methods
by Piraner and Walters [5-8] and the DRC portions of the method are based on
work published by Tanner et al. [2, 3]. The isotopes measured by this method
include zinc (m/z 64), copper (m/z 65) and selenium (m/z 78) and the internal
standard gallium (m/z 71). Serum samples are diluted 1+1+28 with water and
diluent containing gallium (Ga) for multi-internal standardization.

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 9 of 73

2) Limitations of Method; Interfering Substances and Conditions
a. Interferences Addressed by This Method
i. Correction & Elimination of Interferences (64Ni, 36Ar14N2) on Zinc (64Zn).
1. Mathematical Correction for Nickel (64Ni) Interference:
The correction equation (-0.035297* Ni60) is used in the “Equations” tab of
the method to correct the counts observed as m/z 64 to exclude counts
due to 64Ni.
2. Elimination of 36Ar14N2 Interference Using DRC: The dynamic reaction cell
of the ELAN ICP-DRC-MS is used in this method to eliminate interference
from 36Ar14N2 onto zinc at m/z 64. See Section 1.b for an explanation of
this process.
ii. Elimination of Interferences (40Ar25Mg, 36Ar14N21H) on Copper (65Cu) Using
DRC. The dynamic reaction cell of the ELAN ICP-DRC-MS is used in this
method to eliminate the interference 40Ar25Mg, 36Ar14N21H on copper at m/z 65.
See Section 1.b for an explanation of this process.
iii. Correction & Elimination of Interferences (78Kr, 38Ar40Ar, 38Ar40Ca) on Selenium
(78Se).
1. Mathematical Correction for Krypton (78Kr) Interference:
The correction equation (-0.030461*Kr83) is used in the “Equations” tab of
the method to correct the counts observed as m/z 78 to exclude counts
due to 78Kr.
2. Elimination of 38Ar40Ar, 38Ar40Ca Interference Using DRC: The dynamic
reaction cell of the ELAN ICP-DRC-MS is used in this method to eliminate
interference from 38Ar40Ar, 38Ar40Ca onto selenium at m/z 78. See Section
1.b for an explanation of this process.
b. Limitations of Method (Interferences Remaining in Method)
i.

Ca16O1H Interference on Copper (65Cu):
It has been determined that a small interference remains at m/z 65 when the
serum matrix contains very high calcium levels. Even at extreme calcium
levels, this interference has not been found to be significant (< 1%).

ii. Time between dilution of serum materials and analysis:
Selenium is not stable in the diluted sample for more than 7 hours. Diluted
serum must be analyzed within 7 hours of preparation (see Appendix A, test 5
for details).

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 10 of 73

3) Procedures for Collecting, Storing, and Handling Specimens; Criteria for
Specimen Rejection; Specimen Accountability and Tracking
a. Procedures for Collecting, Storing, and Handling Specimens: Specimen handling
conditions, special requirements, and procedures for collection and transport are
discussed in the division (DLS) Policies and Procedures Manual [9]. Copies are
available in branch, laboratory, and special activities specimen-handling offices.
An electronic copy is available at:
http://inside.nceh.cdc.gov/dls/pdf/policiesprocedures/Policies_and_Procedures_
Manual.DLS.2006mod.pdf In general, if more than one vacutainer of blood is to
be drawn from an individual, the trace metals tube should be drawn second or
later. Draw the blood through a stainless steel needle into a pre-screened 7 mL
vacutainer. Allow the blood in the stoppered vacutainer clot for 30-40 minutes,
but not longer than 60 minutes. Without opening the vacutainer, centrifuge it for
10 minutes at 2400 rpm. Use a pre-screened serum separator to remove the
serum from the clot. Under a laminar flow hood, pour the serum in the serum
separator into pre-screened polyethylene vials. Serum specimens should be
transported and stored at  4oC. Once received, they can be frozen at  -20oC
until time for analysis. Portions of the sample that remain after analytical aliquots
are withdrawn should be refrozen at  -20oC. Samples thawed and refrozen
several times are not compromised.
i. No fasting or special diets are required.
ii. Specimen type - serum
iii. Acceptable containers include pre-screened polyethylene vials and prescreened 7 mL vacutainers should be used for specimen acquisition.
iv. Specimen stability has been demonstrated for several months at approximately
-20°C or at approximately -70°C for several years.
b. Criteria for Specimen Rejection: Specimen characteristics that may compromise
test results are indicated above. Reasons for rejection of a sample for analysis
include
i. Low volume: Optimal amount of serum is 1-2 mL, minimum is about 0.8 mL.
The volume of serum used for one analysis is 0.15 mL.
ii. Contamination: Improper collection procedures or collection devices can
contaminate the serum by contact with dust, dirt, etc.
In all cases, request a second serum specimen.

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 11 of 73

c. Transfer or Referral of Specimens; Procedures for Specimen Accountability and
Tracking: Location, status, and final disposition of the specimens will be tracked
at least by paper document in the “Study Folder” (created before analysts receive
the samples). Apart from this specimen tracking form, this folder will also contain
the paper print outs of results from analysis of the specimens. Maintain records
for a minimum of 3 years. Use only numerical identifiers for samples within the
laboratory (e.g., case ID numbers) in order to safeguard confidentiality. Only the
medical supervisor (MS) or project coordinator (PC) i.e. non CDC personnel
should have access to the personal identifiers.
4) Safety Precautions
a. General Safety
i. Observe all safety regulations as detailed in the Division (DLS) Safety Manual.
Additional information can be found in your lab’s chemical hygiene plan.
ii. Observe Universal Precautions when working with serum.
iii. Wear appropriate gloves, lab coat, and safety glasses while handling all
solutions.
iv. Exercise special care when handling and dispensing concentrated nitric acid.
Add acid to water. Nitric acid is a caustic chemical that is capable of causing
severe eye and skin damage. If nitric acid comes in contact with any part
of the body, quickly wash the affected area with copious quantities of
water for at least 15 minutes.
v. Use secondary containment for containers holding biological or corrosive
liquids.
vi. The use of the foot pedal on the Micromedic Digiflex™ is recommended
because it reduces analyst contact with work surfaces that have been in
contact serum and also keeps the analyst’s hands free to hold the specimen
cups and autosampler tubes and to wipe off the tip of Micromedic Digiflex™.
vii. Training will be given before operating the ICP-DRC-MS, as there are many
possible hazards including ultraviolet radiation, high voltages, radio-frequency
radiation, and high temperatures. This information is also detailed in the
PerkinElmer ELAN® ICP-DRC-MS System Safety Manual.
viii. Ammonia gas cylinders (either in use or on storage) should be placed in a
cabinet which is well ventilated to the house exhaust. Ammonia cylinders in
use should not be placed on their side as the cylinder valve can become
“frozen” in place as a result of the cooling capacity of expanding ammonia gas.

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ix. Wipe down all work surfaces at the end of the day with bleach-rite spray or
freshly prepared 10% (v/v) sodium-hypochlorite solution.
b. Waste Disposal: Operators of this method should take the CDC-OHS Hazardous
Chemical Waste Management Course (initial and yearly refreshers).
i. Waste to be Placed in Biohazard Autoclave Bags & Pans:
1. All biological samples and diluted specimens (after analysis run).
2. All disposable plastic and paper which contact serum (autosampler tubes,
gloves, etc.). Pipette tips can be placed in either autoclave pans or sharps
containers.
3. Used non-glass/quartz ICP-MS consumables (i.e. probes, tubing, cones,
ion lenses).
ii. Waste to be Placed Into Sharps Containers: Broken glass or quartz instrument
consumables (broken spray chambers, torches, nebulizers, etc. . .). Pipette
tips can be placed in either autoclave pans or sharps containers. Large broken
glass which will not fit in the sharps container should be placed in a separate
autoclave pan from other waste and labeled as “broken glass” (see the
“Autoclaving” section of the CDC safety policies and practices manual located
in the laboratory).
iii. Liquid Waste
1. Waste discarded down sink: Only liquid waste from the ICP-DRC-MS
instrument can be discarded at the sink. Flush the sink with copious
amounts of water.
2. Waste to be Picked up by Hazardous Waste Program: Submit request for
hazardous waste removal of all other liquid waste.

5) Instrument & Material Sources
a. Sources for ICP-MS Instrumentation
i. ICP-MS: Inductively Coupled Plasma Dynamic Reaction Cell Mass
Spectrometer (ELAN® 6100 DRCPlus or ELAN® DRC II) (PerkinElmer Norwalk,
CT, www.perkinelmer.com).
ii.Recirculating chiller / heat exchanger for ICP-MS: Refrigerated chiller
(PolyScience 6105PE for ELAN® 6100 DRCPlus instruments) or heat exchanger

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(PolyScience 3370 for ELAN® DRC II instruments) (PerkinElmer Norwalk, CT,
www.perkinelmer.com).
iii. Autosampler: ESI SC-4 autosampler (Elemental Scientific Inc., Omaha, NE) or
equivalent.
b. Sources for ICP-MS Parts & Consumables
NOTE: The minimum number of spares recommended before reordering (if
owning one instrument) are listed as “# Spares =” in the descriptions below.
i. Adapter, plastic: 1/4-28 female threads on one side, 1.8mm barb adapter on
the other. Connects ¼-28 nut at flanged tubing connection to 0.045” i.d.
peristaltic pump tubing. Use part # B019-3342 (“Type A” adapter, PerkinElmer
Norwalk, CT, www.perkinelmer.com) or equivalent. # Spares = 4.
ii. Adapter, PEEK: Securely connects 1.6mm O.D. PFA tubing to 0.03” I.D.
peristaltic tubing. Composed of three PEEK parts.
1. Female nut for 1.6mm O.D. (1/16”) tubing. Like part P-420 (Upchurch
Scientific, Oak Harbor, WA, www.upchurch.com).
2. PEEK ferrule. Like part P-260x (10pk SuperFlangeless ferrule, Upchurch
Scientific, Oak Harbor, WA, www.upchurch.com).
3. Conical Adapter Body. Like part P-692 (Upchurch Scientific, Oak Harbor,
WA, www.upchurch.com).
iii. Coolant, for Polyscience chiller or heat exchanger: Only PerkinElmer part #
WE01-6558 (PerkinElmer Norwalk, CT, www.perkinelmer.com) is approved for
use by PerkinElmer. # Spares = 6.
iv. Cone, sampler: Both platinum and nickel cones have been used successfully.
For platinum cones, Spectron part # SC2013-Pt (Spectron, Ventura, CA,
www.spectronus.com) or equivalent. For nickel cones, PerkinElmer part #
WE021140 (PerkinElmer Norwalk, CT, www.perkinelmer.com) or equivalent. #
Spares = 4.
v. Cone, skimmer: Both platinum and nickel cones have been used successfully.
For platinum cones, Spectron part # SC2014-Pt (Spectron, Ventura, CA,
www.spectronus.com) or equivalent. For nickel cones, PerkinElmer part #
WE021137 (PerkinElmer Norwalk, CT, www.perkinelmer.com) or equivalent. #
Spares = 4.
vi. Connector (for tubing): Use to connect 1/8” I.D. PVC tubing to 0.125” I.D
peristaltic pump tubing. Use part # 3140715 (PerkinElmer Norwalk, CT,
www.perkinelmer.com) or equivalent. # Spares = 4.
vii. Detector, electron multiplier: Like part # N8125001 (PerkinElmer Norwalk, CT,
www.perkinelmer.com). Available direct from manufacturer (part # 14210,

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SGE Incorporated, Austin, Texas, http://www.etpsci.com) or various
distributors. # Spares = 1.
viii. Hose, for connection to chiller: Push on hose. I.D. = ½”, O.D. = ¾”. Use part
# PB-8 (per inch, Georgia Valve and Fitting, Atlanta, GA, www.swagelok.com)
or equivalent. Do not normally need spare hose (unless moving instrument
into a new location).
ix. Hose, for exhaust of ELAN: Available as part of ELAN installation kit from
Perkin Elmer (PerkinElmer Norwalk, CT, www.perkinelmer.com). Available
direct from manufacturer as part # S-LP-10 air connector (Thermaflex,
Abbeville, SC, www.thermaflex.net). Equivalent part may be substituted. #
Spares = 10 feet of 4” diameter and 10 feet of 6” diameter hose.
x. Injector, quartz with ball joint: I.D. = 2.0 mm. PerkinElmer part # WE023948
(PerkinElmer Norwalk, CT, www.perkinelmer.com). Available direct from
manufacturer as part # 400-30 (Precision Glass Blowing, Centennial, CO,
www.precisionglassblowing.com) or from various distributors. # Spares = 2.
xi. Injector support (for pass-through injector): PerkinElmer part # WE023951
(PerkinElmer Norwalk, CT, www.perkinelmer.com). Available direct from
manufacturer as part # 400-37 (Precision Glass Blowing, Centennial, CO,
www.precisionglassblowing.com) or from various distributors. # Spares = 2.
xii. Ion Lens: PerkinElmer part # WE018034 (PerkinElmer Norwalk, CT,
www.perkinelmer.com). # Spares = 3.
xiii. Nebulizer, quartz concentric: Initial work using this method has used the
standard Type A, 3mL/min nebulizer. Alternatively, the Type C, 1mL/min
nebulizer may be used to improve sensitivity and precision. The ELAN
supplies 30psi argon to the nebulizer. Variations of these nebulizers may be
substituted with or without quick connects for the gas and liquid ports. Quartz
nebulizers are used to avoid potential contamination from borosilicate glass
(i.e. barium, uranium). # Spares = 2.
1. Type A, Standard ELAN 3mL/min nebulizer: PerkinElmer part #
WE024371 (PerkinElmer Norwalk, CT, www.perkinelmer.com). Available
directly from manufacturer as part # TQ-30-A3 (Meinhard Glass Products,
Golden, CO, www.meinhard.com) or from various distributors. The
flangeless nut and ferrule assembly has been used for liquid sample backend connection to this nebulizer.
2. Type C, 1 mL/min nebulizer with quick disconnects for liquid and gas ports:
One example is part # 500-70QQDAC (Precision Glass Blowing,
Centennial, CO, www.precisionglassblowing.com). This nebulizer is
designed to use quick disconnects part # 500-QD (liquid) and # 500-AC
(argon).
xiv. Nebulizer Connections (gas): (for nebulizer argon side-arm).
1. If not using quick disconnection fitting, insert nebulizer argon side-arm into
the 1/8” i.d. vinyl tubing and secure the connection with a hose clamp for

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¼” o.d tubing (like part # EW-06832-01, Cole Palmer Instrument Company,
Vernon Hills, Illinois, www.colepalmer.com). # Spares = 2.
2. Quick disconnection fitting: Like part # 500-AC (Precision Glass Blowing,
Centennial, CO, www.precisionglassblowing.com). # Spares = 2.
xv. Nebulizer Connections (liquid): (for nebulizer 4mm o.d. liquid sample backend).
Can use quick disconnect or flangeless nut and ferrule assembly.
1. Quick Disconnect: Like Part # 500-QD (Precision Glass Blowing,
Centennial, CO, www.precisionglassblowing.com). # Spares = 2.
2. Flangeless nut and ferrule assembly: An assembly such as part # FIT KIT
3 (Meinhard Glass Products, Golden, CO, www.meinhard.com) or
equivalent. Individual pieces of FIT KIT #3 can be purchased as follows.
a. Nut, flangeless, 1/16”, ¼-28, Delrin® (Acetal), red. Part # P202x (10pk,
Upchurch Scientific, Oak Harbor, WA, www.upchurch.com). # Spares
= 10.
b. Ferrule, flangeless, 1/16”, Tefzel® (ETFE), blue. Part # P-200x (10pk,
Upchurch Scientific, Oak Harbor, WA, www.upchurch.com). # Spares
= 10.
c. Adapter, 1/4-28 internal to 5/16-24 internal, PEEK™. Part # P-135
(Upchurch Scientific, Oak Harbor, WA, www.upchurch.com). # Spares
= 2.
d. Nut, 4mm ID PEEK. Part of fit kit 3 for concentric nebulizers. Part # S1050 (Meinhard Glass Products, Golden, CO, www.meinhard.com). #
Spares = 2.
e. Ferrule, 4mm ID green Delrin. Part of fit kit 3 for concentric nebulizers.
Part # S-1121 (Meinhard Glass Products, Golden, CO,
www.meinhard.com). # Spares = 2.
xvi. Nut: (for flanged connections of 1.59mm (1/16”) o.d. PFA tubing) Flanged, for
1/16” o.d. tubing, 1/4-28 threads. Use part # P-406x (pkg. of 10, Upchurch
Scientific, Oak Harbor, WA, www.upchurch.com) or equivalent. Use a Tefloncoated Viton o-ring with this nut instead of the stainless steel washer that
comes with part # P-406x). # Spares = 10.
xvii. Nut: (for bottom port of autosampler rinse station) 10-32 UMC threads for
1/16” tubing. Such as part # M653x (Upchurch Scientific, Oak Harbor, WA,
www.upchurch.com) or equivalent. # Spares = 2.
xviii. Nut and Ferrule set, 1/8” Swagelok: Such as part # SS-200-NFSET (stainless
steel) or part # B-200-NFSET (brass) (Georgia Valve and Fitting, Atlanta, GA,
www.swagelok.com) or equivalent. For part numbers listed here a quantity of 1
means 1 nut, 1 front ferrule, and 1 back ferrule. Spares = 20.
xix. Nut and Ferrule set, 1/4” Swagelok: Such as part # SS-400-NFSET (stainless
steel) or part # B-400-NFSET (brass) (Georgia Valve and Fitting, Atlanta, GA,

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www.swagelok.com) or equivalent. For part numbers listed here a quantity of 1
means 1 nut, 1 front ferrule, and 1 back ferrule. Spares = 20.
xx. Oil, Welch Directorr Gold: For roughing pumps. Available direct from
manufacturer as part # 8995G-15 (1 gallon, Welch Rietschle Thomas, Skokie,
IL, www.welchvacuum.com) or from various distributors. Equivalent oil may be
substituted. # Spares = 4.
xxi. O-ring: (for sampler cone) PerkinElmer part # N8120511 (pkg. of 5,
PerkinElmer, Shelton, CT, www.perkinelmer.com) or equivalent. # Spares = 20
o-rings.
xxii. O-ring: (for skimmer cone) PerkinElmer part # N8120512 (pkg. of 5,
PerkinElmer, Shelton, CT, www.perkinelmer.com) or equivalent. # Spares = 20
o-rings.
xxiii. O-ring: (for flanged connections of 1.59mm (1/16”) o.d. PFA tubing) Tefloncoated Viton o-ring, i.d. = 1/16", thickness = 1/16”, o.d. = 3/16”. Such as part #
V75-003 (O-rings West, Seattle, WA, www.oringswest.com) or equivalent. #
Spares = 20.
xxiv. O-ring: (for injector support).
1. Internal o-rings: ID = ¼”, OD = 3/8”, thickness = 1/16”. Need 2 o-rings per
injector support to setup. PerkinElmer part # N8122008 (PerkinElmer,
Shelton, CT, www.perkinelmer.com) or equivalent (such as part # V75-010,
O-rings West, Seattle, WA, www.oringswest.com). # Spares = 20.
2. External o-rings: ID = 3/8”, OD = 1/2”, thickness = 1/16”. Need 2 o-rings
for each injector support setup. PerkinElmer part # N8122009
(PerkinElmer, Shelton, CT, www.perkinelmer.com) or equivalent (such as
part # V75-012, O-rings West, Seattle, WA, www.oringswest.com). #
Spares = 20.
xxv. O-ring: (for inside spray chamber at nebulizer port) Such as part # 120-56
(Precision Glass Blowing, Centennial, CO, www.precisionglassblowing.com).
Additional o-rings can sometimes be obtained free of charge or at reduced
price when acquired while purchasing spray chambers. # Spares = 20.
xxvi. O-ring: (for inside of torch mount): Part # WE017284 (PerkinElmer, Shelton,
CT, www.perkinelmer.com). Do not substitute. The PerkinElmer o-ring is
special metal impregnated to minimize RF leakage though the torch mount. #
Spares = 2.
xxvii. Photon Stop: PerkinElmer part # WE018278 (PerkinElmer, Shelton, CT,
www.perkinelmer.com). Alternate “snap in” lens assembly requires
PerkinElmer part # W1013361 (PerkinElmer Norwalk, CT,
www.perkinelmer.com). # Spares = 1.
xxviii. Plugs, Quick Change for Roughing Pump Oil: These plugs will only work on
the Varian roughing pumps which come standard on ELAN DRC II ICPMS
instruments. These plugs will not fit the Leybold pumps which come standard

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on the ELAN DRC Plus instruments. Part # W1011013 (PerkinElmer, Shelton,
CT, www.perkinelmer.com). No spares typically needed.
xxix. Probes: (for ESI autosampler) Teflon, carbon fiber support, 0.8mm i.d., blue
marker, 1/4-28 fittings. Like part number SC-5037-3751 (Elemental Scientific
Inc., Omaha, NE., www.elementalscientific.com). # Spares = 2.
xxx. RF coil. PerkinElmer part # WE02-1816 (PerkinElmer, Shelton, CT,
www.perkinelmer.com) or equivalent. # Spares = 2.
xxxi. Screw, for Torch Mount: PerkinElmer part # WE011870. (PerkinElmer,
Shelton, CT, www.perkinelmer.com) or equivalent. # Spares = 3.
xxxii. Spray chamber, quartz concentric: PerkinElmer part # WE025221
(PerkinElmer, Shelton, CT, www.perkinelmer.com) or equivalent. Available
direct from manufacturer as part # 400-20 (Precision Glass Blowing,
Centennial, CO, www.precisionglassblowing.com) or from various distributors.
# Spares = 2.
xxxiii. Torch, quartz: PerkinElmer part # N812-2006 (PerkinElmer, Shelton, CT,
www.perkinelmer.com) or equivalent. Available direct from manufacturer as
part # 400-10 (Precision Glass Blowing, Centennial, CO,
www.precisionglassblowing.com) or various distributors. Damaged torches
can often be repaired for substantially lower cost than purchasing a new one by
companies such as Wilmad LabGlass (Buena, NJ, www.wilmad-labglass.com)
or Precision Glass Blowing (Centennial, CO, www.precisionglassblowing.com).
# New Spares = 2.
xxxiv. Tubing and adapter, for SC autosampler rinse station drain: Tygon tubing and
adapter to attach to back of SC autosampler for draining rinse station waste
(like part # SC-0303-002, Elemental Scientific Inc., Omaha, NE.,
www.elementalscientific.com).
xxxv. Tubing and adapters, for SC autosampler rinse station filling: Teflon tubing
and adapters (to attach to back of SC autosampler for filling rinse stations and
to attach to rinse containers). Like part # SC-0302-0500, Elemental Scientific
Inc., Omaha, NE., www.elementalscientific.com).
xxxvi. Tubing, argon delivery to instrument: I.D. = 1/8”, O.D. = ¼”. Such as part # C06500-02 (pkg. of 100ft, polypropylene, Fisher Scientific International,
Hampton, NH, www.fishersci.com) or equivalent. # Spares = 50ft.
xxxvii. Tubing, peristaltic, 0.03” i.d. (sampling): Standard PVC, 2-stop (black / black)
peristaltic pump tubing, i.d. = 0.03”. PerkinElmer part # 09908587
(PerkinElmer, Shelton, CT, www.perkinelmer.com) or equivalent. # Spares = 6
packs of 12 tubes.
xxxviii. Tubing, peristaltic, 0.125” i.d. (spray chamber drain): Standard PVC, 2-stop
(black / white) peristaltic pump tubing, i.d. = 0.125”. PerkinElmer part # N8122012 (PerkinElmer, Shelton, CT, www.perkinelmer.com) or equivalent. #
Spares = 6 packs of 12 tubes.
xxxix. Tubing, PFA: I.D. = 0.5mm, O.D. = 1.59mm (1/16”). Used to transfer liquid

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1. possibly used between nebulizer and peristaltic pump tubing (if quick
connection is not used for liquid sample delivery)
The Perfluoroalkoxy (PFA) copolymer is a form of Teflon®. Such as part #
1548 (20ft length, Upchurch Scientific, Oak Harbor, WA, www.upchurch.com)
or equivalent. # Spares = 20ft.
xl. Tubing, PVC, i.d. = 1/8”, o.d. = 3/16”. Used to transfer liquid
1. between spray chamber waste port and peristaltic pump
Like part # 14-169-7A (pkg. of 50ft, Fisher Scientific International, Hampton,
NH, www.fishersci.com) or equivalent. # Spares = 20ft.
xli. Tubing, Stainless Steel, o.d. = 1/8”, wall thickness = 0.028”: Used to connect
DRC gas cylinders to ELAN DRC gas ports. Also used to replace plastic
tubing in the DRC gas path within the ELAN. Like part # SS-T2-S-028-20 (20ft,
Georgia Valve and Fitting, Atlanta, GA, www.swagelok.com) or equivalent.
Spares = 20ft.
xlii. Tubing, Teflon, corrugated, ¼” o.d.: Connects to the auxiliary and plasma gas
side-arms of the torch. Part # WE015903 (PerkinElmer, Shelton, CT,
www.perkinelmer.com). # Spares = 2.
xliii. Tubing, Tygon, i.d. = 3/16”, o.d. = 5/16”: Used to transfer liquid between rinse
station drain port and liquid waste jug. Like part # EW-06409-15 (50 ft, Cole
Parmer, Vernon Hills, Illinois, www.coleparmer.com) or equivalent. # Spares =
20ft.
xliv. Tubing, vinyl (argon delivery to nebulizer): Vinyl Tubing, 1/8" ID x 1/4" OD.
Like part # EW-06405-02 (Cole Parmer, Vernon Hills, Illinois,
www.coleparmer.com) or equivalent. Equivalent tubing material may be
substituted. # Spares = 10ft.
xlv. Union Elbow, PTFE ¼” Swagelok: Connects argon tubing to torch auxiliary
gas sidearm. Like part # T-400-9 (Georgia Valve and Fitting, Atlanta, GA,
www.swagelok.com) or equivalent. Spares = 2.
xlvi. Union Tee, PTFE, ¼” Swagelok: Connects argon tubing to torch plasma gas
sidearm and holds igniter inside torch sidearm. Like part # T-400-3 (Georgia
Valve and Fitting, Atlanta, GA, www.swagelok.com) or equivalent. Spares = 2.
c. Sources for ICP-MS Maintenance Equipment & Supplies
i. Anemometer: Like digital wind-vane anemometer (Model 840032, SPER
Scientific LTD., Scottsdale, AZ, www.sperscientific.com) or equivalent. Use to
verify adequate exhaust ventilation for ICP-MS (check with hoses fully
disconnected).
ii. Pan, for changing roughing pump oil: Like part # 53216 (United States Plastics
Corporation, Lima, OH, www.usplastic.com) or equivalent. # On hand = 1.

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iii. Container, to hold acid baths for glassware: Polypropylene or polyethylene
containers with lids (must be large enough for torch, injector, or spray chamber
submersion). May be purchased from laboratory or home kitchen supply
companies. # On hand = 4.
iv. Cotton swabs: Any vendor. For cleaning of cones and glassware.
v. Cutter (for 1/8” o.d. metal tubing): Terry tool with 3 replacement wheels. Like
part # TT-1008 (Chrom Tech, Inc., Saint Paul, MN, www.chromtech.com) or
equivalent.
vi. Getter Regeneration Kit: Part # WE023257 (PerkinElmer, Shelton, CT,
www.perkinelmer.com). Use this as needed (at least annually) to clean the
getter in the pathway of channel A DRC gas.
vii. Magnifying glass: Any 10x + pocket loupe for inspection of cones and other
ICP-MS parts. Plastic body is preferred for non-corrosion characteristics. Like
part # 5BC-42813 (Lab Safety Supply, Janesville, WI, www.labsafety.com).
viii. Screw Driver, for Ion Lens Removal: Screw driver with long, flexible shaft, and
2mm ball-Allen end for removal of ion lens screws, part # W1010620. Extra
2mm bits, part # W1010598 (PerkinElmer, Shelton, CT,
www.perkinelmer.com).
ix. Toothbrush: Any vendor. For cleaning ion lens and glassware.
x. Ultrasonic bath: Like ULTRAsonik™ Benchtop Cleaners (NEYTECH,
Bloomfield, CT, www.neytech.com) or equivalent.
d. Sources for General Laboratory Consumable Supplies
i. Bar Code Scanner: Like Code Reader 2.0 (Code Corporation, Draper, UT,
www.codecorp.com) or equivalent. For scanning sample IDs during analysis
setup. Any bar code scanner capable of reading Code 128 encoding at a 3 mil
label density can be substituted.
ii. Carboy (for preparation of serum quality control pool and waste jug for ICPMS
sample introduction system): Polypropylene 10-L carboy (like catalog # 02960-20C, Fisher Scientific, Pittsburgh, PA, www.fischersci.com) or equivalent.
Carboys with spouts are not advised due to potential for leaking.
iii. Containers for diluent and rinse solution: Two liter Teflon™ containers (like
catalog# 02-923-30E, Fisher Scientific, Pittsburgh, PA., www.fishersci.com)
and 4L polypropylene jugs (like catalog# 02-960-10A, Fisher Scientific,
Pittsburgh, PA, www.fishersci.com) have both been used. Acid rinse before
use. Equivalent containers may be substituted.
iv. Gloves: Powder-free, low particulate nitrile (like Best CleaN-DEX™ 100%
nitrile gloves, any vendor). Equivalent nitrile or latex gloves may be
substituted.

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v. Paper towels: For general lab use, any low-lint paper wipes such as
KIMWIPES®EX-L Delicate Task Wipers or KAYDRY®EX-L Delicate Task
Wipers (Kimberly-Clark Professional, Atlanta, GA, www.kcprofessional.com).
For sensitive applications in cleanrooms, a wipe designed for cleanroom use
may be desired such as the Econowipe or Wetwipe (Liberty, East Berlin, CT,
www.liberty-ind.com).
vi. Pipette (for preparation of serum dilutions to be analyzed): Micromedic DigiflexCX Automatic™ pipette equipped with 10.0-mL dispensing syringe, 2 mL
sampling syringe, 0.75-mm tip, and foot pedal (Titertek, Huntsville, AL,
http://www.titertek.com/).
vii. Pipettes (for preparation of intermediate stock working standards & other
reagents): Like Brinkmann Research Pro Electronic pipettes (Brinkmann
Instruments, Inc., Westbury, NY, http://www.brinkmann.com/home/). 5-100 L
(catalog #4860 000.070), 20-300 L (catalog #4860 000.089), 50-1,000 L
(catalog #4860 000.097), 100-5,000 L (catalog #4860 000.100). Note:
pipette catalog numbers are without individual chargers. Can purchase
individual chargers (pipette catalog numbers will differ) or a charging stand that
will hold four pipettes (catalog #4860 000.860). When purchasing pipette tips
(epTips), purchase one or more boxes, then “reloads” for those boxes after
that: 5-100 L (box catalog # 22 49 133-4, reload catalog # 22 49 153-9), 20300 L (box catalog # 22 49 134-2, reload catalog # 22 49 154-7), 50-1,000 L
(box catalog # 22 49 135-1, reload catalog # 22 49 155-5), 100-5,000 L (box
catalog # 22 49 138-5, reload catalog # 22 49 198-9, bulk bag catalog # 22 49
208-0). Equivalent pipettes and tips can be substituted.
viii. Tubes for sample analysis (for autosampler): Like polypropylene 15-mL
conical tubes, BD Falcon model #352097 (Becton Dickinson Labware, Franklin
Lakes, NJ, www.bd.com). Equivalent tubes may be substituted which are
shown by lot screening to be free of trace metal contamination. Clear plastics
tend to have lowest trace metal contamination. Blue colored caps have also
been used successfully for this method.
ix. Tubes for storage of intermediate working stock standards: Like polypropylene
50-mL centrifuge tubes, Corning Incorporated #430290 (Corning, NJ, 14831.
www.scienceproduct.corning.com). For use in storage of intermediate working
stock standards. Equivalent tubes may be substituted which are shown by lot
screening to be free of trace metal contamination. Clear plastics tend to have
lowest trace metal contamination. Orange colored caps have also been used
successfully for this method.
x. Votexer: Like MV-1 Mini Vortexer (VWR, West Chester, PA, www.vwr.com).
Used for vortexing serum specimens before removing an aliquot for analysis.
Equivalent item can be substituted.
xi. Water purification system: Like NANOpure DIamond Ultrapure Water System
(Barnstead International, Dubuque, Iowa, www.barnstead.com). For ultra-pure
water used in reagent and dilution preparations. An equivalent water

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purification unit capable of producing >18 Mega-ohm·cm water may be
substituted.
e. Sources of Chemicals, Gases, and Regulators
i. Acid, Hydrochloric acid: Veritas™ double-distilled grade, 30-35% (GFS
Chemicals Inc. Columbus, OH, www.gfschemicals.com). This is referred to as
“concentrated” hydrochloric acid in this method write-up. It is approximately 12
molar in concentration. For use in preparation of intermediate working stock
standards. An equivalent hydrochloric acid product may be substituted, but it
must meet or exceed the purity specifications of this product for trace metals
content.
ii. Acid, Nitric acid: Veritas™ double-distilled grade, 68-70% (GFS Chemicals Inc.
Columbus, OH, www.gfschemicals.com). For use in diluent, rinse solution,
intermediate working stock standards, and QC pool preparations. This is
referred to as “concentrated” nitric acid in this method write-up. It is
approximately 16 molar in concentration. An equivalent nitric acid product may
be substituted, but it must meet or exceed the purity specifications of this
product for trace metals content.
iii. Ethyl Alcohol (C2H5OH), USP dehydrated 200 proof (Pharmco Products, Inc.)
or equivalent.
iv. TritonX-100™ (“Baker Analyzed,” J.T. Baker Chemical Co. [www.jtbaker.com],
or any source whose product is low in trace-metal contamination).
v. Argon Gas (for plasma & nebulizer) and Regulator: High purity argon
(>99.999% purity, Specialty Gases Southeast, Atlanta, GA, www.sgsgas.com)
for torch and nebulizer. Minimum tank source is a dewar of liquid argon (180250L) but bulk tank for total building needs is preferred.
1. Regulator for argon (at dewar, if used): Stainless steel, single stage,
specially cleaned regulator with 3,000 psig max inlet, 0-100 outlet pressure
range, CGA 580 cylinder connector, and needle valve shutoff on delivery
side terminating in a ¼” Swagelok connector. Part number
KPRAFPF415A2AG10 (Georgia Valve and Fitting, Atlanta, GA,
www.swagelok.com). An equivalent regulator from an alternate vendor may
be substituted. # Spares = 1.
2. Regulator for argon (between bulk tank and PerkinElmer filter regulator):
Single Stage 316SS Regulator, with 0-300 psi Inlet Gauge, 0-200 psi
Outlet Gauge, Outlet Spring Range, 0-250 psi, ¼” Swagelok Inlet
Connection, ¼ turn Shut off Valve on Outlet with ¼” Swagelok Connection
and Teflon Seals. Part number KPR1GRF412A20000-AR1 (Georgia Valve
and Fitting, Atlanta, GA, www.swagelok.com). An equivalent regulator from
an alternate vendor may be substituted. # Spares = 1.
3. Regulator for argon (PerkinElmer filter regulator on back of ELAN): Argon
regulator filter kit. Catalog number N812-0508 (PerkinElmer, Shelton, CT,
www.perkinelmer.com).

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vi. Ammonia: Anhydrous ammonia (>99.99%) for DRC channel A is typically
purchased in cylinder size LB (2”x12”) (Matheson Tri-Gas, Montgomeryville,
PA, 18936. www.mathesontrigas.com).
1. Regulator for ammonia: Stainless steel, two stage, specially cleaned
regulator with 3,000 psig max inlet, 2-30 outlet pressure range, cylinder
connector CGA 180 (for lecture bottle cylinder) or CGA 705 (for Airgas
cylinder size 200), and needle valve shutoff on delivery side terminating in
a ¼” Swagelok connector. Like part number 3813-180 or 3813-705
(Matheson Tri-Gas, Montgomeryville, PA, www.matheson-trigas.com). An
equivalent regulator from an alternate vendor may be substituted. #
Spares = 1.
vii. Disinfectant, for work surfaces: Bleach-rite spray (any distributor). On-site
dilutions of bleach (1part bleach + 9 parts water) may be substituted, but must
be re-made daily after dilution.
viii. Standard, Gallium: Like 1,000 mg/L, item # PLGA2-2Y. (SPEX Industries,
Inc., Edison, NJ, www.spexcsp.com). Used as an internal standard in diluent.
Any vendor whose standards are traceable to the National Institute for
Standards and Technology may be substituted. The standard must have low
trace metal contamination.
ix. Standard, multi-element intermediate stock standard: Item number SM-2107013 (High Purity Standards, Charleston, SC, http://www.hps.net/). This is a
custom mix solution (see Table 3 p.49 for concentrations). This solution is
diluted to prepare the intermediate stock working standards, which are in turn
diluted to prepare the working calibrators. This solution can be prepared inhouse from NIST traceable single element stock solutions if necessary.
x. Triton X-100™ surfactant: Like “Baker Analyzed” TritonX-100™ (J.T. Baker
Chemical Co., www.jtbaker.com). Another source may be substituted, but it
must be free of trace-metal contamination.
6) Preparation of Reagent and Materials.
a. Diluent
i. Purpose: All samples (blanks, calibrators, QC, or patient samples) are
combined with the diluent during the sample preparation step before analysis.
This is where the internal standards are added which during the analysis will
compensate for instrumental variations on the analyte signal.
ii. Contents: An aqueous solution of 10 g/L Ga, 2% v/v double-distilled nitric
acid, 5% Ethyl Alcohol, 0.01% Triton X-100TM.
iii. Preparation (4L) & storage: This solution does not have to be made up in a
volumetric flask. The important thing about the concentration of the internal
standards is that they be consistent within all samples in one run. To prepare

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different volumes of diluent, add proportionally larger or smaller volumes of the
solution constituents.
1. Acid-rinse a 4 L container (material may be polypropylene (PP),
polymethylpentene (PMP), or Teflon™).
2. Partially fill (i.e. 70-80% full) the 4 L container with >18 Mega-ohm·cm
water.
3. Carefully add 80 mL double-distilled, concentrated nitric acid and mix.
4. Carefully add 200 mL Ethyl Alcohol and mix.
5. Add 4 mL each of 10 mg/L Ga.
6. Add 20 mL 2% Triton X-100TM stock solution and mix.
7. Make up to volume (approximately 4 L) with >18 Mega-ohm·cm water.
8. Store at room temperature and prepare as needed.
9. Label should include “10 µg/L Ga, 2% (v/v) HNO3, 5% Ethyl alcohol, 0.01%
Triton X-100TM“, “Store at room temperature”, preparation date, expiration
date (1 year from prep), and preparer’s initials.
b. Base Serum
i. Purpose: This serum pool material will be mixed with the intermediate working
calibrators just prior to analysis to matrix-match the calibration curve to the
serum matrix of the unknown samples.
ii. Contents: A mixture of multiple human serum sources purchased from
Tennessee Blood Services, 807 Poplar Ave., Memphis, TN 38105. These
serum were collected from different anonymous donors are used to
approximate an average serum matrix.
iii. Screening serum: Screen all sources of serum for metal content before mixing
together to make the base serum pool. Keep serum at ≤ -20C whenever
possible to minimize microbial growth. Analyte concentrations in the final base
serum pool should be in the low-normal population range (see Table 2, p. 49).
iv. Preparation & Storage:
1. Once screened, mix the serum collections together in a larger container
(i.e. acid washed polypropylene (PP), polymethylpentene (PMP), or
Teflon™) and stir for 30+ minutes on a large stir plate (acid wash large
Teflon™ stir bar before use).
2. For short term storage, store at 2-4°C. For long-term storage, dispense
into smaller-volume tubes (i.e., 10 mL acid-washed or lot screened
polypropylene tubes) and store at ≤ -20°C.
3. Labels on 10 mL tubes should include “Base Serum for Multi-Element
Method”, “Store Long Term at ≤ 20° C”, “Store Short Term at 2-4° C”,
preparation date, expiration date 3 years from prep date, and preparer’s
initials.

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c. ICP-DRC-MS Rinse Solution
i. Purpose: Pump this solution into the sample introduction system between
samples to prevent carry-over of the analytes of interest from one sample
measurement to the next. For this method, we also need to pump >18 Megaohm·cm water into the sample introduction system for 30 minutes after each
run to prevent the clog of the probe and tubing.
ii. Contents: An aqueous solution of 0.01% Triton X-100™ and 2% (v/v) doubledistilled nitric acid solution, 5% Ethyl Alcohol, 0.5% v/v Hydrochloric acid.
iii. Preparation & Storage:
1. Intermediate Triton X-100 Solution: To avoid the process of dissolving
pure Triton X-100 on a daily basis, prepare an intermediate 2% Triton X100™ / 5% (v/v) double-distilled, nitric-acid solution for daily use.
a. To prepare 2 L of Intermediate Triton X-100 Solution:
i.

Partially fill a 2 L acid-washed bottle (PP, PMP, or Teflon™) with
>18 Mega-ohm·cm water (approximately 1-1.5 L). Use of
volumetric flask is not required.

ii.

Add 20 mL of Triton X-100™ and stir until completely dissolved.
Use a Teflon™ stir bar and stir plate if necessary (acid wash stir
bar before use).

iii.

Carefully add 100 mL of double-distilled, concentrated nitric acid.

iv.

Fill to 2 L and stir thoroughly.

Label should include “2% Triton X-100™ / 5% (v/v) HNO3”, “Store
at room temperature”, preparation date, expiration date 1 year from
preparation date, and preparer’s initials.

2. Final Rinse Solution:
a. To Prepare 4 L of the Final Rinse Solution:
i.

Partially fill a 4 L acid-washed bottle (PP, PMP, or Teflon™) with
>18 Mega-ohm·cm water (approximately 2-3 L). Use of volumetric
flask is not required.

ii.

Carefully add 80 mL of double distilled concentrated nitric acid and
mix well.

iii.

Carefully add 200 mL ethyl alcohol and mix.

iv.

Carefully add 20 mL double distilled concentrated hydrochloric
acid.

Add 20 mL of the 2% Triton X-100™ / 5% (v/v) double-distilled,
nitric-acid intermediate stock solution and mix well.

vi.

Fill to 4 L using >18 Mega-ohm·cm water and mix well.

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vii.

Store at room temperature and prepare as needed. To prepare
volumes other than specified here, add proportionally larger or
smaller volumes of the solution constituents.

viii.

Label should include “2% v/v HNO3, 5% Ethyl alcohol, 0.01%
Triton X-100TM, 0.5% v/v Hydrochloric acid”, “Store at room
temperature”, preparation date, expiration date (1 year from prep),
and preparer’s initials.

d. Standards and Calibrators
i. Multi-Element Intermediate Stock Standard (calibrators and calibration
verification)
1. Purpose: This is the master solution from which all working calibrators will
be prepared. It will be diluted to prepare intermediate working calibrators
which are in turn diluted and included in each analytical run on the ICPDRC-MS. This same stock standard will be diluted to prepare the
intermediate working calibration verification solution which will be in turn
diluted and analyzed at least every 6 months for calibration verification
purposes (and as needed by supervisor request).
2. Contents: An aqueous solution containing all 3 elements of interest for this
method (does not include the internal standards). The concentrations of
the 3 elements in the intermediate stock standard are listed in Table 3
p.49. The matrix is 2% v/v HNO3 in >18 Mega-ohm·cm water.
3. Preparation (Purchase) & Storage:
a. Purchasing from vendors: The intermediate stock standard solution
may be purchased as a custom mixture from any vendor which
prepares multi-element solutions that are traceable to the National
Institute for Standards and Technology (NIST) for their accuracy
b. Current vendor & preparation process: Currently it is purchased from
High Purity Standards (Charleston, SC, part number SM-2107-013).
Details of the HPS preparation of the multi-element stock standard is
as follows (per statement on their literature):
“Sub-boiled high purity acids were used to put the high purity
metal, salts, or oxides into solution and to stabilize the
standard. The solution matrix is 2% (v/v) nitric acid in >18
Mega-ohm·cm water. The standard was made
gravimetrically by weighing the reference material to 5
significant figures. Volumetric glassware was calibrated
gravimetrically to 5 significant figures.”
c. In-house Preparation: If outside laboratories were not available to
prepare the intermediate stock standard solution, it is also possible to
make it in the laboratory from single element standards which are NIST
traceable.

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d. Storage: Store the solution at room temperature. Label these bottles
from HPS with additional information such as “store at room
temperature”, date received, date opened, and initials of person to first
open.
ii. Multi-Element Intermediate Working Calibration Standards
1. Purpose: Use the intermediate working standard solutions 1-5 each day of
analysis to prepare the final working calibrators that will be placed on the
autosampler of the ELAN® ICP-DRC-MS.
2. Content: The intermediate working standard solutions used in this method
are aqueous dilutions of the multi-element intermediate stock standard
solution in 2% (v/v) double-distilled nitric acid.
3. Preparation & Storage: To prepare different volumes, add proportionally
larger or smaller volumes of the solution constituents.
a. Cleaning flasks: Acid-rinse five 100-mL and one 2L volumetric flasks.
Check their cleanliness by comparing the counts observed on the ICPDRC-MS for 2% (v/v) HNO3 before and after contact with the flasks.
Mark each of the flasks according to how they will be used. These
flasks should be dedicated to this use in this method, and not used for
other purposes.
b. HNO3 Diluent Preparation: In the cleaned 2L volumetric flask, add 11.5L of >18 Mega-ohm·cm water, 40 mL high purity concentrated
HNO3. Fill to the mark and mix thoroughly. Use this diluent to fill the
remaining flasks during preparation of the intermediate working
standards.
c. Dilutions & Storage:
i.

Partially fill the 100 mL flasks with the HNO3 diluent (50-75% full).

ii.

Using the volumes listed (Table 4 p.49) pipette the appropriate
volume of the multi-element intermediate stock standard solution
into each of the five volumetric flasks. Dilute each solution to the
mark with the HNO3 diluent using a pipette for the final drops. Mix
each solution thoroughly. The final concentrations of the 5
elements are listed in Table 4 p.49.

iii.

Once mixed, transfer to acid-cleaned, labeled, 50-mL containers
(PP, PMP, or Teflon™) for storage. Labels should include
information such as “Multi-Element Serum Working Calibrators”,
“2% (v/v) HNO3”, date of preparation, expiration date (1 year from
date of preparation), “store at room temperature”, initials of
preparer, and concentrations for each element.

iv.

Store at room temperature.

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iii. Working Multi-Element Calibrators
1. Purpose: The working multi-element calibrators are dilutions of the
intermediate working standards. Analysis of these calibrators provides
each run with a signal to concentration response curve for each analyte in
the method. The concentration of an analyte in a patient serum sample
dilution is determined by comparing the observed signal from the dilution of
the patient serum sample to the response curve from the working multielement calibrators.
2. Content: The working multi-element calibrators are 1:30 dilutions of the
corresponding five intermediate working standards.
3. Preparation & Use: The working multi-element calibrators are made
immediately prior to analysis when the intermediate working standards are
mixed with base serum (Section 6.b) and diluent (Section 6.a) using a
Digiflex automatic pipetter. See Table 7 p.51 in section 8.b.ii for details of
sample preparation.
iv. Multi-Element Intermediate Working Calibration Verification Standards
1. Purpose: Use the intermediate working calibration verification standard to
satisfy calibration verification requirements for the method (see section
8.a.ii).
2. Content: The intermediate working standard calibration verification
solution used in this method is an aqueous dilution of the multi-element
intermediate stock standard solution (same as that used to prepare the
intermediate stock calibration standards) in 2% (v/v) double-distilled nitric
acid.
3. Preparation & Storage: To prepare different volumes, add proportionally
larger or smaller volumes of the solution constituents.
a. Cleaning flasks: Acid-rinse one 100-mL and one 2L volumetric flask
(the same 2L flask as was used in preparing the intermediate working
calibration standards can be used here). Check their cleanliness by
comparing the counts observed on the ICP-DRC-MS for 2% (v/v)
HNO3 before and after contact with the flasks. Mark the flasks
according to how they will be used. This flask should be dedicated to
this use in this method, and not used for other purposes.
b. HNO3 Diluent Preparation: In the cleaned 2L volumetric flask, add 11.5L of >18 Mega-ohm·cm water, 40 mL high purity concentrated
HNO3. Fill to the mark and mix thoroughly. Use this diluent to fill the
remaining flasks during preparation of the intermediate working
standards.
c. Dilutions & Storage:
i.

Partially fill the 100 mL flask with the 2% v/v HNO3 diluent (50-75%
full).

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ii.

Using the volumes listed (Table 4 p.49) pipette the appropriate
volume of the multi-element intermediate stock standard solution
into the 100mL volumetric flask. Dilute the solution to the mark
with the 2% v/v HNO3 diluent using a pipette for the final drops.
Mix thoroughly. The final concentrations of the 5 elements are
listed in Table 4 p.49 (also Table 8).

iii.

Once mixed, transfer to acid-cleaned, labeled, 50-mL containers
(PP, PMP, or Teflon™) for storage. Labels should include
information such as “Multi-Element Serum Working Calibration
Verification Standard”, “2% (v/v) HNO3”, date of preparation,
expiration date (1 year from date of preparation), “store at room
temperature”, initials of preparer, and concentrations for each
element.

v. Working Multi-Element Calibration Verifcation Standards
1. Purpose: The working multi-element calibration verification standard is a
dilution of the intermediate working calibration verification standard.
Analysis of this standard meets the calibration verification requirements
detailed in section 8.a.ii.
2. Content: The working multi-element calibration verification standard is a
1:30 dilution of the corresponding intermediate working calibration
verification standard.
3. Preparation & Use: The working multi-element calibration verification
standards are made immediately prior to analysis when the intermediate
working calibration verification standards are mixed with base serum
(Section 6.b) and diluent (Section 6.a) using a Digiflex automatic pipetter.
See Table 7 p.51 in section 8.b.ii for details of sample preparation. Store at
room temperature.
vi. Internal Quality Control Materials (“Bench” QC)
1. Purpose: Internal (or “bench”) quality control (QC) materials are used to
evaluate the accuracy and precision of the analysis process, and to
determine if the analytical system is “in control” (is producing results that
are acceptably accurate and precise). They are included in the beginning
and at the end of each analytical run. These pools will need to be
prepared periodically, as supply indicates, by spiking base serum.
Preparation of new pools should be made far enough in advance so that
both old and new pools can be analytes together for a period time
(preferably at least 20 runs) before switching to the new quality control
materials.
2. Content: The internal (or “bench”) quality control (QC) materials used in
this method are pooled human serum and may have been spiked to reach
a desired concentration. The analyte concentrations in the “low QC” are in
the low-normal concentration range. The analyte concentrations in the
“high QC” are in the high-normal concentration range.

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3. Preparation & Storage: Quality control materials can be either prepared by
and purchased from an external laboratory or prepared within the CDC
laboratories. Quality control must always be traceable to the National
Institute for Standards and Technology (NIST). The CDC laboratory
currently prepares its own bench QC materials using the following
procedures:
a. Collection of serum: Human serum can be purchased from blood
services companies such as Tennessee Blood Services, 807 Poplar
Ave., Memphis, TN 38105.
b. Screening serum: Screen different bottles for metal content before
mixing together to make 2 separate base serum pools (for preparing
the low and high bench QC materials).
i.

Keep serum at ≤ -20C whenever possible to minimize microbial
growth.

ii.

Analyte concentrations in the final serum pool to be spiked for the
low bench QC pool should be in the low-normal population range
(see Table 2, p. 49). Analyte concentrations in the final serum pool
to be spiked for the high bench QC pool should be less than some
pre-selected target concentration values in the high normal
population range.

c. Spiking of serum
i.

Analyze a sample of each serum pool. Record these results for
future recovery calculations.

ii.

Use these results to determine target analyte concentrations
possible for the pools

iii.

Calculate the volume of single element standards needed to spike
each pool to the desired concentrations.

iv.

While stirring the pools on large stir plates, spike each pool with
calculated volumes of single element standards (all spiking
standards used must be traceable to NIST).

Continue to stir pools for 30+ minutes after spiking, then reanalyze.

vi.

Repeat steps 4 and 5 until all analytes reach target concentrations
keeping track of the total volume of spiking solution added to each
serum pool.

d. Dispensing and Storage of serum
i.

Container Types: Dispense serum into lot screened containers
(i.e. 2 mL polypropylene cryovials). If possible, prepare tubes of
QC which have only enough volume for one typical run + 1 repeat
analysis. This allows for one vial of QC to be used per day of
analysis, reducing chances of contamination of QC materials due
to multi-day use.

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ii.

iii.

Dispensing: Dispensing can be accomplished most easily using a
Digiflex automatic pipette in continuous cycling dispense mode.
This process should be done in a clean environment (i.e. a class
100 cleanroom area or hood).
1. Allow serum pool to reach room temperature before
dispensing (to prevent temperature gradients possibly causing
concentration gradients across the large number of vials being
dispensed and to prevent condensation problems during
labeling of vials).
2. Replace the tubing attached to the dispensing syringe (left
when looking at front of Digiflex) with a length of clean
Teflon™ tubing long enough to reach into the bottom of the
carboy while it is sitting on the stir plate.
3. Check cleanliness of Digiflex before use by analyzing 1-2%
(v/v) HNO3 which has been flushed through the Digiflex with a
portion of the same solution which has not been through the
Digiflex.
4. Approximately one hour before dispensing begins,
a. With the large stir plate close to the left side of the Digiflex,
begin stirring the serum pool to be dispensed.
b. Also during this time, flush the Digiflex with serum from the
pool to be dispensed. Place the ends of the tubing
attached to both the sample and dispensing syringes into
the carboy of serum so that serum won’t be used up during
this process. Be sure to secure both ends of tubing in the
carboy with Parafilm so they will not come out during the
flushing process.
5. After dispensing the serum into the vials, cap the vials and
label them. Placing labels on vials after capping minimizes the
chance for contamination during the process.

iv.

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for the purpose of looking for trends in concentrations. Once
dispensed and homogeneity has been shown to be good
throughout the tubes of a pool, store tubes at ≤ -20°C and pull
tubes out as needed for analysis.
v.

Storage: Serum pools should be stored long term at ≤ -20°C.
Short term storage (several days) at refrigerator temperature (~ 24°C).

7) Analytical Instrumentation & Parameters
(see Section 5 for details on hardware used, including sources)
a. Instrumentation & Equipment Setup:
i. ICP-DRC-MS: Inductively Coupled Plasma Dynamic Reaction Cell Mass
Spectrometer ELAN® 6100 DRCPlus or ELAN® DRC II.
1. Modifications made to ICP-DRC-MS
a. All plastic tubing for DRC reaction gases have been replaced with 1/8”
O.D. stainless steel.
2. Sample introduction system setup:
(See Figure 1 in the Appendix for diagram of generic sample introduction
system. Adjustments of connections for the ESI SC4 autosampler are
described below. See figures 3a through 3e in Appendix B for other default
autosampler settings).
a. Concentric quartz nebulizer (quick connect arrangement for liquid and
gas connections available from some vendors).
b. Quartz cyclonic spray chamber.
c. Quartz injector, 2 mm ID, ball joint end (not shown in Figure 1).
3. Configuration of tubing for liquid handling:
(See Figure 1 in the Appendix for diagram of tubing setup. This is a
recommended setup, but other similar arrangements are usable. See
Section 5.b. for part numbers and ordering details.)
a. Tubing for liquid sample uptake:
i.

Probe-to-peristaltic pump tubing: PFA tubing from ESI SC4
autosampler probe connects either directly to sample peristaltic
tubing or through a connection adaptor.

ii.

Nebulizer-to-peristaltic pump tubing:

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1. 3mL/min nebulizer (TQ-30-A3): Hold square-cut end of 0.5mm
x 1.59mm PFA tubing against the inside tapered nebulizer
capillary using a flangeless nut and ferrule assembly. Anglecut opposite end of tubing before inserting into end of black /
black peristaltic pump tubing.
2. 1mL/min nebulizer (500-70QQDAC): Quick connect fitting fits
inside back side of nebulizer. Use a PEEK adapter to securely
connect the PFA tubing to the peristaltic tubing. Higher backpressure from the 1mL/min nebulizer is likely to cause tubing
become disconnected if the PFA tubing is merely inserted into
the peristaltic pump tubing.
b. Tubing for autosampler rinse solution:
i.

SC autosampler setup for non-FAST applications:
See Appendix B, Figure 1b for generic autosampler flow diagram.
Differences to Figure 1b for the ESI SC4 autosampler include
1. Autosampler Probe: (SC4 probe has built-in PFA tubing
extending from the Teflon-coated probe, so no nut and flanged
tubing connection is necessary).
2. Rinse station filling: ESI SC4 autosampler may have a built-in
vacuum pump which pumps rinse solution from the rinse jug to
the rinse station ports. If so, rinse solution will not need to be
routed through the peristaltic pump.
3. Rinse station waste: ESI SC4 autosampler liquid waste may
be setup to drain by gravity (see comment below).

ii.

Tubing connection between autosampler rinse station and rinse
solution reservoir: Tubing of different inner diameters can be
obtained from Elemental Scientific, their distributors, or custom
built in the lab to optimize the rinse station fill rate between
samples. Rinse station should not go empty at any point.

iii.

Tubing for autosampler rinse station waste removal: Use minimum
drain tubing to make this connection. If this tube is too long, the
rinse station will not drain properly.

iv.

Rinse solution jug: Leave one of the caps on the top of the rinse
jug loose to allow air venting into the jug as liquid is removed.
Otherwise the jug will collapse on itself as the liquid is removed
and a vacuum is created inside. Use secondary containment tray
and label appropriately (see solution preparation instructions).

Waste solution jug: Use secondary containment tray and label
appropriately (see solution preparation instructions).

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c. Configuration of tubing for spray chamber waste removal:
i.

Chamber-to-peristaltic pump tubing: Connect 1/8” i.d. x 1/4 inch
o.d. PVC tubing directly to the waste port on the spray chamber.
Connect other end of PVC tubing to the white / black peristaltic
pump tubing using a tubing connector (PerkinElmer item #
B3140715).

ii.

Waste Jug-to-peristaltic pump tubing: Connect 1/8” i.d. x ¼” o.d.
PVC tubing to the white / black peristaltic pump tubing using a
tubing connector (PerkinElmer item # B3140715). Place the free
end of the PVC tubing through the lid of the waste jug (be sure it is
secure). Waste jug should be sitting in a secondary containment
tray in case of overflow.

4. Cones used
Nickel or platinum cones from either PerkinElmer or Spectron have been
used successfully. Platinum cones are preferred for durability.
5. Gases & Regulators setup:
a. Argon: Argon stored as liquid in a dewar (180-250L) or bulk tank.
Gaseous argon used for plasma and nebulizer.
i.

ii.

Step down regulator (if source of argon is a bulk tank): Place this
single stage regulator in the lab so that incoming argon pressure
can be monitored and adjusted. Set delivery pressure to 70-100
psig. See Section 5.e. for part numbers and details.

iii.

Regulator at ICP-DRC-MS: Single stage “argon regulator filter kit”
supplied with the ICP-DRC-MS. If the delivery pressure gauge
range is 0-60psi, set the delivery pressure to 52±1 psig. If the
delivery pressure gauge range is 0-100psi, set the delivery
pressure to 60±1 psig. See Section 5.e. for part numbers.

b. Ammonia gas for DRC channel A.
i.

Regulator for NH3 gas: Set delivery pressure to 5-7 psig. See
Section 5.e. for part numbers and details.

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6. Chiller / Heat Exchanger: Refrigerated chiller (for ELAN® 6100 DRCPlus
instruments) or heat exchanger (for ELAN® DRC II instruments). For
refrigerated chiller, set temperature control to 18°C.
ii. Computer: Dell Optiplex GX150, GX270, or GX280 have all been used.
Processors used have included Pentium III (1 GHz) through Pentium IV (2.8
GHz). Recommend 512Mb - 1Gb RAM. External hard disk drive for nightly
backups of data connects via USB port. Software used includes Windows XP
Professional, service pack 2 and ELAN v3.3.
iii. Autosampler: ESI SC4 autosampler without FAST sample introduction. Rack
calibration, tubing ID for rinse supply, additional rinse time, probe movement
speeds, and probe depth is optimized per autosampler (see Table 1 in
Appendix B for default settings).
b. Parameters for Instrument and Method: See Table 1 pp 46-48 for a complete
listing of the instrument and method parameters. Also, see Figures 2a-2g for
images of the ELAN method screens.
8) Method Procedures
a. Quality Control: Quality control procedures implemented in this method are
defined by the Division Procedures and Practices Guidelines and include two
types of QC systems which are both subjected to the complete analytical
process. The data from these materials are then used to estimate
methodological imprecision and to assess the magnitude of any time-associated
trends. The concentrations of these materials should cover the expected
concentration range of the analytes for the method. Before QC materials can be
used to judge patient analytical runs, acceptable QC concentration limits must be
calculated from the concentration results observed in at least 20 characterization
runs. During the 20 characterization runs, previously characterized QCs or pools
with target values assigned by outside laboratories should be included to
evaluate the analysis. The process of limits calculation is performed using the
laboratory database and the SAS division QC characterization program.
i. Types of Quality Control:
1. “Bench QC”: The bench QC pools used in this method comprise two levels
of concentration spanning the “low-normal” and “high-normal” ranges of the
analyte of interest. The intent of bench QC is for the analyst to evaluate
the performance of the analytical system on the day of analysis. The
analyst inserts both the “low” and the “high” bench QC specimens two
times in each analytical run (a set of consecutive assays performed without
interruption) so that judgments may be made on the day of analysis. The
first analysis of the two bench QC pools is done after the calibration
standards are analyzed but before any patient samples are analyzed (so
that judgments on the calibration curves may be made before analysis of
patient samples). The second analysis of the two bench QC pools is done

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at the end of the run (approximately 20 patient samples total). If more
patient samples are analyzed on the same calibration curve after the
second run of the bench QC, both the low-normal and high-normal bench
QC must be reanalyzed before and after the additional samples. For
example, the schemes shown in Table 5 p.50 are both acceptable ways to
analyze multiple consecutive “runs”.
2. “Blind QC”: When possible, “blind” QC samples are QC materials placed in
vials, labeled, and processed so that they are indistinguishable from the
subject samples handled by the analyst. Ideally, the supervisor decodes
and reviews the results of the blind specimens without the analyst knowing
of their presence in the runs. When it is not possible to have blind QC
materials processed so that they are indistinguishable by the analyst from
the patient samples, it is acceptable for the analyst to randomly insert into
the run a QC material which only the QC reviewer knows the acceptable
concentration limits for. At least one low-normal concentration and one
high-normal concentration QC material should be kept in the laboratory for
this purpose.
3. External Reference Materials: Materials produced by laboratories outside
of the CDC which have assigned target concentrations can be helpful in
verifying method performance. Some examples include Standard
Reference Materials (SRM) from the National Institute of Standards and
Technology (NIST) (i.e. SRM 1598a) and samples from previous
challenges of proficiency testing programs (i.e. Centre de Toxicologie du
Quebec (CTQ)). However, only the results for the bench and blind QC
materials are used to determine if the run results can be used.
ii. Calibration Verification:
1. Bi-annual tests as defined in the DLS Policy and Procedures manual:
CLIA requires the verification of accuracy of instrument response to analyte
concentration be completed at least every 6 months. NIST traceable
calibrators are analyzed in each run to define this response up to the
concentration of the highest calibrator in the run. To verify accuracy of
instrument response at concentrations higher than the highest calibrator in
each run, analyze a NIST traceable standard with very high concentrations
(see Table 8 p.52 in the Appendix for concentrations) at least every 6
months. Prepare the Calibration Verification Standard for analysis just as a
working calibrator is prepared. Use the “Serum Blank” as the blank when it
is analyzed. If the observed concentrations for the Calibration Verification
Standard are not within 10% of the target value (see Table 8 p.52 in the
Appendix) the lab supervisor should be notified and the issue should be
investigated. Do not substitute external reference materials (i.e. biological
samples from a PT program) for the Calibration Verification Standard when
performing this. Solutions needed for the Calibration Verification checks
can be purchased from standards vendors (i.e. SPEX, High Purity
Standards, etc . . .) or prepared in-house from NIST traceable single

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element standards. Always verify that normal background levels have
been re-achieved through adequate rinse time following analysis of
elevated standards for calibration verification.
2. As-needed confirmations (per supervisor discretion): When a sample
result is greater than the highest calibrator in the run by more than 10%,
the supervisor may request that the result be confirmed in an analysis run
which includes a standard or external reference material with equivalent
(within 10%) or greater concentration than the sample. In order to avoid
needless contamination of the instrument with high concentrations of
analytes, the analyst should use the lowest appropriate calibration
verification solution concentrations to meet the need.
For infrequent verification needs, the calibration verification stock solutions
can be used to prepare verification standards to appropriate
concentrations. This will, however, introduce elevated concentrations of all
elements in the method to the sample introduction system. Frequent
measurement of these very high concentrations can result in high
background levels in the instrument which are difficult to rinse out and
which may limit the ability to measure low concentrations.
For frequent verification needs (i.e. when certain studies have many
elevated results on particular elements) or when a concentration higher
than those shown in Table 8 p.52 needs to be verified, use NIST-traceable
single element stock standards to prepare single element verification
standards. This will limit the exposure of the instrument to elevated
concentrations of only the elements needing verification.
Always verify that normal background levels have been re-achieved
through adequate rinse time following analysis of elevated standards for
calibration verification.
An external reference material (i.e. historical proficiency testing sample)
can be used to verify the linearity of calibration within a run in these
situations IF
a. The target value has been assigned by an external source (i.e. NIST,
or the proficiency testing program).
b. The concentration of the external reference material is within 10% or is
higher than the concentration of the material you need it to confirm.
c. There is confidence that there is no contamination of previously used
external reference material.
d. A note to file is made that this was done.
e. If the observed concentrations are not within 10% of the target value
the lab supervisor should be notified and the issue should be
investigated.

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b. Daily Analysis of Samples
i. Preparation of the Analytical Equipment
For further details on any part of this description, see the ITN Daily Startup
SOP for ELAN ICPMS instruments.
1. Power on the computer, printer, peristaltic pump, and autosampler, and log
into the operating system.
2. Peristaltic pump: Set up the peristaltic pump tubing with proper tension for
the sample rinse station.
a. If using an external peristaltic pump, after lighting the plasma go to the
DEVICES window of the software and press the “Connect” button to
establish communication between the computer and the autosampler.
Next, start the peristaltic pump by pressing the appropriate arrow in the
DEVICES window (make sure that the rotational direction is correct for
the way the tubing is set up in the peristaltic pump). Set the pump
speed to 10 rpm in the DEVICES window.
b. If using the on-board ICP-MS peristaltic pump, start the peristaltic
pump by pressing the appropriate arrow in the DEVICES window
(make sure that the rotational direction is correct for the way the tubing
is set up in the peristaltic pump). Set the pump speed to a slow flow
rate (6 to 10 rpm) in the DEVICES window.
3. Software: Starting the ESI software before starting the ELAN software may
improve stability of software.
4. Daily Pre-Ignition Maintenance Checks: Perform daily maintenance checks
as described in the ITN Daily Startup SOP for ELAN instruments (i.e., Ar
supply pressure, interface components cleanliness and positioning,
interface pump oil condition, vacuum pressure, etc.). Make appropriate
notes in the Daily Maintenance Checklist and Instrument Log Book.
5. Start the Plasma: In the INSTRUMENT window of the software (or on the
front of the ELAN), press the “Start” button to ignite the plasma.
6. Send Probe to Rinse Station: Through the METHOD/SAMPLING window
in the software, press the “Probe” button, then the “Go to Rinse” button to
lower the autosampler probe into the rinse solution.
7. Start the peristaltic pump:
8. Warm-up time: Allow at approximately 30 to 45 minutes warm-up time for
the ICP-DRC-MS after igniting the plasma. This warm-up time is for the
RF generator. There will be another “Stability time” for the DRC later in
this procedure.
9. Optimizations and Daily Performance Check: After this warm-up time,
perform a daily performance check and any optimizations necessary (as
described in the ITN Daily Startup SOP for ELANs). Include Be (m/z 9) in

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the daily performance check. Fill in the Daily Maintenance Checklist
according to the optimization procedures performed.
a. Magnesium (24Mg) may have high RSDs due to the use of Triton-X100
in the rinse solution. Avoid this problem by either temporarily using
non-Triton-containing rinse solution during the daily check, or repeating
the daily check multiple times in succession with no rinse time
between.
i.

Saving the Files: Save new tuning (mass calibration) parameters
to the file “default.tun.” Save new optimization parameters (i.e.,
detector voltages, autolens values, nebulizer gas flow rate) to the
file “default.dac.” monthly, or any time large changes are made in
optimization parameters, save a separate copy of these
optimization files under a different name (i.e. –
default_070706.dac).

10. Software setup for Analysis:
a. Workspace (files & folders): Click on “Open Workspace” from the
“File” menu. Select the workspace file “CDC_Serum multielement.wrk” (or one customized for user preferences). Select
“Review Files” from the “File” menu. Verify & set up the correct files
and data directories for your analysis (See Table 1 p.47-49 “File
Names & Directories“).
b. Samples / Batch Window: Update the window to reflect the current
sample set. The only fields which need to be filled in include the
autosampler location, sample identification (id), measurement action,
method, sample flush time, sample flush speed, read delay time, read
delay & analysis speed, wash time, wash speed. Use a bar code
scanner to input data whenever possible. See Table 1 pp 47-49 for
times and speeds. Save the Sample window file and re-use it on other
days by simply replacing the sample IDs for the patient samples.
1. DRC Stability Time: Best analyte-to-internal standard ratio
stability is obtained after 1 hrs of analysis of serum samples
using the DRC method. Analyze enough base serum sample
dilutions prior to any DRC analysis run to fill at least one hours
of analysis time. If analyzing the full set of method analytes, 10
samples will be sufficient. See Table 5 p.50 for example of
setup in the Samples / Batch window.
2. Serum vs. Aqueous Method Files:
a. The difference: There are two method files for this one
method (see Table 1 p.47-49). It is necessary to use both
to accomplish each run because the current PerkinElmer
software will not allow for more than one blank per method
file. The ONLY DIFFERENCE between these two files is

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on the Sampling tab where one lists the autosampler
positions of the serum blank and serum calibrators (the
“sblk” method file) and the other lists the autosampler
position of the aqueous blank (the “aqblk” method file).
b. Use: The ONLY TIME when it matters which of these files
is used is when the measurement action includes “Run
blank” or “Run standards”. When the measurement action
is only ‘run sample’, it does not matter whether the “sblk” or
“aqblk” method file is used. Analysts typically follow the
pattern below, however, for the sake of consistency and as
a reminder of which blank must be used for which type of
sample. See Table 6 p.50.
i. The “sblk” method file: Use to analyze the initial serum
blank (blank for the calibration curve), the serum
calibrators, and the serum blank checks (sblkchk1 &
sblkchk2) at the very beginning of the run. The serum
blank method (set up for a ESI SC4 autosampler
defines the serum blank in autosampler location 109
and the serum calibration standards 1-5 in autosampler
locations 101-106, respectively.
ii. The “aqblk” method file must be used to analyze all QC
materials and patient samples. The aqueous blank
method (set up for a ESI SC4 autosampler) defines the
aqueous blank in autosampler location 109.
3. Notation of Dilutions: To designate an extra dilution of a
sample, edit the sample ID to reflect the level of dilution being
performed (i.e., A 1:2 dilution of sample 1 would be reflected in
the sample ID “sample 1 (2x dilution)”. This sample ID will be
edited during the data-import process to the database so that
it is recognized as the appropriate sample. Do not use the
ELAN® software to automatically correct for sample dilutions.
Extra dilution is performed on serum samples whose
concentration is greater than the concentrations listed in Table
8 p.52 in the Appendix (linearity of the method has been
documented up to these concentrations).
ii. Preparation of Samples for Analysis (See Table 7 p.51)
1.

Thaw the frozen serum specimens; allow them to reach ambient
temperature.

Prepare diluted serum for analysis during the DRC stability period. A 40minute DRC stability period will consume approximately 36mL of
solution. Prepare the necessary volume according to the “patient
sample constituent proportions listed in Table 7, p. 51. This can be

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prepared in a 50mL polypropylene tube or a wide-mouth bottle (which
can be put on the autosampler in place of one of the tube trays).
NOTE: Selenium is not stable in the diluted sample for more than 7
hours. Diluted serum must be analyzed within 7 hours of
preparation (see Appendix A, test 5 for details)
3.

Set up a series of 15-mL polypropylene tubes corresponding to the
number of blanks, standards, QCs, and patient samples to be analyzed.

Prepare the following solutions in the 15-mL falcon tubes using the
Micromedic Digiflex™ (see Table 3 p.49 for a summary).
a. Aqueous Blank: Prepare two aqueous blanks consisting of 300 L of
>18 Mega-ohm·cm water and 4,200 L of diluent (2 x 2100 L). One
will be the actual aqueous blank and the other will be a backup
(“Aqueous Blank Check”) in case the original aqueous blank gets
contaminated….
b. Serum Blank: Prepare three serum blank dilutions consisting of 150
L of base serum (same material used to prepare the serum calibration
standards), 150 L of >18 Mega-ohm·cm water, and 4,200 L of
diluent (2 x 2100 L). One of these serum blanks will be the blank for
the calibration standards; the others will be analyzed after standard 5
as sblkchk1 and sblkchk2, respectively. Results from sblkchk1 and
sblkchk2 will be stored for periodic verification of the method limit of
detection.
c. Calibrators or Calibration Verification Standards: Prepare the working
calibration standards or the working calibration verification standards
as 150 L of the appropriate aqueous intermediate working solution,
150 L of base serum, and 4,200 L of diluent (2 x 2100 L). To avoid
carryover from working calibration standards and the working
calibration verification standards to other samples, rinse tip of digiflex
once with concentrated nitric acid.
d. Patient & QC Samples: Before taking an aliquot for analysis, mix the
sample so that no particulates remain on the bottom of the tube.
Prepare serum sample dilutions as 4,200 L of diluent (2 x 2100 L),
150 L of the serum sample and 150 L of >18 Mega-ohm·cm water.
e. Cap all of the blanks, standards, and samples and mix them well.
Uncap them and place them in the autosampler of the ELAN® ICPMS
in the order that was entered in the Samples / Batch window of the
ELAN software.

iii. Specimen Storage and Handling during Testing: Specimens may be left at
room temperature during analysis in case confirmation analyses must be
made. Take stringent precautions to avoid external contamination by the
metals to be determined. Specimens may be stored short term at refrigerated
temperatures, but should be stored long term (>4 weeks) at ≤ -20 °C.

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iv. Starting the Analysis: To begin analysis, highlight (click and drag with the
mouse) the table rows of the samples that should be included in the run, and
then click on “Analyze Batch.”
v. Monitoring the Analysis: Initiate work in a timely manner so that the run may
be monitored. Make every effort to complete analysis within the work day so
that the entire run can be monitored. If it is not possible to complete the
analysis by the end of the work day, the run may be left to complete itself
unattended as long as appropriate planning is made for either overnight
operation or Auto Stop (see below).
Monitor the analysis for the following:
1. DRC stability (analyte / internal standard ratio stability)
After the analysis of the DRC stability base serum samples, these results
can be reviewed to determine if sufficient stability of the analyte-to-internal
standard ratio has been reached before beginning analysis. Importing data
into an MS Excel template file is useful to simplify this procedure.
2. Proper operation of the instrument.
3. Contaminated blanks.
4. Linear calibration curves.
a. Typical correlation coefficients will be 0.999 to 1.000.
b. The ELAN software generates a “simple linear” calibration curve (using
a least squares calculation) for each of the 3 elements in this method.
The curves are generated using the results from analysis of the serum
blank and the 5 external serum calibrators whose concentrations are
defined in the Calibration tab of the Method file. Specifically, the
software plots the “net intensity” (y-axis) versus the analyte
concentration (x-axis). The “net intensity” is the blank subtracted ratio
of the measured intensity for the analyte to the measured intensity of
the associated internal standard and is calculated as follows:

net int ensity 

Analyte Meas Intensity sample
Internal Std Meas. Intensity sample



Analyte Meas Intensity Blank
Internal Std Meas Intensity Blank

`
5. Bench QC results within the acceptable limits.
If an analyte result for the beginning QC material(s) falls outside of the 3SD
(i.e. 99 percentile) limits, then the following steps are recommended:
a. If a particular calibration standard is obviously in error, remake a new
dilution at the Digiflex of that working calibrator, reanalyze it, and
reprocess the sample analyses using this new result as part of the
calibration curve.

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b. Prepare a fresh dilution of the failing QC material and reanalyze it.
c. Prepare fresh dilutions at the Digiflex of all of the calibration standards
(working serum multi-element standards) and reanalyze the entire
calibration curve using the freshly prepared standards.
If these three steps do not result in correction of the out-of-control values
for QC materials, consult the supervisor for other appropriate corrective
actions. Do not report analytical results for runs that are not in statistical
control.
6. Good precision among replicates. If “air” was sampled into the system, the
precipitated serum protein might be coated within the probe, tubing and the
introduction system which might cause the bad precision among replicates
and/or reduced sensitivity of the instrument. Use >18 Mega-ohm·cm water
to rinse the system for recovering the instrument performance.
7. Consistent measured intensities of the internal standards.
Some sample-to-sample variations are to be expected. However the
intensities should be within a few percent of one another, and should
fluctuate around an average value (not drift continuously in one direction).
8. Elevated patient results.
vi. Records of Results: Run results will be documented daily in both electronic
and paper form.
1. Electronic Records:
a. Transfer of Results to the Laboratory Information System / Database:
Transfer data electronically between computers or software to reduce
errors. When keyboard entry must be used, proofread transcribed
data after entry.
b. Long-Term Storage of ELAN software files: Files used and produced
by the ELAN software in analyzing samples will be backed up long
term on compact disk and kept a minimum of three years.
2. Paper Records: The paper copy of the results from the run should be put
into the study folder(s) and should include
a. A summary of the calibration curve statistics.
b. A printout of analysis of each measurement made during the run.
c. Optional, but helpful, is a printout of the DRC stability check
measurements in graphical form.
d. On the front sheet of the printed records, write the following
i.

Analyst initials

ii.

Instrument ID

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iii.

Date of Analysis

iv.

Run # for the day on this instrument

Study ID and Group Number

vi.

Database batch ID (Not known until the run is imported into the
database)

vii. Transfer of Results to the Laboratory Database: Every analytical run
performed for the analysis of patient samples should be entered into the
laboratory results database unless the run is not useable for obvious reasons
(i.e. the run is stopped for some reason before ending QC is analyzed, no
internal standard spiked into the diluent, etc. . . ).
1. Data Export Process (from ELAN® software to .TXT file): If the data file
was not created during the initial analysis, reprocess the data of interest
either with “original conditions” option, or by loading the files and folders
used during the analysis. In the ELAN® ICP-DRC-MS software, select
“Review Files” from the “File” menu. From this window, you must open the
files and directories that were used when collecting the data of the run that
you wish to export. (If the analysis has just ended, all of these files and
directories will still be open.) NOTE: A second copy of the ELAN®
software can be run as an Edit/Reprocess copy without affecting an
ongoing analysis by the first copy of the software running in Windows.
After you open the relevant files, go to the “Report” page in the METHOD
window. Deselect the box that prints a paper copy of data and select the
box that sends data to a file. Select the “Report Options Template” named
“CDC_Database Output.rop” and type in a report filename using a format
such as “2006-0714a_group55.txt” to designate data from analysis of
group 55 from July 14, 2006, run #1. Under “Report Format”, choose the
“Use Separator” option, and under the “File Write” section choose
“Append.” Finally, reprocess the data of interest. (See PerkinElmer
ELAN® ICPMS Software Manual.) Make sure you apply the aqueous
blank to all sample and quality control material analyses.
2. Data Import Process (from .TXT file to Microsoft Access™ database):
a. Move the .TXT file to the appropriate subdirectory on the network drive
where exported data are stored. Directories for data storage are
named according to instrument \ year \ month\, such as
I:\Instruments\ELANDRCC\2006\07\.
b. Using the ITN Database Frontends, import the instrument file into the
database. On the GoTo window, click on “Add Sample Results to
Database”, then “Import Instrument Data File”.
c. Enter the appropriate information to identify the instrument, assay,
analysis date & time, run number, analyst, calibrator lot number and
prep date used (use the “IS Lot Number” field) and study. If other than
default values for Method LOD, High Calibrator, Rep Delta Limit, and

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units were used in the run, document what was used by clicking on the
“View/Set Batch Parameters” button, changing the appropriate values,
and then clicking “Back”.
d. Press the “Import” button and then browse to the correct network folder
to select the file which contains the results from the run. Select the file
and click “OK”.
e. In the “Import Instrument Results” table, pressing the “Find X’s” button
will show only those samples whose sample ID is not recognized as a
valid QC pool ID or sample ID for this study. (Sample IDs are set up
when the study is logged into the database.) Corrections to sample
IDs and dilution factors can be made in this table (e.g., correction of
transcription errors and adjustment for level of dilution). If samples
were diluted for analysis, both the sample ID and the dilution factor
need to be edited in this table before the values are transferred to the
database (the Replace command under the Edit window is helpful in
this case). When corrections to sample IDs are made, press the
“Check IDs” button to re-evaluate the sample IDs. Any sample or
analyte row marked “Not Recognized” will not be transferred to the
database when the “Transfer” button is pressed. Once transferred
into the database, the data should be evaluated for QC pass / fail, then
set with the appropriate settings for QC accept / reject, final value
status, and comment(s). See the database programmers for more
detail on working in the database.
viii. Analyst Evaluation of Run Results:
1. Bench Quality Control: After completing a run, and importing the results
into the database, export the QC results to the SAS program where the run
will be judged to be in or out of control. The QC limits are based on the
average and standard deviation of the beginning and ending analyses of
each of the bench QC pools, so it will not be possible to know if the run is
officially accepted or rejected until it is completed.
a. Quality Control Rules: The SAS program applies the division QC rules
to the data as follows:
i.

If both QC run means (low & high bench QC) are within 2Sm limits
and individual results are within 2Si limits, then accept the run.

ii.

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If one of the 4 QC individual results is outside a 2Si limit - reject
run if:
1. R 4S Rule – Within-run ranges for all pools in the same run
exceed 4Sw (i.e., 95% range limit)

If only one analyte of the three fails bench QC, then the other two
which passed bench QC may be reported.

ii.

If two analytes of the three fail bench QC, then none of the results
from the run should be used for reporting. The cause of the QC
failures should be investigated and then the entire run should be
repeated.

2. Patient Results:
a. Results Outside the Normal Range: The normal range of
concentrations observed for these elements in serum is listed in Table
10.
i.

Boundaries Requiring Confirmatory Measurement:
1. Results Lower than the First Lower Boundary (1LB) or Higher
than the First Upper Boundary (1UB): Concentrations
observed less than the “first lower boundary” (defined in the
laboratory database as the “1LB”) or greater than the “first
upper boundary (defined as the “1UB” in the laboratory
database) should be confirmed by repeat analysis of a new
sample preparation. The concentration assigned to the 1LB
for an element is determined by study protocol but default 1LB
concentrations for elements in this method can be found in
Table 9 p.52 in the Appendix. Report the original result, as
long as the confirmation is within 10% of the original. Continue
repeat analysis until a concentration can be confirmed.

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IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 46 of 73

2. Results Greater than Highest Calibrator: When a sample
result is greater than the highest calibrator in the run, the
supervisor may request that the result be confirmed in an
analysis run which includes a standard or external reference
material with equivalent (within 10%) or greater concentration
than the sample.
3. Results Greater than Range of Linearity Tested: Perform an
extra dilution on any serum sample whose concentration is
greater than those listed in Table 8 p.52 in the Appendix (the
linearity of the method has been documented up to these
concentrations). See Table 7 p.51 for description of sample
preparation with extra dilution.
ii.

Analyst Reporting of Abnormally Low or Abnormally High Results:
Concentrations observed lower than the “second lower boundary”
(defined in the laboratory database as the “2LB”) or greater than
the “second upper boundary” (defined in the laboratory database
as the “2UB”) should be reported to the QC reviewer as an
“abnormally low result” or an “elevated result”, respectively. The
concentration assigned to the 2LB and 2UB for an element is
determined by study protocol but default concentrations are in
Table 9 p.52 in the Appendix. There is no routine notification for
elevated levels for the metals determined in this method. The
protocol for supervisors reporting elevated results to medical
personnel is defined according to the study protocol.

b. Inadequate Precision Within One Measurement: If the range of the
three replicate readings (maximum replicate concentration value minimum replicate concentration value) for a single sample analysis is
greater than the criteria listed in Table 9 p.52 in the Appendix and the
range of the three replicate readings is greater than 10% of the
observed concentration, do not use the measurement for reporting.
Repeat the analysis of the sample. This type of inadequate precision
is noted in the database by an ‘X’ in the “>Lim Rep Delta” field.
ix. Submitting final work for Review: Once results have been imported, reviewed,
and set as final in the database by the analyst,
1. Submit an email to the QC reviewer informing them of the readiness of the
data for final review. The email should include
a. Instrument ID, run Date, run number, study ID, group ID.
b. Any bench QC failures (include reasons if known).
c. Any patient sample result less than the 2LB or greater than the 2UB
(see Table 9 p.52 in the Appendix).

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IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 47 of 73

d. Anything out of the ordinary about this analytical work which could
have a bearing on the availability (i.e. insufficient sample to analyze),
accuracy, or precision of the results.
2. Include all items called for by the study folder cover sheet in the study
folder (i.e. printouts from the ICP-MS, bench QC evaluation) together in the
study folder before submitting the folder for review when analysis is
complete.
x. Overnight operation or Using Auto Stop: Make every effort to complete
analysis within the work day so that the entire run can be monitored. If it is not
possible to complete the analysis by the end of the work day, the run may be
left to complete itself unattended as long as appropriate planning is made for
either overnight operation or Auto Stop.
1. 24 hrs / day operation in DRC mode:
a. To reduce startup time in the mornings, the analyst is encouraged to
operate the ELAN in DRC mode 24hrs/day during the work week. This
eliminates the need for daily 45 minute RF generator warm-up, and
possibly the need for DRC stability time (if the DRC gas is not off for
extended periods of time before analysis). To maintain the instrument
in DRC mode when not analyzing patient samples, setup multiple
sample rows in the Samples / Batch window with autosampler position
n zero (rinse station of autosampler) and wash time of 1800s (30
minutes). Repeat this sample row enough times to keep the
instrument in analysis mode overnight (1 sample with 15 minute wash
will take ~ 20 minutes).
2. AutoStop: If 24 hrs / day ELAN operation is not desired, the instrument
can shut the plasma off unattended after analysis. Setup this as follows:
a. On the “Auto Start / Stop” tab of the Instrument window, enable the
Auto Stop feature.
b. Press the “Change” button within the Auto Stop box and set the
Delayed shutdown time to 5 minutes. This will rinse the sample
introduction system of serum matrix before turning off the plasma.
c. It will be necessary to replace the sample peristaltic pump tubing the
next day since it will have been clamped shut overnight.
c. Equipment Maintenance: Analysts are expected to follow a 4-day analysis / 1day maintenance schedule in the laboratory.
i. ICPMS Maintenance: On the maintenance day, perform all maintenance per
the Inorganic Toxicology and Nutrition Branch ELAN ICP-MS Weekly
Maintenance SOP. All equipment maintenance should be documented in the
instrument logbook. For this method we can not use straight ethanol to rinse
the sample introduction system, otherwise the probe and tubing will be clogged
because of the precipitation of the serum protein. Use the >18 Mega-ohm·cm
water to rinse the whole system whenever it is necessary.

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IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 48 of 73

ii. Data Backup: Data on the ELAN computer will be backed up via two backup
routines.
1. Daily Backups to External Hard Drive: Automatic backups of the “elandata”
directory and all subdirectories should be programmed to occur each night
onto an external hard disk.
2. Weekly Backup to CD: Backup all files in the active “elandata” directory
and all subdirectories onto one recordable compact disc during the weekly
maintenance SOP. When the active “elandata” directory on the ICP-DRCMS computer hard drive becomes too large to fit onto a single recordable
compact disk, the oldest data can be removed from the computer to make
it easier to backup the entire directory weekly. This can usually be done
annually.
a. Backup the oldest data on the hard drive to two duplicate compact
disks and verify that the files on the CD are readable
b. Label them with the name of the instrument, the date range of the data,
the current date, your name, and “Copy 1 of 2” or “Copy 2 of 2”
c. After verifying that the CDs are readable, the oldest, backed up data
can be deleted from the ICP-MS computer hard drive.
d. It is best to not store duplicate copies in the same location.
9) Interpretation of the Results
a. Reportable Range: Serum multi-element values are reportable in the range
between the method LOD and the highest concentration verified accurate by biannual calibration verification tests (see Table 8 p.52 in the Appendix). For
example, if a serum Se value is less than the method LOD, report it as < “LOD”
g/L where “LOD” is the numerical LOD. Above the highest concentration
verified, extra dilutions are made of the serum sample to bring the concentration
within the verified range.
b. Reference Ranges (Normal Values): In this method the normal reference
ranges (see Appendix, Table 10 p.53) for these elements in serum fall within the
range of the calibrators.
c. Action Levels: There is no routine notification for levels of every analyte
determined with this method. The protocol for supervisors reporting elevated
results to medical personnel is defined according to the study protocol.
10) Method Calculations
a. Method Limit of Detection (LODs): The detection limits for elements in serum
specimens are based on 3 times the concentration standard deviation of serum
blanks (named sblkchk1 or sblkchk2) analyzed in at least 20 separate runs.
Method LODs are re-evaluated periodically.

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IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 49 of 73

b. Method Limit of Quantitation (LOQ): The Division of Laboratory Sciences does
not currently utilize limits of quantitation in regards to reporting limits [9].
c. QC Limits: Quality control limits are calculated based on concentration results
obtained in at least 20 separate runs. It is preferable to perform separate
analyses on separate days and using multiple calibrator lot numbers,
instruments, and analysts to best mimic real-life variability. The statistical
calculations are performed using the SAS program developed for the Division of
Laboratory Sciences (DLS_QC_compute_char_stats.sas).
11) Alternate Methods for Performing Test and Storing Specimens If Test System
Fails:
If the analytical system fails, the analysis may be setup on other ELAN DRC
instruments in the laboratory. If no other instrument is available, store the
specimens at ≤ 4°C until the analytical system can be restored to functionality. If
interruption longer than 4 weeks in anticipated, then store serum specimens at
≤ -20°C.

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 50 of 73

Appendix A. Ruggedness Testing Results.
Parameter Test#1: Evaluate the impact on analysis results if the set RF power is
increased to 1600W (instrument maximum) or decreased to 1150W (by 20%) for the
analytical run.
Test Details:
1. Three different PF power settings were tested in separately prepared, consecutive
runs on the instrument without turning off the plasma. At least 15 minutes stabilization
time was allowed between each run after the RF power was changed. “Junk urine”
samples (20) were analyzed between the beginning and ending QC of each run. All
other method parameters were kept per method.
2. Run #1 (method default, 1450W).
3. Run #2 (decreased RF power by 20% to 1150W).
4. Run #3 (increased RF power to instrument maximum, 1600W).
5. Run #4 (increased RF power to instrument maximum, 1525W).
Parameter Test 1 Results.
Test performed 4/2-5/17/2010; by Gulchekhra Shakirova.
QC
RF Power Tested
Zn (µg/dL)
Pool ID
Characterized Mean
50.7
2SD Range
41.9 - 59.5

Cu (µg/dL)

Se (µg/L)

64.9
61.9 – 67.9

75.0
66.7 – 83.3

1150W (Reduced)

52.5

63.1

75.6

1450W (Per Method)

49.0

62.7

70.1

1525W (Increased)

43.3

63.4

75.7

1600W (Increased)

54.1

63.9

75.6

Characterized Mean
2SD Range

175
142 – 209

203
191 – 215

144
130 – 157

1150W (Reduced)

178

197

145

1450W (Per Method)

168

196

146

1525W (Increased)

157

201

145

1600W (Increased)

178

199

149

LS-03601b

HS-03601b

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 51 of 73

Appendix A. Ruggedness Testing Results (continued).
Parameter Test#2: Evaluate the impact on analysis results if the Cell Gas Flow Rate is
increased or decreased by 20% for the analytical run.
Test Details:
1. Three different Cell Gas Flow Rates were tested in separately prepared, consecutive
runs on the instrument without turning off the plasma. At least 15 minutes stabilization
time was allowed between each run after the axial field voltage was changed. “Junk
urine” samples (20) were analyzed between the beginning and ending QC of each
run. All other method parameters were kept per method.
2. Run #1 (method default = 0.5mL/min).
3. Run #2 (decreased Cell Gas Flow Rate by 20% to 0.4mL/min).
4. Run #3 (increased Cell Gas Flow Rate by 20% to 0.6mL/min).
Parameter Test 2 Results.
Test performed 5/6/-5/17/2010 by Gulchekhra Shakirova.
QC
Cell Gas Flow Rate Tested
Zn (µg/dL)
Pool ID

LS-03601b

Cu (µg/dL)

Se (µg/L)

Characterized Mean
2SD Range

50.7
41.9 - 59.5

64.9
61.9 – 67.9

75.0
66.7 – 83.3

0.40 mL/min (Reduced)

49.5

65.3

80.7

0.50 mL/min (Per Method)

49.5

67.6

73.8

0.60 mL/min (Increased)

47.8

63.4

75.6

Characterized Mean
2SD Range

175
142 – 209

203
191 – 215

144
130 – 157

0.40 mL/min (Reduced)

167

204

152

0.50 mL/min (Per Method)

169

205

146

0.60 mL/min (Increased)

171

203

147

HS-03601b

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 52 of 73

Appendix A. Ruggedness Testing Results (continued).
Parameter Test#3: Evaluate the impact on analysis results if the RPq is increased or
decreased by 20% for the analytical run.
Test Details:
1. Three different RPq settings were tested in separately prepared, consecutive runs
on the instrument without turning off the plasma. At least 15 minutes stabilization time
was allowed between each run after the axial field voltage was changed. “Junk urine”
samples (20) were analyzed between the beginning and ending QC of each run. All
other method parameters were kept per method.
2. Run #1 (method default DRC RPq: 0.56).
3. Run #2 (decreased DRC RPq 20%: 0.70).
4. Run #3 (increased DRC RPq 20%: 0.84).
Parameter Test 3 Results.
Test performed 5/6-5/17/2010 by Gulchekhra Shakirova.
QC
RPq Tested
Zn (µg/dL)
Pool ID

LS-03601b

HS-03601b

Cu (µg/dL)

Se (µg/L)

Characterized Mean
2SD Range

50.7
41.9 - 59.5

64.9
61.9 – 67.9

75.0
66.7 – 83.3

DRC RPq:0.56
(Reduced by 20%)

52.5

63.1

70.8

DRC RPq:0.70
(Per Method)

49.0

62.7

70.1

DRC RPq:0.84
(Increased by 20%)

54.1

63.9

75.9

Characterized Mean
2SD Range

175
142 – 209

203
191 – 215

144
130 – 157

DRC RPq:0.56
(Reduced by 20%)

178

197

129

DRC RPq:0.70
(Per Method)

168

196

131

DRC RPq:0.84
(Increased by 20%)

178

199

145

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IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 53 of 73

Appendix A. Ruggedness Testing Results (continued).
Parameter Test#4: Evaluate the impact on analysis results if the axial field voltage
(AFV) is increased or decreased by 20% for the analytical run.
Test Details:
1. Three different DRC AFV were tested in separately prepared, consecutive runs on
the instrument without turning off the plasma. At least 15 minutes stabilization time was
allowed between each run after the axial field voltage was changed. “Junk urine”
samples (20) were analyzed between the beginning and ending QC of each run. All
other method parameters were kept per method.
2. Run #1 (method default DRC AFV = 450).
3. Run #2 (decreased DRC AFV to 360)
4. Run #3 (increased DRC AFV to 500)
Parameter Test 4 Results.
Test performed 5/6/-5/17/2010 by Gulchekhra Shakirova.
QC
Pool ID

Axial Field
Voltage Tested

Zn (µg/dL)

Cu (µg/dL)

Se (µg/L)

Characterized Mean
2SD Range

50.7
41.9 - 59.5

64.9
61.9 – 67.9

74.9
66.7 – 83.3

AFV-360
(Reduced)

46.9

61.5

72.8

AFV-360
(Per Method)

47.2

64.0

75.0

AFV-500
(Increased)

48.6

63.5

74.1

Characterized Mean
2SD Range

175
142 – 209

203
191 – 215

144
130 – 157

AFV-360
(Reduced)

163

195

142

AFV-360
(Per Method)

170

205

147

AFV-500
(Increased)

168

200

146

LS-03601b

HS-03601b

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 54 of 73

Appendix A. Ruggedness Testing Results. (continued)
Parameter Test #5: Method descriptions and SOP assume preparation and analysis on
same day. Evaluate the impact on analysis results if the analytical run is prepared to
analyze but circumstances do not allow for analysis to occur until 24 or 48 hours later.
Test Details (Part 1):
1. Three separate run sets (A, B, and C) were prepared at one sitting from the same
starting materials. Set ‘A’ was analyzed immediately. Set’s ‘B’ and ‘C’ were stored
at room temperature for 24 and 48 hours, respectively before analysis. “Junk serum
samples (20) were analyzed between the beginning and ending QC of each run,
making each a normal length run. All other method parameters were kept per
method. Results in table are average of beginning and ending QC.
2. On day two, a fresh run set (“D”) was prepared and analyzed immediately for
comparison to results from set “B” (Run 2 of the day. Results not shown).
3. On day three, another fresh run set (“E”) was prepared and analyzed immediately for
comparison to results from set “C” (Run 2 of the day. Results not shown).

HS-03602b

LS-03601b

Parameter Test 5 Results (Part 1). Test performed 3/11/2010 to 3/15/2010
by Gulchekhra Shakirova using ELAN DRC-2R.
QC
Time from
Zn (µg/dL)
Cu (µg/dL)
Se (µg/L)
ID
Preparation
Characterized Mean
50.7
64.9
74.9
±2SD Range
41.9 – 59.5
61.9 – 67.9
66.7 – 83.3
37.5 – 63.9
60.4 – 69.4
52.5 – 87.4
±3SD Range
Fresh Preparation

47.2

65.7

74.7

After 24 hours

51.9

67.1

After 48 hours

51.0

66.2

102
(150, 54.5)
57.1
(123, -8.8)

Characterized Mean
±2SD Range
±3SD Range

175
142 – 209
126 - 225

203
191 – 215
185 - 221

144
130 – 157
124 – 164

Fresh Preparation

160

203

143

After 24 hours

167

203

145

After 48 hours

174

207

130
(98.2, 161)

Note: The serum ICP-MS method is rugged for Zn and Cu to delays in analysis of
samples after preparation for up to 48 hrs. and not rugged for Se to delay in analysis of
samples after preparation for even 24 hrs. Suggested maximum amount of time from
sample prep to end of the run is 450 min, which consists of 3 analytical runs.

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 55 of 73

Appendix A. Ruggedness Testing Results. (continued)
Parameter Test #5:
Test Details (Part 2): Due to the observations in test one for selenium, a shorter time
frame was examined in part two of this test.
1. Seven preparations of the low bench QC serum material were made at the
beginning of the experiment. Each of these seven preparations were 4x the
normal preparation volume (4 preparations into each vial).
2. Four consecutive runs of the serum method were then carried out. Each run
included
a. blanks, calibrators, and run judge QC (beginning and ending) which were
prepared immediately prior to the beginning of each run.
b. Seven preparations of the low bench QC which were prepared
immediately prior to the beginning of each run.
c. Measurements of the seven preparations of the low bench QC pool which
were prepared before the first run (these were alternated with the freshly
prepared low bench QC sequentially throughout the run).
Parameter Test 5 Results (Part 2).
Test performed 5/6/2010 to 5/17/2010 by Gulchekhra Shakirova, DRC2-R.
QC
Pool ID

LS-03601b

Axial Field
Voltage Tested

Zn (µg/dL)

Cu (µg/dL)

Se (µg/L)

Characterized Mean
2SD Range
3SD Range

50.7
41.9 – 59.5
37.5 – 63.9

64.9
61.9 – 67.9
60.4 – 69.4

74.9
66.7 – 83.3
52.5 – 87.4

Run 1
(up to 139 min elapsed)

41.8

58.3

72.5

Run 2
(up to 303 min elapsed)

43.1

60.0

71.3

Run 3
(up to 427 min elapsed)

51.4

65.9

71.0

Run 4
(up to 576 min elapsed)

43.5

59.1

54.7

Note: The serum ICP-MS method is rugged for Zn and Cu to delays in analysis of
samples after preparation for up to 48 hrs (see part 1). The method is only rugged to
delays in analysis for selenium for up to approximately 7 hours (one 90 patient sample
run, or two 40 patient sample runs).

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 56 of 73

Appendix B. Tables and Figures.
Table 1. Instrument and Method Parameters
Instrument: PerkinElmer ELAN DRCPlus or DRC II ICP-MS
CETAC ASX 500 series autosampler (tray B)
Optimization Window Parameters
RF power
1.45 KW
Plasma Gas Flow (Ar)
15 L/min
Auxiliary Gas Flow (Ar)
1.2 L/min
Nebulizer Gas Flow (Ar) 0.90 – 1.0 L/min (optimized as needed for sensitivity)
Ion Lens Voltage(s)
AutoLens (optimized as needed for sensitivity)
QRO, CRO, CPV,
Optimized per instrument by service engineer, or advanced
Discriminator Threshold user.
Parameters of x-y alignment, nebulizer gas flow, AutoLens voltages, mass calibration,
and detector voltages are optimized regularly. Optimization file name = default.dac.
Configurations Window Parameters
Cell Gas Changes
Pressurize Delay (From Standard to DRC mode) = 60
Pause Times
Exhaust Delay (From DRC to Standard mode) = 60
Flow Delay (Gas changes while in DRC mode) = 25
Channel Delay (Gas channel change in DRC mode) = 25
File Names & Directories
Method file names
Serum panel 1_methITS005A_sblk.mth
Dataset
Sample File
Report file name

Tuning
Optimization
Calibration
Polyatomic
Report Options
Template (transferring
results to the database)

Serum panel 1_methITS005A_aqblk.mth
Create a new dataset subfolder each day. Name as “20060718” for all work done on July 18, 2006
Create for each day’s work
For sample results printouts
cdc_quant comprehensive.rop
For calibration curve information
CDC_Quant Comprehensive (calib curve info).rop
Default.tun
Default.dac
N/A
elan.ply
CDC_Database Output.rop
Report Format Options: select only “Use Separator”
File Write Option: Append
Report File name: include date, instrument, and group
being analyzed in file name (i.e. 20060724a_DRCC_HM0364.txt)

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IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 57 of 73

Method Parameters
Method Parameters: Timing Page (see Figure 1 in the Appendix)
Sweeps/reading
90
Readings/replicate
1
Replicates
3
Enable QC Checking
Off
Isotopes Monitored
use 71Ga as an internal standard
64
and Internal Standard
Zn (63.9291), 65Cu (64.9278), 71Ga (70.9247), 78Se
Associations
(77.9173)
(Exact Mass)
Dwell Times
30 ms for 64Zn, 65Cu, 71Ga (70.9247), 78Se (77.9173)
Scan Mode
Peak Hopping for all isotopes (1 MCA channel)
DRC channel A Gas
Ammonia (5-7 psig delivery pressure)
Flow Rate
0.5 L/min *
(*Optimized per instrument, every 6-12 months)
RPa
0 for all isotopes
RPq
0.7 for all isotopes
Method Parameters: Processing Page (see Figure 2 in the Appendix)
Detector mode
Pulse
Process Spectral Peak
N/A
AutoLens
On
Isotope Ratio Mode
Off
Enable Short Settling
Off
Time
Blank subtraction
After internal standard
Measurement units
Cps
Process Signal Profile
N/A
Method Parameters: Equations Page (see Figure 3 in the Appendix)
Equations
On 64Zn, use “-0.035297 * Ni60”
On 78Se, use “-0.030461 * Kr83”
Method Parameters: Calibration Page (see Figure 4 in the Appendix)
Calibration Type
External Std.
Curve type
Simple Linear
Sample units
g/L
Calibration Standard
Zn: 30, 90, 300, 900, 3,000
Cu: 30, 90, 300, 900, 3,000
Concentrations (g/L)
Se: 3, 9, 30, 90, 300
Method Parameters: Sampling Page (see Figure 5 in the Appendix)
“Peristaltic Pump Under On
Computer Control”
Sample Flush
~35s at 24 rpm (optimize time so that solution reaches
nebulizer before Read Delay begins)

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1
Read Delay
Wash
Autosampler Locations
of Blanks and Standards

Page 58 of 73

40s at 18 rpm
60s at 24 rpm
For calibration curve (points to serum blank)
Serum panel 1_methITS005A_sblk.mth
Serum Blank and Calibration Stds 1 – 5 in autosampler
positions 101 – 106.
For QC & patient sample analysis (points to aqueous blank)
Serum panel 1_methITS005A_aqblk.mth
Aqueous Blank in autosampler position 109.
See figures 3a through 3e in Appendix B for other default
autosampler settings.

Table 2. Suggested Maximum Analyte
Concentrations for Base Serum.
Analyte
Concentration (µg/L)
Zn
400 (40 ug/dL)
Cu
500 (50 ug/dL)
Se
70

Table 3. Concentrations of Analytes in the Multi-Element Intermediate Stock
Standard from High Purity Standards.

Analyte
Cu
Zn
Se

Intermediate Stock Standard
Concentrations (mg/L)
High Purity Standards
Item # SM-2107-013 (2% HNO3)
300
300
30
V and Mn are also in the mix at 1 and 2 mg/L for future R&D work

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 59 of 73

Table 4. Preparation of Multi-Element Intermediate Working Standards
(for calibrators and calibration verification).
1

Calib
Verification

100

0.010

0.030

0.100

0.300

1.00

3.00

Concentrations
ug/L
30
90
300
900
3,000
Zn
ug/dL*
3
9
30
90
300
ug/L
30
90
300
900
3,000
Cu
ug/dL*
3
9
30
90
300
Se
ug/L
3
9
30
90
300
* Use ug/dL units for Zn and Cu in the ELAN software and for reporting.

9,000
900
9,000
900
900

Standard #
Vol of Flask
(mL)
Vol Spike of
Int. Stock
Std. (mL)

Units

Table 5. Acceptable ways to perform two consecutive analytical runs,
bracketing with bench quality control samples.
Setup 2 (typical)*
Setup 1*
Run #1
Run #1
Calibration Standards
Calibration Standards
Low Bench QC
Low Bench QC
High Bench QC
High Bench QC
patient samples
patient samples
Low Bench QC
Low Bench QC
High Bench QC
High Bench QC
Run #2
Low Bench QC
High Bench QC
patient samples
Low Bench QC
High Bench QC
* Use >18 Mega-ohm·cm water to
rinse the system for 30 min.
between the two runs.

Run #2
Calibration Standards
Low Bench QC
High Bench QC
patient samples
Low Bench QC
High Bench QC
* Use >18 Mega-ohm·cm water to
rinse the system for 30 min.
between the two runs.

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 60 of 73

Table 6. A typical SAMPLE/BATCH window.
AS
Sample ID
Measurements Action
Method
Location*
5
DRCstability1
Run sample
. . ._sblk.mth
5
DRCstability2
Run sample
. . ._sblk.mth
5
DRCstability3
Run sample
. . ._sblk.mth
5
DRCstability4
Run sample
. . ._sblk.mth
Continue DRC stability samples . . .
5
DRCstability9
Run sample
. . ._sblk.mth
5
DRCstability10 Run sample
. . ._sblk.mth
100
Sblkchk1
Run blank, standards, and sample ** . . ._sblk.mth
101
Sblkchk2
Run sample
. . ._sblk.mth
¥
127
Aq Blk Check
Run blank and sample
. . ._aqblk.mth
138
L Bench QC
Run sample
. . ._aqblk.mth
134
H Bench QC
Run sample
. . ._aqblk.mth
46
Sample 1
Run sample
. . ._aqblk.mth
47
Sample 2
Run sample
. . ._aqblk.mth
48
Sample 3
Run sample
. . ._aqblk.mth
.
.
.
.
.
.
.
.
.
.
.
.
140
Blind QC
Run sample
. . ._aqblk.mth
139
L Bench QC
Run sample
. . ._aqblk.mth
135
H Bench QC
Run sample
. . ._aqblk.mth
* The exact autosampler positions of QCs and patient samples do not have to be those
shown above, but the order in which these are run should be as shown above.
** When executing this row, the ELAN will first analyze the serum blank at AS position 101,
then standards 1-5 at autosampler positions 102-106, then the “sblkchk1” sample at A/S
position 100. The sampling information about AS positions 101-106 are stored in the “sblk”
method file.
When executing this row, the ELAN will first analyze the aqeous blank at AS position 109,
then the “Aq Blk Check” at AS position 20. The sampling information about AS positions
109 is stored in the “aqblk” method file.
¥

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Page 61 of 73

Table 7. Preparation of Multi-Element Intermediate Working Standards
Dilution ID
AQ Blank
Serum Blank and
sblkchk
Working Calibrators
or
Working Calibration
Verification Standards
Patient Serum or
Serum-Based QC
Patient Serum
2x Dilution H
Patient Serum
10x Dilution H
H

AQ Intermediate
Working Standard
(L)
-

Patient or QC
Serum sample
(L)
-

150

4,200

150

4,200

150

4,200

225

4,200

570

8,400

Water
(L)

Base Serum
(L)

300

Diluent *
(L)
4,200

Extra dilution is performed on serum samples whose concentration is greater than the concentrations listed in
Table 8 in the Appendix (linearity of the method has been documented up to these concentrations). Any extra
level of dilution can be prepared as long as the 14:15 ratio of diluent to total dilution volume is maintained. Use
of the lowest possible dilution level is preferred because matrix differences may lead to different observed
concentration results as the sample dilution becomes greater (i.e. 2x dilution is preferred over 10x if 2x is
sufficient to dilute analyte into the documented linearity range).

* Dispense diluent using the Digiflex as 2 portions which add to the total volume required. For example, when
preparing a serum blank above, do the preparation in 2 steps. Step 1: 150 L water + 2100 L diluent. Step
2: 150 L base serum + 2100 L diluent. This method of dispensing helps flush the smaller volume being
added from the pipette with diluent.

Table 8. Range of Reporting and Calibration Verification Requirements.
Highest Conc. (g/L) Verified in
Analyte
Calibration Verification
(“Range of Linearity Tested”, or “RLT”) *
Zn
9,000 (900 ug/dL)
Cu
9,000 (900 ug/dL)
Se
900
* If observed results are not within 10% of target, investigate the problem with
the involvement of the lab su ervis r.

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Page 62 of 73

Table 9. Boundary Concentrations for Serum.
1st Lower
1st Upper
2nd Upper
Range
2nd Lower
Analyte
Boundary Boundary
Boundary
Boundary
Maximum
(units)
(“Lim Rep Delta”) †
(“2LB”)**
(“1LB”)*
(“1UB”) *
(“2UB”) **
35
35
120
240
17
Zn (g/dL)
10
10
300
600
20
Cu (g/dL)
45
45
165
330
20
Se (g/L)
st
* Typically, the 1 upper boundaries (1LB and 1UB) are based on percentiles of nonweighted, non-creatinine corrected concentration results from NHANES. In the absence of
that data, these boundaries can be based on normal ranges reported in the literature. The
concentrations assigned to these boundaries is determined by study protocol but default
concentrations are listed in this table. Report the original result, as long as the confirmation is
within 10% of the original. Continue repeat analysis until a concentration can be confirmed.
** These 2nd boundaries (2LB and 2UB) are set to 0.5x the 1LB and 2x the 1UB, respectively.
The concentrations assigned to these boundaries is determined by study protocol but default
concentrations are listed in this table. Regardless of the study, the analyst should specifically
address patient results confirmed to be less than the 2LB or greater than the 2UB to the QC
reviewer as unusually low or high results.
† Range maximum is the range of the three replicate readings for a single sample analysis.
This value is also called the “Lim Rep Delta” in the database which handles data for the
Inorganic Toxicology and Nutrition Branch. If the range of replicate readings is greater than
the range maximum, and represents greater than a 10% relative standard deviation for the
measurement, do not use the measurement for reporting.

Table 10. Reference Ranges for Serum Concentrations Se in
g/L; Zn and Cu in g/dL.
Analyte
Reference Ranges
Zn
70-120 [10]
Cu
20-302 [10]
Se
95-165 [11], [14]

M.
N.
O.
P.
Q.
R.
S.

Rinse Station (Modified)
Nut, 10-32 threads, 1/16” tubing
Tubing (PFA), 0.5mm x 1.59 mm T.
Nut, ¼-28 thread, 1/16” tubing
U.
O-ring, 1/16” ID x 3/16” OD
Adapter, ¼-28 screw to 1.8 mm barb
V.
Tubing (Peristaltic), 0.045” ID

K.
L.
R.
S.

Inert counterweight
Container for rinse solution
Tube (PP, lot screened), 15 mL
Probe, corrosion resistant
Tubing (Peristaltic), 0.03” ID
Quick Connect for liquid (can be
done using Upchurch adapter)
PEEK adapter (1.6mm OD PFA to
0.03” ID peristaltic tubing)

Figure 1. Suggested configuration of
tubing and devices for liquid handling.

Tubing (Vinyl), 1/8” x ¼”
Argon Quick Connect
Quartz Concentric Nebulizer
Spray Chamber O-rings
Quartz Cyclonic Spray Chamber
Tubing (PVC), 1/8” x 3/16”
Tubing Connector
Tubing (Peristaltic), 0.125” ID
Carboy for Liquid Waste (10L)
Tubing (PVC), 3/16” x 5/16”

IRAT-DLS Method Code: ICPDRCMS-3006.1

A.
B.
C.
D.
E.
F.
G.
H.
I.
J.

Serum Multi-Element ICP-DRC-MS
Page 63 of 73

Appendix (continued): Figures

IRAT-DLS Method Code: ICPDRCMS-3006.1

V and Mn are only Research and
Development. Not for Clinical
Reporting. Remove these rows
when analyzing any clinical
samples (non-Proficiency Testing).

Optimize
Per
Instrument

Serum Multi-Element ICP-DRC-MS
Page 64 of 73

Appendix B (continued)
Figure 2a. ELAN ICP-DRC-MS Method Screen Shots (timing page).

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 65 of 73

Appendix B (continued)
Figure 2b. ELAN ICP-DRC-MS Method Screen Shots (processing page).

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 66 of 73

V and Mn are only Research and
Development. Not for Clinical
Reporting. Remove these rows
when analyzing any clinical
samples (non-Proficiency Testing).

Appendix B (continued).
Figure 2c. ELAN ICP-DRC-MS Method Screen Shots (equation page).

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 67 of 73

V and Mn are only Research and
Development. Not for Clinical
Reporting. Remove these rows
when analyzing any clinical
samples (non-Proficiency Testing).

Appendix B (continued).
Figure 2d. ELAN ICP-DRC-MS Method Screen Shots (calibration page).

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 68 of 73

Appendix B (continued).
Figure 2e. ELAN ICP-DRC-MS Method Screen Shots (sampling page).

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 69 of 73

Appendix B (continued).
Figure 2f. ELAN ICP-DRC-MS Method Screen Shots (report page).

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 70 of 73

Appendix B (continued).
Figure 3a. ESI SC4 Autosampler Screen Shots used (Main page).
Additional flush times and “Max Rinse Time” are default, but can be optimized for best
reduction of elemental carry-over between samples. Tray types can be changed to
allow for different volumes of diluted sample digests. ‘FAST control’ should be
unchecked. Rinse and additional flush times for eliminating carry-over from one sample
to the next while using the minimum amount of rinse solution.
A rinse time of -1 causes the rinse station to be skipped.
A rinse time of 0 causes the probe to only dip into the station, but spends no time there.
Additional flush times can be optimized to keep the rinse station full while not using too
much rinse solution. The inner diameter size of the tubing providing the rinse solution to
the rinse station determines how quickly the station will fill. Various sizes are available
for purchase or can be made in the laboratory.

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 71 of 73

Appendix B (continued).
Figure 3b. ESI SC4 Autosampler Screen Shots used (Configuration).
Appendix B (continued).
“High Speed” option is to only be used for ‘High Speed’ models of the SC4 (look for
“HS” in serial number). Speeds and accel / decel values can be optimized per analyst
preference and to minimize droplet splatter off of probe.

Figure 3c . ESI SC4 Autosampler Screen Shots used (“Communication” page).
Communication ports will differ depending on available ports on instrument control
computer.

Serum Multi-Element ICP-DRC-MS
IRAT-DLS Method Code: ICPDRCMS-3006.1

Page 72 of 73

Appendix B (continued).
Figure 3d. ESI SC4 Autosampler Screen Shots (5x12 Rack Setup window).
Settings are approximate. To be sure the loop is filled, the probe should go down close
to the bottom of the cup, but not touch. Optimize retraction speed for least droplet
splatter.

Figure 3e. ESI SC4 Autosampler Screen Shots (50mL Tube Rack Setup window).
Settings are approximate. To be sure the loop is filled, the probe should go down close
to the bottom of the cup, but not touch. Optimize retraction speed for least droplet
splatter.

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Page 73 of 73

References
1.
2.
3.
4.
5.
6.
7.
8.
9.

10.
11.
12.
13.
14.

Thomas, R., Practical Guide to ICP-MS (Practical Spectroscopy). 2003, New
York, NY: Marcel Dekker. 336.
Tanner, S.D., Baranov, Vladimir I, Theory, Design, and Operation of a Dynamic
Reaction Cell for ICP-MS. Atomic Spectroscopy, 1999. 20(2): p. 45-52.
Tanner, S.D., V.I. Baranov, and D.R. Bandura, Reaction cells and collision cells
for ICP-MS: a tutorial review. Spectrochimica Acta Part B-Atomic Spectroscopy,
2002. 57(9): p. 1361-1452.
PerkinElmer SCIEX Instruments, ELAN DRC II Hardware Guide. 2001, Canada.
Piraner, O., Serum vanadium ICPDRCMS_ITS004A. 2003, Centers for Disease
Control and Prevention.
Piraner, O., Serum manganese ICPDRCMS_ITS003A. 2003, Centers for
Disease Control and Prevention.
Piraner, O., Serum selenium ICPDRCMS_ITS002A. 2004, Centers for Disease
Control and Prevention.
Walters, P. J., Serum Copper Zinc ICP-DRC-MS_ITS001A. 2004, Centers for
Disease Control and Prevention.
Office of Health and Safety in the Division of Laboratory Sciences, Policies and
Procedures Manual. 2002, Division of Laboratory Sciences (DLS), National
Center for Environmental Health, Centers for Disease Control and Prevention,
Public Health Service, Department of Health and Human ServicesCenters for
Disease Control and Prevention.
Centers for Disease Control and Prevention (CDC) Radiation Safety Committee,
CDC/ATSDR Occupational Health and Safety Manual (Radiation Safety chapter).
Centers for Disease Control and Prevention, Public Health Service, Department
of Health and Human ServicesCenters for Disease Control and Prevention.
Tietz Textbook of Clinnical Chemistry, Third Edition, edited by Burtis C. A.,
Ashwood E. R., 1999
Agency for Toxic Substance and Disease Regidtry (2000). Toxicological Profile
for Selenium. Atlanta, GA: U. S. Department of Health and Human Services,
Public Health Service.
Lauwerys R.R. Chapter II: Biological monitoring of exposure to inorganic and
organometallic substances, In: Industrial Chemical Exposure: Guidelines for
Biological Monitoring, Biomedical Publications, pp. 9-50, 1983
Handbook on Metals in Clinical and Analytical Chemistry, edited by Seiler H.G.,
Sigel A., Sigel H., Marcel Dekker, Inc, 1994
Niskar AS, Paschal DC, Kieszak SM, Flegal KM, Bowman B, Gunter EW, Pirkle
JL, Rubin C, Sampson EJ, McGeehin M. Serum selenium levels in the US
population: Third National Health and Nutrition Examination Survey, 1988-1994.
Biol Trace Elem Res 2003 Jan; 91(1): 1-10

Navajo Birth Cohort Study Protocol for Cellular immunological laboratory protocol
Flow cytometry protocol for lymphocyte phenotypization
1. For each sample, label six 12 x 75 mm or 15 ml Falcon tubes, A through F. Also label
each tube with sample identification number or barcode.
2. Place 20 μl of reagent A into tube A, 20 μl of reagent B into tube B, 20 μl of reagent C
into tube C, 20 μl of reagent D into tube D, 20 μl of reagent E into tube E, and 20 μl of
reagent F into tube F.
3. For each sample, use a fresh micropipette tip and add 100 μl of anticoagulated whole
blood sample into the bottom of each of the six labeled tubes. The required WBC
concentration is 3,500-9,800 cells/ μl blood. Vortex thoroughly at low speeds for 3
seconds and incubate for 15 to 30 minutes at room temperature (20-25 oC). During this
incubation protect samples from direct light. Prevent blood from running down the side of
the tube, pipette directly into the staining reagents.
4. Dilute 10X Lysing Solution to 1X following the instructions for Reagent G. The 1X
solution is stable at room temperature for 1 month. Add 2 mL of this 1X lysing solution to
each tube. Immediately vortex them at low speed for 3 seconds and incubate for 10 to
12 minutes at room temperature in the dark. Do note exceed 12 minutes as the lysis can
destroy the stained lymphocytes as well.
5. Immediately after incubation, centrifuge tubes at 300x g for 5 minutes at room
temperature.
6. Aspirate the supernatant, leaving approximately 50 μl of residual fluid in each tube to
avoid disturbing the pellet.
7. Vortex thoroughly at low speed to resuspend the cell pellet in the residual fluid and then
add 2 mL PBS to each tube. Vortex thoroughly at low speed for 3 seconds. Centrifuge at
200 x g for 5 minutes at room temperature.
8. Aspirate the supernatant, leaving appr. 50 μl of residual fluid in the tube to avoid
disturbing the pellet.
9. Vortex thoroughly at low speed to resuspend the cell pellet in the residual fluid and then
add 0.5 mL of 1% paraformaldehyde to each tube. Vortex thoroughly for 3 seconds.
Make sure that the cells are mixed well with the fixing solution.
10. The cells are ready to be analyzed on the flow cytometer. Cap or cover tubes and store
them at 2- 8 oC in the dark till analysis. Analyze the fixed cells within 24 hours after
staining.
Principles of procedure
When the monoclonal antibody reagents are added to human whole blood, the flurorochromelabeled antibodies bind specifically to antigens on the surface of leucocytes. The stained
samples then treated with Lysing Solution to lyse erythrocytes and washed prior to flow
cytometry analysis.
An aliquot of the stained participant sample is introduced into the flow cytometer and passed in
a narrow stream through the path of a laser beam. The stained cells fluoresce when excited by
the laser beam and the emitted light is collected and processed by the flow cytometer. The use
of two fluorochromes permits simultaneous two-color analysis because each fluorochrome emits
5/2011

light at different wavelength when excited at 488 nm by an argon-ion laser. The FITC-stained
lymphocytes emit yellow-green light (maximum 515 nm) while the PE-stained lymphocytes emit
red-orange light (580 nm).
For each sample, the lymphocyte acquisition gate set with LeucoGATE (tube A) and the
fluorescence markers determined using the Control (tube B) are used to analyze the
subsequent tubes (C through F). The software uses quadrant correction option; the lymphocyte
subpopulations in tube C through F are enumerated and then expressed as percentages of
lymphocytes in the acquisition gate.
Reagents
Reagent A – LeucoGATE (CD45/CD14) LeucoGATE is used to define and evaluate the lightscatter gate that distinguishes lymphocytes from granulocytes, monocytes, unlysed or nucleated
red blood cells and debris. The reagent contains FITC-labeled CD45 for identification of
leucocytes, and PE-labeled CD14 for identification of monocytes.
Reagent B – Control This isotype IgG control is used to set the lowest quadrant markers around
unstained (negative) lymphocytes. This reagent helps to establish nonantigen-specific antibody
binding, in particular that caused by Fc receptors.
Reagent C – CD3/CD19 This reagent is used to enumerate T and B lymphocytes. It contains
FITC-labeled CD3 for identification of T lymphocytes and PE-labeled CD19 for identification of B
cells.
Reagent D – CD4/CD8. This stain is used to simultaneously characterize helper/inducer and
suppressor/cytotoxic lymphocytes. It contains FITC-labeled CD4 for identification of
helper/inducer T lymphocytes and PE-labeled CD8 for identification of suppressor/cytotoxic T
lymphocytes.
Reagent E – CD+/Anti-HLA-DR This reagent is used to enumerate T lymphocytes, DR+ non-T
lymphocytes (mostly and primarily B cells) and activated T lymphocytes. It contains FITClabeled CD3 for identification of T lymphocytes and PE-labeled anti-HLA-DR for identification of
DR+ non-T cells and activated T lymphocytes.
Reagent F – CD3/CD16+CD56 This stain is applied to identify T and NK lymphocytes. It
contains FITC-labeled CD3 for identification of T lymphocytes. It also contains PE-labeled CD16
and PE-labeled CD56 for identification of NK cells as well T-lymphocyte subsets.
Reagent G – 10X Lysing Solution. This reagent contains 10X buffered Lysing Solution, with less
than 50% diethylene glycol and less than 15% formaldehyde. When stored at room temperature
this solution is stable until expiration date.
The reagents are under the US Patent No. 4,895,796.
Detection: fluorescence activity will be detected by flow cytometer, Becton Dickinson FACScan
machine BD Catalog #34001010 or Accuri portable flow cytometer machine, Accuri, Ann Arbor,
MI).

Humoral immunological measurements
Serum cytokine measurements
We will address inflammatory and/or hypersentitivity conditions among NBC participants
applying comprehensive multiplexing technology and Luminex 100™ detection system
(Luminex Co. Austin, TX) for serum cytokine production.
Cytokines play a central role in mediating both cellular and humoral immune responses against
invading pathogens and tumor cells. Many of them also control the growth differentiation,
effector function and survival of all cells in the body. Human 10-plex high sensitivity
cytokine/chemokine panel (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, INF-γ, TNF-α, and GM-CSF)
will be used to detect presence or absence of inflammatory cytokines, with special attention paid
to uncovering potential imbalances in Th1/Th2 immune responses.

Assay methodology Cytokine measurements using multiplexing technique
Figure of the assay of determination of cytokines using flow cytometry technique

This methodology is the most advanced technology of using very small amount of
patient samples. The assay requires only 50 µl of serum samples and measures the
entire panel of cytokines parallel from only 1 sample. This is not only saves a lot of
smaples, requires less blood donation from participants, but also the mosts ensitve
technique. The serum cytokines deetcted at the pg/ml detection limits.

Assay detection
At the UNM Flow Cytopmetry Core Facility xMAP multiplexing technology is readily
available for this project. The UNM Research Team will use Luminex 100™ detection
system to quantitate immonoassays in a 96 well format.

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