ICP-MS Determination of Mercury in Urine

Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 1 of 27

Laboratory of Inorganic and Nuclear Chemistry
Division of Environmental Health Sciences
Wadsworth Center
Department of Health
State of New York
NYS CLEP Laboratory ID 1067
CLIA Laboratory ID 33D0654341

Standard Operating Procedure

Mercury in Urine by Inductively Coupled Plasma Mass
Spectrometry (ICP-MS)

Approved: ___________________________________________________
Laboratory Director

Patrick J. Parsons, Ph.D.

Approved: ___________________________________________________
Quality Assurance Officer

Heidi Dillenbeck

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Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 2 of 27

Distribution
Laboratory Copy (Ivory), maintained by Laboratory Supervisor
Laboratory Bench Copy, maintained by Laboratory Staff
Master Copy (original signatures), Heidi Dillenbeck, Quality Assurance Officer
Electronic copies.
//trel/trel/quality assurance
Dr. Patrick J. Parsons, Chief
Laboratory of Inorganic and Nuclear Chemistry
//pern/dedps
Heidi Dillenbeck, Quality Assurance Officer
Division of Environmental Health Sciences

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Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 3 of 27

Revision Record
Rev

Date
Responsible
Person

Description of Change

1

4/26/04

Christopher Palmer

Initial release.

2

5/18/04

Christopher Palmer

Major revision.

3

11/03/06

Michael Minnich

Major revision.

4

1/29/08

Amy Steuerwald

Sulfamic acid solution preparation,
addition to clinical specimens and
diluent revised. Change data back
up from Lead server to trel.

5

2/29/08

David Bellis

Addition of criteria for assessing
repeat measurements.

6

4/21/08

David Bellis

Quality control data updated.

7

9/1/08

David Bellis

Major revision.

8

1/9/09

Amy Steuerwald

Minor revisions.

9

12/13/10

Amy Steuerwald

Minor revisions.

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Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 4 of 27

The following laboratory staff have read this Manual.
A copy of this page will be distributed to the employee training record file.

Name

Title

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Date

Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

1.0

Rev. No. # 9
Page 5 of 27

Method capabilities

This method is used to achieve rapid and accurate quantitative determination of
mercury (Hg) in urine. Protocols for specimen collection were developed based on
Centers for Disease Control and Prevention (CDC) guidelines (1). The method is
suitable for assessing exposure in occupationally and non-occupationally exposed
subjects. This method has been used successfully in several biomonitoring studies
of mercury exposure (2, 3).
2.0

Safety precautions

Before operating the instrument, read the information in the PerkinElmer® ELAN®
ICP-MS System Safety Manual.
All personnel must successfully complete the Hazard Communication / Lab Safety
Training annually, either on-line or in-person. All personnel must abide by the
regulations set forth in the Policies and Procedures Manual issued by the
Wadsworth Center Safety and Security Office.
Wear gloves, a lab coat, and safety glasses while handling all human urine.
Observe universal precautions. Place in a biohazard autoclave bag disposable
plastic, glass and paper (e.g., pipette tips, autosampler tubes, gloves, etc.) that
contact urine. Dispose of all biological samples and diluted specimens in a
biohazard autoclave bag at the end of the analysis. Keep these bags in appropriate
containers until they are sealed and sent to be autoclaved.
Exercise special care when handling and dispensing concentrated nitric acid.
Always remember to add acid to water. 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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Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

3.0

Rev. No. # 9
Page 6 of 27

Standard operating process and procedures

START

Specimen/base urine
collection and storage
and storage

Ye
s

Analyte
above repeat
threshold?

Prepare reagents

Defrost specimens/
samples/QC

Ye
s

No

QC within
limits?

Prepare standards/
specimens/samples/
QC

Process results

ICP-MS readiness
performed

Perform analysis

No

3.1

ICP-MS in
control?

Ye
s

Specimen/base urine collection and storage

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No

Report and archive

EN
D

Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 7 of 27

3.1.1 Specimen collection and storage
This method does not require any special instructions for the patient such as
fasting or special diets. The specimen type is random spot urine. A sulfamic acid
preservative solution (section 3.2.1) is required to prevent endogenous inorganic
mercury loss in urine specimens as previously described (4).
The optimal amount of submitted specimen is 4–7 mL; the minimum amount of
submitted urine is approximately 2 mL. The analysis requires at least 500 µL (0.5
mL) for a single assay. Typical specimen collection involves addition of 50 µL of
sulfamic acid preservative solution for every 5 mL of urine collected. Acceptable
containers include wide mouth specimen containers with screw lids (Kendall
Precision Industries or equivalent), lot-screened 15-mL polypropylene centrifuge
tubes, the Urine-Monovette® (Sarstedt Inc., Newton, NC) or Nalgene® cryovials.
Use of sterile collectors for specimen acquisition is desirable, but not mandatory.
The criteria for unacceptable specimens are (a) insufficient volume (<0.5 mL), (b)
lack of sulfamic acid preservative solution or (c) suspected contamination due to
improper collection procedures or devices. Specimen characteristics that may
compromise test results include contamination of urine by contact with dust, dirt,
etc., from improper handling. A fresh urine specimen should be collected if the
original specimen is believed to be unacceptable or compromised.
The laboratory protocol for urine collection and handling outlines specimen
handling conditions. Collection, transport and special requirements are discussed.
In general, urine specimens may be transported at ambient temperature,
refrigerated or frozen and packed in dry ice during shipment. Once received, store
long term at ≤-20°C until the analysis can proceed.
Short-term storage at approximately 2–4°C is acceptable. Refreeze at ≤-20°C
portions of the sample that remain after analytical aliquots are withdrawn. Thawing
and refreezing has not been found to compromise Hg determination in urine when
the sulfamic acid preservative solution is added to specimens during collection.
For further information refer to the CDC instructional DVD, “Responding to a
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Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 8 of 27

chemical emergency, collection and handling of clinical specimens”.
On receipt of a sample, check if “chain of custody” form is included. Sign and date
form if present and file.
Accession samples (i.e., record in the order of acquisition) in the “Laboratory
accessioning log book”. Record pertinent specimen information including identifiers
and the date of receipt. Provide and record a unique New York State Identification
number for each specimen.
3.1.2 Base urine collection and storage
The base urine used in this method is a pool of human urine collected from
volunteer donors. The pooled urine is acidified to 1% (v/v) using double-distilled
nitric acid and 1% (v/v) sulfamic acid preservative solution (section 3.2.1). The
base urine is frozen at ≤-20ºC, defrosted and centrifuged to remove precipitated
urate salts. The supernatant urine is used to prepare matrix-matched calibration
standards and quality control (QC) materials (section 3.4).
Collect urine in lot-screened or acid-rinsed sample collection cups or 24-hour urine
collection containers. Once the urine is collected from donors, it should be
analyzed to ensure that the Hg concentration is below the suggested maximum
analyte concentration (Table 1). For short-term storage, store at approximately 2–
4°C. For long-term storage, store at ≤-20°C.
Table 1. Suggested maximum analyte concentration for the matrix-match base
urine.
Analyte

Concentration (µg/L)

Hg

<0.11

3.2

Prepare reagents

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Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 9 of 27

Reagents should be prepared under class 100 clean room conditions (i.e., in a
Clean Room or Biological Safety Cabinet) using ≥18 MΩ·cm double-distilled
deionized water (Barnstead Nanopure; Millipore Corporation, Bedford, MA or
comparable) and high purity acids that are purchased from a manufacturer or
generated in-house through use of an acid still. Certified and periodically calibrated
pipettors should be used at all times. Label reagents with contents, date prepared,
date of expiration and initials.
3.2.1 Reagent preparation
(1) Triton X-100™
A solution of 10% Triton X-100™ is made by adding 10 mL of concentrated Triton
X-100™ stock (t-octylphenoxypolyethoxyethanol, SigmaUltra; Sigma-Aldrich
Company, St. Louis, MO) to 90 mL of ≥18 MΩ·cm water. Mix the solution for
several hours on a rotator mixer (Orbitron Rotator 1, Boekel Scientific, or similar)
until the Triton X-100™ has dissolved. Store at room temperature and prepare as
needed.
(2) Sulfamic acid preservative solution
To prepare, acid rinse or use a dedicated 125 mL Teflon™ container. Use an acid
rinsed 100 mL plastic graduated cylinder to fill the Teflon™ container with 90 mL of
≥18 MΩ·cm water. Weigh 20 g of sulfamic acid (99.3% ACS reagent; Sigma–
Aldrich Company, St. Louis, MO) into a weigh boat and add to the Teflon™
container. Dissolve the solid completely by agitating overnight on a rotator mixer or
heating in warm water. Add 10 mL of concentrated Triton X-100™ stock solution.
Mix thoroughly until completely dissolved. Store at room temperature and prepare
as needed. Preservative solution shelf-life is 1 month.
(3) Diluent
The diluent used in this method is an aqueous solution of 1% (v/v) double-distilled
nitric acid, 1% (v/v) sulfamic acid preservative solution, 0.005% Triton® X-100™,
1 mg/L Au and 10 µg/L Ir as an internal standard. This solution is used to prepare
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Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 10 of 27

the 1+19 dilution of all specimens, standards and QC materials. To prepare, acid
rinse or partially fill a dedicated 2 L Teflon™ container with ≥18 MΩ·cm water. Add
20 mL double-distilled, concentrated nitric acid and 20 mL of sulfamic acid
preservative solution. Add 20µL of 1000 mg/L Ir, 200 µL of 10000 mg/L Au and 1
mL of 10% Triton X-100™. Dilute to volume (2 L) with ≥18 MΩ·cm water. Store at
room temperature and prepare as needed. Diluent expires 1 month from
preparation.
(4) ICP-MS Rinse solution
The rinse solution used in this method is an aqueous solution of 2% (v/v) doubledistilled nitric acid, 1000 µg/L Au, and 0.005% Triton X-100™ solution. This
solution is used to prevent carry-over of analyte between samples. To prepare,
acid-rinse or partially fill a dedicated 2 L Teflon™ container and with ≥18 MΩ·cm
water. Add 40 mL double-distilled, concentrated nitric acid. Add 1 mL of 10%
Triton X-100™ to the rinse solution and 200 µL of 10000 mg/L Au. Dilute to 2 L
using ≥18 MΩ·cm water. Store at room temperature and prepare as needed.
3.3

Defrost specimens, samples and QC materials

All specimens, samples and QC materials should be stored at ≤-20°C until batched
analysis can be arranged. Place all frozen urine specimens, previously prepared
and frozen base urine for calibration curve matrix matching, QC materials and
samples for analysis on a rotator mixer. Start rotation and allow specimens to
reach ambient temperature.
3.3.1 Internal QC materials
Internal QC materials may be prepared by spiking prepared base urine (section
3.1.2) with Hg, stirring for 24 hours, aliquoting and freezing. QC materials should
be prepared under Class 100 Clean Room conditions.
3.3.2 External QC samples
Samples certified for the analytes of interest (e.g., NIST Standard Reference
Material (SRM) 2670a Toxic Elements in Urine (National Institute of Standards and
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Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 11 of 27

Technology, Gaithersburg, MD)) may be used for method validation and should be
analyzed periodically to ensure method accuracy. External Quality Assessment
(EQA) scheme (e.g., L’Institut National de Santé Publique du Québec (INSPQ),
Centre de Toxicologie du Québec (CTQ), Canada) or Proficiency Testing (PT)
samples (i.e., New York State Department of Health, Albany, New York) may also
be used during validation and for ongoing QC procedures.
3.4

Prepare standards, specimens, samples and QC materials

Standards should be prepared under class 100 clean room conditions (i.e., in a
Clean Room or Biological Safety Cabinet) using ≥18 MΩ·cm double-distilled
deionized water and high purity acids that are purchased from a manufacturer or
generated in-house through the use of an acid still. Caps should only be removed
from standards, specimens, samples and QC under class 100 clean room
conditions. Certified and periodically calibrated pipettors should be used at all
times. Label reagents with contents, date prepared, date of expiration and initials.
3.4.1 Standard preparation
(1) Hg Stock standard
The stock standard solution is a NIST traceable aqueous solution of 1000 mg/L of
inorganic Hg. This solution is a single element ICP standard prepared by GFS
Chemicals (GFS Chemicals, Powell, OH). The shelf life of the stock standard is
certified by the manufacturer and should be stored according to manufacturer
instructions. The Hg stock standard is used to prepare the Hg intermediate stock
standard.
(2) Hg intermediate stock standard
The Hg intermediate stock standard is an aqueous solution of 8 mg/L Hg in 1%
(v/v) nitric acid and 1% (v/v) sulfamic acid preservative solution. Prepare by acid
rinsing or using a dedicated 100 mL polypropylene (PP) volumetric flask and
partially filling it with ≥18 MΩ⋅cm water. Add 1 mL of double-distilled, concentrated
nitric acid and 1 mL of the sulfamic acid preservative solution. Add 800 µL of the
1000 mg/L Hg stock solution. Bring to volume with ≥18 MΩ⋅cm water. Table 2
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Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 12 of 27

shows the Hg intermediate stock standard preparation scheme.
Table 2. Hg intermediate stock standard preparation.
Reagent

Volume (µL)

Hg stock standard (1000 µg/L)

800

Concentrated HNO3

1000

Sulfamic acid preservative solution

1000

≥18 MΩ·cm water

Balance to 100 mL final volume

(3) Hg Intermediate working standards
The intermediate working standard solutions used in this method are a series of six
aqueous dilutions of the Hg intermediate stock standard solution and one aqueous
blank in 1% (v/v) double-distilled nitric acid and 1% (v/v) sulfamic acid preservative
solution. These solutions are used each day of analysis to prepare the final
working standards. To prepare, acid-rinse or partially fill seven dedicated 100 mL
PP volumetric flasks and partially fill them with ≥18 MΩ·cm water. To each 100 mL
flask, add 1 mL of double-distilled, concentrated nitric acid and 1 mL of sulfamic
acid preservative solution. Add the appropriate aliquot of the Hg intermediate stock
standard solution (Table 3) and bring to volume with ≥18 MΩ·cm water. One of the
volumetric flasks will not have an aliquot of the intermediate stock standard and
serves as the 1% (v/v) nitric acid and 1% (v/v) sulfamic acid blank. Store solutions
at room temperature in the dedicated 100 mL volumetric flasks. Intermediate
working standards expire 1 week from the preparation date. The final
concentration of Hg in each standard is listed in Table 4.

Table 3. Preparation of Hg single element intermediate working standards.
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Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Hg Intermediate

Rev. No. # 9
Page 13 of 27

Volume of Hg Intermediate Stock Standard (µL)

Stock Standard

8 mg/L

Blank

Std. 1

Std. 2

Std. 3

Std. 4

Std. 5

Std. 6

0

25

63

125

250

375

500

Table 4. Concentration of the intermediate working standards.
Analyte

Intermediate Working Standard Concentration (µg/L)
Std. 1

Std. 2

Std. 3

Std. 4

Std. 5

Std. 6

Hg*
2.0
5.0
10
20
30
40
*
Ir-193 internal standard.
Note: Enter the Table 4 concentrations into the method calibration page of the
ELAN® software.
3.4.2

Standard, specimen, sample and QC dilution

Class 100 clean room conditions are required for dilutions. Prepare test samples,
including urine specimens, QC materials and working calibration standards with the
Digiflex dispenser (ICN Biomedicals Inc., refer to manufacturer’s handbook for
more operational guidelines) or similar. Table 5 provides a summary of the sample
preparation process.

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Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 14 of 27

Table 5. Preparation scheme for the NYS urine Hg method.
Volume (µL)
1% HNO3 /
Base
Intermediate Patient or
1% Sulfamic
Urine
Working
QC Urine
Acid
Standards

Diluent

Reagent
blank

1000

0

0

0

2x4500

Urine blank

500

500

0

0

2x4500

Standards

0

500

500

0

2x4500

500

0

0

500

2x4500

Specimens/
Samples
and QCs

Note: 9000 µL diluent is best dispensed from the Digiflex™ as two 4500 µL portions
(i.e., when preparing a Standard dilution, dispense 4500 µL diluent + 500 µL
intermediate working standard in one cycle of Digiflex™, then 4500 µL diluent +
500 µL base urine in the next cycle of the Digiflex™ to prepare a 10 mL total
volume dilution).
(1) Reagent blanks
Prepare three reagent blanks consisting of 1000 µL of 1% (v/v) nitric acid, 1%
sulfamic acid preservative solution and 9000 µL of diluent. These will be used as
the blank for any urine based QC materials, external reference samples and patient
specimens.
(2) Urine blank
Prepare one urine blank dilution consisting of 500 µL of base urine (i.e., same
material used to prepare the urine calibration standards), 500 µL of reagent blank,
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Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 15 of 27

and 9000 µL of diluent. The urine blank will be the blank for the calibration
standards.
(3) Working calibration standards
Prepare the working calibration standards as 500 µL of the appropriate aqueous
intermediate working calibration standard, 500 µL of base urine, and 9000 µL of
diluent.
(4) Urine specimens, samples and urine-based QC materials
Prepare the urine specimens, samples and the urine-based QC material by diluting
500 µL of urine with 500 µL of reagent blank and 9000 µL of diluent.
Prepare a single sample from each specimen. For research or biomonitoring
studies, prepare a minimum of 2% random repeats for quality assurance purposes.
If (i) results are needed as-soon-as-possible or (ii) results are likely to exceed the
repeat threshold (Table 9), it is acceptable to prepare specimens in duplicate and
perform two consecutive runs on separate calibration curves.
At least 2 levels of urine QC materials must be analyzed with specimens, one with
typical element concentrations and one with elevated concentrations. QC materials
must be present at the beginning and end of each analytical run. QC materials
should also be analyzed between approximately every 10–20 specimens.
Additional QCs can be prepared if thought necessary.
Cap and mix well all diluted urine materials.
3.5

ICP-MS readiness performed

Total urine Hg (m/z=202) is determined by this method using a Perkin Elmer Sciex
ELAN DRC II inductively coupled plasma-mass spectrometer (PerkinElmer Life and
Analytical Sciences, Shelton, CT) or similar, with analyses performed in standard
mode as previously described (5). The instrument should be equipped with an
automated sample introduction system, such as the CETAC ASX500 autosampler,
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Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 16 of 27

a Meinhard® concentric quartz nebulizer (Meinhard Glass Products, Golden, CO)
and a baffled quartz cyclonic spray chamber (Glass Expansion, Pocasset, MA).
Comparable hardware may be substituted.
Start the Elan software and ensure that the instrument is in ready mode. Check
argon supply, that water circulator is on and that vacuum pressure is within limits.
Inspect sample introduction tubing, nebulizer, spray chamber and injector. Clean
or replace any sample introduction parts if necessary. Inspect sampling and
skimmer cones for accumulation and clean or replace as needed.
In the case of serious problems, contact a supervisor. Telephone Perkin-Elmer
Technical support: 1-800-762-4000) if necessary.
Turn on plasma and allow at least 30 minutes stabilization prior to optimization.
Follow the optimization steps below using the Tuning Solution as described in the
ELAN Software Guide.
1. Optimization of nebulizer gas flow rate for 3% oxides (Section 4-72)
2. Optimization of lens voltage and autolens (Sections 4-60 and 4-61)
3. Daily Performance (Section 4-39), see Table 6 below for typical
acceptable criteria.
3.6

ICP-MS in control?

Generate “Daily performance report”.
Inspect report to see if it meets the requirements given in Table 6.
If performance is acceptable, then proceed. File the printed “Daily performance
report” in the appropriate folder and fill in “Daily performance summary” document.
If performance is unacceptable, take the recommended corrective actions.

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Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 17 of 27

Table 6. Assessment of daily performance results.
Parameter

Acceptable level

Possible corrective actions

Sensitivity of Mg

>1,000 cps

Make new Daily performance solution
Perform x-y adjustment

Sensitivity of In, U

>10,000 cps

Adjust nebulizer gas flow
Clean lens
Optimize detector voltage

Oxide CeO/Ce

≤0.03

Adjust nebulizer gas flow
Etch spray chamber

Lens voltage (In)

<10

Clean lens

If problems persist contact a supervisor.
support: 1-800-762-4000 if necessary.
3.7

Telephone Perkin-Elmer Technical

Perform analysis

Refer to the manufacturer handbook and for further guidelines on ICP-MS
operation.
Open the workspace “analysis.wrk”.
Open method template“xxxxxx NYS Urine Hg.mth”.
Save method as “mmddyy NYS Urine Hg.mth”, where mmddyy represents the date
of the analysis.
In the method-report window, define report filename as mmddyy NYS Urine Hg.
Create new sample file and save as “mmddyy NYS Urine Hg.sam”.
Create new dataset folder “mmddyy NYS Urine Hg” and load.
Open Report Template file: “NYS Report.rop.”
Load Tuning file: “default.tun” (Note: The setting in the method file used during
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Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 18 of 27

analysis will supersede your selection here).
Load Optimization file “default.dac” (Note: The setting in the method file used
during analysis will supersede your selection here).
No Calibration is file needed.
Load Polyatomic file: elan.ply.
Review and save all files.
Typical ICP-MS parameters are listed in Table 7.
Table 7. ICP-MS settings.
Parameter

Setting

RF power

1100-1400 W

Nebulizer gas flow rate

0.67 – 0.99 L/min

Sweeps/reading

30

Readings/replicate

1

Replicates

3

Autolens

On

Detector mode

Dual

Measurement units

Counts per second (cps)

Blank subtraction

After internal standard

Curve type

Simple linear

Dwell time

50 ms

Sample units

µg/L

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SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 19 of 27

Load prepared standards, specimens, samples and QC materials into the
Autosampler. Autosampler position numbers are dependent on the type of
autosampler being used.
For a CETAC ASX 500 series autosampler (tray B), calibration solutions 1–6 will be
in positions 11–16, the urine blank will be in position 17 and the reagent blanks will
be placed in positions 18-20.
Place start QCs and urine specimens in order from position 21. The run will end
with the end normal and elevated QC materials.
In the sample window, enter the Autosampler locations in ascending order and
input sample name, analysis method file, and peristaltic pump speeds (Table 8).
For the first sample (i.e., position 17 Urine Blank), select “Run Standards and
Sample” in the measurement action field. For all further samples select “Run
Sample”.
Table 8. Pump speed and duration for sample analysis and sample rinse-out.
Action

Pump Speed (rpm)

Duration (seconds)

Sample Flush

-18

90

Read Delay and Analysis

-18

30

Wash

-24

0-240

In the method window, check that the information entered for calibration standards
(i.e., concentration level, Autosampler position, pump speeds) are correct.
In the sample window, select the sample rows to be analyzed.
Select the “Analyze Batch” operation to start measuring the calibration standards
and samples.

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SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 20 of 27

Monitor the calibration curves to check linearity. If 2 or more calibration points are
not co-linear, the run can be stopped at this point for remedial action (i.e., remake
standards and re-start run).
If desired select “auto off” option to shut down plasma after completion of the
samples.
Allow analytical run to complete unless serious problems occur.
3.8

Process results

Examine the calibration curve. If a standard gives anomalous results (i.e., not colinear with the other standards), then that result may be rejected. Select and reject
the point in the calibration window of the Elan software. Reprocess the data to
produce a new results file. No more than 1 calibration point can be rejected.
Refer to the manufacturer’s guidelines for further instructions on Microsoft Excel™
(or similar) operations to process results.
Open the “Report Output” folder and locate file “mmddyy NYS Urine Hg.csv”.
Copy columns A&B from “mmddyy NYS Urine Hg.csv” to columns A&B in the “Raw
Data” worksheet in the “xxxxxx NYS Urine Hg.xls” results template. Save as the
appropriate “mmddyy NYS Urine Hg.xls” file.
The worksheets “Organized data”, “Blank Corrected data”, and “Data corrected for
dilution” will update automatically.
Select and copy the data in “Data corrected for dilution” worksheet.
In the “Paste Special_values, transpose” worksheet, select cell A2, select “paste
special” from edit menu, select “values” and “transpose” and click “OK”.
Select, copy, and paste QC results to QC table starting in column F.

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Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

3.9

Rev. No. # 9
Page 21 of 27

QC within limits?

The analyst and supervisor are responsible for review of QC results. For each QC
pool, the acceptable range is the mean ± 2 standard deviations of the results
determined in at least 20 characterization runs (±2Sm).
The following modified Westgard QC rules are applied to the data to establish
acceptance/rejection:
(a) If the QC means are within 2Sm limits, and individual results are within 2Si limits,
then accept the run (Sw = Within-run standard deviation).
(b) If 1 of the mean QC is outside the 2Sm limit, then reject the run if:
i. Extreme Outlier – Run mean is beyond the characterization mean ±
4Sm
ii. 1 3S Rule – Run mean is outside a 3Sm limit
iii. 2 2S Rule – Both run means are outside the same 2Sm limit
(c) If one of the 4 QC individual results is outside a 2Si limit, then reject the run if:
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.
Sm = Standard deviation of the run means.
Sw = Within-run standard deviation.
If the criteria defined above for evaluating the QC results are not satisfied, then the
results for all patient specimens analyzed during that run are invalid for reporting.
Repeat the analysis using freshly prepared calibration standards and QCs.
3.10

Analyte above repeat threshold?

Refer to the Third National Report on Human Exposure to Environmental
Chemicals for selected percentiles for concentrations of National Health and
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Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 22 of 27

Nutrition Examination Survey (NHANES) elements in urine (6). Table 9 includes
the 95th percentile concentration for Hg in urine for the U.S. population from the
survey years 2001-2002.
If the concentration of Hg is greater than the repeat upper boundary concentration
listed in Table 9, perform a confirmatory analysis. An additional QC material with
similarly high levels of Hg can be included in the repeat analysis if necessary. The
repeat threshold may be changed based on results from research studies to reflect
the 99th percentile of the study population or at the discretion of the Laboratory of
Inorganic and Nuclear Chemistry Chief.
NOTE: If a specimen or sample is believed to have a Hg concentration above the
repeat threshold, then two identical runs may be performed with independent
calibration curves.
If the repeats are in agreement with 4 standard deviations of the long-term
precision of the method as established from the QC plots, then report the mean
value of the repeats (i.e. the repeats lie within 2 standard deviations of the mean).
If the repeats do not meet this criteria perform a third analysis. If agreement is
achieved between 2 of the three analyses, report that mean. If agreement is not
achieved, consult with the Laboratory of Inorganic and Nuclear Chemistry Chief.
Table 9. Analyte reference ranges, and repeat upper boundaries.
Analyte

LODa
(µg/L)

Geometric
Meanb
(µg/L)

95th Percentileb
(µg/L)

Repeat
Thresholdc
(µg/L)

Call
Valued
(µg/L)

Hg
0.09
0.606
3.99
10
20
a
Limit of detection (LOD), calculated according to Clinical Laboratory Standards of
Practice (CLEP), Trace Elements Standard 1, TE1 as 3 times the standard
deviation of a blank or low concentration sample.
b
Third National Report on Human Exposure to Environmental Chemicals, Centers
for Disease Control and Prevention (July 2005), survey years 2001-2002.
c
Set at 10µg/L below the required report value to NYS DOH Heavy Metals Registry
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Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 23 of 27

for elevated reportable metals in urine.
d
Reportable Hg concentration to NYS DOH Heavy Metals Registry as provided in
10NYCRR, Sections 22.6 and 22.7.
3.11

Reporting and archiving

This method is currently fully validated for Hg. Hg can be reported for identifiable
patient specimens, mandatory PT and EQA.
Urine Hg values are reportable in the range between the method limit of detection
(MDL), and the highest calibration standard. If a specimen has a concentration
higher than the highest calibration standard, it can be reported if
(1) the value is within the periodically assessed extended calibration range and (2)
a internal or external QC material with similar concentration is analyzed and within
acceptable limits. Accuracy for results above the highest calibration standard may
also be demonstrated by satisfactory performance in PT and EQA samples at
concentrations above 40 µg/L, the highest Hg standard.
The LOD is calculated according to Clinical Laboratory Standards of Practice
(CLEP), Trace Elements Standard 1, TE1 as 3 times the standard deviation of a
blank or low concentration sample for a minimum of 10 independent runs.
The extended calibration range is the maximum linear range of the method
established annually by preparing additional calibration solutions with high analyte
concentrations.
Documentation of the current LOD and extended calibration range is kept with
these procedures.
Results should be reported using the “Internal Results Sheet Urine Hg.xls”
template.
Note: This form automatically calculates the combined uncertainty of the method
and indicates whether repeats are within acceptable limits.
The uncertainty of the results of sample measurements made by this method
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Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 24 of 27

should be calculated in a manner consistent with the ISO Guidelines for the
expression of uncertainty in measurement (GUM). These guidelines are based on
the principle that uncertainty should be expressed as a standard deviation or
expanded uncertainty that provides the best estimate of all possible sources of
uncertainty in the result. Potential sources of uncertainty in this method are listed
in Table 10. It should be noted that many of these component affect multiple
stages in the method.
Table 10. Uncertainty components for method
Component

Description

Calibration stock solutions

Manufacturers stated uncertainty in
element concentration

Base urine

Uncertainty in the element
concentration

Blanks (reagent and calibration)

Uncertainty in the element
concentration

Pipetting

Uncertainty in the volume dispensed
by pipettes

Dilution into volumetric flasks

Uncertainty in final volume of
intermediate standard solutions

Digiflex dispense/dilute

Uncertainty in the Digiflex procedure,
including uncertainty in dispensed
volume and in diluted volume

Internal standard

Uncertainty in the amount of internal
standard added to samples and
solutions

ICP-MS signal for analyte/internal
standard

Uncertainty in the recorded ICP-MS
signal

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Laboratory of Inorganic and Nuclear Chemistry

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Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 25 of 27

Calibration curve

Uncertainty in the linear regression

Method bias

Uncertainty of any bias in the method

Operator skill

Different operators may perform to
different standards

A typical approach to evaluating uncertainty is to quantify the uncertainty of each
individual component and combine those uncertainties into a ‘combined
uncertainty’ based on the measurement equation. In the case of an externally
calibrated method with multiple standards, defining a single measurement equation
and combing individual uncertainty components is extremely complex.
The uncertainty for this method is thus calculated from (i) the best estimate of the
long-term precision of the method and (ii) the best estimate of method bias.
The best estimate of the long-term precision ( u p ) of the method (i) is gained from
the long term QC data. It is reasoned that the standard deviation of multiple
measurements performed over a long period of time accounts for all possible
variation in the individual uncertainty components.
!
The best estimate of method bias ( ub ), is obtained from repeated measurements
over time of certified reference materials for trace metals in urine (e.g., NIST SRM
2670a or similar). No systematic bias has been established for this method. The
bias uncertainty is thus the uncertainty in the certified values quoted on the
! of certified values for a particular element, the
certificate. In the absence
uncertainty in reference values may be used. In the absence of reference values
for a particular element, scientific judgment can be used to assign an uncertainty.
The combined uncertainty uc is thus calculated as:

uc = u 2p + ub2
END

!

!
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Laboratory of Inorganic and Nuclear Chemistry

SOP: Mercury in Urine

Wadsworth Center, NY State Dept. of Health

Doc. DOH-LINC-412

Division of Environmental Health Sciences

Date: 12/13/10

Rev. No. # 9
Page 26 of 27

REFERENCES
1.
Centers for Disease Control and Prevention. Laboratory Procedure
Manual, Mercury in Urine by Flow Injection Cold Vapor Atomic Absorption
(CVAA). NHANES 2001-2002, August 22, 2001.
2.
Rogers HS, Jeffery N, Kieszak S, Fritz P, Spliethoff H, Palmer CD,
Parsons PJ, Kass DE, Caldwell K, Eadon G and others. 2008. Mercury
exposure in young children living in New York City. Journal of Urban
Health;85(1):39-51.
3.
McKelvey W, Jeffery N, Clark N, Kass D., and Parsons PJ. 2010.
Population-Based Inorganic Mercury Biomonitoring and the Identification of Skin
Care Products as a Source of Exposure in New York City. Environ Health
Perspect. (in press).
4.
Parsons PJ, Palmer CD, Caldwell KL, and Jones RL. 2005. Determination of
total mercury in urine by inductively coupled plasma-mass spectrometry (ICP-MS).
Special Publication-Royal Society of Chemistry (301) Plasma Source Mass
Spectrometry: 59-71.
5.
Minnich MG, Miller DC, and Parsons PJ. 2008. Determination of As, Cd,
Pb, and Hg in urine using inductively coupled plasma mass spectrometry with
the direct injection high efficiency nebulizer. Spectrochimica Acta Part B: Atomic
Spectroscopy;63(2):389-395.
6.
Centers for Disease Control and Prevention, National Center for
Environmental Health, NCEH Pub. No. 05-0570, July 2005.

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