Laboratory Standard Operating Procedures

11.LabSOPcomb_metal_PCB_PFC_PAH.pdf

Biomonitoring of Great Lakes Populations Program III

Laboratory Standard Operating Procedures

OMB: 0923-0056

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Attachment 11
Laboratory Standard Operating Procedures
“Biomonitoring of Urban Anglers in Milwaukee's Area of Concern”
Biomonitoring of Great Lakes Populations Program III

Procedure Ghange Log
Procedure: Blood multi-element analvsis bv ICP-DRC-MS
DLS Method Gode: 3016.8-05
Date

Changes Made

Rev'd
By
(lnitials)

Date
Rev'd

4t1t2011

1UB and 2UB for Mn changed from 15 to 25
uq/L and from 30 to 50 uq/L, respectivelv.
Limit Rep Delta for Mn changed from 1.0 to
2.0.
Clarified matrix
internal standard
intermediate from "dilute HNO3' to "1o/o vlv

JHJS

4t1t2011

JHJS

4t1t2011

JHJS

7128t2011

Changed BMN 1UB (25 ug/L to 20 ug/L) and
2UB (50u9/L
35 ug/L). Supporting
references added.
Added comment to CV standard tables
reqardinq use of qravimetric preparation.
Sample Diluent Preparation: Triton X-100
percentage correction (typo)
DRC Stability Test Preparation: alternate
preparation procedure using the intermediate
workinq calibrators
Preparation of Samples for Analysis: changed
the Blood Blank name from "BldBlkChk" to "WB
Blank" and WB Blank2"
Contaminated Blanks: added clarification on
procedure to follow in the event of
contaminated blanks

JHJS

8t9t2011

JHJS

10t7t2011

napl

JHJS

3t20t2012

napl

JHJS

3t20t2012

napl

JHJS

3t20t2012

napl

JHJS

3t20t2012

3t2ot2o12

Linear Calibration Curves: clarification

napl

JHJS

3t20t2012

droppinq points
Appendix B, Table 1:
Added description for method file names

napl

JHJS

3t20t2012

napl

JHJS

3t20t2012

napl

JHJS

3t20t2012

napl

JHJS

3t20t2012

napl

JHJS

3t20t2012

4t1t2011
7t28t2011

HNOs".

8t9t2011

10t7t2011

3t20t2012
3t20t2012

3t20t2012

Method Parameters: updated sample flush
3t20t2012

3t20t2012

3t20t2012
3t20t2012

times and sample wash times
Autosampler Locations: Aq Blank location
Appendix B, Table 3:
Clarification of stock standard preparation
Appendix B, Table 10:
Typical sample/batch window: changed
autosampler location to reflect current
positions
Updated screenshots in Appendix B, Figures
1e, 1f , and 2d
Created Appendix C for "help sheets

3t20t2012

5t03t2012
5t10t2012

9t10t2012
1t22t2013

1t22t2013

Method Procedures:
Types of Quality Control: Removed reference
to blind QC
Sample Diluent Preparation: Changed
concentration of TMAH from 0.25o/o to 0.4o/o
Added Appendix A, Experiment 6: Validated
extra dilutions up to 20x. Updated Reportable
Range and Table 6 (descriptions of sample
preparation).
Sample Rinse Preparation: Changed
concentration of TMAH from 0.25o/o to 0A%
Extended calibration range S0-S8 adding a
third bench QC level. Changed to weighted
linear reqression and dual detector mode.
Clarified and updated handling elevated
concentrations, Tables 8 - 11, Sections 7
11 and references. Added Fiqures 1 and 4
Added description of solutions for DRC and
dual detector optimizations.
Updated reference ranqe Tables
Added detail of potentíal MoOz interference

napl

JHJS

3120t2012

napl

JHJS

5103t2012

EMU2

JHJS

5t10t2012

napl

JHJS

9t10t2012

JHJS

KLCT

1t22t2013

JHJS

KLCT

1t22t2013

JHJS

KLCT

1t22t2013

JHJS
JHJS

KLCT
KLCT

1t22t2013
1t22t2013

Updated action levels
Updated evaluating calibration curves
language
Updated help sheets re: calibration std prep

JHJS

'1t2212013

napl

KLCT
JHJS

3t20t2013

napl

JHJS

4t16t2013

Replaced references to urine with references
to blood in Table of Figures, Section 7.c.ä,
and Section 12. Updated reference from
"Section 10a" to "Section 1 1a" in Section 10a.
References to Tables 5, 10, and 1 1 updated.
Clarified method details (esp. references to
urine methods and solutions preparations).
Bldblkchk to be made with S0 instead of

JHJS

KlcT

512012013

JHJS

KlcT

9t15t2014

KLC

12t9t2015

KLC

12t9t2015

KLC

12t9t2015

KLC

12t9t2015

1t22t2013
1t22t2013
1t22t2013

On 130Te

1t22t2013
3t20t2013
4116120',13

511512013

9t15t2014

water.
12t08t2015 Changed method name from Blood Metals
Panel 3 (BMP3) by ICP-DRC-MS to Blood
multi-element analysis by ICP-DRC-MS
12t08t2015 Updated Title page to new DLS template

12t08t2015 Updated Section 3 to specify not to freeze
blood in blood collection tubes (esp. glass)
12t08t2015 Clarified comments, updated examples,
corrected typos: lncreased use of active
voice (eliminated 'may' and 'shall'). Clarified
comments in Tables 8 and 9. Renamed
second "Figure 29" to "Figure 2h". Correct
table references in Section 10.

Minor equipment updates: References to
Digiflex pipette changed to Hamilton Microlab
625 benchtop automatic pipette and updated
Table 8 volumes. Updated regulator part
numbers for methane and oxygen
compressed qases.
12t08t2015 Updated instructions related to very elevated
results. Set criteria to confirm proper washout
after an elevated sample to t 3SD limits of
low bench QC wash check (Section 8.b.iv).
Set criteria to confirm samples potentially
affected by insufficient washout to +10o/o or
t3SD of the low bench QC, whichever is
greater (Section 8.b.vii.2.a). Updated
extended wash details in Table 1. Added
highest validated washout concentrations to
Table 9. Updated Figure 4 (Flow Chart for
handlinq an elevated result).
12t08t2015 Added 2011-2012 NHANES reference level
data to Table 10 and replaced statement
about blood lead >10 ¡rg/dl with statement
about 5 uq/dl reference level.
12t08t2015 Updated record retention in section Section
9.c to match DLS policv (3 years to 2 vears)
03t02t2016 Left justified text. Updated references.
Removed "(esp. glass)" regarding do not
freeze blood in blood tubes. Referenced
highest calibrator and max extra dilution
tables in reportable range section. Updated
description of disinfectant. Changed "working
cal ibration sta nda rd " to "worki n g ca ibrator"
throughout. Updated references to high purity
water.
12t08t2015

KLC

12t9t2015

KLC

12t9t2015

KLC

12t9t2015

KLC

12t9t2015

RLJ

03t02t2016

Laboratory Procedu re Man ual
Environmenlal Heallh

Anarytes:

Gadmiuffi, Lead, Manganese,
Mercury, and Selenium

Matrix: Whole Blood
Method:

blood multi-element analysis by ICP-DRC-MS

Method No: DLS 3016.8-05
As pefformed

by: lnorganic and Radiation Analytical Toxicology Branch
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]

James L. Pirkle, M.D., Ph.D.
Director, Division of Laboratory Sciences

lmportant lnformation for Users
The Centers for Disease Control and Prevention (CDC) periodically refines these
laboratory methods. lt 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 multi-element analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page

I of94

Table of Contents

Gross reference to DLS CLIA and Policy and Procedures . .

lndex of tables

lndex of figures

1) Clinical relevance & summary of test principle

a. Clinical relevance
b. Test principle .

6
10

2) Limitations of method; interfering substances and conditions

a.
b.

lnterferences addressed by this method . . .

Limitations of method (interferences remaining in method)

3) Procedures for collecting, storing, and handling specimens; criteria for
specimen rejection

a. Procedures tor collectrng, stoflng, ancl handlrng spectmens
b. Criteria for specimen rejection . . .
c. Transfer or referral of specimens; procedures for specimen accountability
.

13
14

andtracking...

4) Safety precautions
a. General safety

o.
I

vvasre qrsposar

l^,

¡:

5) lnstrument & material sources
a. Sources for ICP-MS instrumentation
b. Sources for ICP-MS parts and consumables

Sources for ICP-MS maintenance equipment and supplies . .

d.
e.

Sources for general laboratory equipment and consumables .
Sources for chemicals, gases, and regulators

16
16

22
.

23
24

6) Preparation of reagents and materials

a. lnternal standard intermediate mixture
b. lntermediate Triton X-100 solution
c. Sample diluent and carrier . .
d. ICP-MS rinse solution .
e. Standards, calibrators, base blood and QC
.

26
27
27
28
29

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8-05

Page 2 oÍ 94

Optimization solutions. . .

7) Analytical instrumentation setup

a. lnstrumentation and equipment setup
b. lnstrument and method parameters (see Table 1) . . . . .
8)

The

a.
b.

Bench QC, reference materials and calibration verification

Perform, evaluate and report a run

run: quality, execution, evaluation, and reporting

9) Routine equipment maintenance and data backups
a. Equipment maintenance .
b. Parameter optimizations

Data backup . .

47
47
47

0) Reporting thresholds

a. Reportable range
b. Reference ranges (normal values)
c. Action levels

48
48
48

111 Method calculations

a. Method limit of detection (LOD)
b. Method limit of quantitation (LOQ)
c. QC limits

48
48
48

12) Alternate methods for performing test and storing

specimens if test system fails

Appendix A (ruggedness test results)

Appendix B (tables and figures)

Appendix C (help sheets)

References

blood multi-element analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8-05

Page 3 of 94

Cross reference to DLS CLIA and Polic van dP rocedures nolicv
1

2
3

I
I
10
44

Summary of Test Principle and Clinical Relevance
l) 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 lnstrumentation
5) a. i i¡ ¡¡¡ b. 6) a. b. c. d. e.7) a. b. c. d. 8) c- i ii
Calibration and Calibration Verification Procedures
8) ¡¡
Procedure Operating l nstructions; Calculations; l nterpretation of Results

8)b.¡ii ivvx

Reportable Range ot Results
9) a.
Quality Control (QC) Procedures

8)a.i
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Limitations of Method; lnterfering Substances and Conditions

2la.b
13
14
15
16
17
18

Reference Ranges (Normal Values)
s) b.
Critical Call Results ("Panic Values")
9) c.
Specimen Storage and Handling During Testing
8) b. ¡¡¡
Alternate Methods for Performing Test or Storing Specimens lf Test System Fails
a al
I t,
Test Result Reporting System; Protocol for Reporting Critical Calls (lf Applicable)
9) c.
Transfer or Referral of Specimens; Procedures for Specimen Accountability and
Tracking
3) c.
References

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page 4 oi 94

lndex of tables
Table

lnstrument and method parameters . . . .

Potential Emergency Response Modifications

2. Suggested analyte concentrations for base blood . .
Table 3. Stock standard concentrations
Table 4. Preparation of intermediate stock standard
Table 5. Preparation of intermediate working standards
Table 6. Acceptable ways to perform two consecutive analytical runs, bracketing
Table

63
63

64
65

A typical SAMPLE/BATCH window

Table

Preparation of samples, working calibrators, and QC materials for
analysis

with bench quality control samples

Table

Boundary concentrations (1U8, 2UB, and Lim Rep Delta)

66
67

Table 10. Reference ranges for blood concentrations (from the Fourth National
Report on Exposure to Environmental Chemicals)

Table 11. References concentrations from published literature for
blood manganese and blood selenium

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lndex of figures
Figure

Configuration of tubing and devices for liquid handling

a.
Figure

ESI SC4DX-FAST autosampler. .

ICP-MS method screen shots (3016, 5 elements)

a. Timing page
b. Processing page.

Equations page.

d. Calibration page.
e. Sampling page (AqBlank

method)

Sampling page (BldBlank method)

Report page

h. QC/samplepage....
Figure

Figure

ESI SC4 FAST autosampler screen shots

a. Main page
b. 5x12 rack setup

c. 50mL tube rack setup.

d.
e.

Configurq autosampler page . .

Communication page

FAST method control page . . .

Rinse station rack setup

83
.

Flow Chart for handling an elevated result

84
85

blood multi-element analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8-05
1

Page 6 of 94

Clinical relevance & summary of test principle

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.9., air, soil, water, dust, or food) combined, rather than
the amount that gets into them. The laboratory method described here is a multielement 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 is 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 chemicalform. In the general population, total blood
mercury is due mostly to the dietary intake of organic forms which are formed
through microbíal 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.9. dental
amalgams or occupational exposures) is particularly reflected in urine excretion
rather than blood. Psychic and emotional disturbances are the initial signs of
chronic intoxícation by elemental mercury vapors or salts. Those exposed are at
increased risk for parasthesia, neuralgias, renal disease, digestive disturbances,
and ocular lesions [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,

blood multi-element analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

7 of94

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. Blood mercury reference
ranges for the U.S. population are listed in Table 10 in Appendix B.

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.9. stained glass), commercial products (e.9. batteries, lead-containing jewelry),
home remedy medicines containing lead compounds and non-Western
cosmetics. Soil contains 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
<5 ¡rg/dl [6-10]. Acute, elevated lead exposure is associated with anorexia,
dyspepsia, and constipation followed by diffuse paroxysmal abdominal pain.
When lead exposure is high, particularly in children, the person is at increased
risk for encephalopath.v [11]. The alkyl lead species are highly toxic to the central
nervous system[12]. The primary screening method for lead exposure is blood
lead, which primarily reflects recent exposures (excretory half-life in blood is
approximately 30 days)[13]. Lead in blood is primarily (99%) in the red blood
cells. Blood lead reference ranges for the U.S. population are listed in Table 10
in Appendix B. The CDC now uses a reference level of 5 pg/dl to identify
children with blood lead levels that are much higher than most children's levels.
This new level is based on the U.S. population of children ages 1-5 years who
are in the highest2.So/o of children when tested for lead in their blood. This
reference value is based on the 97.5th percentile of the National Health and
Nutrition Examination Survey (NHANES)'s blood lead distribution in children.
CDC will update the reference value every four years using the two most recent
NHANES surveys [14].
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. ln 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. ln 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

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8-05

Page

of 94

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 - a ¡rg/L[15]. Newborn babies are
practically free of Cd[16]. 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[11]. Principal symptoms reported were
respiratory distress due to chemical pneumonitis and edema. lt has been
estimated that 8 hrs. exposure to 5 g Cd/m3 will be lethal[11]. lngestion of high
amounts of Cd puts a person at increased risk 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 princípally indicative of recent
exposure(s) to cadmium rather than whole-body burdens 117-201. Urine
cadmium levels primarily reflect total body burden of cadmium, although urine
levels do respond somewhat to recent exposure[21]. Blood cadmium reference
ranges for the U.S. popufation are listed in Table 10 in Appendix B.
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 1221.l,Aanganese is also a known
neurotoxin but little information exists about levels of manganese that cause
toxicity. Symptoms of manganese toxicity arc similar to Parkinson's Disease and
can also include disorientation, memory impairment, anxiety and compulsive
behavior [23]. There is much concern for the levels of manganese in humans
whom are occupationally exposed to it [24-30]. Recently, there are growing
concerns over exposure due to contamination of drinking water with manganese
[31-33] and as a result of methylcyclopentadienyl mangangese tricarbonyl (MMT)
used as an anti-knocking additive in gasoline [34-40]. Populations suffering from
iron deficiencies are at an increased risk to manganese toxicity because iron
deficiency can result in an accumulation of manganese in the central nervous
system [37]. 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 [41]. 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 [25, 41-431. Manganese in blood or urine are
useful in detecting groups with above-average current exposure, but
measurements of manganese in these body fluíds in individuals are sometimes
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

blood multi-element analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8-05

9of90

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 144,451. Thus, levels of manganese in blood
or urine are not expected to be the most sensitive indicators of exposure [46].
Typical blood manganese concentrations in humans which have been reported in
the literature are listed in Table 11 of Appendix B.
Selenium is an essential element that is required to maintaín good health but
both selenium deficiency and excessive levels of selenium are associated with
several disorders[47, 48J. 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. ln 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 increase risk of chronic diseases such as cancer and
heart disease[48, 49]. Other selenoproteins help regulate thyroid function and

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the U.S. but is seen in other countries where soil concentration of selenium is
low[54]. There is evidence that selenium deficiency increases the risk of a form of
heart disease, hypothyroidism, and a weakened immune system[55, 56]. There is

also evidence that seleníum 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[57]. 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[47]. Selenium can be detected ín 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[47]. Typical
blood selenium concentrations in humans which have been reported in the
literature are listed in Table 11 of Appendix B.

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 (Hg), manganese (Mn) and selenium
(Se) in whole human blood. Use this method to screen blood when people are
suspected to be acutely exposed to these elements or to evaluate chronic
environmental or other non-occupational exposure.

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8-05

Page

l0 of 94

Test orinci ple
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.9. Pb) are known to be associated
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. lf 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). Clotted samples are not
analyzed by this method due to the inhomogeneity concerns (i.e. all results for
the sample are processed as "not reportable").
Dilution of the blood in the sample preparation step prior to analysis is a símple
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, 0A% vlv)
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 (0.01%) 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 (lCP) ionization source. The liquid diluted blood sample is
forced through a nebulizer which converts the bulk liquid into small droplets in an

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Metñod Gode: 3016.8-05

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of94

argon aerosol. The smaller droplets from the aerosol are selectively passed
through the spray chamber by a flowing argon stream into the lCP. 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
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-s 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. ln 'vented' (or'standard')
mode the cell is not pressurized and ions pass through the cell to the quadrupole
mass filter unaffected. ln '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. ln 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 signai. ln this methoci, the instrument is operateci in DRC mode
when analyzing for manganese, mercury, and selenium. For selenium, the DRC
is pressurized with methane gas (CH+, 99.999%) which reduces the signal from
40Arz* while allowing the 80Se* ions to pass relatively unaffected through the DRC
on toward the analytical quadrupole and detector. Manganese and mercury arc
both measured when the DRC is pressurized with oxygen gas (Oz, 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 iÎ
different gas flows were used for the two analytes. The oxygen reduces the ion
signalfrom several interfering ions (37C118O+, 40Ar15N+, 38Ar1601H+, 54FerH*) while
allowing the Mn* ion stream to pass relatively unaffected through the DRC on
toward the analytical quadrupole and detector. ln 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

blood multi-element analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page 12 ol 94

based on the method by Lutz et al [58]. The DRC portions of the method are
based on work published by Tanner et al. [59, 60].

2l Limitations
a.

of Method; lnterfering Substances and Conditions

lnterferences addressed bv this method
i.

40Ar2+

is a polyatomic ion formed in the plasma as a result of a
reaction between the plasma gas (Ar) and itself. The dynamic reaction cell of
the ICP-MS is used to reduce ion signals from polyatomic ions via ionmolecule reaction chemistry [60, 61]. ln the reaction cell, methane (CH+)
molecules react with 40412+ ions through a charge transfer reaction. The
products of the reaction are aoAr* (ion at a different mass) and aoAr (neutral).
The background ion signal atmlz 80 is reduced by six orders of magnitude
because of this reaction.

DRC-MS:

ii. Reduction of aroon nitride (0Ar15 N*). arqon hvdroxide l38Ar1601H+)
interference on ma noa nese l55Mn) usino ICP_DRC_MS. 40Ar15N* and
38A11601H* are polyatomic ions formed in the plasma as a result of reactions
between the plasma gas (Ar) and atmospheric gases (Nz, Oz) or the solvent
(HzO). The dynamic reaction cell of the ICP-MS is used to reduce ion signals
from polyatomic ions via ion-molecule reaction chemistry [60, 61]. In the
reaction cell, oxygen molecules react with 40Ar15N* and 38Ar1601H* 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). ln either
case, attenuation of the background ion signal atmlz 55 occurs.
iii.

usinq ICP-DRC-MS' 37Cl18O+, 3sK160+, 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 (HzO). Due to the high concentrations of
Cl, K, and Fe in the blood matrix the resulting ion signals of 37Cl18O+, 3eK160+,
and 5aFe1H* interfere with the measurement of 55Mn* atmlz 55. The dynamic
reaction cell of the ICP-MS is used to reduce ion signals from polyatomic ions
via ion-molecule reaction chemistry [60, 61]. ln the reaction cell, oxygen
molecules react with 37Cl18O+, 3eK160+, 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). ln either case, attenuation of the
background ion signal atmlz 55 occurs.

blood multi-element analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8.05

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l3 of 94

Limitations of method (interferences remaining in method)

i. MoOz interference on 130Te: Molybdenum will combine with oxygen in the DRC
conditions used in this method for Hg analysis to form a polyatomic ion,
sBMo1602+, which interferes with the measurement of the internal standard
130Te+. lncreased signal atmlz 130 (due to measuring both 130Te+ and
e8Mo16O2+) results in an erroneously low net intensity for Hg (net intensity
=
measured intensity for analyte isotope / measured intensity for internal
standard isotope). lf this interference occurs during the measurement of the
calibration standards (i.e. a multi-element calibration stock standard includes
high levels of Mo) it can result in a positive bias for observed mercury
concentrations as a consequence of a nonlinear calibration curve having an
artificially low slope. lf this interference occurs during the measurement of an
unknown sample, the reduced net intensity observed can result in reporting an
erroneously low Hg result. This interference has been verified to be of concern
(>5o/o effect negative bias) at blood molybdenum concentrations greater than
15 ug/L. However, typical levels of molybdenum in whole blood (0.2- 4.6 ug/L
[62, 63]) are below this. Also, levels of molybdenum in whole blood after acute
exposures have been observed to be s15 ¡rg/L [62]. Molybdenum
concentrations below 5 ¡rg/mL in stock calibration standard solutions do not
produce an observable intefference.

3) Procedures for collecting, storing, and handling specimens; criteria for
specimen rejection; specimen accountability and tracking

Procedures for collectinq. storing. and handling specimens: Specimen handling
conditions, special requirements, and procedures for collection and transport are
discussed in the Division of Laboratory Science's (DLS) Policies and Procedures
Manual [64]. In general,

i. No fasting or special diets are required before collection of blood
ii. Specimen type

- whole blood

iii o ptimal amount

of specimen is 1* mL. Request a minimum volume of 0.25
m L. Volume for one analytical measurement is 0.05 mL.

iv. Verify sample collection devices and containers are free of significant
contamination ("pre-screened") before use.

v. Draw the blood through a stainless steel needle into a pre-screened
vacutainer.

vi. Do not freeze blood in blood collection tubes due to risk the tubes cracking
Transfer to plastic, pre-screened cryovials before freezing.

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page 14o194

vii. Once received, store blood collection tubes at refrigerated temperatures (2-8
oC). Transfer to plastic, pre-screened cryovials before freezing. Specimen
stability has been demonstrated for over 1 year at < -20 "C.

Criteria for specimen rejection: The criteria for an unacceptable specimen
include:

i. Contamination: lmproper 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 0.25 mL. Volume for one
analytical measurement is 0.05 mL.
ln all cases, request a second blood specimen
c.

trackinq: 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.9., case lD numbers) in order to safeguard confidentiality. Access
to personal identifiers for samples will be limited to the medical supervisor or
project coordinator (e.9. non-CDC personnel).

Safety precautions

General safetv
i. Observe all safety regulations as detailed in the Laboratory Safety Manual and
the Chemical Hygiene Plan. 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. Take special care when handling and dispensing bases and concentrated
acids. Use additional personal protective equipment which protects face, neck,
and front of body. lf 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.

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8-05

Page 15 of 94

v. Use secondary containment for containers holding biological or corrosive
liquids.

vi. The use of the foot pedal on the benchtop automatic pipette 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 benchtop
automatic pipette.
vii. There are many potential hazards on an operating ICP-MS instrument
including ultraviolet radiation, high voltages, radio-frequency radiation, and
high temperatures. This information is detailed in the ICP-MS System Safety
Manual.
viii. Transport and store compressed gas cylinders with proper securing
harnesses. For compressed oxygen gas, use regulators which are oil-free and
are equípped with a flash arrestor.
lx. WIpe down all work surtaces at the end ot the day wrth cltsrntectant.
Disinfectant may be either daily remake of diluted bleach (1 part household
bleach containing 5.25o/o sodium hypochlorite + 9 parts water) or an equivalent
disinfectant

Waste disposal:
i. Autoclavinq: All diluted biological specimens, original biological specimens

being disposed, or consumables which come into contact with biological
specimens (even diluted or aerosolized). Use sharps containers or special
autoclave pans for broken glass I quariz or items which puncture autoclave
bags (e.9. pipette tips).
ii. Other liquid waste
1. Waste discarded down sink: Only non-corrosive liquid waste (EPA defines
as pH >2 and pH<12.5,40CF R 5261 .22) from the ICP-MS instrument can
be discarded at the sink. Flush the sink with copious amounts of water.
ô
¿-

ê,.L*i+I ilt -^^,,^^+
\JlltJ¡
I CLlUCÐt

for hazardous waste removal of all other liquid waste generated in the CDC
laboratory for this method.

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page 16 of 94

5) lnstrument & material sources

Sources for ICP-MS instrumentation

i. ICP-MS: lnductively Coupled Plasma Mass Spectrometer with Dynamic
Reaction Cell Technology (ELAN@ DRC ll) (PerkinElmer Norwalk, CT,
www. perki nel mer. com ).

ii. Recirculatinq chiller / heat exchanqer for ICP-MS: Refrigerated chiller
(PolyScience 6105PE) or heat exchanger (PolyScience 3370) (PerkinElmer
Nonryalk, CT, vvww. perkinelmer. com)
iii. Autosampler: ESI SC4-DX autosampler (Elemental Scientific lnc., Omaha, NE)
or equivalent.

iv. Computer: Computer controller provided or recommended by ICP-MS
manufacturer is recommended to ensure proper communication between
computer and ICP-MS. Recommend 1-2 Gb RAM and secondary internal hard
disk for nightly backups (if network backups are not possible).

v. FAST sample introduction system (optional): Standard peristaltic pump on
ICP-MS replaced by DX|-FAST micro-peristaltic pump / FAST actuator and
valve combination unit. Like part # DXI-54-P4-F6. lf DX|-FAST upgrade on
ICP-MS is not used, a separate FAST actuator (built-in option on ESI SC4-DX
autosampler or stand-alone FAST actuator) will be necessary to complete the
FAST sample introduction system.

Sources for ICP-MS parts & consumables

NOTE: The minimum number of spares recommended before reordering (if
owning one instrument) are listed as "# Spares = X amount" in the descriptions
below.

Adapter, PEEK: Securely connects 1.6mm O.D. PFA tubing to 0.03" l.D.
peristaltic tubing. Composed of three PEEK parts.

1. Female nut for 1.6mm O.D. (1/16") tubing. Like pad P-420 (Upchurch
Scientific, Oak Harbor, WA, www.upchurch.com).

2. PEEKferrule. Like part P-260x (1Opk SuperFlangeless ferrule, Upchurch
Scientifíc, Oak Harbor, WA, www.uochurch.com

3. Conical Adapter Body. Like part P-692 (Upchurch Scientific, Oak Harbor,
WA, wr¡¡w.uochurch.com )

ii. Bottles (for rinse solution): Four liter screw-cap polypropylene container with
built-in luer connections (2) designed for use with FAST sample introduction

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page 17 of 94

system (like catalog# SC-0305-1, Elemental Scientific lnc., Omaha, NE.,
e leme ntalscientific. com).

www.

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 #7BE25126, Lab Safety Supply, Janesville, Wl, www.lss.com) with cap assembly
like part # N0690271 (PerkinElmer, Nonrualk, CT, www.perkinelmer.com) with
tubing connections built into the cap for addition of liquid waste.

iv.

: Only PerkinElmer part #
WE01-6558 (PerkinElmer Nonrualk, CT, www.perkinelmer.com) is approved for
use by PerkinElmer. # Spares = 6.

v. Cones: Platinum or Nickel cones have been used and tested to be
comparable in performance from either PerkinElmer or Spectron. Platinum
cones are more expensive, but will last longer, can be refurbished (often for
free by the manufacturer), and will frequently yield higher sensitivity.
1. Sampler (nickel/platinum): PerkinElmer part # WE021 140 lWE0278O2
(PerkinElmer Nonrualk, CT, www.perkinelmer.com). # Spares = 4.
2. Skimmer (nickel / platinum): PerkinElmer part #WE021137 1WE027803
(PerkinElmer Nonrualk, CT, www.perkinelmer.com). # Spares = 4.
vi. Connector (for tubinq): Use to connect 118" l.D. PVC tubing to 0.125" LD
noricialfi¡ hrrrrlrr írrhinn iica naÉ il?iâ,ît7iÃ íParkintrirnar ñnn¡rnik
trvrrrt
I t
v1-.iwww.perkinelmer.com) or equivalent. # Spares = 4.
vii. Detector. electron multiplier: Like part # N8125001 (PerkinElmer Norwalk, CT,
wmry.perkinelmer.com). # Spares = L
viii. FAST accessories
1. Valve: CTFE High-flow valve head for SC-FAST (uses %-28 fittings). Like
part # SC-0599-1010 (Elemental Scientific lnc., Omaha, NE.,
www. e lem e nta lscie ntific. com ).

2. Stator: CTFE Stator for 6 port SC-FAST high flow valve (%-28 fittings).
Like part # SC-0599-1010-01 (Elemental Scientific Inc., Omaha, NE.,
www. eleme ntalscie ntific. com).

3. Rotor: Composite rotor for 6 port SC-FAST high flow valve (%-28 fittings)
Like part # SC-0599-1010-05 (Elemental Scientific lnc., Omaha, NE.,
)

4. Sample Loop: 1 mL Teflon, white connector-nuts for high flow valve
head(%-29 fíttings). Like part # SC-0315-10 (Elemental Scientific lnc.
Omaha, NE., www. elementalscientif

ic.

com )

5. Pfebe, Autosampler: Teflon , carbon fiber support, 0.8mm i.d., blue marker,
114-28 fittings. Like part number SC-5037-3751 (Elemental Scientific lnc.,
Omaha, NE., un¡rw.elementalscientific.com). # Spares = 2.

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page 18 of 94

6. Probe. Carrier Solution: Teflon, carbon fiber support, 0.Smm i.d., orange
marker, 114-28 fittings. Like part number SC-5037-3501 (Elemental
Scientific Inc., Omaha, NE., www.elementalscientific.com). # Spares = 2.

7. Tubinq. 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 lnc., Omaha, NE.,
www. elementa lscientific. com ).

8. Tubinq, connects nebulizer to valve: See "Nebulizer, PolyPro-ST micro
flow"

ix. Hose, for connection to chiller: Push on hose. l.D. = y2", O.D. = T+". Use part
# PB-8 (per inch, Georgia Valve and Fitting, Atlanta, GA, www.swaqelok.com)
or equivalent. Do not normally need spare hose (unless moving instrument
into a new location).

x. Hose, for exhaust of ICP-MS: Available as part of ICP-MS installation kit from
Perkin Elmer (PerkinElmer Nonryalk, CT, www.oerkinelmer.com ). Available
direct from manufacturer as part # S-LP-10 air connector (Thermaflex,
Abbeville, SC, www.thermaflex.net), or equivalent. # Spares = 10 feet of 4"
diameter and 10 feet of 6" diameter hose.
xi. lniector, quartz with ball ioint: l.D. = 2.0 mm. PerkinElmer part # WE023948
(Perkin Elmer Norwa lk, CT, www. perkinelmer. com). Available d irect from
manufacturer as part # 400-30 (Precision Glass Blowing, Centennial, CO,
www.precisionqlassblowinq.com) or from various distributors. # Spares = 2.
xii. lon lens: PerkinElmer part # WE018034 (PerkinElmer Nonrualk, CT,
WWW
nelmer.com # Spares = 3.
xiii. Nebulizer: PolyPro-ST micro flow polypropylene nebulizer with external l14-28
threaded connector for liquid delivery, low pressure version or equivalent. Like
part # ES-4040-7010 (Elemental Scientific lnc., Omaha, NE.,
www.elementalscientific.com). # Spares =
Different nebulizers are
acceptable, however, the nebulizer gas flow rate, sample flush time, read delay
time, loop fill time, loop size, blood sample dilution preparation volume, and
sample-to-sample carry-over must be evaluated and optimized.

1. Gas connection:

a. Teflon tubinq: 4mm o.d.,2.4mm i.d. Teflon tubing (like part # ES2502, Elemental Scientific lnc., Omaha, NE.,
www.elementalscientific.com). # Spares = l.
b. Adapter kit: Plastic adapters to connect Teflon tubing (2.4 mm i.d) to
To" male Swagelok (compression) port on ICP-DRC-MS. Parts can
be obtained as components in a "gas fittings kit for microflow
nebulizer", kit like part # ES-2501-1000 (Elemental Scientific lnc.,
Omaha, NE., www.elementalscientific.com). # Spares = L

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8-05

Page

l9 of 94

2. Liquid connection: Connects nebulizer to port #3 of high flow FAST valve
head with green, 114- 28 fitting. Like part # SC-0317-0250 (Elemental
Scientific lnc., Omaha, NE., www.elementalscientific.com). # Spares = 2.
xiv. Nut: (for flanged connections of 1.59mm (1/16') o.d. PFA tubing) Flanged, for
1116" o.d. tubing,l14-28 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.
xv. Nut and ferrule set, 1/8" Swaqelok: Such as part # SS-2O0-NFSET (stainless
steel) or part # B-200-NFSET (brass) (Georgia Valve and Fitting, Atlanta, GA,
www.swaqelok.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" Swaqelok: Such as part # SS-4OO-NFSET (stainless
steel) or part # B-4O0-NFSET (brass) (Georgia Valve and Fitting, Atlanta, GA,
www.swaqelok.com) or equivalent. For part numbers listed here a quantity of
1 means 1 nut, 1 front ferrule, and 1 back ferrule. Spares = 20.
----:: rvril 1^- -^- --L:^-.
^:t tul luul¡lliltlJ fJuiltlJìt.
Ãvil.

1. Welch Directorr Gold For roughing pumps. Available direct from
manufacturer as part # 8995G-15 (1 gallon, Welch Rietschle Thomas,
Skokie ll r¡rnruw welnhvaeuum eomì or eouivalent # Snares = 4
.-r-

2. Fomblin Y14l5 fluid: PerkinElmer part # N8122265 (1 kg bottle,
PerkinElmer, Shelton, CT, www.perkinelmer.com) or equivalent. # Spares
=1 per instrument.

xviii. O-rinq: (for sampler cone) PerkinElmer paft # N8120511 (pkg. of 5,
PerkinElmer, Shelton, CT, www.perkinelmer.com) or equivalent. # Spares =
20 o-rings.
xix. O-rinq: (for skimmer cone) PerkinElmer part # N8120512 (pkg. of 5,
PerkinElmer, Shelton, CT, rnrurrw.perkinelmer.com) or equivalent. # Spares
20 o-rings.

xx. O-rinq: (for flanged connections of 1.59mm (1116") o.d. PFA tubing) Tefloncoated Viton o-ring, i.d. = 1/16", thickness = 1116", o.d. = 3116". Such as part #
V75-003 (O-rings West, Seattle, WA, wwur.orinqswest.com) or equivalent. #
Spares = 20.
xxi. O-rinq: (for injector support).
1, lnternal o-rinqs: lD = yo", OD = 3/8", thickness = 1116". 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, un¡rw.orínqswest.com). # Spares = 20.

2. External o-rings: lD = 3/8", OD = 112", thickness = 1116". Need 2 o-rings
for each injector support setup. PerkinElmer part # N8122009
(PerkinElmer, Shelton, CT, www.perkinelmer.com) or equivalent (such as

blood multi-element analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page 20 of 94

part # V75-012, O-rings West, Seattle, WA, www.orinqswest.com). #
Spares = 20.

xxii. O-rino lfor inside nebulizer oort on sta ndard PerkinElmer cvclonic quartz sÞrav
chamber for the E[,AN): Such as part # 120-56 (Precision Glass Blowing,
Centennial, CO, www. precisionqlassblowing.com). Additional o-rings can
sometimes be obtained free of charge or at reduced price when acquired while
purchasing spray chambers. # Spares = 20.
xxiii. O-rinq: (for inside of bayonet torch mount): Part # WEO17284 (PerkinElmer,
Shelton, CT, www.perkinelmer.com). Do not substitute. The PerkinElmer oring is specially metal impregnated to minimize RF leakage though the torch

mount. #Spares=2.
xxiv. Photon stop: PerkinElmer part # WE018278 (PerkinElmer, Shelton, CT,
wrnrw.perkinelmer.com). # Spares = L
xxv. Pluqs, quick chanqe for rouqhinq pump oil: These plugs will only work on the
Varian roughing pumps which come standard on ELAN DRC ll ICPMS
instruments. These plugs will not fit the Leybold pumps which come standard
on the ELAN DRC Plus instruments. Part # W101 101 3 (PerkinElmer, Shelton,
CT, www.perkinelmer.com). No spares typically needed.
xxvi. Probes
1. for ESI autosampler:Teflon, carbon fiber support, 0.8 mm i.d., blue marker,
114-28 fittings. Like part number SC-5037-3751 (Elemental Scientific lnc.,
Omaha, NE., www.elementalscientific.com). # Spares = 2.

2. for carrier solution of FAST sample introduction svstem: Teflon , carbon
fiber support, 0.5mm i.d., orange marker, 114-28 fittings. Like part number
SC-5037-3501 (Elemental Scientific Inc., Omaha, NE.,
www.elementalscientific.com). # Spares = 2.
xxvii. RF coil: PerkinElmer part #W802-1816 (PerkinElmer, Shelton, CT,
www.perkinelmer.com) or equivalent. # Spares = 2.
xxviii. Sprav 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.precisionqlassblowinq.com) or from various distributors.
# Spares = 2
xxix. Torch, quartz PerkinElmer part # N812-2006 (PerkinElmer, Shelton, CT,
www.perkinelmer.com) or equivalent. # New Spares = 2.
xxx. 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 lnc., Omaha, NE.,
www. elementa lscientific. com ).

xxxi. Tubing and adapters. for SC autosampler rinse station fillinq: Teflon tubing
and adapters (to attach to back of SC autosampler for filling rinse stations and

blood multiclement analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8-05

Page 21 ol 94

to attach to rinse containers). Like part # SC-0302-0500, Elemental Scientific
lnc., Omaha, NE., www.elementalscientific.com).
xxxii. Tubinq and nut, for FAST carrier solution: 0.5 mm i.d. Teflon tubing (orange
marker) with red %-28 male nut. Connects to high flow FAST valve head, port
#2. Like part # SC-0316-0500 (Elemental Scientific lnc., Omaha, NE.,
www. elementa lscientific. com).
xxxiii. Tubinq. 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, N E., www.elementalscientific.com).
xxxiv. Tubing, main arqon delivery to instrument: l.D. = 1/8" , O.D. = T¿". Like part #
C-06500-02 (pkg. of 100ft, polypropylene, Fisher Scientific lnternational,
Hampton, NH, www.fishersci.com) or equivalent. # Spares = 50 ft.
xxxv. Tubinq. PFA: LD. = 0.5 mm, O.D. = 1.59 mm (1116'). Used to transfer
liquidbetween rinse solution jug and peristaltic pump tubing

The Perfluoroalkoxy (PFA) copolymer is a form of Teflon@. Like part # 1548
(20ft length, Upchurch Scientific, Oak Harbor, WA, www.upchurch.com) or
equivalent.# Spares = 20ft.

xxxvi.

: use either

1. Standard PVC, 2-stop (black / black) peristaltic pump tubing, i.d. = 0.03".
PerkinElmer part # 09908587 (PerkinElmer, Shelton, CT,
wwur.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.
xxxvii. Tubinq, 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 (PerkinEltner, Shelton, CT,
wrmry.perkinelmer.com) or equivalent. # Spares = 6 packs of 12 tubes.

2. Standard Santoprene, 3-stop (grey/ greylgrey) 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.
xxxviii. Tubinq, PVC, i.d. = 1/8". o.d. = 3116". Used to transfer I iquid

1. between spray chamber waste port and peristaltic pump
2. between peristaltic pump and liquid waste jug
Like part # 14-169-74 (pkg. of 50 ft, Fisher Scientific International, Hampton,
NH, www.fishersci.com) or equivalent. # Spares = 20ft.

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8-05

Page 22 ol 94

xxxix. Tubino. Stainless Steel. o.d. = 1 /8", wall thickness = 0.028": Used to connect
gas cylinders to NexIONUCT gas ports. Like part # SS-T2-S-028-20 (20ft,
Georgia Valve and Fitting, Atlanta, GA, www.swagelok.com) or equivalent.
Spares = 20 ft.
xl. Tubino. Teflon. corru qated, To" o.d Connects to the auxiliary and plasma gas
side-arms of the torch. Part # WEO15903 (PerkinElmer, Shelton, CT,
www.perkinelmer.com) or equivalent. # Spares = 2.
xli. Tubinq, vinyl (arqon delivery to nebulizer): Vinyl Tubing, 1/8" lD x114" OD
Like part # EW-06405-02 (Cole Parmer, Vernon Hills, lllinois,
www. coleparmer.com) or equivalent. # Spares = 10 ft.

xlii. Union elbow, PTFE %" Swaqelok (ELAN bayonet mount): Connects argon
tubing to torch auxiliary gas sidearm on bayonet mount NEXION ICP-MS
instruments. Like part # T-400-9 (Georgia Valve and Fitting, Atlanta, GA,
www.swagelok.com) or equivalent. Spares = 2.
xliii

lok ELAN ba
et mount Connects argon tubing
Yo'
plasma
gas
to torch
sidearm and holds igniter inside torch sidearm on bayonet
mount NEXION ICP-MS instruments. Like part # T-400-3 (Georgia Valve and
Fitting, Atlanta, GA, www.swaqelok.com) or equivalent. Spares = 2.

c. \Sources for ICP-MS maintenance equipment & supplies
i. Anemometer: Like digital wind-vane anemometer (Mode|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 chanqinq rouohins pump oil: Like part # 53216 (United States Plastics
Corporation, Lima, OH, www.usplastic.com) or equivalent.
iii. Container, to hold acid baths for qlassware: Polypropylene or polyethylene
containers with lids (must be large enough for torch, injector, or spray chamber
submersion). Available from laboratory or home kitchen supply companies.
iv. Cotton swabs: Any vendor. For cleaning of cones and glassware.
v. Cutter (for 1/8" o.d. metal tubinq): Terry tool with 3 replacement wheels. Like
part # TT-1008 (Chrom Tech, lnc., Saint Paul, MN, www.chromtech.com) or
equivalent.
vi. Getter reqeneration 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. Maqnifvinq qlass: 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, Wl, www.labsafety.com).

blood multi+lement analysis by ICP-DRC-MS
Page 23 ol 94

IRAT-DLS Method Code: 3016.8-05

viii. Ultrasonic bath: Like ULTRAsonikrM Benchtop Cleaners (NEYTECH,
Bloomfield, CT, www.nevtech.com) or equivalent.

nt and consumables

i. Bar code scanner: Like Code Reader 2.0 (Code Corporation, Draper, UT,
www.codecorp.com) or equivalent. For scanning sample lDs duríng analysis
setup. Any bar code scanner capable of reading Code 128 encoding at a 3 mil
label density can be substituted.
ii. Carbov (for preparation of blood qualitv control oool and waste iuo for ICPMS
sample introduction system): Polypropylene 10-L carboy (like catalog # 02960-20C, Fisher Scientific, Pittsburgh, PA, unryw.fischersci.com) or equivalent.
Carboys with spouts are not advised due to potentialfor leaking.
iii. Containers for diluent and rinse solution: Two liter TeflonrM containers (like
catalog# O2-923-30E, Fisher Scientific, Pittsburgh, PA., www.fishersci.com, or
equivalent) and 4L polypropylene jugs (like catalog# 02-960-104, Fisher
.fishersci.com or equivalent) have both been
Scientific, Pittsburgh, PA,
used. Acid rinse before use.
iv. Gloves: Powder-free, low particulate nitrile (like Best C|eaN-DEXTM 100%
nitrile gloves, any vendor).

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, w¡rur.kcprofessional.com).
For sensitive applications in cleanrooms, use a wipe designed for cleanrooms
such as the Econowípe or Wetwipe (Liberty, East Berlin, CT,
ind.com).

vi.

Like the Microlab 625 advanced dual syringe diluter (Hamilton, Reno, NV,
http://www.hamilton.com/) equipped with a 5.0 mL left syringe, a 250 ¡rL right
syringe, a 12 gauge Concorde CT probe dispense tip, the Microlab cable
management system and a foot pedal. Alternatives are acceptable, including
the Micromedíc DigiflexrM (Titertek, Huntsville, AL, http://www.titertek.com/)
equipped with 10.0-mL dispensing syringe, 200 ¡rL sampling syringe, 0.75-mm
tip, and foot pedal.
vii.

reaqents): Like Brinkmann Research Pro Electronic pipettes (Brinkmann
lnstruments, Inc., Westbury, NY, http://rnnruw.brinkmann.com/home/). 5-100 ¡rL
(catalog #4860 000.070),20-300 pL (catalog #4860 000.089), 50-1000 ¡rL
(catalog #4860 000.097), 100-5000 pL (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 fÉ4860 000.860). When purchasing pipette tips (epTips),
purchase one or more boxes, then "reloads" for those boxes after that: 5-100

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8-05

Page 24 ol 94

pL (box catalog # 22 49 133-4, reload catalog # 22 49 153-9), 20-300 ¡rL (box
catalog # 22 49 134-2, reload catalog # 22 49 154-7),50-1000 ¡rL (box catalog
# 22 49 135-1 , reload catalog # 22 49 155-5), 100-5000 ¡rL (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,
Franklinlakes, NJ, www.bd.com) or equivalent. 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 workinq stock standards Like polypropylene
50-mL conical tubes, BD Falcon model #352098 (Becton Dickinson Labware,
Franklinlakes, NJ, unryw.bd.com) or equivalent. For use in storage of
intermediate working stock standards. 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.

Sources of chemicals. qases, and regulators

acid: VeritasrM double-distilled grade, 30-35% (GFS
Chemicals Inc. Columbus, OH, www.gfschemicals.com) or equivalent. This is
referred to as "concentrated" hydrochloric acid in this method write-up. For use

i. Acid. hvdrochloric

in preparation of intermediate working stock standards.
ii. Acid, nitric acid: VeritasrM double-distilled grade,68-70Vo (GFS Chemicals lnc.
Columbus, OH, www.qfschemicals.com). For use in cleaning any bottles,
vials, tubes, and flasks. This is referred to as "concentrated" nitric acid in this
method write-up.

iii. Blood. whole lhuman or bovine Bags of human blood can be purchased from
various sources such as American Red Cross (http://www.redcross.orq) or
Tennessee Blood services (Memphis, TN,
Request that human blood be screened
for infectious diseases such as Hepatitis B and HlV. Source for bovine blood
includes the Wisconsin State Laboratory of Hygiene (WSLH, Madison, Wl,

)

http ://un¡¡w. sl h. wisc. ed u).

iv. Ethanol (EtOH): USP dehydrated 200 proof (Pharmco Products, lnc.) or
equivalent.
v. Ammonium pyrrolidine dithiocarbamate. laboratory grade (Fisher Scientific,
Fairlawn, NJ) or equivalent.

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8-05

Page 25 of 94

vi. Arqon qas (for plasma & nebulizer) and requlator: High purity argon
(>99.999% purity, Specialty Gases Southeast, Atlanta, GA, r¡n¡¡w.sqsqas.com)
for torch and nebulizer. Minimum tank source is a dewar of liquid argon (180250 L). Bulk tank (1500.1 is preferred).

1. Requlator for arqon (at dewar): Stainless steel, single stage, specially
cleaned regulator with 3000 psig max inlet, 0-200 outlet pressure range,
CGA 580 cylinder connector, and needle valve shutoff on delivery side
termínating in a %" Swagelok connector. Part number
"KPRCGRF415A21AG1O-AR1" (Georgia Valve and Fitting, Atlanta, GA,
www.swaqelok.com) or equivalent. # Spares = l.
2. Requlator for arqon (between bulk tank and PerkinElmer filter requlator):
Single Stage 31655 Regulator, with 0-300 psi lnlet Gauge, 0-200 psi
Outlet Gauge, Outlet Spring Range, 0-250 psi, To" Swagelok lnlet
Connection, lqturn Shut off Valve on Outlet with T¿" Swagelok Connection
and Teflon Seals. Part number KPR1GRF412A20000-AR1 (Georgia Valve
and Fitting, Atlanta, GA, www.swagelok.com) or equivalent" # Spares = l.
3. Requlator for arqon (filter requlator on back of ICP-MS): Arqon requlator
filter kit. Catalog number N812-0508 (PerkinElmer, Shelton, CT,
www. pe rki nel mer. com).

vii. Disinfectant, for work surfaces Daily remake of diluted bleach (1 part
household bleach containing 5.25% sodium hy"pochloi'ite + I paits watei'), oi'
an equivalent disinfectant.
viii. Methane: Methane (Research Grade 5.0, 99.99% purity), for DRC channelA.
Typically purchased in cylinder size 200 (part # ME R200, Airgas South,
Atlanta, GA, www.airqas.com).
1. Requlator for methane: 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
aTa" Swagelok connector. Like part number KCYADPF412A2AD10
(Georgia Valve and Fitting, Atlanta, GA, www.swagelok.com), or

equivalent.#Spares=L
2. Flash Arrestor: Like part # 6104a (Matheson Tri Gas, Montgomeryville,
PA, www.mathesontriqas.com) or equivalent.
ix. Oxvqen: Oxygen ("Research Grade Research Grade 5.0", 99.9999% purity)
for DRC channel B. Like part # OX R33A (Airgas South, Atlanta, GA,
www.at
com)

1. Requlator for oxyqen: Stainless steel, two stage regulator for use with high
purity oxygen (cleaned to be free of all oils). Maximum inlet pressure
3600-5000 psi. lnlet gauge pressure 0-5000 psi (no oil in gauge).
Maximum delivery pressure 50-100 psi with a 0-30 psi outlet gauge (no oil
in gauge). CGA 540 cylinder connector on inlet side and an angle pattern
(90 degree) stainless steel needle valve on the delivery side terminating in

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page 26 of 94

a llS" stainless steel Swagelok connector. Like part #
GEORG/KCYCFR/ORS2/540 (Georgia Valve and Fitting, Atlanta, GA,
www.swaqelok.com), or equivalent.

2. Flash arrestor: Like part # 6104A (Matheson Tri Gas, Montgomeryville, PA,
www.mathesontrigas.com), or equivalent. # Spares = 1.
x. Standard, iridium: Like 1,000 pg/ml, item #CGlR1-1 (lnorganicVentures,
Ch ristiansbu rg, VA http://www. inorqan icventu res.com). Used as an internal
standard in diluent. Standard must be traceable to the National lnstitute for
Standards and Technology.

xi. Standard, multi-element stock calibration standard: ltem number SM-21070a2 (High Purity Standards, Charleston, SC, http://www.hps.neU). Standard
must be traceable to the National lnstitute for Standards and Technology.
xii. Standard, rhodium: Like 1,000 mg/L, item # PLRH3-2Y. (SPEX lndustries,
lnc., Edison, NJ, www.spexcsp.com). Used as an internal standard in diluent.
Standard must be traceable to the National lnstitute for Standards and
Technology.
xilt Standard. sinole element stock stand ards for oreoaration of calibrators and

blood qualitv control pools: National lnstitute of Standards and Techno logy
(NIST) Standard Reference Materials (SRMs): 3108 (Cd), 3132 (Mn), 3128
(Pb), 3133 (Hg), 3149 (Se). (Gaithersburg, MD, www.nist.sov). Standard
must be traceable to the National lnstitute for Standards and Technology.

xiv. Standard, tellurium: Like 1,000 mg/L, item #CGTE1-1 (lnorganicVentures,
Ch ristiansbu rg, VA http://www. inorgan icventu res.com). Used as an internal
standard in diluent. Standard must be traceable to the National lnstitute for
Standards and Technology.
xv. Tetramethylammonium hvdroxide,2so/o w/w, or equivalent (AlfaAesar, 30 Bond
St., Ward Hill, MA 01835).
xvi. Triton X-100rM surfactant. Like "Baker Analyzed" TritonX-100rM (J.T. Baker
Chemical Co., www.itbaker.com).

6) Preparation of reagents and materials
a. lnternal standard intermediate mixture
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 20 mglL Rh, lr, Te in

1o/o

vlv HNOs:

1. lf not previously dedicated to this purpose, acid wash a 50 mL volumetric
flask (PP, PMP, or TeflonrM). For example, with 1% (vlv) HNO¡ and >18
Mohm'cm water (at least 3 times each) and verify cleanliness through
analysis of rinsate.

blood multi+lement analysis by IGP-DRC-MS
Page 27 ol 94

IRAT-DLS Method Code: 3016.8-05

2. Partially fill the 50 mL volumetric flask with

1Vo

vlv HNOg (approximately

25-40 mL).
3. Add 1 mL of 1,000 ¡rg/ml Rh standard, 1 mL of 1,000 pg/ml lr standard,
and 1 mL of 1,000 pg/ml Te standard. lf initial Rh, lr, or Te standard
concentration is d ifferent, adj ust vol u me proportional ly.

4. Fill to mark (50 mL) with

1o/o

vlv HNOs and mix thoroughly.

5. Store at room temperature and label appropriately. Expiration is 1 year
from date of preparation.

Intermediate Triton X-100@ solution:

i. Purpose: To ease daily preparation of the diluent and rinse solutíons by first
preparing an intermediate Triton X-100@ solution.
ii. Preparation: To prepare 1 L o120o/o Triton x-100@

1. lf not previously dedicated to this purpose, acid wash a 200 mL volumetric
flask (PP, PMP, or TeflonrM). For example, with 1o/o (vlv) HNOs and >18
Mohm.cm water (at least 3 times each) and verify cleanliness through
analysis of rinsate.
2. Add 200 mL of Triton X-100@ to the 1L container that is partially filled with
:,1
O i¡l^L*
/ I (J
^* I l ..,^+^lVl(J¡ ll ¡ l-\-l
ClIEI

3. Fill to 1 L with >18 Mohm'cm water and mix until the Triton X-100@ has
completely dissolved into solution (overnighQ. A magnetic stirring plate
can be used to assist mixing by adding an acid-washed Teflon@coated
stirring bar to the bottle.

4. Store at room temperature and label appropriately. Expiration is 1 year
from date of preparation.

Sample diluent and carrier
i. Purpose: This solution will be used in the preparation of all samples and
calibrators during the dilution process prior to analysis. Make all samples,
standards, blanks, QC, etc. . . in a run 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), use the same solution for the the 'carrier' and sample diluent.
The diluent is an aqueous solution of 5 ¡rg/L internal standard mixture (Rh, lr,
Te), in O.4o/o v/v tetramethyl ammonia hydroxide (TMAH),1o/o ethyl alcohol,
0.01o/o APDC, and 0.05% v/v Triton X-100@. Larger volumes of these solutions
can be prepared by adjusting component volumes proportionally.

ii. Preparation: To prepare 2L of 5 ¡rg/L Rh, lr and Te, 0.01o/o APDC in 0.4o/o vlv
TMAH, 1% ethanol, and 0.05% v/v Triton X-100:

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page 28 o1 94

1. lf not previously dedicated to this purpose, acid wash a 2L container (PP,
PMP, or TeflonrM). For example, with 1o/o (vlv) HNOs and >18 Mohm.cm
water (at least 3 times each) and verify cleanliness through analysis of
rinsate.

2. Parlially fill the 2L container with >18 Mohm'cm water.
3. Add 0.2 g of APDC , 8 mL of
of 20o/o Triton X-100@.

25o/o

v/v TMAH, 20 mL

of ethanol, and 5 mL

4. Dilute to volume (2L) with >18 Mohm'cm water.
5. Spike 500 ¡rL of 20 mg/L Rh, lr, Te to the final diluent.
6. lnvert bottle a few times to insure thorough mixing. Allow to sit for several
hours or overnight before using.
7. Store at room temperature and label appropriately. Expiration is 1 year
from date of preparation.

ICP-MS rinse solution
i. Purpose: The rinse solution used in this method is an aqueous solution of
0.01% APDC in 0A% v/v TMAH, 1% ethanol, and 0.05% v/v 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 0.01% APDC in 0.4o/o v/v TMAH,
and 0.05% v/v Triton X-100:

170

ethanol,

1. lf not previously dedicated to this purpose, acid wash a 4L container (PP,
PMP, or TeflonrM). For example, with 1o/o vlv HNOg and >18 Mohm'cm
water (at least 3 times each) and verify cleanliness through analysis of
rinsate.

2. Parlially fill the 4 L bottle with >18 Mohm'cm water (approximately 2-3

L).

Use of volumetric flask is not requíred.

3. Add 0.4 g of APDC

4. Add 16 mL of TMAH
5. Add 40 mL of ethyl alcohol,
6. Add 10mL of 20% Triton X-100@, (See Section 6.b for details on
preparation)
7

. Fill to 4 L using >18 Mohm.cm water.

8. 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.

blood multi-element analysis by ICP-DRC-MS
Page 29 of 94

IRAT-DLS Method Code: 3016.8-05

9. lnvert bottle a few times to ensure thorough mixing. Allow to sit for several
hours or overnight before using.
10.Store at room temperature and label appropriately. Expiration is 1 year
from date of preparation.

Standards. calibrators. base blood and QC

i. Multi-element stock calibration standards
1. Purpose: This multi-element stock standard will be used to prepare the
intermed iate working calibration standards.

2. Purchase & Storaqe:
a. Purchasinq from vendors: Whether purchased or prepared in-house,
the starting materials must be NIST-traceable. Matrix and
concentrations of Pb, Cd, Hg, Mn and Se are listed in Table 3 of
Appendix B.

b. Storaqe: Store at room temperature and label appropriately.
Expiration is as defined by the manufacturer or 1 year from date of
opening, whichever comes first.

ii. Diluent for intermediate calibration standard preparations:
1. Purpose: This diluent is used to dilute stock and intermediate stock
calibration standards, not to prepare working calibrators or blood samples
for analysis.

2. Preparation: To prepare 2L of 3o/o v/v HCI:
a. lf not previously dedicated to this purpose, acid wash a 2L container
(PP, PMP, or TeflonrM). For example, with 3% HCI and >18 Mohm'cm
water (at least 3 times each) and verify cleanliness through analysis of
rinsate.

b. ln the 2 L flask, add

1-1

.5L >18 Mohm'cm water.

c. Add 60 mL high purity concentrated HCl.

d. Fill to the mark and mix thoroughly.
e. Store at room temperature and label appropriately. Expiration is 1 year
from date of preparation.
iii. Multi-element intermediate stock calibration standard

1. Purpose: This multi-element intermediate stock standard will be used to
prepare the intermediate working calibration standards.

2. Preparation: To prepare

3o/o v/v HCI solutions containing Cd, Pb, Hg, Se,
and Mn with concentrations listed in Table 4 of Appendix B:

a. Acid-rinse one 100 mL, PP (or PMP) volumetric flask. For example,
with 3% HCI and >18 Mohm'cm water (at least 3 times each) and verify

blood multi-element analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8-05

Page 30 of 94

cleanliness through analysis of rinsate. Mark flask according to
intended use. Dedicate to purpose.

b. Partially fill (50-75% full) the 100 mL flask with the

3o/o

(vlv) HCI diluent

prepared in Section 6.e.ii.

c. Using the volume listed in Table 4 of Appendix B, pipette the
appropriate volume of the multi-element stock calibration standard
solution into the volumetric flask. Dilute to the volumetric mark with the
3% HCI (v/v) diluent using a pipette for the final drops. Mix each
solution thoroughly. Final concentrations are listed in Table 4 of
Appendix B.

d. Once mixed, transfer to acid-cleaned, labeled, 50 mL containers (PP,
PMP, or TeflonrM) for storage.

e. Store at room temperature and label appropriately. Expiration is 1 year
from date of preparation.
iv. lntermediate workinq calibration standards

1. Purpose: Used each day of analysis to prepare the final working
calibrators that will be placed on the autosampler.

2. Preparation: To prepare3o/o v/v HCI solutíons containing Cd, Pb, Hg, Se,
and Mn with concentrations listed in Table 3 of Appendix B:

a. Acid-rinse eight 100 mL, PP (or PMP) volumetric flasks and one 2 L
PP (or PMP) volumetric flasks. For example, wíth 3% HCI and >18
Mohm'cm water (at least 3 times each) and verify cleanliness through
analysis of rinsate. Mark each flask according to intended use.
Dedicate to purpose.

b. Fill each 100 mL flask 50-75% with the 3% (v/v) HCI diluent prepared
in Section 6.e.ii.

c. Using the volumes listed in Table 5 of Appendix B, pipette the
appropriate volume of the multi-element intermediate stock calibration
standard solutions into each of the volumetric flasks. Dilute each to
the volumetric mark with the 3% HCI diluent using a pipette for the final
drops. Mix each solution thoroughly. Final concentrations are listed in
Table 5 of Appendix B.

d. Once mixed, transfer to acid-cleaned, labeled, 50 mL containers (PP,
PMP, or TeflonrM) for storage.
e. Store at room temperature and label appropríately. Expiration is 1 year
from date of preparation.

Pour aliquots of each standard into clean 15mL polypropylene tubes
and label for daily use.

blood multi-element analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 301 6.8-05

Page3l of94

v. Workinq calibrators

1. Purpose: The working calibrators 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
(elemenUinternal standard) from the dilution of the patient blood sample to
the signal ratio response curve from the working calibrators.
2. Content: Dilutions (1:50) of the corresponding eight intermediate working
calibration standards with base blood and sample diluent.
3. Preparation: Mix with base blood and diluent (Section 6.c) using a
benchtop automatic pipette to make 1:50 dilutions of the corresponding
eight intermediate working calibration standards immediately prior to
analysis (see Table I of Appendix B).
vi. Base blood
1. 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.

2. Preparation: To prepare a mixture of multiple blood sources collected from
anonymous donors to approximate an average blood matrix:

a. Purchase several bags of whole blood.
b. Screen each individual bag of blood for concentration of analytes of
interest. See Table 2 in Appendix B for minimum acceptable values

c. Once screened, mix the acceptable blood together in a larger container
(i.e. acid washed polypropylene (PP), polymethylpentene (PMP), or
TeflonrM) and stir for 30+ minutes on a large stir plate (acid wash large
TeflonrM stir bar before use).

d. Store long-term as smaller portions for daily use (e.9. 2 mL cryovials)
according the same storing and handling criteria described in Section
3.

vii. lnternal oualitv control materials (" bench " oc)
1. Purpose: lnternal (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. Preparation: To prepare pooled animal or human blood at low-normal and
high-normal concentrations:
Both purchased or in-house prepared quality control materials are suitable
for this purpose if volumes, concentrations meet method requirements and

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page 32 of 94

any spikes of elemental levels are traceable to the National lnstitute for
Standards and Technology (NIST).

3. Screeninq 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
grovuth.

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. Select blood for the low bench QC pool which has analyte
concentrations in the low-normal population range. Select blood for
the high and elevated bench QC pools which has analyte
concentrations less than some pre-selected target concentration
values in the high normal population range. See Table 2 in Appendix
B for recommended concentration ranges.

4. Combininq collected blood: The goal is for combiníng 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 ínto 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.

5. Spikinq of blood

a. Analyze three samples of each blood pool. Record these results for
future recovery calculations.

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.

blood multi+lement analysis by ICP-DRG-MS
IRAT-DLS Method Code: 3016.8-05

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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.

6. Dispensinq and storaoe of blood
a. Container types: Dispense blood into lot screened containers (i.e. - 2
mL polypropylene tubes). lf 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. lnclude at least the
name of QC pool (text and bar code), date of preparatíon, and a vial
number on the labels.

c. Dispensinq: Dispensing can be accomplished most easily using

benchtop automatic pipette in continuous cycling dispense mode.
Dispense the pools in a clean environment (i.e. a class 100 cleanroom
area or hood).
1. Allow blood to reach room temperature before dispensing (to
nrcrrcnf
temnerafr rrc orarlicnts nossihlv ear rsinn enneentratinn
rlvlY.'v--.
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 the benchtop automatic pipette) with a
length of clean TeflonrM 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 the benchtop automatic pipette before
use by analyzing 1-2o/o (v/v) HNOs which has been flushed
through the benchtop automatic pipette with a portion of the
same solution which has not been through the benchtop
automatic pipette.
4. Approximately one hour before dispensing begins,
a. With the large stir plate close to the left side of the
benchtop automatic pipette, begin stirring the blood poolto
be dispensed.

b. Also during this tíme, flush the benchtop automatic pipette
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

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page 34 of 94

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.

d. Homoqeneitv test: Check homogeneity of analyte concentrations

pool aliquots.

e. Storage: Store long-term as smaller portions for daily use (e.9. 2 mL
cryovials) according the same storing and handling criteria described in
Section 3.

Ootimization solutions
i. DRC optimization

1. Purpose: For periodic testing of the DRC cell parameters. Procedure
requires at a minimum a blank (i), an analyte solution (ii), a blank with
interference (iii), and an analyte and interference containing solution (iv).
For Se, only the blank (i), an analyte solution (ii) are needed because the
interference on Se is plasma based.

2. Content:
Diluent in this section refers to sample diluent (5 pg/L internal standard
mixture (Rh, lr, Te),0.4o/o v/v tetramethyl ammonia hydroxide (TMAH),
1% ethyl alcohol, 0.01% APDC, and 0.05% v/v Triton X-100@ as
described in Section 6c.

a. Solutions for testing elimination of 54Fe1H interference on

i.
ii.
iii.
iv.

Base
Base
Base
Base

blood in diluent
blood in diluent
blood in diluent
blood in diluent

(1
(1
(1
(1

+ 49)
+ 49) + 4.5 ¡rg/L Mn
+ 49) + 500 ¡rg/L Fe
+ 49) + 4.5 ¡rg/L Mn + 500 pg/L Fe

b. Solutions for testing elimination of

i.
íi.

55Mn:

a0Arz

interference on 80Se

Base blood in diluent (1 + 49)
Base blood in diluent (1 + 49) + 90 pg/L Se

3. Préparation & storaqe: Prepare different volumes, if needed, by adding
proportionally larger or smaller volumes of solution constituents.
lnterference concentrations can be prepared higher as needed by
adjusting the volume of this spike. Keep interference spike volume small
(<0.3 mL) using a high concentration stock solution (i.e. 1000 mg/ml).
Analyte concentrations can be made higher if needed for sensitivity
reasons by preparing a higher concentration calibrator.

blood multi-element analysis by ICP-DRC-MS
Page 35 of 94

IRAT-DLS Method Code: 3016.8-05

a. Solutions for testing elimination of

i.
ii.
iii.

iv.

54Fe1H interference

on 55Mn:

Base blood in diluent (1 + 49)
1. ln a 50 mL lot screened or acid-washed polypropylene tube,
prepare a 50 mL portion of working calibrator 0 as described
Table 6 (multiply volumes by 5).
Base blood in diluent (1 + 49) + 4.5 pg/L Mn
1. ln a 50 mL lot screened or acid-washed polypropylene tube,
prepare a 50 mL portion of working calibrator 2 as described
Table 6 (multiply volumes by 5).
Base blood in diluent (1 + 49) + 500 pg/L Fe
1. In a 50 mL lot screened or acid-washed polypropylene tube,
prepare a 50 mL portion of working calibrator 0 as described
Table 6 (multiply volumes by 5).
2. Add 0.025 mL of 1000 mg/ml Fe.
Base blood in diluent (1 + 49) + 4.5 ¡rg/L Mn + 500 pg/L Fe
1. ln a 50 mL lot screerted <¡r acicl-washed polypropylene tube,
prepare a 50 mL portion of working calibrator 2 as described
Table 6 (multiply volumes by 5).
) Add ô fì2Ã rnl nf lôl'ìô rnn/rnl Fa

b. Solutions for testing elimination of 40Arz interference on

80Se:

Base blood in diluent (1 + 49)
1. ln a 50 mL lot screened or acid-washed polypropylene tube,
prepare a 50 mL portion of working calibrator 0 as described in
Table 6 (multiply volumes by 5).
ii. Base blood in diluent (1 + 49) + 90 ¡rg/L Se
1. ln a 50 mL lot screened or acid-washed polypropylene tube,
prepare a 50 mL portion of working calibrator 2 as described in
Table 6 (multiply volumes by 5).
c. Store at room temperature and prepare as needed.
d. Label appropriately (see Section 6.f .i.2\, "Store at room temperature",
preparation date, expiration date one year from preparation date, and
preparer's initials

ii. Dual detector calibration:
1. Purpose: Use as necessary to perform the dual detector calibration.

2. Content: Aqueous dilutions of single element stock standard solutions
2% (vlv) nitric acid. Current solution in use contains: Pb with a final
concentration of 200 ug/L.

blood multi-element analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8-05

Page 36 of 94

3. Preparation & storaoe: Prepare different volumes, if needed, by adding
proportionally larger or smaller volumes of solution constituents.
a. To prepare a total of 50 mL: ln a 50 mL lot screened polypropylene
tubes, spike ín 0.01 mL of 1000 mg/ml single element stock solution
for each element desired in the final solution.
b. Dilute to the 50 mL mark with2o/o (v/v) nitric acid.
c. Store at room temperature and prepare as needed.
d. Label appropriately, e.g. "200 ug/L Pb in 2% (vlv) HNO3", "Store at
room temperature", preparation date, expiratíon date one year from
preparation date, and preparer's initials.

7) Analytical instrumentation setup
(see Sectíon 5 for details on hardware used, including sources)
a

lnstrumentafi on and eouioment setuo
i. Confisuration for liquid handlinq

1. FAST valve setup: See Appendix B, Figure 1 for diagram and Section 5.b
"FAST / ESI SC4-DX autosampler accessories" for source information.
a. Port 1: sample loop (white nut).
b. Port 2: 0.5 mm lD probe (red nut) for carrier solution.
c. Port 3: nebulizer line (green nut) for transfer of liquid to nebulizer.
d. Port 4: sample loop (white nut).
e. Port 5: 0.8 mm lD probe (blue nut) for diluted samples.
f. Port 6. vacuum line (black nut).

2. Carrier solution uptake. Use peristaltic pump to control uptake flow rate of
carrier solution to the SC-FAST valve. Use of a 'peristaltic to Teflon tubing
adapter'for prevents damage to small i.d. tubing when making connections
(see consumables descriptions in Section 5.b).

3. Spray chamber waste removal
Use of a 'peristaltic to Teflon tubing adapter' for prevents damage to small
i.d. tubing when making connections (see consumables descriptions in
Section 5.b).

a. Between sprav chamber and peristaltic tubinq:
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.

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 301 6.8-05

Page 37 of 94

i¡.

Sprav chambers without threaded connection: Use of specialized
push-on connectors available from various vendors (like UFT-075
from Glass Expansíon, Pocasset, MA) are preferred for safety
reasons to direct connection of PVC tubing (e.9. 1/8" i.d. xTq" o.d.).
b. Between peristaltic pump tubinq and waste container: Connect 1/8"
i.d. xTq" o.d. PVC tubing to the white / black peristaltic pump tubing
using a tubing connector (PerkinElmer item # 83140715). Place the
free end of the PVC tubing through the lid of the waste jug (be sure it is
secure). Place waste container in a deep secondary containment tray
in case of overflow.

4. Rinse solution for autosampler:
a. Rinse solution iuq: 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.
b. Rinse solution uptake to autosampler rinse station: Use tubing of
different lengths and inner diameters between the rinse solution
container and the autosampler rinse station to control uptake rate of
rinse solution. These can be obtained from the autosampler
manufacturer, their distributors, or custom built in the lab. Optimize
these factors along with fill time in the software so that waste of rinse
solution is minimized and rinse station does not go empty.
c. Autosampler rinse station waste removal: Gravity drain of waste to the
waste container is sufficient. Use minimum drain tubing to make this
connection. lf this tube is too long, the rinse station will not drain
properly.
ii. Gas delivery and requlation

1. ICP-MS modifications:
a. Plastic tubing between mass flow controllers and dynamic reaction cell
have been replaced with stainless steel. Stainless steel tubing is
preferred between the reaction gas cylinder / regulator and the back of
the ICP-MS instrument.
b. A second mass flow controller will be needed (channel B) that does not
send the DRC gas through a 'getter'.
2. Argon qas: Used for various ICP-MS functions including plasma and
nebulizer.

a. Requlator for arqon source (if a dewar): Set delivery pressure of this
regulator at least 10 psi higher than the delivery pressure of the step-

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page 38 of 94

down regulator to allow for pressure drop across tubing that stretches to
the instrument.
b. Step down requlator (if source of arqon 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 10 psig above the
delivery pressure of the filter regulator on the ICP-MS.
c. Filter requlator at ICP-MS: Single stage "argon regulator filter kit"
supplied with the ICP-DRC-MS. Set the delivery pressure depending
on the instrument setup:
ELAN with a 0-60psi qauge on the filter requlator: 52t1psi when
plasma is running (need 0-150 psi regulator if using a PolyPro or
PFA nebulizer made by Elemental Scientific lnc).
ELAN with a 0-150psi qauqe on the filter requlator: 90-100 psi
when plasma is running.

ii.

3. Methane (99.99%) qas: Used for dynamic reaction cell interference
removal from selenium isotopes.
a. Connect to DRC channelA
b. Set the delivery pressure of regulator to 5-7 psig when gas is flowing
See section 5.e for part numbers and details.

4. Oxyqen (99.999*%) qas: Used for dynamic reaction cell interference
removal from manganese isotopes.
a. Connect to DRC channel B.
b. Set the delivery pressure of regulator to 5-7 psig when gas is flowing.
See Section 5.e for part numbers and details.
c. Use a brass flash arrestor on outlet side of regulator. See Section 5.e
for part numbers and details.
iii. Chiller / heat exchanqer: lf using refrigerated chiller, set temperature control to
approximately 18'C.

lnstrument and method parameters: See Tables and Figures in Appendix B for a
complete listing of the instrument and method parameters and software screen
shots.

8) The run: quality, execution, evaluation, and reporting
c,

Bench OC reference materials and calibration verification

i. Bench "QC": Analysis of bench QC permits assessment of methodological
imprecision, determination of whether the analytical system is 'in control' during
the run, and assessment of time-associated trends. Before QC materials can
be used in the QC process, they must be characterized by at least twenty (20)
analytical runs to determine appropriate QC parameters.

blood multi-element analysis by ICP-DRC-MS
Page 39 of 94

IRAT-DLS Method Code: 3016.8-05

Bench QC pool analyte concentrations in this method span the analyte
concentration range of the calibrators including "low-normal" ('Low QC'), "highnormal" ('High QC'), and "above-normal" ('Elevated QC') concentrations.
In each analytical run the analyst will test each of the three bench QC samples
two times, subjecting them to the complete analytical process. Bench QC pool
samples are analyzed first in the run after the calibration standards but before
any patient samples are analyzed. This permits making judgments on
calibration linearity and blank levels prior to analysis of patient samples. The
second analysis of the bench QC pools is done after analysis of all patient
samples in the run (typically 40-50 patient samples totalwhen analyzing for all
elements in the method) to ensure analytical performance has not degraded
across the time of the run. lf more patient samples are analyzed on the same
calibration curve after the second run of the bench QC, all bench QC must be
reanalyzed before and after the additional samples. For example, the schemes
shown in Table 6 in Appendix B are both acceptable ways to analyze multiple
consecutive "runs".

ii. Reference materials: Use standard reference materials (SRM) from the
National lnstitute of Standards and Technology (NIST) (i.e. SRM 955c Levels
1-4) to verify method accuracy. Use previously characterized samples from
proficiency testi ng p rog ram or com mercia ly-prod uced reference materia ls
I

...L--

lllôT

ôntt-

.^:t^Lt-

wl!E!! ttlù ! Ðñ_lvl.- alg LlMvallalrtE.

iii. Calibration verification: The test system is calibrated as part of each analytical
run with NIST-traceable calibration standards. These calibrators, along with
the QCs and blanks, are used to verify that the test system is performing
properly.

Perform. evaluate and report a run
r. Startinq the equi oment for a run

1. Power on the computer, printer, and autosampler, and instrument
computer controller.

2. Peristaltic pump: Set proper tension on peristaltic pump tubing
3. Software: Start software for the ICP-MS and autosampler control.
4. Dailv pre-iqnition maintenance checks: Perform and document daily
maintenance checks (e.9., Ar supply pressure, interface components
cleanliness and positioning, interface pump oil condition, vacuum pressure,
etc.).

5. Place probe in adequate volu me of carrier or rinse solution lf using an ESI
FAST, manually place carrier probe into carrier solution. lf not, send the
autosampler probe to a rinse solutíon (e.9. autosampler rinse station).
6. Start the plasma

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8-05

Page 40 of 94

7. Start the peristaltic pump: Start the pump running slowly, making sure that
the rotational direction is correct for the way the tubing is set up.
8. Warm-up time: Allow warm-up time suggested by the manufacturer for the
ICP-MS (e.9. RF generator) after igniting the plasma. There will be
another warm-up time (or "stability time") for the DRC later in this
procedure.

9. Dailv performance check: Perform and document a daily performance
check and any optimizations necessary.

Save new parameters to the "default.tun" and "default.dac" files.
10. DRC stability

time: Best analyte{o-internal standard ratio stability is

typically observed after 1-1.5 hours of analysis of diluted blood samples
using the DRC mode method (-15 measurements of the 5 element panel
can be made in t hour). Prepare 50mL* of a calibration standard (e.9.
standard 2) to be analyzed repeatedly before the beginning of the run to
achieve a stable analyte-to-internal standard ratio. Time to reach stability
is instrument-specific and learned from performance of runs. See Table 7
in Appendix B for example of setup in the Samples / Batch window and
Table 8 in Appendix B for details of making a working standard.
11. Readyinq the instrument for quick-start analysis: Leave the plasma
running to eliminate the need for an initial instrument warm-up period and I
or a DRC stabilization period as long as appropriate planning is made for
sufficient solution supply and waste collection. Analysis of conditioning
samples (diluted blood matrix) can also be scheduled to occur at roughly a
predetermined time. Accomplish this by setting up multiple sample
analyses with extended rinse times (e.9. one 5 element analysis with a
1500s rinse tíme will take approximately 30 minutes to complete). lnitial
samples would be non-matrix, while final samples would be diluted matrix
for conditioning. lf running a DRC-only method during these scheduled
analyses, the ICP-MS will remain in DRC-mode for approximately 45
minutes without depressurizing the cell.
for analysis:
a. Workspace (files & folders): Verify & set up the correct files and data
directories for your analysis (See Table 1 in Appendix B for defaults).

12. Software setup

b. Samples / batch window: Update the software to reflect the current
sample set. Use a bar code scanner to input data whenever possible.
See Table 1 in Appendix B for times and speeds.

1. Blood vs. aqueous method files:
a. The difference: There are two method files for this one
method (see Table 1 in Appendix B). lt is necessary to use
both to accomplish each run because the current
PerkinElmer software will not allow for more than one blank

blood multi-element analysis by ICP-DRC-MS

Page4l of94

IRAT-DLS Method Code: 3016.8-05

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-based
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 fíles
is used is when the measurement action lncludes "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 7 in Appendix B.

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 at the very
beginning of the run. The blood blank method defines
the autosampler location of the blood blank and the
blood calibration standards.
ttanhll)'
ii
Tha
11. I I l¡t Crt,ll./rÃ

ma{lra¡{
lila
I I ll-Ll ltJL¡ llll-

mrra*
I I lL¡itl

}ra
¡Jtv rraaÄ
uatl-V

{a anal(7 h¡za
lY49
lv Gll

all fìl^
Ctil \{V

materials and patient samples. The aqueous blank
method defines the aqueous blank in autosampler
location.

ii. Preparation of samples for analysis (See Table 6 in Appendi¡ B)

1.
2.

Thaw blood samples; allow them to reach ambient temperature.
Prepare the following solutions into pre-labeled containers using the
benchtop automatic pipette or other volumetric sample transfer device.
See Table 8 in Appendix B for a summary.

a. Aqueous Blank: Prepare a minimum of two aqueous blanks. One will
be the actual aqueous blank and the other will be a backup ("Aqueous
Blank Check") in case the original aqueous blank is unusable.

b. Calibrators: Prepare the working calibrators (S0-S8). Prepare S0 in
triplicate. One of these S0 preparations will be the zero calíbrator
(blood blank) for the calibrators; the other two will be analyzed twice
after the last calibrator to collect run blank data that can be used in
calculating method limit of detection (LOD).

c. Patient & QC Samp/es: Before taking an aliquot for analysis,
homogenize the sample thoroughly.

After preparation, mix and cover. Place prepared dilutions on the
autosampler of the ICP-MS in the order corresponding to the sequence
setup in the ICP-MS software.

blood multi-element analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page 42 ol 94

Room temperature is acceptable for the original samples for the work
day.

NOTE: Samples must be analyzed within 24 hours of preparation to
obtain valid resu/fs 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 results in Appendix A for
details.
iii. Start the analysis using the ICP-MS software.

iv. Monitor the analvsis in real-time as much as possible. lf necessary, leave the
run 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. Verify proper operation of the instrumenf (proper loop filling, sample
reaching nebulizer in correct timing, autosampler arm moving properly, etc
)

2. Verify that background signal from instrument and reagents are low.
Helpful checks when diagnosing high background problems include:
a. Water to be used ín Aq Blank Checks and dilutions.
b. Diluent before and after being flushed through the benchtop automatic
pipette.

lf contamination is observed from the pipette, flush the pipette with
>500 mL of nitric acid solution (< 5o/o v/v HNOs) and retest.

c. Comparison with other instruments.
3. Verify analyte / internal standard ratio stability
The net intensity (analyte / internal standard ratio) of the measurements
made while stabilizing the DRC can be evaluated to determíne the
readiness of the system to begin analysis. Continual trending in this ratio
indicates that unwanted instrument drift will occur within the run.

4. Verify calibration curues meet R2 requirements (minimum of 0.98, typically
0.99 to 1.000).

5. Verify bench QC resulfs are within the acceptable limits.
lf an analyte result for the beginning QC material(s) falls outside of the +
3SD limits, then the following steps are recommended:

a. Evaluate the blank results.
b. Evaluate the reproducibility of the 3 replicates wíthin the
measurements.

c. Evaluate the consistency of the internal standard across the
measurements (esp. the calibrators).

blood multi-element analysis by ICP-DRC-MS
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Page 4Íl of 94

d. Evaluate calibration curves. lf a particular calibration standard is
obviously in error, it can be re-analyzed as a sample (old or new
dilution) and incorporated into the curve through data reprocessing as
a calibrator. As a last resort, a single calibration point per analyte
between or including 52 and 57 can be removed from the curve (Do
not drop S0, S1 or S8). Follow up problems with calibration standards
with appropriate corrective actions (e.9. re-preparation of intermediate
working standards or troubleshooting instrument parameters).

e. Prepare a fresh dilution of the failing QC material (same vial) and
reanalyze it to see if the QC dilution was not properly made.

f. Prepare a fresh dilution of the failing QC material (unused vial) and
analyze it to see if the QC vial had become compromised.

g. Prepare and analyze new working calibrators.
h. Test a different preparation of intermediate working calibration
standards.
lf these steps do not result in correction of the out-of-control values for QC
materials, consult the supervisor for other appropriate corrective actions.

6. Verify good precision among replicates of each measurement.
7. Veriñ.t consrsfenf measured intensities of the internal standards.
Some sample-to-sample variations are to be expected, however,
intensities drifting continuously in one direction resulting in failing results
for ending QC indicate the instrument needs additional pre-conditioning
before the run or environmental conditions are changing too much
around the instrument.

8 Verify elevated patient resulfs.
Refer to Figure 4 in Appendix B for flowchart.

a. Confirminq an elevated concentration: Repeat for confirmation any
sample having a concentration greater than the 1UB threshold. See
Table 9 in Appendix B.

b. Dilution of a sample to within the calibration ranqe: Repeat in duplicate
with extra dilution any sample having a concentration greater than the
highest calibration standard to bring the observed result within the
concentration range of the calibrators. See Table 7 in Appendix B for
validated extra dilutions.
c. Confirminq proper washout atter an elevated sample: When monitoring
the analysis in real-time, if a sample concentration is greater than the
highest concentration validated for washout (see Table 9 of Appendix
B), do the following to verify that the run is still in control for low
concentration samples before proceeding with analysis.

í.

Stop run following elevated sample

blood multi-element analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

ii.

Page

ol 94

Verify that the run is still in control for lower concentration samples
before proceeding with analysis. Analyze 2 blood blank checks
followed by a low bench QC washout check. lf the low bench QC
wash check is not in control (within t 3SD limits), repeat these 3
check samples until washout is verified before proceeding with
analysis.
Example:
3016 BldBlkChk Washl
3016 BldBlkChk Wash2
LBXXXXX Wash

ii¡. lf the run is not verified in-control for low concentration

samples
before the next samples are analyzed, see Section 8.b.vii.2. for
directions.

v. Overniqht operation or usinq auto stop: The run may be left to complete itself
unattended as long as appropriate planning is made (e.9. sufficient solution
supply and waste collection). Turn on the AutoStop feature of the ICP-MS
software. Delay the shutdown at least 10 minutes (use peristaltic pump speed
approximately that of the method wash) to rinse the sample introduction
system of blood matrix before turning off the plasma. lt will be necessary to
replace the sample peristaltic pump tubing the next day since it will have been
clamped shut overnight. Enable "Auto Start / Stop" is on the "AutoStop" tab of
the lnstrument window.
vi. Records of results: Run results will be documented after each run in both
electronic and paper form.
1. Electronic records: Transfer data electronically to the laboratory
information system. When keyboard entry must be used, proofread
transcribed data after entry.

a. Export data from the ICP-MS software using "original conditions" or
files and folders used during the analysis. Use descriptive report
filenames (e.9. 2005-0714a_group55.txt). ln the ICP-MS software
under "Report Format" (METHOD window, REPORT tab) choose the
"Use Separator" option, and under the "File Write" Section choose
"Append."

b. Move the generated .TXT data file to the appropriate subdirectory on
the network drive where exported data are stored prior to import to the
laboratory information management system.

c. lmport the instrument file into the laboratory information system with
appropriate documentation (e. g. instrument lD, analyst, calibration
standards lot number, and run or sample specific comments).

2. Paper records: Printed run sheets must be documented with

Analyst initials

blood multi+lement analysis by ICP-DRG-MS
IRAT-DLS Method Gode: 3016.8-05

ii.
iii.

Page 45 of 94

lnstrument lD
Date of analysis and run # for the day

vii. Analvst evaluation of run results:

1. Bench qualitv control: After completing a run, and ímporting the results into
the laboratory information system, evaluate the run bench QC according to
laboratory QC rules. 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 in control until
statistically reviewed.
a. Rules for bench qualitv control evaluation The following are the CDC
DLS QC rules for three QC pools per run with two or more QC results
per pool.

lf all three QC run means are within 2Sm limits and individual
results are within 25¡ limits, then accept the run.

i¡.

lf one of the three QC run means is outside a 2Sm limit - reject run
if:

1. Extreme Outlier
mean +

4St

ñ--t-

^
./

.'t.-ì
^^
KlllÊ

- l{lln

- Run mean is beyond the characterization
__-_:_

meen

_--a_:-t_

lS fllllsll-le

t:-^^:a

â^ .1.-lm
^^
llffllT

; ;; ;; _;" ;;;";;il;;;;;,e
25'

ou,side the same

limit

4. 10 X-bar Rule - Current and previous 9 run means are

same side of the characterization mean

iii.

lf one of the QC individual results is outside a 25¡ limit - reject run
if:

1. Extreme Outlier - One individual result is beyond the
characterization mean + 4Sm

2. R 45 Rule - 2 or more of the within-run ranges in the same run
exceed 45* (i.e. ,95Vo range limit)
Note: Since runs have multiple results per poo! for
R 45 rule is applied within runs only.

pools, the

Abbreviations:
S¡ =

Standard deviation of individual results.

Sm =

Standard deviation of the run means.

Sw =

Within-run standard deviation.

b. lmplications of QC failures: lf the DLS 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.

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page 46 of 94

For 1 or 2 analytes: ONLY the analytes which were "out of control"
are invalid for reporting from the run.

ii.

For 3 or more analvtes: All results, regardless of analyte, are
invalid for reporting from the run.

2. Patient results:
a. Elevated concentrations: Refer to Figure 5 in Appendix B for flowchart.

Boundaries reouirino confi rmatory measurement:
Results oreater tha n the first (1UB) or second (2UB) uooer
1
boundaries
The concentrations assigned to 1UB and 2UB for an element
is determined by study protocol but default concentrations are
in Table 9 in Appendix B.

a. Results qreater than the first upper boundary (1UB).
Confirm by repeat analysis of a new sample preparation
concentrations observed greater than the "first upper
boundary" (defined in the laboratory database as the
"1UB'). Report the first analytically valid result, as long as
the confirmation is within 10o/o. Continue repeat analysis
until a concentration can be confirmed.
b. Analyst reportinq of elevated results: Report any patient
results confirmed to be greater than the second upper
boundary (2UB) as an "elevated result".
2. Results qreater than highest calibrator: Samples that exceed
the high calibrator must be prepared with minimum extra
dilution in duplicate to bring the observed result within the
calibration range (< SB). Report the first analytically valid
result (i.e. the first one within the calibration range), as long as
the confirmation is within 10%. Continue repeat analysis until
a concentration can be confirmed.

ii.

Concentrationsreouiri
verification of washout: Following a result
greater than the highest concentrations validated for washout (see
Table 9 of Appendix B) do the following:

1. lf the run was determined to be in-control for low concentration
samples before the next samples were analyzed, no further
action is required.

2. lf the run was not determined to be in-control for low
concentration samples before the next samples were analyzed
confirm by re-analysis the results for the 2 samples
immediately following the elevated sample. Report the results
if they confirm the initial results within t10o/o or +3SD of the low
bench QC, whichever is greater.

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8-05

Page 47 ol 94

b. Unacceptable reproducibilíty: lf the range of the three replicate
readings (maximum replicate concentration value - minimum replicate
concentratíon value) for a single sample analysis is greater than the
range maximum criteria listed ín Table 9 in Appendix B andthe range
of the three replicate readings is greater than 1 O% of the observed
concentration, do not use the measurement for reporting. Repeat the
analysis of the sample.

viii. Submittinq finalwork for review: All analyses must undergo quality control and
quality assurance review. After appropriately documenting the run in the
laboratory information system (e.9. sample and run QC, and run and sample
comments), inform the first level reviewer of the completed work and submit
any printed documentation.

9) Routine equipment maintenance and data backups
Maintenance activities will be documented in the instrument logbook

Equipment maintenance: Analysts are expected to regularly evaluate the need
for, and when necessary perform, cleaning, replacement, or re-positioning of
components in ICP-MS the sample introduction system, interface, ion optics
region, and equipment required resources (e.9. autosampler, exhaust,
compressed gases, and coolant). Frequency of equipment maintenance will be
dependent on instrument throughput.

Parameter optimízations: Analysts are expected to optimize instrument
parameters.

i. Dual detector calibration: Perform dual detector calibration regularly for any
element exceeding 1,000,000 cps for calibration standard 8. This is typically
only Pb. Dual detector calibration solution is described in Section 6.f.ii.
Frequency of dual detector calibration is typically monthly when throughput
requires multiple analytical runs per week, or as needed for optimized linearity.
ii. DRC optimízations: DRC conditions (cell gas flow rate and RPq value) can be
verified by analyzing the DRC optimization solutions (see Section 6.f.i) as
needed to ensure proper reduction of potential ICP-MS interferences.

Data backup: Data on the instrument computer will be backed up via two backup
routines. Files used and produced by the ICP-MS in analyzing samples will be
backed up and kept a minimum of two years after analysis.

i. Dailv backups to secondary hard drive: Program automatic backups of the
relevant computer files to occur each night onto a secondary hard drive to
prevent loss of data from failure of primary hard drive.
ii. Weeklv backup: Backup relevant computer files weekly either to secondary
hard drive which is remote to the laboratory or to removable media which will
be placed remote to the laboratory for retrieval in the case of catastrophic data
loss elsewhere.

blood multi-element analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05
I 0) Reporti ng

Page 48 of 94

thresholds

Reportable ranqe: Blood elemental concentrations are reportable in the range
between the method LOD and the highest calibrator (see'calibrator
concentrations' in Table 1) times the maximum validated extra dilution (see Table
8). Above the highest concentration verified, extra dilutions are made of the
blood sample to bring it within the reportable range.
b
Reference ranqes (normal values): ln this method the 95% reference ranges
(see Appendix B, Table 10) for these elements in blood fall within the range of
the calibrators.
c.

Action levels: Report concentrations observed greater than the "second upper
boundary" (defined in the laboratory database as the "2U8") 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 9 in
Appendix B. The protocolfor supervisors reporting elevated results to medical
personnel is defined according to the study protocol. But typically,
i. Lead: Levels of lead in blood of children ages 1-5 are considered elevated
above 5 pg/dl and chelation treatment is recommended at blood lead levels
>45 ¡rg/dL[65]. The Occupational Safety and Health Administration regulations
use a blood lead level of 40 pg/dL as cause for written notification and a
medical exam, and a blood lead level of 60 ¡rg/dl as cause for medical
removal from exposure[66].
ii. Cadmium: Levels of concern for cadmium in blood is >5 prg/L[67, 68].
iii. Mercurv: The American Conference of Governmental lndustrial Hygienists has
a biological exposure index (BEl) of 15 ¡rg/L for inorganic mercury in blood
(end of shift at end of work week)t681.

iv. Manoanese: lnsufficient data to establish an action level

v. Selenium: >500 ¡rg/L [69, 70]
111 Method Calculations

Method limit of detection (LODs): The method detection limits for elements in
blood specimens are defíned as 3 times so, where so is the estimate of the
standard deviation atzero analyte concentration. So 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.

Method limit of ouantitation (LOO) The Division of Laboratory Sciences does not
currently utilize limits of quantitation in regards to reporting limits [71]

C Limits: Qual ity control limits are calculated based on concentration results
obtained in at least 20 separate runs. lt is preferable to perform separate
analyses on separate days and using multiple calibrator lot numbers,

li,rr,

l,,l,,i , rt

iit

l, f lrii 'r lr iii trl i[ , i'tv,'

l(r

:ì 'ìir)i .ri:r.

i¡\[

)

instruments, and analysts to best mimic real-life variability. The statistical
calculations are performed us¡ng the SAS program developed for the Division of
La bo ratory Scien ces (D LS_QC_com p ute_cha r_stats. sas).

l2) Alternate methods for perform¡ng test and stor¡ng specimens if test system
fails:
lf the analytical system fails, the analysis may be setup on other ICP-MS instruments
in the laboratory. lf no other instrument is available, store the specimens at -4 "C
until the analytical system can be restored to functionality. lf interruption longer than
4 weeks in anticipated, then store blood spec¡mens at < -20'C.

blood multi-element analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8-05

Page 50 of 94

Appendix A: Critical parameter test results
Critical parameter test #1: Testing scenario of something preventing a set of prepared
samples from being analyzed immediately.
Test details:
Day 1: Prepared a set of dilutions (calibrators, blanks, reference material, fake samples)
for analysis in triplicate. Analyzed set 1 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
Day 3: Prepared run set 5 and analyzed it sequentially with run set 3
Table l. Ruggedness testing results: Evaluating the significance of time from preparation to analysis
on sample stability. Test performed 1216-8110 by Deanna Jones. Results are the average of the
beginning and ending QC results for each analytical run.
Time, prep to
ID
Hs (ps/L)
Pb (ug /dL)
Cd (¡rg /L)
Mn (us /L)
Se (pg /L)

Hg
J
@

Þs
F=
U)

ËË

õ5
ct)

analysis
target mean
and 3SD ranqe

0hr
24 hr (fresh)
48 hr
target mean
and 3SD ranqe

0hr
24hr
48 hr
target mean
and 3SD ranqe

0.585
0.318 - 0.852
0.418
0.504 (0.522\
0.396 (0.418)
6.19
5.74 - 6.63
5.86
5.46 (5.7)
2.64 (5.9\

2.12
1.99 - 2.25
2.03
1 .99 (2.18)
2.04 e.æ\
10.1

9.73 10.4
10.0
9.5 (10.7)
9.2 (10.0)

0.488
0.353 -0.623
0.399
0.419 (0.47\
0.509 (0.40)
3.14
2.84 - 3.44
3.03
2.85 (3.17\
2.79 (3.03)

7.98
6.38 - 9.59
6.09
7.06 (7.88)
7.82 (6.09)
14.9
12.8 - 17.1
12.5
13.6 (4.7)
13.5 (12.5)

0hr
24 hr
48 hr
target mean
and 2SD ranqe

0hr

ÉH

24 hr
48 hr
*samples purchase from Le centre de toxicology du
Quebec (Quebec, Canada)

-o"

228
206 - 251
192
202 Q17\
56 (192)
239
215 - 253
212
221 (238\
62 (212)

Conclusion: Samples whích have been diluted 1+1+48 for analysis up to one (1) day
previously can still be analyzed.

blood multi-element analysis by ICP-DRC-MS

PageSl of94

IRAT-DLS Method Gode: 301 6.8-05

Appendix A: Critical parameter test results (continued)
Critical parameter test #2: This test evaluated the s ignificance of the RF Power setting
of the ICP when analyzing blood samples for whole blood metals
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

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 averaqe of the beqinninq and endinq QC results for each analytical run
Mn (uq /L) Se (us /L)
ID
RF power (W)
Hq (uq /L)
Pb (uq /dL) Cd (uq /L)
target mean
7.98
2.12
0.488
0.585
0.398 - 0.578 6.91 - 9.05
0.407 - 0.763 2.03 -2.21
and 2SD range
=
l7.35
0.517
2.09
0.432
1150W
o
l1450 W
@
203
0.369
6.76
0.512
o
(default)
MN
Jco 1600 w
717
2.02
0.418
0.529
target mean
14.9
10.1
3.14
6.19
2.94 - 3.34
13.5 - 16.4
5.89 - 6.48
9.84 - 10.3
and 2SD ranqe
=
@
2.93
13.7
10.0
5.90
1150 W
o
fr
1450 W
@
12.8
10.2
2.90
6.23
o
(default)
dl o,¡
I

-co
@

fl¿"

>q
oó

1600 w
target mean
and 2SD range

5.99

10.1

3.07

13.3

1150W
1450 W

(default)
1600 w
target mean
anci 2SD ranqe
o
U)
1150 W
1450 W
(default)
1600 w
*samples purchase from Le centre de toxicology du Quebec (Quebec, Canada)

fra
>q
oó

293
273 - 313
269
288
314
1 65

i 54

179
147

146

Conclusion: Results are not compromised by changes in RF power within the range of
1 150W to 1600W.

blood multi-element analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page 52 of 94

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 oxygen (Oz) and the per method setting is 1.2 mUmin. The cell gas
flow rate for Se is methane (CH+) and the per method setting is 0.84 ml/min.
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.

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 (ug /L)
Pb (ps /dL) Cd (ug /L)
Mn (pg /L) Se (ug /L)
flow rate
N

cfl

=
l-

o
f-

=
æ
o
f@

o
m

target mean
and 2SD range
0.96 ml/min Oz;
0.7 ml/min CH¿
1.2 mLlmin Oz;
0.84 ml/min CH¿
1.44 mLlmin Oz;
1.0 ml/min CH¿
Target Mean
and 2SD Ranqe
0.96 ml/min Oz;
0.7 ml/min CH¿
1.2 mLlmin Oz;
0.84 ml/min CH¿
1.44 mLlmin Oz;
1.0 ml/min CH¿

0.585
0.407

2.12

0.763

2.O3

-2.21

0.488
0.398

0.578

7.98
6.91

0.457

2.10

0.471

8.49

0.479

2.10

0.438

8.15

0.555

2.11

0.457

8.12

6.19
5.89

10.1

6.48

9.84

10.3

3.14
2.94 -3.34

14.9
13.5

4.71

10.0

3.19

14.4

5.45

2.92

15.2

5.34

10.3

3.04

14.6

9.05

See

ïable 4

- 16.4

Conclusion: Accuracy of Mn and Hg results are not compromised by changes in cell
gas flow rate within the range tested (0.96 - 1.44 ml/min).

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page 53 of 94

Appendix A: Critical Parameter Test Results (Continued)
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
Mn (ps /L) Se (ps /L)
Hs (ps /L)
Pb (r¡g /dL)
Cd (pg /L)
flow rate
target mean
157
*
O)
- 168
146
oI and 2SD range
m
0.96 ml/min Oz;
187
lo 0.7 ml/min CH¿
Ø
1 2 ml/min Oz;
186
o
0.84 mL/min CH¡
I.JJ
1.44 mLlmin Oz;
(^l
1I1
1.0 ml/min CH¿
See
Table 3, Appendix A
293
target mean
+
N
273 - 313
o and 2SD ranqe
m
0.96 ml/min Oz;
328
@
o 0.7 ml/min CH¿
I

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LUB

Report the first analytically

valid result.

yes
no
> 2UB?

Repeat elevated result for
confirmation, and rcport the
first analytically valid result.

yes

no
> s8?

Repeat elevated result for
confirmation, and report the

first analytically valid result
as >2U8.

yes

> highest

concentration
validated for
washout?

Repeat elevated result for
confirmation by dilution in
duplicate, and report the first
analytically valid result as >2UB

yes

Repeat elevated result for confirmation
by dilution in duplicate, and report the
first analytically valid result as >2U8.

run verified in
control for
low conc
samples?

yes

repeat elevated result for
confirmation by dilution in
duplicate, and report the first
analytically valid result as >2UB

Confirm by re-analysis the results for the
2 samples immediately following the
elevated sample. Report first analytically
valid result when it is confirmed within
llOo/o or 13SD of the low bench QC,
whichever is larger,

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page 86 of 94

Appendix C: Help Sheets

Reaqent Preparation (paqe

I of 3)

NOTE:
mg/L = ppm
ug/L = ppb
ug/ml = ppm

Rinse solution
1. Partially fill a 4 liter bottle with >18 Mohm'cm water.
2. Add 0.4 grams of APDC.
3. Add 16 mL of TMAH (Tetramethylammonium hydroxide,2So/o w/w ((CH3)4NOH)
4. Add 40 mL of ethyl alcohol (C2H5OH ,200 proof)
5. Add 200 mL of 1% Triton X-100 (OR add 10mL of 2}%Triton X-100).
6. Add enough >18 Mohm'cm water to bring to 4 liter mark.
7. Mix well by gently inverting several times.
Sample diluent
1. Partially flll a 2liter bottle with >18 Mohm.cm water.
2. Add 0.2 gram of APDC.
3. Add 8 mL of TMAH.
4. Add 20 mL of ethyl alcohol.
5. Add 500 uL of a 20 mg/L stock solution of Te, Rh, and lr.
8. Add 100 mL of 1o/o Triton X-100 (OR, if using a20o/o Triton X-100 solution, add 5mL)
9. Add enough >18 Mohm.cm water to bring to 2 liter mark.
10. Mix well by gently inverting several times.

0.5% HNO3
(Garrier solution for optimization)

1. Partially fill a2liter bottle with >18 Mohm'cm water.
2. Add 10 mL of conc. HNOg.
3. Add enough >18 Mohm'cm water to bring to 2 liter mark

Mix well by gently inverting several times.

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Appendix

Page 87 of 94

Help Sheets (continued)

Reaqent Preparat¡on (page 2 of 3)
1o/o

vlv HNO¡

1. Partially fill a 10 liter bottle with >18 Mohm'cm water.
2. Add 100 mL of conc. HNOg.
3. Add enough >18 Mohm'cm water to bring to 10 liter mark.

Mix well by gently swiding several times.

vlv HNO¡
1. Partially fill a 2liter bottle with >18 Mohm'cm water.
2. Add 100 mL of conc. HNO¡.
3. Add enouqh >18 Mohm'cm water to bring to 2 liter mark.
5o/o

Mix well by gently inverting several times.

20% Triton X-í00
1. Partially fill a 1 liter bottle with >18 Mohm'cm water.

2. Add 200 mL of Triton X-100.
3. Add enough >18 Mohm'cm water to bring to 1 liter mark.
4. Allow to dissolve overnight (or add a Teflon magnetic stirring bar and stir on stirrer
until dissolved).
Mix well by gently inverting several times.

1o/o

Triton X-100

Partially fill a 1 liter bottle with >18 Mohm'cm water.
2. Add 10 mL of Triton X-100.
3. Add enough >18 Mohm'cm water to bring to 1 liter mark.
4. Allow to dissolve overnight (or add a Teflon magnetic stirring bar and stir on stirrer
until dissolved).
5. Mix well by gently inverting several times.
1

rnal standard solution
20 ppm Rh. Te and lr i
1. Partially fill an acid rinsed, 50 mL flask with 1o/o vlv HNOs.
2. Add 1mL of Rh from 1000ppm stock standard.
3. Add 1mL of Te from 1000ppm stock standard.
4. Add 1mL of lr from 1000ppm stock standard.
5. Add enough 1o/o vlv HNOo to fill to 50mL mark.
6. Mix well by gently inverting several times.
7. Pour the standard solution over into an appropriately labeled 50mL polypropylene
tube.

blood multi-element analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page 88 of 94

Appendix C: Help Sheets (continued)

Reaoen t Pre D aration (paqe 3 of 3)
Dailv solution (l ppbl in 2% v/v HNOg
1. Partially fill a 1 liter volumetric flask with >18 Mohm.cm water.
2. Add 1mL of High Purity Standard: SM-21 07-018 (or current lot #)
3. Add 20mL of concentrated HNOg
4. Add enough >18 Mohm.cm water to bring to 1 liter mark.
5. Mix well by gently inverting several times.

Stabilitv test soluti n (1 liter bulk preo)
1. Use a 1 liter bottle dedicated to stability test solution preparation
2. Add 960 mL of Sample Diluent
3. Add 20 mL of 'Junk" whole blood
4. Add 20 mL of Intermediate Working Calibration Standard (may use 51 or 52)

5.
6.

OR add 1.5mL of lntermediate Stock Calibration Standard.
Mix well by gently inverting several times.
Store in the refrigerator (when not using).

blood multi-element analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8-05

Appendix

Page 89 of 94

Help Sheets (continued)

Standard Preparat¡on (paqe 1 of 1)
(from single element stock standards)
Prepare 3% HCI v/v solution:

1. Partially fill a clean 2liter bottle with >18 Mohm'cm water.
2. Using a clean 50 mL polypropylene tube to measure, add 60 mL of high purity
3.

concentrated HCl.
Add enough >18 Mohm'cm water to bring to 2 liter mark.
Gently invert to mix.

Prepare intermediate stock standard (see Table 4 in Appendix B):

1. Partially fill a 100 mL volumetric flask with 3o/o vlv HCI solution
2. Label as: "HgPbCdMnSe lntermediate Stock Std"
3. Add 2 mL of HgPbCdMnSe multi-element stock solution.
4. Add enough 3% v/v HCI to bríng to 100 mL mark.
5. Mix well by gently inverting several times.

Prepare intermediate workinq standards (see Table 5 in Appendix B):

1. Partially fill each of eight, 100 mL volumetric flasks with 3% v/v HCI solution.
2. Label as: Intermediate Working Std "S1", "S2", "S3" and "S4", "S5", uS6", "S7"
and "S8".

3. For "S1 lntermediate
4. For "S2 Intermediate
5. For "S3 lntermediate
6. For "S4 lntermediate
7. For "S5 lntermediate

Working Std": add 50 uL of the lntermediate Stock Std.
Working Std": add 150 uL of the lntermediate Stock Std.
Working Std": add 350 uL of the lntermediate Stock Std.
Working Std": add 500 uL of the lntermediate Stock Std.
Working Std": add 1mL of the lntermediate Stock Std.
"56
For
lntermediate Workíng Std": add 50 uL of the Multi-Element Stock Std.
For "ST lntermediate Working Std": add 150 uL of the Multi-Element Stock Std
10. For "S8 lntermediate Working Std": add 400 uL of the Multi-Element Stock Std
1 1 . Add enough 3o/o vlv HCI solution to bring to 100 mL mark.
12.Mix well by gently inverting several times.
13.These intermediate working standards may be poured over into clean 15 mL
Falcon tubes for daily use (NOTE: "S0 lntermediate Working Std" is 3% HCI

L
L

only).

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

Page 90 of 94

References
1

2.
3.

4.
5.

6.
7.
8.
9.

10
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12
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15
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Mahaffey, K.R. NHANES 1999 - 2002 Update on Mercury. in Northeast Regional
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Bellinger, D.C., Low-level lead exposure, intelligence and academic
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Lauwerys, R., et al., Cadmium - Exposure Markers as Predictors of Nephrotoxic
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blood multi+lement analysis by ICP-DRC-MS

Page9l of94

IRAT-DLS Method Gode: 3016.8-05

21.
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Chiswell, B. and D. Johnson, Manganese, in Handbook on Metals in Clinical and
Analytical Chemistry, A.S. Hans G. Seiler, Helmut Sigel, Editor. 1994, Marcel
Dekker: New York. p. 467-478.
Smargiassi, A., et al., Peripheral Markers of Catecholamine Metabolism among
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77(1-3): p. 329-333.
Roels, H.A., et al., Assessment of the Permissible Exposure Levelto Manganese
in Workers Exposed to Manganese-Dioxide Dust. British Journal of lndustrial
Medicine, 1992. a9(1): p.25-34.
Cowan, D.M., et al., Manganese exposure among smelting workers: blood
manganese-iron ratio as a noveí iooi ior manganese exposuie assess¡¡le¡rÍ.
Biomarkers, 2009. M(1\ p. 3-16.
Gennart, J.P., et al., Feñility of Male Workers Exposed to Cadmium, Lead, or
ílllanaanaaa
,frgttvqr
r9ù9,

Âmariaan
^rlrvlrvqrr

la',¡nal
vvurrrqr

nf Eni¿.lamialnar¡
vl
LlJrvvrrrrvrvvt,

4OOt
tev1,

4)ñ9-4)40
'l?Ã/41\'
tl. n
tÊtv!vv\r
ì¿. rlvv

Bader, M., et al., Biomonitoring of manganese rn blood, urine and axillary hair
following low-dose exposure during the manufacture of dry cell batteries.
lnternational Archives of Occupational and Environmental Health, 1999. 72(8): p.
521-527.
Lauwerys, R., et al., Ferfility of Male Workers Exposed to Mercury-Vapor or to
Manganese Dust - a Questionnaire Study. American Journal of lndustrial
Medicine, 1985. 7(2): p. 171-176.
Standridge, J.S., et al., Effect of Chronic Low Level Manganese Exposure on
Postural Balance: A Pilot Study of Residenfs rn Soufhern Ohio. Journal of
Occupational and Environmental Medicine, 2008. 50(12): p. 1421-1429.
Woolf, 4., et al., A child with chronic manganese exposure from drinking water.
Environmental Health Perspectives, 2002. 110(6): p. 613-616.
Wasserman, G.A., et al., Water manganese exposure and children's intellectual
function in Araihazar, Bangladesh. Environmental Health Perspectives, 2006.
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Ljung, K.S., et al., Maternal and Early Life Exposure to Manganese in Rural
Bangladesh. Environmental Science & Technology, 2009. a3Q): p. 2595-2601.
Bazzi,4., J.O. Nriagu, and A.M. Linder, Determination of toxic and essentiai
elements in children's blood with inductively coupled plasma-mass specfrometry.
Journal of Environmental Monitoring, 2008. 10(10): p. 1226-1232.
Rollin, H.B., et al., Examining the assocrafion between blood manganese and
lead levels in schoolchildren in four selected regions of South Africa (vol 103, pg
160, 2007). Environmental Research, 2008. 106(3): p.426-426.
Rollin, H., et al., Blood manganese concentrations among first-grade
schoolchildren in two South African cities. Environmental Research,2005. 97(1):
p. 93-99.

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Code: 3016.8-05

37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
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55.

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Aschner, M., Manganese: Brain transporf and emerging research needs.
Environmental Health Perspectives, 2000. 108: p. 429-432.
Yokel, R.A., Brain uptake, retention, and efflux of aluminum and manganese.
Environmental Health Perspectives, 2002. I l0: p. 699-704.
Davis, J.M., Methylcyclopentadienyl manganese tricarbonyl: Health risk
uncertainties and research directions. Environmental Health Perspectives, 1998.
106: p. 191-201.
Davis, J.M., et al., The EPA health rsk assessment of methylcyclopentadienyl
manganese tricarbonyl (MMT). Risk Analysis, 1998. 18(1): p. 57-70.
Roels, H., et al., Relationship Between External and Internal Parameters of
Exposure to Manganese rn Workers From a Manganese Oxide and Salt
Producing Plant. American journal of industrial medicine, 1987. 1 f (3): p. 297305.

Jarvisalo, J., et al., Urinary and blood manganese in occupationally nonexposed
populations and in manual metal arc welders of mild-sfeel. International archives
of occupational and environmental health, 1992.63(7): p. 495-501 .
Smyth, L., et al., Clinical manganism and exposure to manganese in the
production and processrng of ferromanganese alloy. Journal of occupational
medicine, 1973.15(2): p. 101-9.
Klaassen, C., Biliary-Excretion of Manganese in Rafs, Rabbits, and Dogs.
Toxicology and applied pharmacology, 1974.29(3): p. 458-468.
Malecki, E., et al., Biliary manganese excretion in conscious rats is affected by
acute and chronic manganese intake but not by dietary fat. The Journal of
nutrition, 1996. 126(2): p. 489-498.
Agency for Toxic Substances and Disease Registry, Toxicological Profile for
Manganese, ATSDR, Editor. 2000: Atlanta, GA.
Agency for Toxic Substances and Disease Registry, Toxicological Profile for
Selenium. 2003: Atlanta, GA.
Goldhaber, S.8., Trace element nsk assessment: essentiality vs. toxicity.
Regulatory Toxicology and Pharmacology., 2003.38: p. 232-242.
Combs, G.F. and W.P. Gray, Chemopreventive agents. Pharmacology and
Therapeutics, 1998. 79: p. 179-192.
Arthur, J.R., Ihe role of selenium in thyroid hormone metabolism. Can J Physiol
Pharmacol, 1991. 69: p. 1648-1652.
Corvilain, 8., et al., Selenium and the thyroid: How the relationship was
established Am J Clin Nutr, 1993. 57 (2 Suppl): p.2445-2485.
Levander, O.A., Nutrition and newly emerging viral diseases; An overuiew. J
Nutr, 1997. 127: p.9485-9505.
McKenzie, R.C., T.S. Rafferty, and G.J. Beckett, Se/enium: an essentialelement
for immune function lmmunol Today, 1998. l9: p. 342-345.
Ellis, D.R. and D.E. Salt, Plants, selenium and human health. Curr Opin Plant
Biol, 2003. 6: p. 273-279.
Combs, G.F., Food system-based approaches to improving micronutrient
nutrition: the case for selenium. Biofactors, 2000. 12 p.39-43.

blood multi+lement analysis by ICP-DRC-MS
Page 93 of 94

IRAT-DLS Method Code: 3016.8-05

56.
57.
58.
59.
60.
61.
o¿.
^^

vv.
^a

64.
65.
66.
67.
68.
69.
70.
71.

Zimmerman, M.B. and J. Kohrle, The impact of iron and selenium deficiencies on
iodine and thyroid metabolism: biochemistry and relevance to public health.
Thyroid, 2002. 12: p.867-878.
Beck, M.4., O. Levander, and J. Handy, Selenium deficiency and viral infection.
Journal of Nutrition, 2003. 133: p. 14635-14675.
Lutz, T.M., P.M.V. Nirel, and B. Schmidt, Whole-blood analysis by ICP-MS.
Applications of Plasma Source Mass Spectrometry, ed. G. Holland and A.N.
Eaton. 1991, Cambridge: Royal Soc Chemistry. 96-100.
Tanner, S.D., Baranov, Vladimir l, Theory, Design, and Operation of a Dynamic
Reaction Cellfor ICP-MS. Atomic Spectroscopy, 1999. 20(2): p.45-52.
Tanner, S.D., V.l. 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.
Tanner, S.D. and V.l. Baranov, Theory, design, and operation of a dynamic
reaction cellfor ICP-MS. Atomic Spectroscopy, 1999. 20(2): p.45-52.
r r
a -t
Fr^L^-^^:^
^-^-L-^--^L--a--tt-,-----t
-L^---t:--slJeÇuurileuy
trtecuotneilnat
aK)tÍilu
apsutpuun
E urguef a, J.L., e[ at.,
determination of molybdenum in whole blood. Spectrochimica Acta Part B-Atomic
Spectroscopy, 2002. 57(3): p. 561-569.
Iarra{t
gqrrvtt,

I l\/l af
al Lttttttttgatttv
Elíminalinn
v.lYr.,
v( qa.,

mahth¡lanttm
l,tv,rvvv,tuttt

nwi¡la r.r.vt.vtvttvv
inlarFaran¡a
v^tvv

..in

ttrirta

¡a¡lrt¡ittt>'t

biomonitoring using ICP-DRC-MS. Journal of Analytical Atomic Spectrometry,
2008. 23(7): p. 962-967.
Division of Laboratory Sciences, Divrsion of Laboratory Sciences Policies and
Procedures Manual.2015, Centers for Disease Control and Prevention: Atlanta,
GA.
Centers for Disease Control and Prevention, CDC Response to Advisory
Committee on Childhood Lead Poisoning Prevention Recommendations in "Low
Level Lead Exposure Harms Children: A Renewed Call of Primary Prevention"",
Department of Health and Human Services, Editor. 2012: Atlanta, GA.
Occupational Safety and Health Administration, Occupational Safety and Health
Standards, in 29 CFR part 1910, Subpart Z, Standard number 1910.1025,
"Lead",. 1989,
Occupational Safety and Health Administration, Cadmium (OSHA 3136-06R

2004).2004.
American Conference of Governmental lndustrial Hygienists, Tlvs and Beis 2007:
Based on the Documentation for Chemical Substances and Physical Agents &
B iolog ic al Exposu re I nd ice s. 2007 : American Conference of Govern menta I
I ndustrial Hygienists.
Baselt, R.C., D,'sposition of Toxic Drugs and Chemicals in Man,.2011, Seal
Beach, CA: Biomedical Publications.
Nuttall, K., Evaluating selenium poisoning. Annals of clinical & laboratory
science, 2006. 36(a): p.409-420.
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, .

blood multi+lement analysis by ICP-DRC-MS
IRAT-DLS Method Gode: 3016.8-05
72

74
75
76

Page 94 of 94

Centers for Disease Control and Prevention, Foutth National Report on Human
Exposure to Environmental Chemicals, February 2015 Update. 2015, CDC:
Atlanta, GA.
Carson, 8.L., H.V.E. lll, and J.L. McCann, Se/enr¿.rm,in Toxicology and biological
monitoring of metals in humans., B.L. Carson, H.V.E. lll, and J.L. McCann,
Editors. 1986, Lewis Publishers, lnc.: Chelsea, Michigan. p.213-218.
Fell, J.M.E., et al., Manganese toxicity in children receiving long-term parenteral
nutrition. Lancet, 1996. 347(9010): p. 1218-1221.
Henn, 8.C., et al., Early Postnatal Blood Manganese Levels and Children's
N e u rodevelop me nt. Epidem íol ogy, 20 1 0. 21 $): p. 433-439.
lkeda, M., et al., Cadmium, chromium, lead, manganese and nickel
concentrations in blood of women in non-polluted areas in Japan, as determined
by inductively coupled plasma-secfor field-mass spectrometry.lnternational
Archives of Occupational and Environmental Health, 2011. 8,aQ): p. 139-150.

Division of Laboratory Sciences
Laboratory Protocol
Analytes:

Polybrominated diphenyl ethers (PBDES),
polybrominated biphenyls (PBBs), polychlorinated
biphenyls (PCBs) and persistent pesticides.

Matrix:

Serum / Plasma

Method:

Liquid-Liquid Extraction (LLE) / Silica-sulfuric ac¡d
cleanup and Gas Chromatography/lsotope Dilution High
Resolution Mass Spectrometry (GC/IDHRMS) Analysis

Method code: 6701.04

Branch:
Prepared By:

Organic Analytical Toxicology Branch (OATB)
Richard Jones

Author's name

nature

Author's name

Supervisor.

Date

Andreas Siodin
Supervisor's name

Branch Chief:

Date

¿/n /ø

Antonia Calafat
Branch Chiefs name

Date current version of method first used in lab:
Date

Director's Sign
Reviewed:

Date

Block

J
Signature

Pt¿

(t,

?
Date

Date

Procedure Ghange Log
Procedure:
Date

@a
Changes Made

DLS Method Gode: 6701.04

Rev'd
By

(lnitials)

Date
Rev'd

CDC
Environmenlal Health

Laboratory Proced ure Man ual
Analyte:

Polybrom¡nated diphenyl ethers
(PBDES), Polybrominated Biphenyls
(PBBs), Polychlorinated biphenyls
and Persistent Pesticides (PPs)

Matrix: SgfUm
Method:

lsotope dilution High resolution
Mass Spectrometry (IDHR-MS)

Method No: 6701.04

Revised: June 17, 2016

as pertormed by:
Organic Analytical Toxicology Branch
Division of Laboratory Sciences
National Center for Environmental Health
contact:
Dr. Andreas Sjodin

Phone: 770-488-4711
Fax: 770-488-0142
Email: ASiod [email protected]

James L. Pirkle, M.D., Ph.D.
Director, Division of Laboratory Sciences

lmportant lnformation for Users
CDC periodically refines these laboratory methods. lt 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.

BFRs, PCBs and PPs in Human Serum or Plasma
DLS Method

Gode:6701.03

DLS-OATB

Tablg Of GOntentS

Clinical Relevance and Summary of Test Principle

1.1

1.2

Clinical Relevance
Test Principle...

Safety Precautions..

2.1
2.2

... ... .:.
Biohazards. . . .
Chemical hazards
Hazardous waste handling

4
4

Computerization; Data System Management..

Data Entry and Transfer... .
Routine Computer Hard-Drive Maintenance....
Data Backup and Schedule of Back-ups... ...

5
5
5

2.3
3.
3.1

3.2
3.3

4.
5

Reagents and consumables.
Rinsing of expendables prior to use...
lnternal standards
Recovery standard.
GC/l DH RMS calibration standard
lnstrumentation.
Gilson 215 liquid handler.
Rapid Trace@, SPE work station.
Procedures for preparing quality control material.....

L
8.2
8.3
8.4
8.5
8.6
8.7
8.8
8.9

Procedures for Collecting, Storage and Handling of Specimens;
Criteria for Specimen Rejection
Procedures for Microscopic Examinations; Criteria for Rejecting
Inadequately Prepared Slides... ...
Preparation of Reagents, Calibration Materials, Control Materials,
and all Other Materials; Equipments and
Instrumentation

6.1.
6.1.1
6.1.2
6.1.3
6.1.4
6.2
6.2.1
6.2.2
6.3.
7.
7.1.
7.2.
7.3.
7.3.

8.1

::::::::

6
12

't¿
12
14
14
15
15
16
16
16
18

Calibration and Calibration Verification

Calibration of Mass Spectrometer
Creation of Calibration Curve... ...
Calibration Verification... ...
Standard concentrations and target isotopic ratios

19
19

20
20

Procedure Operation lnstructions; Calculations; lnterpretation of
Results.

Sending aliquot of serum for lipid determination.,
Thawing samples and weighing in samples.
Sample pretreatment, using Gilson 215 liquid handler... ..
Liquid-Liquid Extraction, using Gilson 215 Liquid Handler
Cleanup, using Rapid Trace SPE workstation.
Evaporation and transfer to final GC-vial.
GC/IDHRMS analysis of PBDEs and PBB
Final preparation of GC vial for PCB analysis
GC/IDHRMS analysis of PCBs/PPs....

20
21
21

22
23
25
24
26
26

BFRs, PCBs and PPs in Human Serum or Plasma
DLS Merhod Gode:

9.
10
11

12.
13.

14.
15.
16.

17
18
19

DLS.OATB

620r.03 Table Of GOntentS

Reportable Range of Results.....
Quality Assessment and Proficiency Testing... ..
Remedial Action if Calibration or QC Systems Fail to Meet
Acceptable Criteria... ..
Limitations of Method, lntefering Substances and Conditions
Reference Ranges (Normal Values)
Critical Call Results ("Panic Values")...
Specimen Storage and Handling During Testing.
Alternate Methods for Performing Test or Storing Specimens if
Test System Fails.
Test Result Reporting System; Protocol for Reporting Critical Calls
(if Applicable)
Transfer or Referral of Specimens; Procedures for Specimen
Accountability and Tracking.
References...

26
29
30
30
30
31
31
31
31

32
30

BFRs, PGBs and PPs in Human Serum or Plasma

DLS.OATB

DLS Method Gode: 6701.04

Page

1.
1.1

Clinical Relevance and Summary of Test Principle
Glinical Relevance
Organohalogen compounds may be characterized as halogen substituted
hydrocarbons, neutral and lipophillic organic compounds that are only very slowly
degraded or transformed under environmental conditions. According to the United
Nations Environmental Program, 12 polychlorinated compounds or compound groups
have been defined as persistent organic pollutants (POPs), including polychlorinated
biphenyls (PCBs) and2,2-bis(4-chlorophenyl)-1,1,1-trichloroethane (DDT) [1]. The
physicochemical properties of such man-made chemicals have led to their
accumulation in fatty tissues of wildlife and humans. This behavior of POPs was
basically unknown at the time of World War ll, when the chemical industry developed
these substances and made them available in increasing quantities. Organohalogen
compounds were commercially produced for use in agricultural, industrial and/or
household applications, while others were formed unintentionally during municipal
waste incineration, in other combustion and thermal processes or as by-products in
the chemical industry. For example, PCB products are industrial chemicals that were
rrca¿.|
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q9
qùvv
slvlvvlrrv

an¡{ haaf-av¡han¡ra
r¡vql
v^v¡rqrlvv

qrrv

flrri¡le
ac caalanfe
anr{ rrrrr¡h rnr-ìrÂ [?l
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applied as a pesticide, in agriculture and household applications [3].
The environmental implications first of DDT and later of PCB were not realized until
the 1960s, when DDT and also PCBs were detected at high concentrations (several
hundred to a few thousand ppm) in wildlife from the Baltic Sea region [3;4]. These
high concentrations of DDT; 2,2-bis(4-chlorophenyl)-1,1-dichloroethene (DDE), and
PCB were later found to correlate with toxicological effects observed in e.g. whitetailed sea eagles [5] and seals living in the Baltic Sea region [6-8]. However, the list of
organohalogen compounds present in the environment is long today, including
chemicals such as toxaphene, polychlorinated paraffins (CPs), polybrominated
r:-L^-..r
/ñnñr^\
t^:-t-^-..1^
/l-tl:tEl^\
^aL^-^
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urPilYilyrÐ [rErtr'ù), -^1..^Ll^-:-^1^l
urfJile¡ryr gr.ilers
(rDUEsr,
Pvtyvt lr(.,ililcrruu
(BCPS)
(PCNs),
and
numerous other
sulfone
naphthalenes
bis(4-chlorophenyl)
pesticides and technically applied substances. This illustrates the research needs
about environmental issues and persistent pollutants to hopefully avoid future
problems similar to those caused by PCB and DDT including bioaccumulation and
biomagnifications in fatty tissues.
Polybrominated diphenyl ethers (PBDEs) included in the group of chemicals known as
Brominated Flame Retardants (BFRs), have been and are still heavily used as
additive chemicals in polymers and textiles [9;101. Hence humans may be exposed
though food and/or though contact with flame retarded products [1 1-13]. lncreasing
PBDE levels have been observed in mothers' milk from Sweden [14] as well as in
blood from Germany [15] and Norway [16]. The PBDE levels are in general lower than
that of polychlorinated biphenyls (PCBs) in Europe [13;17]. However, the PBDE
concentrations found in the North Americans are considerably higher compared to
European subjects [11;13;17;181. The PBDEs are dominated by 2,2',4,4'tetrabromodiphenyl ether (BDE-47) 111;13;17;181. Decabromodiphenyl ether (BDE-

BFRs, PCBs and PPs in Human Serum or Plasma
DLS Method Code: 6701.04

DLS.OATB
2

209) is reported both in the general population and in occupationally exposed persons
showing the bioavailability of this high molecular weight compound [1 1 ;18;19]. While
the lower and medium brominated diphenyl ethers are persistent BDE-209 has a fairly
short half-life of approximately two weeks [19].
PBDEs have in pregnant mice been shown to cause neurodevelopmental disorders in
the offspring, as measured by behavioraltest systems 120;211. Neurodevelopmental
disorders in relation to exposure to PBDEs in humans has to date not been assessed,
although, such investigations are currently ongoing
Polybrominated biphenyls (PBBs) are another type of chemicals that in the past has
been used and applied for similar application areas as PBDEs 19.,221. No known
commercial production of PBBs currently exists. HexaBB has in humans been shown
to have a half-life of approximately 30 years [23].
1.2.

Test Principle
The method described in this manual assesses human body burden of BFRs,
specifically PBDEs and PBBs, as well as polychlorinated biphenyls (PCBs) and
persistent pesticides (PPs) in serum and/or plasma. This is done by measuring the
concentration in serum/plasma through the use of automated liquid/liquid efraction
and subsequent sample clean-up. Final determination of target analytes is performed
by isotope dilution gas chromatography high-resolution mass spectrometry
GC/IDHRMS.
Concentrations of target analytes are reported on two different concentration bases,
i.e., (i) fresh weight basis (i.e., pg/g serum) and (ii) lipid weight basis (i.e., ng/g lipid).
Lipid adjusted concentration values are preferable because (i) organohalogen
compounds are lipophillic and hence distribute in the body mainly according to the
tissues lipid content. Lipid adjusted concentrations correlates with the adipose tissue
concentrations of the chemical. Normalization according to lipid content further reduces
variability since differences in individuals serum lipid concentrations are cancelled out.

The samples are extracted using LLE, employing an automated Liquid Handling
instrument (Gilson 215 Liquid Handler@, Gilson, lnc.). Required sample pretreatment
prior to extraction is performed on the Gilson 215 liquid handler, including automated
addition of (r) internal standards, (r) methanolwith a manual vortexing step in-between
each addition. Hydrochloric acid is added manually to denature proteins in the sample
enabling efficient extraction of target compounds. During the extraction step the target
analytes are transferred from a water medium to an organic solvent.
Sample cleanup, i.e., removal of co-extracted lipids, is obtained by elution (5% DCM
in hexane; 10 mL) of the extract through a column containing from the top 0.25 g of
silica and 1 g of silica/sulfuric acid (33% by weight). Serum lipids are during this
procedure degraded in the sulfuric acid layer while cholesterol is removed in the top
layer consisting of activated silica gel. Without the activated silica gel layer cholesterol

BFRs, PCBs and PPs in Human Serum or Plasma

DLS.OATB

DLS Method Gode: 6701.04

Page 3

would eliminate water forming cholestene when coming in contact with the sulfuric
acid. Cholestene is not removed in the silica gel/sulfuric acid layer and would then
interfere in the final HR-MS analyses. The presence of cholestene causes an ion
suppression in the region of 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) and
2,2',4,4',$-pentabromod ip henyl ether (B DE- 1 00).

The lipid removal is automated using the Rapid Trace@ (Caliper Life Sciences). The
samplcs arc evaporated and transferred to GC vials. Evaporization is performed on
the Caliper TurboVap using increased temperature and a stream of nitrogen to aid
evaporization.
Serum concentrations are determined using gas chromatography isotope dilution high
resolution mass spectrometry (GC/IDHRMS), which minimizes or eliminates many
nterferences associated with low-resol ution measu rement of orga noha loge n
compounds. Splitless injection is used employing a short GC column (DB-SHT; 15 m
length, 0.1 pm film thickness, 0.25 mm lD) enabling the determination of high
molecular weight compounds such as decabromodiphenyl ether (BDE-209) having a
i

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most abundant ions in the isotopic cluster (fragment or molecular ion) are monitored
for the target analyte as well as for the 13C-labeled internal-surrogate standard.
Quantification is made against a calibration curve covering the full concentration range
of the target analytes. Serum PCB/PP concentration is also determined using
GC/IDHRMS but using a longer column (DB-5MS, 30 m length, 0.25 pm film
thickness, 0.25 mm lD).

2.
2.1

Safety Precautions
Biohazards
Foiiow Universai Precautions. Wear appropriate gioves, iab coat, anci proiective eye
glasses while handling human serum. Serum may be contaminated with pathogens
such as hepatitis or HIV; hence all safety precautions must be followed as outlined in
the laboratory hazardous chemicals exposure plan. Wear gloves, lab coat and glasses
at all times, and conduct all work in fume hood or biological safety cabinets (BSCs).
Place disposable plastic, glass, and paper (e.9., pipette tips, autosampler tubes, and
gloves) that come in contact with serum 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 serum was handled with a 10o/o (vlv)
sodium hypochlorite solution or equivalent

After an accident the CDC/ATSDR lncident Report must be filed according to
hazardous exposure control plan by supervisor.

BFRs, PCBs and PPs in Human Serum or Plasma

DLS.OATB

DLS Method Gode: 6701.04

Page 4

2.2.

Chemical hazards
Acids and Bases: Exercise caution when handling and dispensing concentrated
sulfuric acid, formic acid and nitric acid. Always remember to add acid to water. Acids
and bases are capable of causing severe eye and skin damage. Wear powder-free
gloves, a lab coat and safety glasses. lf acids or bases come in contact with any part
of the body, quickly wash the exposed area with copious quantities of water for at
least 15 minutes. Use safety shower if exposed area is not limited to hands and/or
arms. Use eye wash station in the event of eye exposure to acids and/or bases. ln the
event of an accident, lab colleagues will contact the clinic by phone or emergency
medical response by dialing 9-911.

Solvents: Solvents may penetrate skin causing long-term adverse health effects.
Exercise caution and always use gloves when handling solvents and other chemicals.
ln the event of spill on gloves immediately change to a new glove since solvents do
penetrate many gloves with time.

After an accident the CDC/ATSDR lncident Report must be filed according to
hazardous exposure control plan by supervisor.
2.3

Hazardous waste handling
Solvent waste: Collect solvent waste in waste bottles (empty solvent bottles may be
used). Clearly write WASTE on bottles, and the solvent(s) the waste bottle contains. lf
possible, always keep different solvents separated in dífferent waste bottles, since this
will make the final disposal of the different solvent wastes easier. When a bottle is
filled, arrange for waste pickup according the Chemical Hygiene Plan.
Serum waste: Dispose of serum waste originating as a waste fraction in the
extraction step on the Gilson Liquid Handler by completing the forms as outlined by
Chemical Hygiene Plan. Also attach a Memorandum stating that the contents of the
bottle are a mixture of hydrochloric acid, water, and serum that is considered to be
biologically inactivated by the acid present.
Sotid urasúes,'Sort solíd waste in three fractions and placed in metal boxes with lid
according to below and Chemical Hygiene Plan:
a

Non-Biogenic Contaminated Reusable Glassware (e.9. beakers, cylinders and
other reusable glassware). When the container is filled, label and return to
Glassware Services according to CDC protocol.

Broken glass includes used Pasteur pipets contaminated with biogenic materials,
or serum bottles and vials that are not reused. When this container is filled (r) add
approximately I L water to container, (,,) place sticker with your name, room and
buildíng number on container, (iií) place autoclave tape over lid and down the side

BFRs, PCBs and PPs in Human Serum or Plasma

DLS.OATB

DLS Method Gode: 6701.04

Page 5

of the box and (iv) bring the container to autoclave located in the loading dock,
building 103.
a

Gloves and other plastic parts contaminated with biogenic material - Place
biohazard bag in metal container before placing any waste in container. When
container is filled (r) add approximately 1 L water to container, (,,) place sticker with
your name, room and building number on container, (iii) place autoclave tape over
lid and down the side of the box, (iv) place autoclave sticker on container and (v)
bring the container to DLS designated handling area.

Computerization; Data System Management

3.1.

Data Entry and Transfer
Sample analysis results generated by this method are stored in SAS and/or Microsoft
ExcelrM software. The analytical 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 (lD), and method identifier.

3.2.

Routine Gomputer Hard-Drive Maintenance
Defragment the computer hard drive regularly by using software such as Norton
UtilitiesrM to maximize computer performance and maintain data integrity for files on
the hard drive.

3.3.

Data Backup and Schedule of Back-ups
GC/IDHRMS: lnstrument raw data files are mirrored through a local network
connection with each HRMS instrument computer to a local share drive Network Path:
\\'1-o2.168.210.3\volume_'1),,^,,hich is mirrored to a net¡rork share dri.,,e (ltlet'*ot'k Path:
\\cdc.qov\project\NCEH OATB HRMS Archive). Between the 1Oth and the 14th of
each month all generated instrument raw data is copied into the folder
X:\LONG_TERM_BACKUP_001 on the network share for compression into a monthly
compressed ZIP-file. The creation of the ZIP-file is an automatic process that runs at
mid-night on the 1sth of each month. The created ZIP-file is called POPLab_YYYYMM-DD where YYYY-MM-DD is a date time stamp. After completion of the monthly
backup all instrument operators will be informed over email that the backup has been
completed and any raw-files from the preceding month should be transferred to a local
archive folder on the instrument computer. After completion of the monthly backup the
compressed ZIP-file will be made available on the lab share in the folder
Z:\_Shared_Folders\_01_BACKUP_GOING BACK 6 MONTHS which is a
synchronized folder between the lab and network share drives. The monthly backup
ZIP-file will be made available on the Lab Share for at a minimum 6 months after
which older backups are accessible on the Network Share drive in the folder
X:\LONG_TERM_BACKU P_00 I \ZipFiles.

BFRs, PCBs and PPs in Human Serum or PIasma

DLS.OATB

DLS Method Gode: 6701.04

Page 6

Procedures for Gollecting, Storage and Handling of Specimens;
Criteria for Specimen Rejection

.
o
o
o

No special instructions for fasting or special diets are required, although, preferably
the sample has been drawn in the morning before breakfast (i.e. fasting).
The specimen type is serum or plasma.
Minimum preferred serum amount is 0.5grams and the minimum acceptable
amount is 0.125 grams.
Acceptable containers for storage are thick-walled glass vials with TeflonrM-lined
caps or cryovials or equivalent container. Rinse contaíners using the same
procedure as for other glassware used in the current method (see section 6.1).
Preferred container is a 10 mL Wheaton glass serum vial.
The criteria for an unacceptable specimen are either a low volume (. 0.t25 g) or
suspected contamination due to improper collection procedures or collection
devices. ln all such cases, request a second serum specimen. The limit of
detection for the minimum acceptable serum amount 0.125 to 2 g of serum is given
in Table 1.
Transport and ship frozen serum specimens on dry ice. Upon receipt, they must
be kept frozen at < -60 oC until time for analysis. Refreeze at < -60 oC any portions
of the sample that remain after analytical aliquots are withdrawn. Samples thawed
and refrozen several times are not compromised.

BFRs, PCBs and PPs in Human Serum or Plasma

DLS-OATB

DLS Method Code: 6701.04

Page 7

Table 1. Method limit of detection (LOD, pg/gram of serum) by target analyte and used sample amount
(gram). The method LOD corresponding to the minimum preferred sample amount of 0.5 grams are colored

in blue, method LODs between the minimum preferred sample amount and the minimum acceptable
sample size are colored in red. Method LODs two and four fold higher than the minimum preferred sample
amount are colored in green. A sample amount greater than the minimum preferred sample amount may be
used to lower the method LOD. Any sample for which the available serum amount for measurement is less

than the minimum acceptable serum amount of 0.L25grams will be reported as QNS (Quantify Not

Sufficient) in reportable data tables.

Class
BFR

Analyte

PBDE17

Serum

Weight (g)
0.125

8.8

BFR

DED
ut t\

BFR

PBDE28

PBDE47

DDñCêC
f uvLwv

PBDE85

PBDE99

0.125

o.25

4.4

0.25

0.375

2.9

0.375

0.5

2.2

0.5

L.L

0.55

BFR

0.125

5
lq,

2
BFR

PBDElOO

0.L25

lì 1tr

Âa

n 't(

0.375

4,5

0.375

6.9

0.5

3.4

0.5

5.2

1..-/

2.6

0.85

0.125

BFR

PBDE153

1.3

0.125

0.25

0.2s

0.375

0.5

1.t

5.5
DOñEl tr/t

0.125

r'ì I îE

1ô

0.2s

1.4

0.25

0.375

9.6

0.375

t.¿

0.5

1.2

0.5

5.4

3.6

2.7

1.8

0.125

DED

BFR

PBDE183

i.4

0.125

1000

0.25

8.4

0.2s

520

0.375

5.6

0.375

3s0

0.5

4.2

0.5

260

2.1.

130

1..1.

Method LOD defined the highervalue of

(Taylor, K. T. (1987) ln Quality Assurance of Chemical

Measurements, pp 79-82, Lewis Publishers, Washington, DC) and three times the standard deviation of
blank samples. Method LOD determination based on gennerated measurements during 2015 and Lst and
2nd quarter of 2016.

BFRs, PGBs and PPs in Human Serum or Plaema

DLS-OATB

DLS Method Code: 6701.04

Page

Table 1(Continued). Method limit of detection (LOD, pg/gram of serum) by target analyte and used sample
amount (gram). The method LOD corresponding to the minimum preferred sample amount of 0.5 grams are

colored in blue, method LODs between the minimum preferred sample amount and the minimum
acceptable sample size are colored in red. Method LODs two and four fold higher than the minimum
preferred sample amount are colored in green. A sample amount greater than the minimum preferred
sample amount may be used to lower the method LOD. Any sample for which the available serum amount
for measurement is less than the minimum acceptable serum amount of 0.L25grams will be reported as QNS
ble data tables
Quanti Not Sufficient tn re

Class Analyte
BFR PBDE2O9

BFR

PCB

PBB153

PCB28

PCB66

PCB74

Serum
Weicht

(s)

Method

LOD

0.125

180

Class
PCB

Analyte

PCB99

Method

Serum

Weight

(g)

LOD

(pglg serum) "

o.t2s

0.25

0.375

0.5

0.125

11. PCB

PCB105

3.5

0.125

o.2s

5.6

0.2s

0.375

3.7

0.375

0.5

2.8

0.5

1..4

7.5

o.7

0.125

PCB

PCBLL4

3.8

0.125

0.25

o.25

9.6

0.37.5

0.375

6,4

0.5

4.8

9.4

2.4

4.7

1.2

0.125

0.25

0.375

0.5

8.5

8.6

4.3

0.125

0.2s
0.37s

0.125

PCB

PCBL18

PCB138-1s8

4.3

0.125

t20

0.2s

0.25

0.375

0.5

8.5

4.3

7.5

Method

LOD

(pglg serum)

defined the highervalue of

(Taylor, K. T. (1987) ln Quality Assurance of Chemical

BFRs, PCBs and PPs in Human Serum or Plasma

DLS-OATB

DLS Method Gode: 6701.04

Page 9

Table 1 (Continued). Method limit of detection (LOD, pg/gram of serum) by target analyte and used sample
amount (gram). The method LOD corresponding to the minimum preferred sample amount of 0.5 grams are

colored in blue, method LODs between the minimum preferred sample amount and the minimum
acceptable sample size are colored in red. Method LODs two and four fold higher than the minimum
preferred sample amount are colored in green. A sample amount greaterthan the minimum preferred
sample amount may be used to lowerthe method LOD. Any sample for which the available serum amount
for measurement is less than the minimum acceptable serum amount of 0.125grams will be reported as QNS
(Quantify Not Sufficient) in reportable data tables.
Serum Method LOD
Serum Method IOD

Class Analyte
PCB PCB146

PCB

PCR

PCB153

PCB156

PCR157

Weight

(g)

Class Analyte
5L PCB PCB170

(pglg serum) "

(pglg seruml'

0.125

0.2s

0.37s

0.5

6.4

6.6

3.2

3.3

PCB

0.t25

0.25

0.2s

7.)

0.375

4.8

0.5

3.6

8.8

1.8

4.4

0.9

0.125

PCB

PCB772

0.L25

0.2s

0.25

8.4

0.375

21.

0.375

5.6

0.5

4.2

0.125

PCB177

1.7

2.1.

3.9

1..7

n 1?5

0.25

PCR

PCR17ß

1õ
LJ

n 1?q

o.2s

0.375

a1
LI

0.5

6.4

2
PCB1.67

(g)

o.25

0.L25

0.375

PCB

Weight

J.J

0.r25

PCB

PCB180

5.¿

0.r25

0,25

0.25

0.375

0.5

1.4

0.5

6.8

7.4

3.4

3.1

Method LOD def ined the higher value of

(Taylor, K. T. (1987) ln Quality Assurance of Chemical

BFRs, PCBs and PPs in Human Serum or Plasma

DLS.OATB

DLS Method Gode: 6701.04

Page 10

colored in blue, method LODs between the minimum preferred sample amount and the minimum
acceptable sample size are colored in red. Method LODs two and fourfold higherthan the minimum
preferred sample amount are colored in green. A sample amount greaterthan the minimum preferred
sample amount may be used to lowerthe method LOD. Any sample for which the available serum amount
for measurement is less than the minimum acceptable serum amount of 0.1.25grams will be reported as QNS
(Quantify Not Sufficient) in reportable data tables.

Method LoD.
serlm Method LoD
crass Anaryte -serum
crass
Anaryte
Weight (C) (pglg serum) "
Weight (g) (pglg serum) "

PCB

PCB183

PCB187

PCB189

PCB194

0.L25

0.25

o.2s

0.375

0.5

1.4

0.5

6.8

3.4

PCB196-203

0.725

o.2s

0,375

0.375

0.5

6.7

3.4

LOD

PCB

PCB206

o.L2s

0.r25

o.2s

0.25

o.375

0.375

0.5

PCB209

7.3

6.5

3.7

3.3

PST

0.r25

0.125

L10

0.25

0.375

0.5

1.4

0.5

6.9

1.4

HCB

3.5

100

0.125

PsT

B-HCCH

0.t25

220

0.25

110

0.375

0.5

6.5

1.4

Method

PCB

PCB199

0.25

0.125

PCB

o.125

defined the higher value of

(Taylor, K. T. (1987) ln Quality Assurance of Chemical

BFRs, PCBs and PPs in Human Serum or Plasma

DLS.OATB

DLS Method Code: 6701.04

Page

for measurement is less than the minimum acceptable serum amount of 0.125grams will be reported as QNS
(Quantify Not Sufficient) in reportable data tables.
Serum Method LOD
Serum Method LOD

Class

PST

Analyte

G-HCCH

Weight

(g)

(pglg serum) "

0.125

PST

IDCT
Jr

PST

OXYCHLOR

Ï-NONA

DD ññE

OP-DDT

PST

Analyte

PP-DDT

Weieht

(cl

(pglg serum) "

0.125

140

0.25

0.375

0.5

7.9

o.!25

0.25

0.375

0.5

'1.

7,2

7,5

3.8

PST

Class

0.125

ntq
0.375

3.6

0.r25

0.25

0.375

0.5

8.9

4.5

0.125

1Crì

0.25

130

0.375

ð5

0.5

o.r2s

0.25

0.37s

0.5

Method LOD defined the highervalue of

PST

MIREX

3
Ss

(Taylor, K. T. (1987) ln Quality Assurance of Chemical

Measurements,ppT9-82, Lewis Publishers, Washington, DC) and three t¡mes the standard deviation of
blank samples. Method LOD determination based on gennerated measurements during 2015 and 1st and
2nd quarter of 20L6.

BFRs, PGBs and PPs in Human Serum or Plasma

DLS.OATB

DLS llllethod Code: 6701.04

Page

Procedures for Microscop¡c Examinations; Griteria for Rejecting
lnadequately Prepared SIides
Not Applicable

Preparation of Reagents, Calibration Materials, Control Materials, and
all Other Materials; Equipments and lnstrumentation

6.1

Reagents and consumables
The method has been validated using the chemicals, solvents and consumables listed
in Table 2 and 3. Other manufacturer's products of equivalent purity can be used after
verification of chemicals purity.

BFRs, PGBs and PPs in Human Serum or Plasma

DLS.OATB

DLS Method Gode: 6701.04

Page

Table 2. Solvents and chemicals used for development of current methodology,
equivalent products from other manufacturer may be used with exception to the SPE
sorbent.
Chemical/Solvent
Manufacturer
Grade

Acids
Hydrochloric acid
Sulfuric acid

Aldrich
Aldrich

37%
95-97%

TEDIA
EM Science
TEDIA
TEDIA

Pesticide
min 99%
Pesticide
Pesticide

TEDIA
Siqma
TEDIA

Pesticide
99%
Pesticide

Waters
Sigma

nla

Solvenfs
Dichloromethane
Dodecane
Hexane
Methyl ferf-Butyl
Ether (MTBE)
Methanol
n-Nonane
Water
SPE sorbenfs
OASIS HLB@
Silica gel

100-200
mesh

Table 3. Expendables used for development of current methodology, equivalent
be used
roducts from other manufacturer m
lfam

M¡n¡ rf¡afr ¡¡.arfQ¡rr rr¡a

Glassware and caps
Test tube 16 x 100 mm
Septum for test tube
Open top cap for test tube
Borosilicate GlassPasteur pipette
Boston Round (amber qlass bottle)
V-vial (3 mL) with septum-cap
GC vials and caps

Fisher
Fisher
Fisher
Fisher
Fisher
Fisher
Fisher

Scientific
Scientific
Scientific
Scientific
Scientific
Scientific
Scientific

Others
Label printer (Bradv TLS PClink)
Maqnetic stirrer (heavv dutv, larqe)
Pipette disoenser

Fisher Scientific
Fisher Scientific
VWR

BFRs, PCBs and PPs in Human Serum or Plasma
DLS Method Code: 6701.04

6.1.r

DLS-OATB
14

Rinsing of Gonsumables Prior to Use
PBDEs and other brominated flame retardants are common indoor pollutants. Clean
all glassware including new glassware according to following procedure to eliminate
risk of sample contamination.

Culture tubes and other glassware: Rinse glassware first in dishwasher (Labconco,
Steam Scrubber or equivalent dish washer). Place test tubes in racks and insert them
in the dishwasher. Place detergent in reservoir in the door, and start the dishwasher
using program "Scientific".
After completion of the program, transfer the glassware to the oven located next to the
dishwasher. After a heat cycle of at least 12 hours at >200 oC, the glassware is ready
to be used.
For satellite bottles such as glass tapered-stopper bottles intended for storing for small
volume, everyday use in the BSC the normal large labels are not to be used because
it would interfere with the proper procedure for re-cleaning them. Instead label by
hand using a "Sharpie" pen and affix a small hazard pictogram sticker to the bottle or
alternatively attach a sheet of paper on the fume hood/BSC were relevant chemicals
are listed by name with appropriate pictogram.

Caps and septa: Rinse caps and septums for test tubes prior to use to remove
contaminants. This is done by Soxhelet extraction for five hours using methanol as the
extraction solvent. Alternatively, if the Soxhelet apparatus cannot be used it is also
acceptable to sonicate the items in methanol (20 min x 3 times). After cleaning the
items, allow them to dry on aluminum foil. After the caps are completely dry, place
them in a large glass beaker or in plastic re-sealable bags (not in cardboard boxes) for
safe storage until used.
Gas Ghromatography Vials: Heat GC vials in an oven at >200 oC overnight prior to
use. Store vials in a beaker covered with aluminum foil. The caps for GC vials are
cleaned by Soxhelet extraction, using the same procedure as for caps and septum's

Pasteur Pipets: Place glass Pasteur pipets in oven on aluminum foil and heat the
oven to >200 oC overnight. After completing the heating cycle for at least 12 hours, the
pipets are ready to be used.
6.1.2

lnternal standards (lS)
The current method is validated for BFRs, PCBs, and acid stable persistent pesticides
(PPs). Use three internal standard spiking solutions for quantification of the three
compound classes included. Order these standards pre-made from Cambridge
lsotope Laboratory (ClL). The PBDE standard contains 7.5 pg/pl of 10 different 13Cplabeled PBDE and PBB congeners. The PCB standard contains 7.5 pgl¡tL of 21

BFRs, PCBs and PPs in Human Serum or Plasma

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Page

different PCB congeners and the PP standard contains 11 13C-labeled PPs. CIL
supplies the spiking standard, in 10-mL ampoules.
When opening a new ampoule transfer the standard to a Wheaton 3-mL vial. Label
the vial with "BFR lS", 'PCB lS" or "PP lS' using a computer-generated label.
Note the weight of the container, and the date the ampoule was opened. (The weight
is used to detect any potential evaporation of the standard during storage) One vial of
each standard is consumed in each analytical run on the automated liquid handler.
(See 8.3)
File the certificate of analysis from CIL for each internal standard solution in the SOP
binder located in building 103, room 2103.

6.r.3

Recovery standard (RS)
Use one recovery standard for measurement of recovery. This standard contains
1234-13Cø-TC DD (2. 5 pg/U L), 3C r z-C 8-208 (1 0. Opg/p L) and 3Cr z-BDE-1 39
(10.0p9/pl) in hexane containing 10% nonane and 2Yo dodecane by volume. Add the
standard (100¡rL) to the GC vial during initial liquid handling. Transfer and mix the final
extracted and purified sample with the recovery standard at the end of the procedure.
Nonane and dodecane is present in the standard to act as a "keeper" (solvent that will
not evaporate or evaporate to a lesser degree during subsequent evaporation step) to
reduce evaporation losses during the final evaporation step. (This recovery standard
is ordered pre-made from CIL)
1

When opening a new ampoule the standard is transferred to a Wheaton 3-mL vial,
and the vial is labeled using a computer-generated label. The weight of the container
is noted as well as the date the ampoule was opened. The weight is used to detect
any potential evaporation of the standard during storage. One vial of recovery
standard is consumed in each analytical run on the automated liquid handler. See 8.3
File the certificate of analysis from CIL for the recovery standard solution in the SOP
binder located in building 103, room 2103.

ß4lt

GC/!DHRMS Galibration Sr¿ndard (CS)
The calibration standards includes several calibration levels denoted CSX (X=1
through 10). This standard is prepared by CIL and delivered in ampoules.

When opening a new ampoule, aliquot the standard into GC vials (5-1OuL in each
vial). Label the vials BFRX, PCBX, and PSTX where X corresponds to the calibration
point lthrough 10 using a computer-generated label. Replace the standards used for
calibration of the DFS after completion of every run.

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6.2
6.2.1

lnstrumentation
Gilson 215liquid handler: Liquid handling is automated using the Gilson 215 Liquid

handler, cf. Figure L Place the samples in the auto-mix to the far right in Figure 1. The
probe (moving arm) picks up and dispenses reagents (internal standards, methanol
and water) to the samples according to a predefined sequence with mixing in-between
each type of addition.
Recovery of the internal standards, as a percentage, is an important quality
measurement of the analytical run. ln order to enable recovery measurements, in this
automated procedure, recovery standard will be added to empty GC vials located in a
rack at the far left in Figure 1. These GC vials will be stored capped until the last step
of the sample preparation method in which the purified extract will be transferred to
the GC vials and mixed with the recovery standard.

Figure L Gilson 215 Liquid Handler used for automated additions of internal surrogate standards and
water to the serum samples with mixing by rotation in-between the additions. This equipment also adds
recovery standard to GC vials.

6.2.2 Rapid Trace@, SPE work station: The Rapid Trace@ SPE workstation (Caliper

Life

Sciences) (Figure 2) includes (A) syringe pump for drawing and dispensing solvents and
sample (B) mixing chamber (not used in this method), (C) plunger, compressing SPE
cartage and dispensing liquids through cartridge, (D) cannula used for drawing serum
sample from test tube and (F) rack containing serum samples and collected fractions.
The Rapid Trace@ instrument processes the samples in sequence. Up to 10 samples

BFRs, PCBs and PPs in Human Serum or Plasma

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Page 17

per module for unattended cleanup. Six modules are used for the default batch size of
30 samples, resulting in simultaneous processing of six samples at any one time.

(A)
(B)

(E)
..o

-{
,-o

-o
.-o

tt..t"at

-o

;

a.:..-.

,Ì:ttt¡./

f......

/tr\
\rl

--i

i¡:Ðll

Figure 2. Rapid Trace modular SPE work station up to 10 modules controlled by one computer.
lnstrument includes (A) syringe pump, (B) mixing chamber [not used in current method], (C) plunger,
(D) cannula and (F) sample and fraction collection rack.

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6.3

Procedures for preparing quality control materials
The QC material for this assay is bovine serum in which the concentrations of the
target analytes have been certified. One QC sample is analyzed in every set of 10
samples to ensure comparability and reliability between different sets of samples over
time. ln addition to the QC sample, a bovine blank is analyzed in every set of 10
samples. The method is designed to include several sets of 10 samples to be
analyzed in parallel in one batch. (See Sample preparation below).
Specific predefined rules are applied in order to determine if the QC sample analyzed
in one set is in agreement with previously analyzed QC samples. lf the QC sample is
found to be an outlier that set has to be reanalyzed. Example QC rules are below. All
QC rules are checked by the DLS QC program available in StarLlMS.
The QC determination must not deviate more than 3 times the standard
deviation from the mean value of previous determinations of the same QC
pool, and
No more than ten consecutive QC samples may fall either above or below
the mean value of previous determinations of the same pool after one data
point has fallen outside of +l- 2SD. lf the QC sample fails any of these tests
the set of unknown study samples must be reanalyzed.

(¡)

(ii)

For further details, see data handling section below

Day 1: Rinse the vials (including caps in which the serum will be aliquoted) according
to the procedure outlined in glassware rinsing procedures before use (see section
6.1.2. Label the vials with computer-generated labels.
This label should contain a unique name, constructed from the page number in the
pool note book. For example SERUM:02:03 where 02 is the notebook number and 03
is the page number. State the date of the pool preparation on the label.
Thaw the serum by submerging the container in water (37 oC) until the serum is
completely thawed. Pour the serum into a large beaker (4 L) containing a heavy-duty
stir bar (45-mm length). Spike with native analytes to appropriate concentration level,
e.9., 500 pg/ml, and stir solution overnight using a magnetic stirrer.
Day 2= While still stirring the solution, transfer serum in 6.0 mL aliquots to each of the
vials. Cap the vials and place them in cardboard boxes (e.9., a lid for Xerox paper
boxes) for simple freezer shelf organization. Place one identifying label on the edge of
the cardboard box and place in freezer (-70 oC).

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7.
7.1

Calibration and Galibration Verification
Calibration of Mass Spectrometer
Calibrate and tune the Thermo DFS mass spectrometer using the appropriate
calibration gas (either high boiling PFK (perfluorokerosene) for BFR analysis or FC43
for PCB/PP analysis) according to the instructions in the operator's manual.

Sensitivity Check prior to analytical run:
o BFRs.' After tuning the instrument to 10,000 resolution, a greater than 10:1 signal
to noise ratio for the native ions is required for an injected CS1 standard (0.2p9lul)
except PBDE209 which needs to meet a signal to noise ratio of 10:1 for the CS4
standard (Spg/uL).
o

PCBIPST: After tuning the instrument to 10,000 resolution a 100:1 signalto noise
ratio for the injection of 0.01pg/ul of 2378-tetrachloro-p-dibenzodioxin (TCDD) with
a 2ul injection (20f9 on-column).

Mass Spectrometer gain checks are performed when the multiplier is replaced or as
needed. A Magnetic Calibration (MCAL) is performed during routine PMs and/or as
needed. An Electric Calibration (ECAL) is performed during routine PMs and/or as
needed.
7.2.

Greation of Calibration Curve
A linear calibration curve, consisting of at least five CS standards with concentrations
ranging from 0.5 to 500 pg/1.¡L, is generated using the ratio of the peak area of the
analyte to the labeled internal standard.
The R-squared value of the curve must be equal or greater than 0.995. Linearity of the
standard curve must extend over the entire standard range.
The lowest point in the calibration curve is the lowest reportable level and the highest
point is highest reportable value. The remainder of the points are equally distributed
between the two extreme concentrations (on a log scale).
Generate a new calibration curve with every new set of sarrrples to be an'ralyzerl, using
the certified calibration stancjards from ClL. Before using a new batch of standards
with the current method, verify that the new standards agree with in 2Oo/o of the old
standard, this is accomplished by quantifying the new standard using the old standard
The certified value (pg/Ul) of the new standard must be within 20o/o of the in-house
quantified value (pg/pl). The tolerance of 20o/o between new and older standard is
derived from the certificate of analysis giving a 1Oo/o tolerance of each standard
released by ClL. Due to the fact that the response ratio between a native and 13Cfabeled internal standard is measured, a maximum deviation oÍ 2Oo/o is used. This is
accomplished by quantifying the new standard using the old standard. The certified
value of the new must be within 20o/o of the in-house quantified value.

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7.3.

Calibration Verification
Calibration verification of the test system is done by the inclusion of quality control
samples with a determined concentration in every run of unknown specimens and by
the analysis of Proficiency Testing (PT) samples at least twice per year. See section
10 for further information on PT procedures.

7.4.

Standard concentrations and target isotopic ratios
The specified concentration for analytical standards and target isotopic ratios for all
measured analytes are given in the files MS PARAM SASEG 20YY-MM-DD.xlsx and
STD CONS 20YY-MM-DD.xlsx where YY-MM-DD is the creation date of the file.
These files are located at the DLS share drive at the location:
\\cdc.qov\project\NCEH_OATB SASEG HRMS\LOOKUP TABLES
Standard concentrations are also specified in the manufacturers COA included in the
SOP binder.

Procedure Operation lnstruct¡ons; Galculations; lnterpretation of
Results
Formal training in the use of a high resolution mass spectrometer is necessary for all
GC/HRMS operators. Users are required to read the operation manuals and must
demonstrate safe techniques in performing the method. New operators must be
evaluated after 6 months of initial training by the supervisor to certify that they are
appropriately qualified to perform the assay.

Anyone involved in sample preparation must be trained in sample preparation
equipment, chemical handling, and have basic chemistry laboratory skills. The training
may be delegated to more experienced analyst.

8.1

Sending aliquot of serum for lipid determination
Serum lipid concentration in serum is determined in an aliquot of the sample (100 pl)
using enzymatic methods by the Clinical Chemistry Branch (CCB). Aliquot 100 ¡rl of
each sample into polypropylene vials after mixing the thawed serum samples; use a
new pipette tip for every sample to avoid cross contamination. Label vials for lipid weight
determination with Study name, Study Number and notebook number. A lipid aliquot
may have been drawn upon arrival of the samples to CDC and prior to the samples
being sent to the POPs laboratory in which case no additional lipid aliquot needs to
made prior to analysis.

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lf the available sample amount is low (<1mL of serum) then the entire sample may be
sent to CCB for lipids measurements to minimize losses of serum during aliquoting. ln
this case the sample is returned to the POPs lab upon completion of the lipids
measurements.
8.2.

Thawing and weighing samples
Store samples in a -70 oC freezer before starting analysis. Samples are taken out
from the freezer to thaw completely; this can be done the day before analysis and the
samples placed in a refrigerator overnight. Thoroughly mix the samples by vortex. For
each batch of 30 samples, complete a run sheet. On the run sheet, enter ALL
requested information under heading "Contact lnformation", e.9., analyst's name or
initials, the date and run number.
Print four complefe sets of labels for the samples to be used during the cleanup
procedure.

To ensure optimum performance of the balance (Ohaus Adventure) used for weighing
serum samples, verify the balance calibration using NIST calibration weights spanning
the range 1.000 g and 10.000 g before weighing each batch of samples and document
recorded weights on the run sheet in the "Balance Calibration" section. Calibration
weights are placed on the balance after taring, and the reading is recorded on the run
sheet. The difference from true value may not exceed +/- 0.01 g. lf this limit is
exceeded, any problems must be resolved, such as cleaning the balance tray,
recalibration of balance and/or calling for service of balance. After verifying the
balance calibration, weigh serum samples into 16 x 100 mm test tubes with septumequipped open-top screw caps. Record all sample weights on the run sheet.

8.3.

Sample pretreatment, using Gilson 215 - Liquid handler
Procedure

A. Adjust

the volume of the sample to 2mL if less serum was available for the
measurement.
B. Place new internal standards vials containing the internal standards in the rack
on the Gilson 215.
C, Begin the Gilson Spiking Application in Trilution LH. During the procedure all
samples are fortified with the internal surrogate standards (Approximately 20
minutes).
D. After completion the Gilson spiking application is complete, the samples are
removed from the Gilson and vortexed manually for at least 10 seconds each.
E. Next 0.5mL of 6M hydrochloric acid is added to each sample. All samples are
then vortexed again for at least 10 seconds each.
F. To each sample, add 2.5mL methanol and vortex for at least 10 seconds each.

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8.4.

Liquid-Liquid Extraction, using the Gilson 215 Liquid Handler
The extraction procedure is automated using the Gilson 215 Liquid Handler@

The software controlling the Gilson Liquid Handler is called Trilution LH and a
shortcuUicon is located on the desktop. After launching the software, the main menu is
displayed (Figure 3). For setting up the software for extraction, first click on
"Applications" button in the menu. ln the Application Menu (Figure 4) select the
application named "LLE Methanol Extraction - Neutral Fraction Only". Make sure that
number of samples to be extracted is correct for each method in the application. Then
click the "Run" button to begin the extraction procedure outlined below. After the first
sample transfer step, the samples will be removed from the 818 AutoMix, vortexed,
and centrifuged (3min, @2000rpm) to separate the organic/aqueous phases. Then,
the samples are placed back in the 818 AutoMix and the Application proceeds with the
second transfer of the organic phase.

Tnrlur¡oN LH
SOFTWARE

oo
r¡('t¡jl! tihtil'l¡f l(.:

¡dnilnr5r.¡t !.r ro.ri

¿.l.l

Figure 3. Detail of the Trilution Main Menu. A: The Application Menu button
B
FreUm
Fræù@

h*dlrasÉr

dêru Pàt 1
hp Pd 2 Trânftr

Tst

-.1:
Aúdrh

Hth Ps. frm*

!l'

l;:ll

:15

:l'

Figure 4. Detailof the Application Menu in Trilution LH. A: The Application Run Button. B: The column where the
number of samples to be extracted is entered.

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Check trbf - Extraction
A. Ensure that sufficient quantities of all solvents and reagents are present in
containers under the Gilson 215 instrument and that all solvent lines are kept at
the bottom of each container by an attached weight at the end of the solvent
line.
B. lf necessary, empty waste containers by replacing the container with an empty
one.
C. Place the sample tubes in positions 1-30 in the rack in the 818 AutoMix.
D. Place empty 16x100mm tubes in positions 1-30 in the "Sample Extract" rack on
the tray.
E. Select the application named "LLE Methanol Extraction - Neutral Fraction Only
- Std Rinse Port". Click on the "Run" button.
F. The Gilson will add the hexane/MTBE solution to each sample and then mix the
samples automatically by rotation via the 818 AutoMix for 10 minutes.
G. After mixing the Gilson will prompt the user to remove the samples and
centrifuge them.
H. After centrifuging, the samples are placed back in the rack in the AutoMix and
click on the "OK" button on the prompt window in the software.
The Gilson will then transfer the organic phase from the original sample tube to
the corresponding 16x100mm tube.
J. After transferring all samples, the Gilson will add more hexane/MTBE solution
to each original sample tube. The Application will then pause and prompt the
user to vortex the samples.
K. Remove the samples from the AutoMix and mix by vortexing for at least 10
seconds each.
I Pla¡a fha carnnlac hank in fha ra¡k in fha A¡rfnllir¿ anr{ nlink fha "ôK" hrrtfnn fn
continue the Application.
M. The Gilson will then transfer the organic phase from the original sample tube to
the corresponding 16x100mm tube. Then the Application will end.

8.5

Cleanup, using Galiper Life Sciences, Rapid Trace SPE workstation
The cleanup procedure is automated using the Rapid Trace@ modular SPE system,
cf. section 6,2.2).

Preparation of Silica gel / Silica gel:Sulfuric acid and packing of SPE cartridges
The SPE cartridges packed a with Silica and Silica:Sulfuric acid have a shelf life of 2
days when stored in plastic sealable bag (Ziploc) and hence must be prepared directly
prior to use.

Procedure for preparation of cartridges
A. See section 6.1 for Manufacturer, grade and brand for all chemicals used

BFRs, PCBs and PPs in Human Serum or Plasma

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B. Activate silica gel in oven at >200 oC overnight
C. Using laboratory balance add 6.6 g Silica gelto 50-mL glass tube fitted with
Teflon lined cap and add 3.3 g of concentrated sulfuric acid to the tube with.
After adding the acid, vigorously shake mixture to break up large lumps.
Standard laboratory Personal Protective Equipment must be used, such as lab
coat, safety glasses and gloves. See section 2.2for additional safety
precautions when handling concentrated acids.
D. Allow the mixture to rotate overnight using rotating mixer. After overnight
rotation confirm that no lumps are present in mixture.
E. Press frit to bottom of empty 3-mL SPE
F. Add 1.0 Silica/Sulfuric acid mixture to the cartridge, and place another frit on
top
G. Add 0.25 g activated Silica gel (>200 oC overnight) and place another frit on top
of the silica
H. Store packed cartridges in a reseal-able plastic bag in dessicator untiljust prior
to use

Setting up the Equipment for Processing Samples (Cleanup)
The software controlling the workstation is launched by the RapidTracerM
Development lcon on the desk top. After launching the software the main menu is
displayed (Figure 3). For setting up the software for cleanup click on "Setup Racks",
the menu given in Figure 4 is displayed. Select the modules to be used in lower left
corner in this menu and transfer method CL#lONLY.spe to position "one". Transfer
method CL2to1O.spe to positions 3, 5, 7 and 9. Exit this menu by pressing "OK". Enter
the "Run Monitor Menu" and launch the modules to be used for cleanup, cf. Figure 5.
Check List - Cleanup
A. Evaporate all unknowns and QC samples to dryness and blank samples to
approximately 0.2-0.5mL by placing samples in the Caliper TurboVap
evaporator and using the settings 50deg C water bath temperature and -Spsi.
B. Make certain that sufficient quantitíes of the 5% DCM in Hexane solution are
present in the solvent bottle under the RapidTracerM instrument and that all
solvent lines are kept at the bottom of the container by an attached weight at
the end of the solvent line.
C. lf necessary, empty waste containers by replacing the container with an empty

one.
Place extracts in racks (one rack per module) on the right hand side of the
racks, and remove screw caps.
Place collection tubes on the left hand side of the racks.
Place racks in tray at the bottom of each module.

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G. Assign method to each module by clicking "Setup racks" in the main menu of
the RapidTracerM software and placing method "CL#1ONLY.spe" as sample
one for each module used and method "CL2to1O.spe" for remaining positions.
H. Exit the setup racks menu by pressing OK.
l. Enter the Run Monitor Screen. Wait a few seconds after entering the Run
Monitor Screen to allow the software time to detect all modules present. Press
start on modules to be run.
J. Watch the instrument for a few minutes to ensure that all modules has been
initiated and inspect the modules running during the initial purge to ensure that
all solvents lines are connected properly.

8.6.

Evaporization and transfer to final Gc-vial

Conduct all in a fume hood or BSC or at the Caliper TurboVap evaporator.
B. Samples from cleanup step are evaporated to approximately 0.5 mL using the
eaiiper TurboVap evaporator and starting the evaporization with the tbllowing
settings as a guide: 50deg C water bath temperature and -5psi line pressure. lf
r.s essentral that the samples are not evaporated to dryness at täís step,
since all volatile analytes would be losf.
C. Transfer the sample to the GC vial that was spiked with recovery standard in
section 8.3. MAKE CERTAIN THAT THE SAMPLES ARE TRANSFERRED
TO THE CORRECT VIAL !!!
D. Rinse the sample test tube with -0.5mL of hexane and transfer to the GC-vial
E. Evaporate samples until <1OuL remains using the Caliper TurboVap
evaporator. Start the evaporization with the following settings as a guide. -51Opsi line pressure. Adjust the final volume to 1OuL with nonane.
F. Complete any lab notes, and bring samples to HR-MS operator.

8.7

GC/IDHRMS analysis of BFRs
GC/IDHRMS analysis is performed on a DFS (ThermoFisher, Bremen, Germany)
instrument. The chromatographic separations are carried out on an Trace 1300 gas
chromatograph (GC) (ThermoFisher, Bremen, Germany) fitted with a Rxi 5HT [(15 m
l^^^+L
lçllYl,ll,

n
ît **
W.¿-\) llllll

Il.lJ,
f.ì Cll
^^¡l\¡ n
tJ. 4l.l
',lL|-Plll

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{l-'i^Lnaaa\'DaatalLlllU^ll9ÐÐ/rr l\çÐl,g]\r

trlall{anla
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LrçilllrJllLgr trl/\t
Tt-ll r;dPll16lly

column. Splitless injection is used with an injector temperature of 260oC, the oven is
programmed to increase from 140 "C (1 min) to 320"C (0 min) with a ramp rate of 10
oC/min. The source temperature is 290oC in the electron impact mode using a filament
bias of 45 eV. Refer to the MS PARAM file for all monitored masses. Injections (2uL)
are performed using the TriPlus RSH (ThermoFisher, Bremen, Germany)
autosampler. All wash solutions should be changed at least weekly:

8.8

Final Preparation of GC Vials for PCB Analysis

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A. After analysis for BFRs the samples are returned

to the Controlled-Aír
Environment Clean Room. lf necessary, reconstitute the samples with nonane
to bring the volume back to 1OuL.
B. Recap the samples.
C. Bring samples to HR-MS operator for PCB/PP analysis.

8.9

GC/IDHRMS analysis of PCBs/PPs
GC/IDHRMS analysis is performed on a Thermo DFS (ThermoFinnigan, Bremen,
Germany) instrument. The chromatographic separations are carried out on an Trace
1300 gas chromatograph (GC) (ThermoFisher, Bremen, Germany) fitted with a RxiSsil MS [(30-m length, 0.25 mm l.D. and 0.1O-pm film thickness); Restek, Bellfonte,
PAI capillary column. Splitless injection is used with an injector temperature of 260oC,
the oven is programmed to increase from 140 oC (1 min) to 320oC (0 min) with a ramp
rate of 10 oC/min. The source temperature is 300oC in the electron impact mode using
a filament bias of 40 eV. Refer to the MS PARAM file for all monitored masses.
Injections (2uL) are performed using the TriPlus RSH (ThermoFisher, Bremen,
Germany) autosampler. All wash solutions should be changed at least weekly.

Reportable Range of Results
The linear range of the standard calibration curves determines the highest and lowest
analytical values of an analyte that are reportable. However, samples with a
concentration exceeding the highest reportable limit may be re-extracted using a
smaller volume and re-analyzed, so that the result is in the reportable range or the
extract may be diluted so that the native area counts are less than the corresponding
area count for the highest calibration standard

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a. Linearity Limits
Calibration standards are linear for all analytes through the range of concentrations
evaluated. The linear range for all analytes except p,p'-DDE were 0.5 to 1000 pg/ul.
Calibration curves for p,p'-DDE were extended to 6,000 pg/t¡L, due to higher
concentrations in unknown specimens. Samples exceeding the calibration curve must
be diluted or analyzed using a smaller volume of serum.

Ceftificate of analysis for all standards used are stated in the certificate of analysis as
provided by the manufacturer, Cambridge lsotope Laboratory (ClL).
b. Precision

The precision of the method is reflected in the variance of quality control samples
analyzed over time. The coefficients of variance (CV) of the method are listed in Table
3 below.

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Table 3. Mean Concentration and CV for QC samples (QC identifier SSP:01:08)
Mean

cvN

Analyte

(pclc fw)

PBDE17

462.7

PBDE28

455.7

PBDE47

643.7

5.0
3.9
6.2

PBDE66

448.4

13.9

PBDElOO

474.6

PBDE99

486.0

5.0
4.7

PBDE85

512.4

13.0

88153

425.0

PBDEl 54

427.5

PCB167

401.6

PCB156

412.2

PCBl 57

417.3

PCB178

396.1

PCB187

396.3

PCB183

393.7

PCB177

399.1

PCB172

391.5

PCB180

5.2 29
3.7 29
3.6 29
4.3 39

409.0

16.4

PBDE209

417.0

3.3 29
736
1.7 36
1.9 36
4.5 36
5.2 36
3.8 36
3.4 36
1.8 36
1.8 36
3.4 36
3.6 36
1.9 36
2.1 36
6.7 36
8.8 36
2.2 36
2.4 36

408.9

PCB049

429.8

PCB044

453.8

PCB074

415.4

PCB066

426.2

PCBl 0l

410.7

PCB099

400.7

PCB087

426.0

PCB110

430.3

PCBl18

426.5

PCB105

418.1

PCB151

399.6

PCBl49

378.8

PCB146

398.8

PCB153

443.9

415.8

PBDE203

PCB052

2.7
1.9
1.9

839.0

PCB128

470.3

399.0

PCB138/158

413.7

401.9

fw) cv

PBDEl 53

PCB028

Analyte

PBDE183

PCBo18

Mean

(pclc

1.9
1.7

36
36
36
36

336

429.3

4.4
3.7
3.2
2.2
1.7

PCB170

419.5

1.8

PCB189

392.1

36
36
36
36

PCB199

393.2

1.5

PCB196/203

753.9

2.3

PCB195

414.0

PCB194

383.0

2.8

PCB206

365.4

3.5

PCB209

341.2

2.3

PCB114

PCB123

HCB

438.8

1.3

BHCCH

209.1

3.2

GHCCH

374.4

2.7

OXYCHLOR

243.2

5.5

TNONA

476.6

3.1

PPDDE

1265.2

OPDDT

345.6

4.5

PPDDT

248.0

2.7

MIREX

399.5

1.3

Analytical specificity

lsotope Dilution High Resolution Mass Spectrometry (lD-HRMS) coupled wíth gas
chromatography is used for sample analysis. This instrumentation offers a high mass
resolution (10,000 resolution) measurement which provides excellent specificity. In

BFRs, PGBs and PPs in Human Serum or Plasma
DLS Method Gode: 6701.04

DLS-OATB
29

addition, two ions are monitored for each native analyte and 13C-labeled internal
standard. For each measurement, the ratio between these two ions is verified to be
with +/- 260/ofrom the theoretical isotope ratio. This provides additional confirmation of
the identity of the target analyte.

ln addition, the relative retention time of native compound divided with its 13C-internal
standard is verified for each measurement to eliminate the risk of mistakes during
integration.

10. Quality Assessment and Proficiency Testing
a. Quality Assessment
ln this method, a set of samples is defined as 24 unknown samples, prepared and
analyzed together with 3 analytical blanks and 3 QC sample. Quality control limits are
established by characterizing assay precision with repeated analyses of the QC pool.
For QA/QC purposes measurement of a target analyte in a set of samples is
considered valid only after the QA/QC sample have fulfilled the following criteria as
verified by the Division QC program available in StarLlMS:
(i) lf all of the QC samples are within 2o limits, then accept the run
(ii) lf one or more QC results is outside the 2o limits, then apply the rules below and
reject the run if any conditions are met.

- Extreme outliner: the result is outside the characterization mean by more than 4o
-

13s,

Average of three QCs is outside of the 3o limit.

- 22o, QC results from two consecutive runs are outside of 2o limit on the same side
of the mean.

- R¿o sequential, QC results from two consecutive runs are outside of 2o limit on
cpposite sides of mean.

- l0x seeuential, QC results from ten consecutive runs are on the same side of the
mean
lf the QC result for an analyte is declared "out of control", then the results of that
analyte for all samples analyzed during that run are considered invalid for reporting

Further, every measurement of a set of samples must fulfill the following criteria to be
considered a valid measurement:

BFRs, PCBs and PPs in Human Serum or Plasma

DLS-OATB

DLS Method Code: 6701.04

Page 30

(i)
(¡i)

(iii)

The ratio of the two ions monitored for every analyte and 13C-labelled internal
standard, must not deviate more than 26% from the theoretical value.
The ratio of the retention time of the analyte over its corresponding 13Clabeled internal standard must be within the range 0.99 - 1.01. For analytes
that do not have an identical 13C -labeled lS the ratio to the lS used may not
deviate more than 1o/o from the average of the same ratio of the calibration
standards analyzed in the same analytical run
The measured recovery of the lS must be within the range 10-150o/o.

b. Proficiency testing (PT): Currently the only established PT program for this assay
is the Arctic Monitoring and Assessment program (AMAP) in which our lab
participates. ln this program 3 serum samples are received three to four times per
year and analyzed with respect to PCB/PP/PBDEs. The program provides a report
after each set of PT samples has been reported. ln addition, our lab uses an in house
PT program (as specified in the Division Policy and Procedures manual) where 5
blinded PT samples are measured twice per year.

11. Remedial Action if Calibration or QC Systems Fail to Meet Acceptable
Criteria
lf the calibration or QC systems fail to meet acceptable criteria, suspend all operations
until the source or cause of failure is identified and corrected. lf the source of failure is
easily identifiable, for instance a failure of the mass spectrometer or a pipetting error,
correct the problem immediately. Otherwise, prepare fresh reagents and clean the
mass spectrometer system. Before beginning another analytical run, re-analyze
several QC materials (in the case of QC failure) or calibration standards (in the case of
calibration failure). After re-establishing calibration or quality control, resume
analytical runs. Document the QC failures, review the cases with supervisor to
determine source(s) of problem, and take measures to prevent re-occurrence of the
same problem.

12. Limitations of Method, Interfering Substances and Conditions
This method is an isotope dilution mass spectrometry method, widely regarded as the
definitive method for the measurement of organic toxicants in human body fluids. By
using high resolution mass spectrometry, most interferences are eliminated. Due to
the matrix used in this procedure, occasional unknown interfering substances have
been encountered. lf chromatographic interference with the internal standards occurs,
reject that analysis. lf repeat analysis still results in an interference with the internal
standard, the results for that analyte are not reportable.

13. Reference Ranges (Normal Values)
Reference ranges have been reported for BFRs in the NHANES survey and are
available at www. cdc. qov/exposurereport

BFRs, PCBs and PPs in Human Serum or Plasma

DLS.OATB

DLS Method Code: 6701.04

Page

14. Critical Call Results ("Panic Values")
It is unlikely that any result would be a "critical call", which would only be observed in
acute poisonings. There are no established "critical call" values. Application of this
method to NHANES studies will assist in determining levels of BFRs normally found in
the US populations. Test results in this laboratory are reported in support of
epidemiological studies, not clinical assessments. Data will help determine critical
exposures.

15. Specimen Storage and Handling During Testing
Store serum samples in -70 oC freezer before and after analysis. Keep extracts at
room temperature covered with aluminum foil for storage, due to documented UVsensitivity of target analytes.
After analysis, keep GC vials in Styrofoam boxes for storage at room temperature until
the final analytical data have been reported.

16. Alternate Methods for Performing Test or Storing Specimens if Test
System Fails
Alternate validated methods have not been evaluated for measuring BFRs in human
serum. lf the analytical system fails, refrigerate the samples (at 4 - 8 oC) until the
analytical system is restored to functionality. lf long-term interruption (greater that one
day) is anticipated, then store serum specimens at <-40 oC.
The method is designed to run on a GC/IDHRMS instrument, and is not generally
transferable to other instrumentation. lf the system fails, store sample extracts at
room temperature covered with aluminum foil untilthe analytical system is restored to
functionality.

17. Test Result Reporting System; Protocol for Reporting Critical Calls (if
Applicable)
Study subject data is reported in both concentration units (og/ml serum) and adjusted
based on serum lipids (ng/g lipid).
Once the validity of the data is established by the QC/QA system outlined above,
these results are verified by a DLS statistician, and the report is created. These data
and a cover letter will be routed through the appropriate channels for approval (i.e.
supervisor and/or branch chief, DLS statistician, division director) as outlined in the
DLS Policy and Procedure Manual. After approval at the division level, the report will
be sent to the contact person or principal investigator who requested the analyses
typically in an email.

BFRs, PCBs and PPs in Human Serum or Plasma

DLS.OATB

DLS Method Code: 6701.04

Page 32

18. Transfer or Referral of Specimens; Procedures for Specimen
Accountability and Tracking
lf greater than 0.1 mL of sample remains following successful completion of analysis,
this material must be returned to storage at <-40 oC in case reanalysis is required.
These samples shall be retained until valid results have been obtained and reported
and sufficient time has passed for review of the results.
Standard record keeping formats (e.9., database, notebooks, data files) are used to
track specimens. Specimens will only be transferred or referred to other DLS Branch
laboratories or, if required, to CLIA certified laboratories. Specimens may be stored at
the CDC specimen handling and storage facility (CASPIR).

19. References

C. Baird, Environmental Chemrsfiy (second edition Edn). W.H. Freeman and Company,
Houndmills, Basingstoke (1 999).

2. WHO, Environmental Health Criteria 140. Polychlorinated biphenyls

and terphenyls
(Second Edition).lnternational Program on Chemical Safety, WHO, Geneva, Switzerland
(1ee3).

S. Jensen, The PCB story, Ambio

M. Olsson, Mercury, DDT and PCB in aquatic test organiems. Baseline and monitoring
studíes, field studies on biomagnification, metabolism and effects of some
bioaccumulating substances harmful to the Swedish environment, PhD Thesis Swedish
museum of natural history, Section for Vertebrate Zoology, (1977).

A. Olsson, Applications of various analytical chemical methods for exposure studies of
halogenated environmental contaminants in the Baltic environment, PhD Thesis
Department of Environmental Chemistry, Stockholm University, (1 999).

E. Helle, M. Olsson and S. Jensen, DDT and PCB levels and reproduction in ringed seal
from the Bothnian Bay, Ambio 5, pp. 188-189 (1976).

E. Helle, M. Olsson and S. Jensen, PCB levels correlated with pathological changes in
seal uteri, Ambio 5, pp.261-263 (1976).

A. Bergman and M. Olsson, Pathology of Baltic grey seal and ringed sealfemales with
special reference to adrenocortical hyperplasia: ls environmental pollution the cause of
widely distributed disease syndrome?, Finnish Game Res 44, pp.47-62 (1985).

pp. 123-131 (1972)

WHO, Environmental Health Criteria 162. Brominated diphenyl ethers.lnternational
Program on Chemical Safety, WHO, Geneva, Switzerland (1994).

BFRs, PCBs and PPs in Human Serum or Plasma

DLS.OATB

DLS Method Gode: 6701.04

10. WHO, Environmental Health Criteria 192. Flame

retardants: A general introduction.
International Program on Chemical Safety, WHO, Geneva, Switzerland (1997).

11. A. Sjödin, L. Hagmar, E. Klasson-Wehler, K. Kronholm-Diab, E. Jakobsson and A.
Bergman, Flame retardant exposure: Polybrominated diphenyl ethers in blood from
Swedish workers, Environ Health Perspect 107, pp.643-648 (1999).

12. A. Sjödin, H. Carlsson, K. Thuresson, S. Sjölin, A. Bergman and C. Östman, Flame
retardants in indoor air at an electronics recycling plant and at other work environments,
Environ Sci Technol 35, pp. 448-454 (2001).

13. A. Sjödin, L. Hagmar, E. Klasson-Wehler, J. BjörkandA. Bergman, Influenceof the
consumption of fatty Baltic Sea fish on plasma levels of halogenated environmental
contaminants in Latvian and Swedish men, Environ Health Perspect 108, pp. 1035-1041
(2000).

14. D. Meironyté, K. Norén and A. Bergman, Analysis of polybrominated diphenyl ethers in
Swedish human milk. A time-related trend study, 1972-1997, J Toxicol Environ Health 58
PartA, pp. 329-341 (1999).

15.

C. Schröter-Kermani, D. Helm, T. Herrmann and O. Päpke, The German environmental
specimen bank - Application in trend monitoring of polybrominated diphenyl ethers in
human blood, Organohalogen Comp 47, pp. 49-52 (2000).

16. C. Thomsen, E. Lundanes

and G. Becher, Brominated flame retardants in plasma
samples from three different occupational groups in Norway, J Environ Monit 3, pp. 366370 (2001).

17.

K. Norén and D. Meironyté, Certain organochlorine and organobromine contaminants in
Swedish human milk in perspective of past 20-30 years, Chemosphere 40, pp. 1 1 11-1123
(2000).

18. A. Sjödin, D. G. Patterson Jr and A. Bergman, Brominated Flame Retardants in serum
from U.S. Blood donors, Envlror¡ SclTechnol35, pp. 3830-3833 (2002).

19. A. Sjödin, Occupational and dietary exposure to organohalogen substances, with special
emphasis on polybrominated diphenyl ethers, PhD Thesis Department of Environmental
Chemistry, Stockholm University, (2000).

20.

P. Eriksson, E. Jakobsson and A. Fredriksson, Developmental neurotoxicity of
brominated flame-retardants, polybrominated diphenyl ethers and tetrabromo-bis-phenol
A, Organohalogen Comp 35, pp. 375-377 (1998).

21.

P. Eriksson, H.Viberg, E.Jakobsson, U. ÖrnandA. Fredriksson, PBDE,2,2',4,4',5pentabromodiphenyl ether, causes permanent neurotoxic effects during a defined period
of neonatal brain development, Organohalogen Comp 40, pp. 333-336 (1999).

22. WHO, Environmental Health Criteria 152. Polybrominated

Biphenyls. lnternational
Program on Chemical Safety, WHO, Geneva, Switzerland (1994).

23.

H. M. Blanck, M. Marcus, V. Hertzberg, P. E. Tolbert, C. Rubin, A. K. Henderson and R.
H. Zhang, Determinants of polybrominated biphenyl serum decay among women in the
michigan PBB coho¡t, Environ Health Perspect 108, pp. 147-152 (2000).

Division of Laboratory Sciences
Laboratory Protocol
Analytes: Nine monohydroxy-polycyclic aromatic hydrocarbons: I hydroxynaphthalene, 2-hydroxynaphthalene, 2hyd roxyfl uore ne, 3-hyd roxyfl u orene, I -hyd roxyphena nth re ne, 2& 3-hydroxyphenanthrene, 4-hydroxyphenanthrene, 1 hydroxypyrene
Matrix: Urine
Method: lsotope Dilution Online Solid Phase Extraction- High
Performance Liquid Chromatography-Tandem Mass Spectrometry
(online SPE-HPLC-MS/MS)
Method code: 6705.02

Branch: OAT
Prepared By

2.ë íç

Yuesong Wang
Author's name

Lei Meng

¿ots:

/a"t.r

Author's name

Supervisor.

Xiaoyun Ye
Supervisor's name

Branch

Chief:

Antonia

tlr.lry

lafat

Branch Chiefs name

Date

Date current version of method first used in lab
Date

Director's Signature Block
t5

Reviewed
Signature

Date

Procedure Ghange Log
ic aromatic hvdrocarbons
Procedure: Nine monohvdroxv-polvc
DLS Method Code: 6705.02
Date

Ghanges Made

Rev'd
By

Date
Rev'd

(lnitials)
12t29t2014 1. Changed the configurations, columns,
and gradients for HPLC and SPE
systems.
2. Prepared new pools of standards,
QCs, and PTs.
3. Updated the QC ranges and LODs.

1t2t2015

Environm¡:nl¡¡l Ht alth

Laboratory Procedure Manual
ne monohyd roxy-polycycl ic promatic
hydrocarbons: 1 -hydroxynaphthalene, 2-

Analyte:

hyd roxynaphthalene, 2-hyd roxyfluorenê, 3hydroxyfluorene, 1 -hydroxyphenanthrene, 2- & 3-

hydroxyphenanthrene, 4-hydroxyphenanthrene, 1 hydroxypyrene
Matrix

Urine

Method:

lsotope Dilution Online Solid Phase Extraction High
Perform ance Liq u id G h romatog raphy/Ta ndem Mass
Spectrometry (online SPE-HPLC-MS/MS)

Method

No:

6705.02

As pefformed by:
Organic Analytical Toxicology Branch
Division of Laboratory Sciences
National Center for Environmental Health
Contact:

Antonia Calafat, Ph.D.
770-488-7891
[email protected]

Phone:
Email:

James L. Pirkle, M.D., Ph.D.
Director, Division of Laboratory Sciences
lmportant lnformation for Users
The Centers for Disease Control and Prevention (CDC) periodically refines these laboratory methods. lt 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.

NCEH/DLS/OATB

OH-PAHs in Urine
DLS Method Code: 6705.02

Table of Gontents

1. Glinical Relevance and Summary of Test Principle
a. Clinical Relevance...........
b. Test Principle.
2. Safety Precautions ...........
a. Reagent Toxicity or Carcinogenicity
b. Radioactive Hazards .........

c. Microbiological

1
1
1

2
2
2
2
2
3
3
3

Hazards
Mechanical Ha2ards............
Protective Equipment ..............
f. Training
g. Personal Hygiene
3
h. Disposal of Wastes
4
3. Gomputerization; Data-System Management
4
a. Software and Knowledge Requirements..
4
b. Sample lnformation.........
4
c. Data Maintenance..
4. Procedures for Collecting, Storing, and Handling Specimens; Griteria for
4
Specimen Rejection
4
a. Special lnstructions.........
4
b. Sample Collection
5
c. Sample Handling
5
Sample
d.
Quantity.
5
e. Unacceptable Specimens
5. Procedures for Microscopic Examinations; Criteria for Rejecting lnadequately
.............5
Prepared SIides
6. Preparation of Reagents, Calibration Materials, Control Materials, and all Other
5
Materials; Equipment and lnstrumentation
5
a. Reagents and Sources .....
6
b. Preparation of Reagents
6
1) Sodium Acetate Buffer Solution (-1 mol/L, pH 5.5t0.2)................
g/L)
(-1
0
6
enzyme/Buffer
solution
2) B-glucuronidase/arylsulfatase
6
3) Ascorbic Acid Solution (-12.5 g/L)..........
7
Urine
4) Synthetic
7
c. Preparation of Calibration Materials.............
7
1) lndividual Standards and Mixed Working Standards..
7
2) Calibration Standards.........
3) Working Standard Solution of 13C-labeled Standard Mix and lnternal Spiking
7
Solution (lSS)...
8
d. Preparation of Quality Gontrol Materials
I
1) Quality Control (QC) Materials
I
2) Proficiency Testing Material (PT) .........
I
e. Other Equipment, Materials, and Supplies
I
f. lnstrumentation
9
1) Online SPE.........

d.
e.

2)
3)

HPLC Configuration...........
Tandem Mass Spectrometer (MS/MS) Configuration.

OH-PAHs in Urine
DLS Method Gode: 6705.02

NCEH/DLS/OATB

Table of Contents

7. Calibration and Galibration Verification ..
a. Tuning and Calibration of Mass Spectrometer....
b. Creation of Calibration Curve .............
1)

11
11

Calibration data
Evaluation of Curve Statistics

Calibration Verification............
8. Procedure Operation lnstructions; Calculations; lnterpretation of Results
a. Sample Preparation ............,

1) Enzymatic Hydrolysis
2) Centrifugation and sample transfer

l3
l3

b. lnstrument and software setup for the online SPE-HPLC-MS/MS
1)

Data.

a. Linearity Limits......
b. Limit of Detection......

Preliminary System Setup and Performance Check

2) Runsheet and Batch Setup.......
3) Online SPE-HPLC-MS/MS Analysis Procedure
c. Processing of
d. Replacement and Periodic Maintenance of Key
Reportable Range of Results ......

Precision

d. Analytical Specificity
e. Accuracy

2
13
13

I3
............ 13
.................... 14

Components

................15
...........15
15
16
16
17
17
18
19
19
19

Quality Assessment and Proficiency Testing ....
a. Quality Assessment......
b. Quality Control Procedures............
....... 19
1) lndividual Sample Quality Checks
...................20
2) Establishing QC 1imits.........
20
3) Quality Control Evaluation
21
c. Proficiency Testing (PT)
11. Remedial Action if Galibration or QC Systems Fail to Meet Acceptable Criteria...21
12. Limitations of Method, lnterfering Substances and Gonditions.............. ....22
...........22
13. Reference Ranges (Normal Values)
("Panic
...........22
Values")
l4.Gritical Gall Results
......22
l5.Specimen Storage and Handling During Testing
if
Test
System
Performing
Test
or
Storing
Specimens
Methods
for
l6.Alternate
....23
Fails
17.Test Result Reporting System; Protocol for Reporting Critical Galls (if
......23
Applicable) ............
lS.Transfer or Referral of Specimens; Procedures for Specimen Accountability and
.........23
Tracking
.........24
19. References
.......25
Appendix A - SPE tubing and valve switching system
............26
Appendix B - Ruggedness Testing.
..........31
Results............
Appendix C - Method Comparison
1

NCEH/DLS/OATB

OH-PAH in Urine

Page l1

DLS Method Code: 6705.02

Glinical Relevance and Summary of Test Principle

a. Clinical

Relevance

Polycyclic aromatic hydrocarbons (PAHs) are a class of ubiquitous environmental
contaminants formed during incomplete combustion processes. Many of them have
been identified as suspected human carcinogens (1), but threshold levels for
carcinogenicity have not been determined for most PAHs. Occupational exposure
may occur through work involving diesel fuels and coal tars such as paving and
roofing. Possible environmental exposures include smoking, diet, smog and forest
fires (2). Because of potentialwidespread human exposure and potential risk to
health, biomonitoring of PAHs is relevant for environmental public health (3,4).
Upon exposure, PAHs are metabolized in humans; some of these metabolites are
excreted in urine. lnformation on the concentration of metabolites of PAHs in people
is important for understanding human exposure.

b. Test Principle
The test principle utilizes high performance liquid chromatography-electrospray
ion ization-tandem mass spectrometry (H PLC-ESl-MS/MS) for the q uantitative
detection of several monohydroxylated metabolites of PAHs (OH-PAHs) in urine,
The procedure involves enzymatic hydrolysis of glucuronidated/sulfated OH-PAH
metabolites, centrifugation, dilution and analysis using isotope dilution online solid
phase extraction (SPE) coupled with HPLC-ESI-MS/MS. lon transitions specific to
each analyte and carbon-13 labeled internal standards are monitored, and the
abundances of each ion are measured. The analytes measured in this procedure
are shown in Table 1.
Table 1. Analytes, their parent compounds, and their abbreviations.
No.

Metabolite/Analyte

1-hydroxynaphthalene
2-hydroxynaphthalene
2-hvdroxyfluorene
3-hvdroxvfluorene
1 -hvd roxvphenanthrene

2-hvd roxyp hen a nth rene

3-hvd roxvp hen a nth rene
4-hyd roxyp hen a nth rene

1-hydroxypyrene

2
3
4

Parent PAH

Abbreviation

Naphthalene

1-NAP
2-NAP

Fluorene

Phenanthrene

Pyrene

Note

2-FLU
3-FLU
1-PHE

2-PHE
3-PHE
4-PHE
1-PYR

2-, 3-PHE
measured
together

NCE

OH-PAH in

Page l2

DLS Method Code: 6705.02

Safety Precautions

Reagent Toxicity or Carcinogenicity
Some of the reagents needed to perform this procedure are toxic. Special care must
be taken to avoid inhalation or dermal exposure to these reagents.
B-Glucuronidase is a known sensitizer. Prolonged or repeated exposure to the
sensitizer may cause allergic reactions in certain sensitive individuals.

Note: Material Safety Data Sheets (MSDS) for the chemicals and solvents used in
this procedure can be found at
or
com/enol ish/index cfm. Laboratory personnel are advised
htto://www.msdsxcha
to review the MSDS before using chemicals and solvents

b. Radioactive

Hazards

There are no radioactive hazards associated with this procedure

c. Microbiological

Hazards

Although urine is generally regarded as less infectious than serum, the possibility of
being exposed to various microbiological hazards exists. Appropriate measures
must be taken to avoid any direct contact with the specimen. CDC recommends a
Hepatitis B vaccination series and a baseline serum test for health care and
laboratory workers who might be exposed to human fluids and tissues. Laboratory
workers observe universal precautions to prevent any direct contact with the
specimen. Also, laboratory personnel handling human fluids and tissues are required
to take the "Bloodborne Pathogens Training" course and subsequent refresher
courses offered at CDC to insure proper compliance with CDC safe work place
requirements.

Mechanical Hazards
There are only minimal mechanical hazards when performing this procedure using
standard safety practices. Laboratory analysts must read and follow the
manufacturers' information regarding safe operation of equipment. Avoid direct
contact with the mechanical and electronic components of the mass spectrometer
unless all power to the instrument is off. Generally, mechanical and electronic
maintenance and repair must only be performed by qualified technicians. Avoid
contact with the heated surfaces of the mass spectrometer (e.9., interface). Multiple
solvent bottles (1L) are located on and around the instruments. Numerous tubing
lines are used to transfer solvents from storage bottles to the instrument and the
waste bottles. Precautions must be used when working in these areas.

in Urine

DLS Method Code: 6705.02

Page l3

Protective Equipment
Standard safety precautions must be followed when performing this procedure,
including the use of a lab coaUdisposable gown, safety glasses, appropriate gloves,
and chemical fume hood. Refer to the laboratory Chemical Hygiene Plan and CDC
Division of Laboratory Sciences safety policies and procedures for details related to
specific activities, reagents, or agents.

Training
Formal training is necessary in the use of the Staccato@ automated sampler, Agilent
1200 HPLC, AB Sciex 5500/6500 MS-MS, and online SPE system. Users are
required to read the operation manuals and demonstrate safe techniques in
performing the method. Laboratorians involved in sample preparation must be
trained for all sample preparation equipment, chemical handling, and have basic
chemistry laboratory skills.

Personal Hygiene
Follow Universal Precautions. Care must be taken when handling chemicals or any
biological specimen. Routine use of gloves and proper hand washing must be
practiced. Refer to the laboratory Chemical Hygiene Plan and CDC Division of
Laboratory Sciences safety policies and procedures for details related to specific
activities, reagents, or agents.

Disposal of Wastes
Waste materials must be disposed of in compliance with laboratory, local, state, and
federal regulations. Solvents and reagents must always be disposed of in an
appropriate container clearly marked for waste products and temporarily stored in a
chemical fume hood. Disposable items that come in direct contact with the
biological specimens are to be placed in a biohazard autoclave bag that must be
kept in appropriate containers until sealed and autoclaved. Needles, pipette tips and
disposable syringes must be discarded into sharps containers and autoclaved.
Contaminated surfaces should be disinfected with a freshly prepared bleach solution
(e.9., -0.5o/o available chlorine, or a 100 mL/L dilution of commercial sodium
hypochlorite solution containing 5o/o available chloride). Any non-disposable
glassware or equipment that comes in contact with biological samples must be
washed with bleach solution before reuse or disposal. Any other non-disposable
glassware must be washed and recycled or disposed of in an appropriate manner.
To insure proper compliance with CDC requirements, laboratory personnel are
required to take annual hazardous waste disposal training courses.

OH-PAH in Urine

NCEH/DLS/OATB

DLS Method Gode: 6705.02

Page l4

Gomputerization; Data-System Management

Software and Knowledge Requirements
Spiking of samples and hydrolysis of conjugates normally take place on a Staccato@
Automated System controlled by the Perkin Elmer ilink and Maestro softwares. The
SPE-HPLC-MS/MS system uses an Agilent 1200 series HPLC pump and AB Sciex
5500/6500 MS/MS, controlled by AB Sciex AnalystrM software, and an iChrom
SymbiosisrM online SPE system, controlled by Sparklink@ software. Analyte
chromatographic peaks are integrated by MultiQuantrM or Analyst@. Results can be
exported from MultiquantrM or Analyst@as text files which are subsequently
processed using Excel, Access or SAS Enterprise Guide (SAS EG). Knowledge and
experience with these software packages (or their equivalent) are required to utilize
and maintain the data management structure,

b. Sample lnformation
lnformation pertaining to particular specimens is entered into the database (Access
or STARLIMS) either manually or electronically using the files received from Sample
Logistics. The result file is transferred electronically into the database. No personal
identifiers are used, only coded sample identifiers.

Data Maintenance
All sample and analytical data are reviewed for overall validity. The database is
routinely backed up locally through the standard practices of the CDC network. The
local area network manager can be contacted for emergency assistance.

Procedures for Gollecting, Storing, and Handling Specimens; Criteria for
Specimen Rejection

Special Instructions
No special instructions such as fasting or special diet are required

b. Sample Gollection
Urine specimens are collected from subjects in standard urine collection cups.
Samples should be refrigerated as soon as possible, and preferably transferred to
specimen vials within 24 hours of collection. lf possible, a minimum of 2 milliliters of
urine is collected and poured into vials (e.9., polypropylene, glass) with screw-cap
tops. The specimens should be labeled,lrozen at or below -20oC, and stored on
dry ice for shipping, Special care must be taken to protect vials from breakage

OH-PAH in Urine
DLS Method Gode: 6705.02

NCEH'DLS/OATB

Page l5

during shipment. At CDC, samples are kept frozen, preferably at -70 oC, until and
after analysis.

c. Sample Handling
Specimen handling conditions are outlined in the Division of Laboratory Sciences
(DLS) protocol for urine collection and handling (e.9., copies available in branch,
laboratory). ln general, urine specimens should be transported and stored frozen.
Once received, they should be frozen, preferably at -70 oC, until time for analysis.
Portions of the sample that remain after analytical aliquots are withdrawn must be
refrozen as soon as possible after use.

Sample Quantity
The regular sample size for analysis is 0.1 mL; the minimum amount of specimen
generally required for a regular analysis is 0.05 mL.

Unacceptable Specimens
Specimens must be frozen when delivered to the lab. The minimum volume
generally required for a single analysis is 0.1 mL. lf either of these criteria is
violated, the specimen may be rejected. Specimens can also be rejected if
suspected of contamination due to improper collection procedures or devices.
Specimen characteristics that may compromise test results include contamination of
urine from improper handling. Samples with visible microbiological growth (e.9.,
mold, bacteria) might be inadequate for analysis. In case of rejected specimens, we
would request a second specimen if possible. A description of reasons for rejecting
a sample must be recorded on the sample transfer sheet (e.9., low sample volume,
leaking or damaged container).

Procedures for Microscopic Examinations; Griteria for Rejecting lnadequately
Prepared Slides
Not applicable for this procedure

Preparation of Reagents, Calibration Materials, Gontrol Materials, and all Other
Materials; Equipment and lnstrumentation

Reagents and Sources
See Table 2.

NCEH/DLS/OATB

OH-PAH in Urine

Page l6

DLS Method Gode: 6705.02

Table 2.

ested manufacturers

ents and su

Reagent

Suqqested Manufacturers"

Water (HPLC grade), acetonitrile, methanol

ThermoFisher Scientific, lnc., Waltham,
MA

B-glucuronidase/arylsulfatase (H-1, powder
enzyme), glacial acetic acid, sodium
acetate, ascorbic acid, formic acid

Sigma-Aldrich Chemical, St. Louis, MO

13Co

1-NAP, 13Co 2-NAP, 13Co 2-FLU, 13Co
3-FLU, 13CO 1-PHE, 13CO 2-PHE, 13CO 3PHE, , 13Co 4-PHE,13Co 1-PYR

Cambridge lsotope Laboratories,
Andover, MA

1-NAP, 2-NAP, 1-PYR
2-FLU, 3-FLU, 1-PHE, 2-PHE, 3-PHE, 4PHE

Sigma-Aldrich Chemical, St. Louis, MO

Cambridge lsotope Laboratories,
Andover, MA

* Products from other manufacturers with similar purity

or specifications may be used

Preparation of Reagents
Buffer Solution

-1 mol/L

H 5.5t0

Weigh sodium acetate and record in logbook. Transfer contents to a clean
glass bottle and add necessary volume of de-ionized water (Dl HzO) to make a
1 mol/L solution. An example solution is 10.25 g sodium acetate diluted with
125 mL of Dl water. Stir on a stir plate until the solid completely dissolves.
Measure pH and record the value in logbook. Adjust the pH to 5.5 with glacial
acetic acid; record final volume in logbook.
2)

lucuronid

sulfatase e

Weigh the needed amount of B-glucuronidase/arylsulfatase (H-1, powder
enzyme) into a glass vial to have a final concentration of -10 g/L. Add 1 mL of
the sodium acetate buffer solution (1 mol/L, pH 5.5) for each 0.01 g of enzyme
and cap the vial (e.9. 0.40 g enzyme to 40 mL buffer). Place vial on a rotating
mixer al -40 rpm until the enzyme is completely dissolved. Store unused
enzyme/buffer solution refrigerated for up to one week.
3)

Ascorbic Acid Solution (-12 5 glL)

Weigh L-ascorbic acid into a glass vial or test tube. Add 80 ¡rL of deionized
water for each milligram of ascorbic acid (e.g. 1 4 mg ascorbic acid to 11.2 mL
water) and cap the vial. Place vial on a rotating mixer at 40 rpm until the solute
is completely dissolved. Store unused ascorbic acid solution at room
temperature for up to one week.

NCEH/DLS/OATB

OH-PAH in Urine

Page l7

DLS Method Code: 6705.02

Svnthetic Urine
For a 1L final volume, add the following chemicals in order, then fill to 1L with
D.l. water. Store the solution in the refrigerator or freezer until use.
¡ 500 mL Water

.
.
o
.
.
.
.
.
.
.
.

c.P

g
g

Potassium Chloride
Sodium Chloride
24.59 Urea
1.03 g Magnesium Sulfate (MgSOn.THzO)
1.03 g Citric Acid
0.34 g Ascorbic Acid
1.18 g Potassium Phosphate, dibasic
1.4
Creatinine
0.64 g Sodium Hydroxide (add slowly)
0.47 g Sodium Bicarbonate
0.28 mL Sulfuric Acid (concentrated.)
3.8
8.5

Calibration Materials
Standard preparation is based on gravimetric and volumetric determination
Actual calculated concentrations based on weight are used in all data
calculation and processrng and the actual preparation and final
concentrations may slightly deviate from the normal procedure or target
concentrations.

lndividual Standards and Mixed Workino Standards
Nine individual monohydroxylated PAH standard solutions were prepared by
dissolving weighted amounts of target analytes in ethanol. The mixed working
standards containing nine analytes were prepared from serial dilution of the
individual stock solutions in 40 % (v/v) ethanol.

Calibration Standards
The calibration standards were prepared from serial dilution of the working
standards in 40 % (v/v) ethanol. The calibration standards were aliquot into
glass vials, capped and kept frozen until use.

Workinq Standard Solution
Solution (lSS)

13C-labeled Standard Mix and lnternal Spikinq

Combine individual 13C-labeled internal standard (1.S.) stock solutions (90
ng/¡rL) to generate the working internal standard solution (WSl). Homogenize
the mixture.

NCEH/DLS/OATB

OH-PAH in Urine
DLS Method Gode: 6705.02

Page l8

Transfer 1 mL of WSI into a 500-mL volumetric flask. Dilute the solution with
HPLC grade water to prepare internal spiking solut¡on (lSS). Aliquot into amber
4-mL standard vials, cap and keep frozen until use.

Preparation of Quality Control Materials

Qualitv Control (QC) Materials
Prepare quality control materials by spiking a known amount of native standard
mixture (in acetonitrile) into 2000 mL of an anonymous filtered urine pool.
Homogenize the QC solutions for at least 3 h. Aliquot into 4-mL vials and store
at -70 oC until use.

Proficiencv Testinq Material (PT)
Prepare proficiency testing materials by spiking 100 mL of an anonymous urine
pool (filtered) with a known amount of working standard solution to achieve the
target concentration (preferably different from those of the QCs). Prepare at
least three urine pools at levels within the linear range of the method.
After spiking the urine pool with a known amount of working standard solution,
homogenize the PT solutions overnight for equilibration. Then, aliquot the PT
solutions into 16 x 100 mm test tubes (2 mL in each tube). PT samples are then
randomized by an external PT administrator, labeled by external lab
technicians, and stored at -70 oC until use.

e. Other Equipment, Materials, and Supplies
Materials / supplies and sources, or their equivalent, used during the development,
validation, and application of this method are listed below.

.
.
.
.
.
.
.
.
.
.
.
.
.

lncubator ovens (Fisher Scientific, INHECO)
pH meter (Corning)
Microbalance (Mettler-Toledo)
Rotarv suspension mixer (Glas-Col)
Stirring/heating plates (Corning)
Miscellaneous glassware (Pyrex, Kimax, Wheaton, Corning)
Repeater Plus Pipette (Eppendorf)
Electronic and Manual Pipettes (Rainin)
Maxi-mix Vodex mixer (Barnstead lnternational)
Amber screw top vials of various volumes (Supelco, lnc)
96-Well plates (Axygen, Eppendorf)
SBS Format Reservoirs (Seahorse Bioscience)
Sample Tubes (Fluidx)

NCEH/DLS/OATB

AH in Urine
DLS Method Gode: 6705.02

Oasis WAX On-Line SPE Cartridges for Symbiosis and Prospekt 2 Systems
(Merck KGaA, Darmstadt, Germany)
Chromolith HighResolution RP-18 endcapped HPLC Column 100x4.6 mm
(Merck KGaA, Darmstadt, Germany)
Chromolith HighResolution RP-18 endcapped Guard Column 5x4.6 mm
(Merck KGaA, Darmstadt, Germany)

Page l9

lnstrumentation
The sample preparation procedure can be fully automated on a Staccato@
Automated System (Perkin Elmer Co.) with the following components:

o
.
.
.
.
.
.
.

Sciclone G3/G3T
Fluidx CESD-24PRO decapper
Fluidx XTR-96-Cryo 2D barcode reader
Turntable/1D barcode reader
Hettich Rotanta 460 centrifuge
ThermoScientificALPS3000sealer
IVD lnheco Incubator Shaker DWP (4)
Mitsubishi robotic arm

The analyses are performed on an iChrom Symbiosis online SPE system, coupled
with an Agilent 1260 HPLC and AB Sciex 5500 or 6500 MS operated under negative
electrospray ionization mode.
1)

Online SPE
The SPE tubing and the valve switching system is used in concurrent
SPE/HPLC mode controlled by the Sparklink software. The method uses both
left and right cadridge clamps, the four switching valves, the high pressure
dispenser, and the autosampler. The left clamp, the left clamp valve (LCV), and
left integrated Stream Switching (lSS1)are used for SPE clean-up while the
right clamp, the right clamp valve (RCV) and right integrated Stream Switching
(lSS2) are used for the HPLC elution.
The SPE run of each sample starts with the conditioning of an Oasis WAX
online cartridge with HPlC-grade acetonitrile (4 mL) and 0.1% formic acid (2
mL). Aftenruard, 50-500 trrL of the sample (- 50 pL of urine in 500 pL sample
solution) is injected into the 1 mL sample loop and loaded onto the SPE column
using 1.5 mL 0.1o/o formic acid. Next, the SPE column is washed with 1.5 mL
Acetonitrile/MethanolAffater (11112, v/v/v). The duration of the SPE procedure
(including injection time) is approximately 11 min. Before stading the clean-up
of the next sample, the cartridge containing the extracted analytes is
transferred by a robotic gripper from the left clamp into the right clamp. While

NCEH/DLS/OATB

OH-PAH in Urine

Page

DLS Method Code: 6705.02

110

the right clamp is used for analyte elution and HPLC-MS/MS acquisition, the
left clamp could be used for the clean-up of the next sample.

HPLC Confiquration

After online SPE, the extract is loaded onto a Chromolith HighResolution RP-18
endcapped Guard Column (5x4.6 mm) with 350 pL methanol at a flow rate of
100 ¡rl/min. The HPLC gradient (Table 4) starts with 1% methanol (with 0.1
mM ammonium fluoride) as mobile phase B at 500 ¡rl/min for the first 3.5
minutes to focus the analytes onto the guard column. After the first 3.5 minutes,
the eluent from the guard column is connected to the waste. Afterwards, the
valve switches and the eluent from the guard column connects to a Chromolith
column.
Column and guard column:
Chromolith HighResolution RP-18 endcapped HPLC Column 100x4.6
mm (Merck KGaA, Darmstadt, Germany)
Chromolith HighResolution RP-'l8 endcapped Guard Column 5x4.6
mm (Merck KGaA, Darmstadt, Germany)

o
o

HPLC Mobile Phase:

o Mobile Phase A: 0
o Mobile Phase B: 0

1 mM ammonium fluoride in Water
1 mM ammonium fluoride in Methanol

Table 4: HPLC Gradient.
Flow Rate (UL/min)
Time (min)
0.0
3.5

500

3.9
4.3

500

5.0
18.0
19.5
20.0
21.0
22.0
24.0
24.5
24.6
27.0

800
800
800
1 000
1 000
1 000
1 000
I 000
I 000
1 000

500
500

A (%)

B (Vo)

99.0
99.0
40.0
40.0
38.0
32.0
30.0
15.0
15.0
10.0
5.0
5.0
99.0
99.0

1.0
1.0
60.0
60.0
62.0

68.0
70.0
85.0
85.0
90.0
95.0
95.0
1.0
1.0

NCEH/DLS/OATB

OH-PAH in Urine

Pag

DLS Method Code: 6705.02

111

Tandem Mass Spectromete r IMS/MS) Confiouration
Detection of the target analytes is conducted on the AB Sciex tandem mass
spectrometer in the negative electrospray ionization mode (Table 5).

Table 5.

resentative Mass s

2-NAP
2-NAP2**
2-NAP-IS
1-NAP
1-NAP2**
1-NAP-IS
3-FLU
3-FLU-IS
2-FLU
2-FLU-IS
2-3PHE***

2-3PHE-lS"**
1-PHE
1-PHE-IS
4-PHE
4-PHE-IS
1-PYR
1-PYR-IS

meters*

Precursor
ion (m/z)

Product ion

(mlzl

Dwell Time
(ms)

143
143
149
143
143
149
181

115

115
115

180

187

186

181

180

187

186

193

165

199

171

193

165

197

168

193

165

197

168

217

189

25
25
25
25
25
25
25
25
25
25
25
25
25
25
25

223

195

115

DP (volts)

(volts)

-120
-260
-120
-120
-260
-120
-1 00
-100

-34
-34
-34
-34
-34
-34
-26
-26

-1

-34
-34
-41
-41
-38
-38
-38
-38
-45

-100

-45

-150

Actual parameters are optimized for different instruments.
** 1-NAP2 and 2-NAP2 results may be used when concentrations are -20-200 ng/ml.
*** 2-PHE and 3-PHE are measured together (2-3PHE).
"

Galibration and Calibration Verification

Tuning and Galibration of Mass Spectrometer
The AB Sciex MS/MS is calibrated and tuned at least once per year using a
polypropylene glycol (PPG) solution according to the instructions contained in the
operator's manual.

OH-PAH in

NCEH/DLS/OATB

flne

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112

b. Creation of Calibration Gurve
1)

Calibration data

At least five calibration standards are analyzed with every analytical run.
Calibration curyes are generated by plotting the analyte area ratios (i.e.,
analyte arealinternal standard area) against the native analyte concentrations
through linear regression analysis with 1/X weight.

Evaluation of Curve Statistics

The R-squared value of the curve must be >0.95. Linearity of the standard
curve must extend over the entire calibration range. Samples with
concentrations of a given analyte exceeding the highest point in the calibration
curve are reanalyzed using less urine volume.

Calibration Verification

Calibration verification is not required by the manufacturer. However, it should be
performed after any substantive changes in the method or instrumentation (e.9.,
new internal standard, change in instrumentation), which may lead to changes in
instrument response, have occurred.

2) All calibration

verification runs and results shall be appropriately documented

3) According to the updated Clinical Laboratory lmprovement Amendments (CLIA)
reg u latio ns f rom 2003 (http ://www. cms. gov/Reg

latio ns-and -

G u id ance/Leg islation/C L lA/down loads/6065b k. pdf), the req u i rement fo r
calibration verification is met if the test system's calibration procedure includes
three or more levels of calibration material, and includes a low, mid, and high
value, and is performed at least once every six months.

All of the conditions above are met with the calibration procedures for this
method. Therefore, no additional calibration verification is required by CLIA

OH-PAH in Urine

NCEH/DLS/OATB

DLS Method Gode: 6705.02

Page

113

Procedure Operation lnstructions; Galculations; lnterpretat¡on of Results

a. Sample Preparation
Sample preparation can be performed manually or on a Staccato@ Automated
System.

EnzymaticHydrolvsis

o
.
.
.
.
.
2)

Allow calibrators (CS), quality control samples (QCs), synthetic urine, and
urine samples to thaw and reach room temperature.
Aliquot 100 ¡rL of specimens into a vial or plate well.
Add 20 ¡L of ascorbic acid solution (12.5 mg/ml) to each vial or well.
Add 50 pL of lSS.
Add 50 pL of 1M sodium acetate buffer (pH 5.5) containing Bglucuronidase/arylsulfatase enzyme from Helix pomatia (1 Omg enzymel 1 mL
buffer) to all aliquots and mix well.
Cap or seal all samples and incubate overnight (-18 hours) at -37"C.

Centrifuqati on and samole transfer

After overnight enzymatic hydrolysis, add at least 175 trrL methanol (total
volume = 220+175 pL) to all samples and mix well to precipitate the enzyme. Cap or
seal all samples and centrifuge for -15 minutes at 3000 - 5000 rpm. Transfer 200 pL
of the supernatant layer to a new sample container containing 350 pL of deionized
HzO and mix well. Transfer the rest of the supernatant layer (around 150 ¡rL) to a
second new sample container containing 350 pL of deionized HzO, mix well and store
it as backup. The backup container may be used for LC-MS analysis, and will be
discarded after a month.

b. lnstrument and software setup for the online SPE-HPLC-MS/MS

Preliminary System Setup and Pedormance Check
The ABSciex 5500/6500 TripleQuad mass spectrometer is calibrated and tuned
periodically using positive and negative polypropylene glycol (PPG) solution
provided by the manufacturer. The instrument sensitivity is checked before
each analytical run by injecting the instrument test solution.

Runsheet and Batch Setu

A typical analytical run consists of 11 calibration standards, 3 blanks, 2 QCLs, 2
QCHs and 78 study samples.

OH-PAH in Urine

NCE

DLS Method Gode: 6705.02

Page

114

Create a runsheet using a MS Excel runsheet template. ln the Excel runsheet,
two batch files, one for the Analyst software and one for the Sparklink software,
are generated for each analytical run. The two batch files are saved as text files
(.txt or .csv) to be imported to the Analyst software and Sparklink software,
respectively.
3)

Online SPE-H PLC-MS/MS Analysis Procedure

.
.
'

Check the basic instrument functions and settings according to the
manufactu rer's instructions.
Check the instrument performance by running an instrument test sample.
ln the SparkLink software, create a new runtable and import the Sparklink
batch text or excel file created from the batch runsheet. The runtable will be
automatically populated with the method, sample name, vial position,
injection volume and SPE cartridge position. Make sure the correct vial
position and SPE cartridge are used. Click the "start" icon on the top of the
runtable.
ln the Analyst software, create a new batch file, then import the Analytst
batch text file and the batch table will be populated with sample name,
sample lD, and data file name. Make sure the correct acquisition method
and quantitation method are selected. Go to the quantitation tab, and make
sure all the levels of the calibration curve are filled in. Go to submit tab.
Highlight the rows of samples to run and click "submit" on the top right
corner. All samples on the Queue Manager should be in "waiting."
Click "Start" on the runtable in SparkLink and the online SPE will start with
the first sample. Then submit the batch in Analyst and click "run." Once the
sample clean-up finishes, the cartridge in the left clamp will be moved by
the gripper to the right clamp for elution, and the mass spectrometer will
promptly start the data collection.

OH-PAH in Urine

Page

DLS Method Gode: 6705.02

115

Processing of Data
2-NAP
l.Oe6

2-3PHE.

e 5 Oe5
ét

2-FLU

I-NAP

1-PYR
3-FL

o.o

1-PHE 4-pr1.6

l5'o

Tin e, ,r,¡rr2o'o
Figure 1. An example chromatogram of the standard (level 6).

25.O

An example HPLC chromatogram ¡s given in Figure 1. Process the data using the
Analyst, MultiQuant or lndigo software.
After peak integration is reviewed, go to the calibration curve page. Review the
calibration curve for each analyte and confirm that R2>0.95.
Export the results table as a text file (.txt), and place a copy of this result text file on
the CDC shared network drive. Run the DLS SAS program to check the batch QCs

Replacement and Periodic Maintenance of Key Gomponents
The instrumentation used is serviced according to the manufacturers'guidance
included in the instruments manuals or based on the recommendations of
experienced analysts/operators after following appropriate procedures to determine
that the instruments perform adequately for the intended purposes of the method.

Reportable Range of Results
The linear range of the standard calibration curve and the method limit of detection
(LOD) determine the highest and lowest reportable concentrations for the target
analytes. However, urine samples with analyte concentrations exceeding the highest
reportable limit may be re-extracted using a smaller volume (e.9., 50 prl or 2 fold
dilution) and re-analyzed so that the result is in the repodable range. Generally,
samples with extremely high values can be diluted up to 100 times.

NCEH'DLS/OATB

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116

Linearity Limits
Calibration curves constructed with the analytical standards are linear for all analytes
through the range of concentrations evaluated (Table 6). The linear range is up to
200 ng/ml for 1-NAP and 2-NAP (using their secondary ions) and up to 25 ng/ml for
the remaining analytes. Urine samples whose OH-PAH concentrations exceed these
ranges must be diluted and re-analyzed using a smaller sample size (up to 100
times dilution).

b. Limit of Detection
The limit of detection (LOD) for each analyte is presented in Table 6
Table

6. Limits of detection (LOD) and linearity limits.
Analyte

LOD (ng/mL)

Upper linearity limit
(ns/mL)
200
200

0.06
1-NAP*
2-NAP*
0.09
25
0.008
2-FLU
10
0.008
3-FLU
10
1-PHE
0.009
20
0.01
2-3PHE**
10
4-PHE
0.007
10
0.07
1-PYR
* Upper limits for 1-NAP and 2-NAP are achieved by using the first transition using
the ABSciex 6500 and the secondary ion using the ABSciex 5500, "1-NAP2" and"2NAP2.'
** 2-PHE and 3-PHE are measured together.

Urine

Page

DLS Method Gode: 6705.02

117

Precision
The precision of the method is reflected in the variance of quality control samples
analyzed over time. The mean concentrations and coefficients of variation (CV) of
QC samples are listed in Table 7. These QC samples were analyzed over 6 weeks
using two online SPE-HPLC-/MS/MS instruments.
Table 7. Mean, standard deviation, and GV for QG samples. The parameters are QG
pool specific.
QCH

QCL

Analyte

Mean

(nglmL)

Between

Within

Day SD

day SD

(nelmL)

cv%

Mean

(nelmL)

Between

Within

Day SD

day SD

(nelmL)

cv%

1-NAP

1-.17

0.09

0.05

6.9o/o

7.65

0.62

o.46

6.9%

2-NAP

1.53

o.t4

0.08

8.O%

8.84

0.65

o.41

6.6%

2-FLU

o.26

0.02

0.01

8.6%

L.L2

0.10

o.o7

8.0%

3-FLU

o.29

0.02

0.01

6s%

L.20

0.08

0.06

s.2%

1-PHE

0.18

0.03

0.01

13.7o/o

0.70

0.09

0.05

L2.2%

2-3PHE

0.39

0.05

0.02

1,L.2%

1.47

0.L5

0.09

4-PHE

o.1-4

o.02

0.01

16.7%

0.55

0.07

0.0s

93%
12.I%

1-PYR

o.37

0.05

0.03

12.8%

o.73

0.L1

0.08

13.8%

d. Analytical Specificity
The analyte peaks are located in well-defined regions of the chromatogram with no
visible interferences and low background.

NCEH/DLS/OATB

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DLS Method Gode: 6705.02

118

e. Accuracy
The accuracy of this method was evaluated by analyzing two NIST Standard
Reference Materials (SRMs) and by comparing the results obtained to their certified
concentrations for the 9 OH-PAHs (Table 8). The smoker SRM 3672 and the nonsmoker SRM 3673 were analyzed 4 times in 2 different runs. Averages of the
measured concentrations are given in Table 8.

Table 8. Measured concentrations (ng/mL) using this method in comparison to
the certified concentrations in two NIST SRMs (5)
SRM 3672 Smoker urine

Analyte

SRM 3673 non-Smoker urine

This

NIST

method

Certified

Accuracy

method

Certified

Accuracy

2-NAP

8.L82

8.730

93.7%

1.438

1,.345

106.9%

1-NAP

36.853

34.400

r07.1%

216.064

211.000

LO2.4%

3-FLU

o.4t4

o.428

96.8%

0.044

0.039

712.0%

2-TLU

0.720

0.870

82.8o/o

0.090

o.ro7

83.7%

2-3PHE

0.201

0.209

96.L%

0.055

0.053

IO3.Oo/o

1-PHE

0.L15

0.136

84.3%

0.048

0.049

98.4%

4-PHE

0.046

0.049

94.2%

o.o2r

0.010

2r3.9%*

1-PYR

0.r77

o.r73

ro2.2%

0.051

0.030

1.69.7%*

*: <3LOD

This

NIST

NCEH/DLS/OATB

OH-PAH in Urine

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DLS Method Code: 6705.02

119

The accuracy of the method was further assessed by repeated analyses (n=7) of
three spiking concentrations in synthetic urine (Table 9).

Table 9. Accuracy
Level -l-

Analyte

Spiked

(nelmL)

Level -2

Accuracy

Spiked

(nelmL)

Level -3

Accuracy

Spiked

(nelmL)

Accuracy

2-NAP

0.708

1,L2.8%

3.845

99.7%

L9.226

99.7%

1.-NAP

0.705

I13.3%

3.826

9s3%

19.130

98.7%

3-FLU

0.168

ro7.2%

0.915

98.6%

4.573

98.1%

2-FLU

o.r76

107.4%

0.956

993%

4.778

IOO.3%

2-3PHE

0.357

IT3.I%

1,.937

99.4%

9.684

101.9%

1-PHE

0.r75

ro9.8%

0.9s1

99.4%

4.753

101,.2%

4-PHE

0.173

r085%

0.939

98.3%

4.697

98.2%

1-PYR

o.t79

I05.5%

0.972

93.6%

4.860

toL.8%

10. Quality Assessment and Proficiency Testing
a. Quality Assessment
Daily experimental checks are made on the stability of the analytical system. Two
QCLs and two QCHs are prepared and placed randomly on each run. The
concentrations of the two QCH and two QCL are averaged to obtain one
measurement of QCH and QCL for each batch.

b. Quality Gontrol Procedures
1)

lndividual Sample

C)u al ifv

c hecks

Each individual sample will be subjected to a number of quality checks:
Auto integrations must be reviewed and integrated manually if needed
The relative retention time (RRT) of each analyte, if detectable, in relation to
its respective lS must be within 0.995 - 1.005 (e.g. 0.12 min difference
between the native and labeled 1-PYR). Check integration if the RRT falls
out of the range. lf a peak is present with its retention time out of the limit in

.
.

OH-PAH in U ne

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120

relation to its lS retention time, an analytical interference may prevent the
correct measurement of the target analyte, and the result for that analyte is
coded as non-repodable (NR)
2)

Establishi

QC limits

Quality control limits are established by characterizing assay precision with
repeated analyses of the QC pools. Different variables are included in the
analysis (e.9. multiple analysts and instruments) to capture realistic assay
variation over time. The mean, standard deviation (within day and between
day), coefficient of variation, and confidence limits are calculated from this QC
characterization data set. lndividual quality control charts for the
characterization runs are created, examined, and quality control limits are used
to verify assay precision and accuracy on a daily basis. QC characterization
statistics for OH-PAH analytes are listed in Table 7. Characterization statistics
are pool specific.
3)

Qualitv Control Evaluation

After the completion of a run, the quality control concentrations are evaluated to
determine if the run is "in control." The quality control rules apply to the
average of the two replicates of each of the QC pools. The quality control
results are evaluated according to a multi-rule QC check (6), and standard
criteria for run rejection based on statistical probabilities are used to declare a
run either in-control or out-of-control.
Two QC pools perrun with two or more QC results perpool

A) lf both QC run means are within
25¡ limits, then accept the run.

25'.n limits and individual results are within

B) lf 1 of the 2 QC run means is outside a 2Sm limit - reject run if
a) Extreme Outlier

4S'

- Run mean is beyond the characterization mean +/-

b) 3S Rule - Run mean is outside a 35' limit
c) 25 Rule - Both run means are outside the same 25' limit
d) 10 X-bar Rule - Current and previous 9 run means are on same side of
the characterization mean

C) lf one of the 4 QC individual results is outside a 25¡ limit - reject run
a) Extreme Outlier
mean +/- 45¡

if:

One individual result is beyond the characterization

AH in Urine

NCEH/DLS/OATB

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121

b) R 45 Rule - Within-run ranges for all pools in the same run exceed 45*
(i.e., 95% range limit). Note: Since runs have multiple results per pool for
2 pools, the R 45 rule is applied within runs only.

Abbreviations
S¡ = 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).
lf the QC result for an analyte is declared "out of control", the results of that
analyte for all study samples analyzed during that run are invalid for reporting

Proficiency Testing (PT)
The in-house proficiency testing (PT) scheme for this method is administered
by an in-house PT coordinator. PT samples are prepared in-house by spiking a
known amount of standard into a well characterized urine pool and blind-coded
by an in-house PT coordinator. PT samples are analyzed twice a year using the
same method described for unknown samples.
ln addition to the in-house PT program, a minimum of once per year, we also
analyze two reference urine samples fodified with 1-NAP, 2-NAP and 1-PYR
(as of 2015) as part of the German External Quality Assessment Scheme (GEOUAS). G-EQUAS is organized and managed by the lnstitute and Outpatient
Clinic for Occupational, Social and Environmental Medicine of the University of
Erlangen-Nuremberg (Erlangen, Germany). The program, evaluation, and
certification are based on the guidelines of the German Federal Medical
Cou ncil (http ://www. g-eq uas.de/).
All proficiency results shall be appropriately documented. lf the assay fails
proficiency testing, then the sample preparation and instrumentation are
thoroughly examined to identify and correct the source of assay error.

11. Remedial Action if Calibration or QG Systems

Fail to Meet Acceptable Griteria

lf the calibration or QC systems fail to meet acceptable criteria, suspend all operations
until the source orcause of failure is identified and corrected. lf the source of failure is
easily identifiable, for instance, failure of the mass spectrometer or a pipetting error, the
problem is immediately corrected. Othenruise, prepare fresh reagents and clean the
mass spectrometer system. Before beginning another analytical run, re-analyze several
QC materials (in the case of QC failure) or calibration standards (in the case of

OH-PAH in Urine
DLS Method Code: 6705.02

NCEH'DLS/OATB
P

age

122

calibration failure). After re-establishing calibration or quality control, resume analytical
runs. Document the QC failures, review the situation with the supervisor and/or his/her
designee to determine source(s) of problem, and take measures to prevent reoccurrence of the same problem.

12, Limitations of Method, lnterfering Substances and Conditions
This is an isotope dilution mass spectrometry method, widely regarded as the definitive
method for the measurement of organic toxicants in human body fluids. By using
tandem mass spectrometry, most analytical interferences are eliminated. However,
unknown endogenous substances may interfere with the chromatographic separation of
certain analytes and/or suppress instrument sensitivity, especially when the urine
samples are non-fasting or may also contain many other potential substances in
addition to the target analytes (e.9., smokers' urine). To overcome inadequate
chromatography or analytical sensitivity, dilute the sample and re-inject ("instrument
rerun"). lf the instrument rerun results are not acceptable, repeat the sample
preparation by using up to 100 times dilution. lf the diluted analysis still results in an
interference that cannot be separated chromatographically or severe suppression of the
target analyte signal and/or its lS, the result for that analyte is coded as not reportable
(NR)

13. Reference Ranges (Normal Values)
Reference range values for the OH-PAH metabolites, established based on the National
Health and Nutrition Examination Survey (NHANES), can be found at
http ://www. cd c. gov/exposu rerepo rt.

14. Critical Call Results ("Panic Values")
lnsufficient data exist to correlate urinary OH-PAH concentrations with serious health
effects in humans. Therefore, no established "critical call" values exist. Test results in
this laboratory are reported in support of epidemiological studies, not for clinical
assessments.

15. Specimen Storage and Handling During Testing
Urine specimens may reach and maintain ambient temperature during analysis. The
urine extracts are stored in a sample collection plate (when prepared using the Staccato
automated method) at -70 "C after analysis. CDC's unpublished data suggest that
these extracts are stable for at least one month.

OH-PAH in Urine
DLS Method Gode: 6705.02

16. Alternate Methods for Perform¡ng

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123

Test or Storing Specimens if Test System Fails

A GC-MS/MS method is available on site if necessary for measuring these PAH
metabolites in urine (3). Furthermore, aliquoting and spiking of the urine can also be
done manually if the Staccato automated system fails. Upon system failures, urine
extracts can be refrigerated for up to a week until the analytical system is restored to
functionality.

17. Test Result Reporting System; Protocol for Reporting Critical Calls (if Applicable)
Study subject data can be reported both in concentration units (e.9., trrg/L, ng/ml, ng/L,
pg/ml) and/or adjusted based on creatinine excretion (e.9., Ug/g creatinine).

c.
d.

The data from each analytical run are initially processed and reviewed by the
laboratory supervisor, his/her designee or Quality Control officer to check
sample Quality Control parameters (e.9., recovery, relative retention time, blank
levels, calibration curve). The supervisor and/or his/her designee can provide
feedback to the analyst and request confirmation of the data as needed.
The Quality Control officer reviews each analytical run, identifies the quality
control samples within each analytical run, and determines whether the
analytical run is pedormed under acceptable control conditions.
One of the Division statisticians reviews and approves the quality control charts
pertinent to the results being reported.
lf the quality control data are acceptable, the laboratory supervisor or his/her
designee generates a memorandum to the Branch Chief, and a letter reporting
the analytical results to the person(s) who requested the analyses to be signed
by the Division Director.
The data are sent (generally electronically by e-mail) to the person(s) who
made the initial request.

Final hard copies of correspondence are maintained in the office of the Branch Chief
and/or his/her designee and/or with the quality control officer.

18. Transfer or Referral of Specimens; Procedures for Specimen Accountability

and

Tracking
Following successful completion of analysis, the urine must be returned to storage at 70 "C in case reanalysis is required. Urine samples shall be retained until valid results
have been obtained and reported and sufficient time has passed for review of the
results. Residual urine may be returned to the study Pl or properly discarded upon
completion of the project.

NCEH/DLS/OATB

OH-PAH in Urine

DLS Method Code: 6705.02

age

124

Standard record keeping (e.9., database, notebooks, data files) is used to track
specimens. Specimens can be transferred or referred to other DLS Branch laboratories
or, if required, to other laboratories. Transfer is normally carried out through the DLS
Samples Logistic Group. Specimens may also be stored at CDC specimen handling
and storage facility (CASPIR).

19. References
1 . IARC. 2010, vol 92 IARC Monographs on the Evaluation of Carcinogenic Risks to
Humans. Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some
Related Exposures. http://monographs.iarc.frlENG/Monographs/vol92/mono92.pdf.

2. ATSDR (1995) Toxicological Profile for Polycyclic Aromatic Hydrocarbons. Agency
for Toxic Substances and Disease Registry. Atlanta, GA.
http ://www. atsd r. cd c. g ov/toxp rof es/tp6 9. pdf .
i

3. LiZ, Romanoff LC, Trinidad DA, Pittman EN, Hilton D, Hubbard K, Carmichael H,
Parker J, Calafat AM, Sjödin A.2014 "Quantification of Twenty-one Metabolites of
Methylnaphthalenes and Polycyclic Aromatic Hydrocarbons in Human Urine", Anal
Bioanal Chem 406(13):31 19-29.

4. LiZ, Romanoff LC, Trinidad D, Hussain N, Porter EN, Jones RS, Patterson Jr DG,
Sjodin A. 2006. "Measurement of Urinary Mono-Hydroxylated Polycyclic Aromatic
Hydrocarbons Using Automated Liquid-Liquid Extraction and lsotope Dilution Gas
Chromatography/High Resolution Mass Spectrometry". Anal Chem 7 8:57 44-57 51
.

5. Schantz MM, Benner BA Jr, Heckert NA, Sander LC, Sharpless KE, Vander Pol SS,
Vasquez Y, Villegas M, Wise SA, Alwis KU, Blount BC, Calafat AM, Li Z, Silva MJ, Ye
X, Gaudreau E, Patterson DG Jr, Sjödin A.2015. "Development of urine standard
reference materials for metabolites of organic chemicals including polycyclic aromatic
hydrocarbons, phthalates, phenols, parabens, and volatile organic compounds", Anal
Bioanal Chem, DOI 10.1007/s00216-014-8441-02015 Feb 5. [Epub ahead of print]

6. Caudill SP, Schleicher RL, Pirkle JL. 2008. Multi-rule quality control for the agerelated eye disease study. Stat Med 27:4094-4106.

Use of trade names is for identification only and does not imply endorsement by the Public
Health Seruice or the U.S. Depañment of Health and Human Services.

OH-PAH in Urine

Page

DLS Method Gode: 6705.02

125

Appendix A - SPE tubing and valve sw¡tching system
Posltiou

Guard colùüu

lß

2 l:

3 rs6-2

3 rÆv

5.,. \l'¡ste

Rc'r¡
4

\l¡rsle
EPD nos

Right chup

Lefr cl¡DÞ

Gur¡d columû

\$
Posltlon 2

2t
3 rs$2

3 t,cv
4

5,' \'aste

LCllos

RCV
4

EPDfos'

Left

Rlgùt cl.Dp

(l¡Ep
Gürrd coloúD

Its
Posltlon 3

3 ¡.cv

$$r
I

LC llow

\llsle

IIPD nos

3 rss2

'\ 4

$¡¡le

r"'

Left GL[p

R¡ght

cl¡np

Gu¡ral coluDD

\ß
Posltlon 4

3 Rc'v

3 tcílt

3 rss.r
4

3 Rcv

rss-2
LC

llor

lv¡sae

Il'¡sae
EPD llo$

Left

clrep

Rlgha

tl¡mp

Position 1: The Cartridge is activated at the left clamp with 4 mL acetonitrile and equilibrated
with 2 mL 0.1o/oformic acid in water. Sample solution (50-500 pL) is injected with
1.5 mL 0.1o/o formic acid in water, followed by wash with 1.5 mL
acetonitrile/methanol/water (25125150, vlvlv) (the actual wash solution is -0.4 mL
by counting the volume of sample loops).
Position 2:The cartridge is moved to the right clamp. Methanol (2 mL) is used to clean the
loop.
Position 3: Methanol (350 pL) is used to elute out the analytes from the cartridge to the guard
column (pre-column). The HPLC flow passes the guard column at the same time to
focus the analytes onto the guard column.
Position 4: Methanol (2 mL) is used to clean the tubing system.

OH-PAH in Urine

NCEH/DLS/OATB

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DLS Method Gode: 6705.02

126

Appendix B - Ruggedness Testing
Procedure
Ruggedness testing was conducted to evaluate 5 parameters: enzyme amount, buffer
strength, buffer pH value, deconjugation time and deconjugation temperature. For each
parameter, 3 or 5 levels were tested, including 1 or 2lower level(s), 1 or 2 higher level(s),
and the method level. An anonymous urine pool was used in the ruggedness experiments.
Samples were run in triplicate. Repoded below are results on the major detectable OH-PAH
analytes.
Results in Tables

Enzyme amount

(mg/sample)

2-NAP

1-NAP

21-PHE
3PHE
averaqe concentration (nq/mL)

3-FLU

2-FLU

4-PHE

PYR

Lower levell

5mq

38.39

32.66

1.77

3.06

3.29

1.70

0.39

1.29

Lower level2

9mq

37.56

33.01

1.76

3.06

3.23

1.68

0.40

1.27

Method

1Omg

37.91

33.09

1.76

3.09

3.24

1.70

0.48

1.27

Hiqher levell

11mq

39.54

33.91

1.77

3.06

3.28

1.70

0.49

1.29

Hioher leve12

20mq

38.38

33.50

1.79

3.06

3.28

1.69

0.49

1.28

standard deviation
Lower level

5mq

0.43

0.02

0.00

0.02

0.03

0.01

Lower level2

9mq

0.25

0.10

0.03

0.02

0.01

0.0'1

0.01

Method

1Omq

0.18

0.65

0.01

0.04

0.03

0.00

0.01

Hiqher levell

11mq

0.28

1.12

0.03

0.04

0.02

0.01

0.02

Hiqher level2

20mq

0.3'1

0.31

0.01

0.02

0.01

0.00

0.03

23PHE
1-PHE
averaqe concentration (nq/mL)

4-PHE

Buffer strength
(M)

2-NAP

1-NAP

3-FLU

2-FLU

PYR

Lower levell

0.5M

35.59

31.60

1.73

3.09

3.21

1.73

0.49

1.22

Lower level2

0.9M

36.07

31.71

1.75

3.03

3.23

1.71

0.45

1.23

35.63

31.29

1.75

3.O7

3.22

1.69

0.44

1.23

Higher levell

1.1M

35.52

31.31

1.75

3.04

3.21

1.71

0.37

1.25

Hiqher leve12

1.5M

36.01

32.17

1.75

3.07

3.20

1.75

0.37

1.23

Method

standard deviation
Lower levell
Lower leve12
Method
Hiqher levell

0.5M

0.53

0.5'1

0.03

0.04

0.02

0.9M

1.43

1.70

o.o2

0.02

0.05

0.06

0.03

0.02

0.58

0.54

0.02

0.05

0.07

0.03

0.02

1.1M

0.87

0.81

0.03

0.04

0.05

0.01

OH-PAH in Urine

NCEH/DLS/OATB

DLS Method Gode: 6705.02

her level2

1.5M

Buffer pH

0.35

2-NAP

1.10

1-NAP

0.08

0.06

0.04

0.02

23PHE
1-PHE
(nq/mL)
averaqe concentration

4-PHE

0.02

3-FLU

2-FLU

age

0.02
1-

PYR

Lower levell

pH4.5

34.66

26.97

1.77

2.94

3.25

1.70

0.44

1.19

Lower level2

pH5.3

35.25

29.12

1.75

3.06

3.20

1.67

0.40

1.29

Method

pH5.5

36.27

29.22

1.75

3.10

3.26

1.70

0.47

1.26

Hiqher levell

pH5.7

35.47

28.72

1.73

3.02

3.22

1.63

0.44

1.21

Hiqher level2

pH6.5

36.46

28.17

1.79

3.11

3.27

1.63

0.44

1.24

Lower levell

pH4.5

1.67

0.18

0.12

0.01

0.02

0.03

Lower leve12

pH5.3

0.72

0.92

0.02

0.03

0.05

0.02

0.05

0.02

Method

pH5.5

0.83

0.9'1

0.0'l

0.05

0.04

0.03

0.00

o.o4

Hiqher level'1

pH5.7

0.51

0.55

0.04

0.08

0.03

0.01

Hiqher level2

pH6.5

0.73

0.76

0.07

0.08

0.05

0.03

3-FLU

2-FLU

1-PHE

4-PHE

standard deviation

De-conjugation
time (hours)

2-NAP

1-NAP

23PHE

PYR

averaqe concentration (nq/m L)
Lower leve12

17 hr

34.33

28.59

1.74

2.98

3.00

1.63

0.43

1.23

Method

18 hr

34.94

28.29

1.72

2.95

3.02

1.60

0.41

1.21

Hiqher levell

'19 hr

34.27

27.46

1.71

2.95

2.99

1.59

0.41

1.20

Hiqher level2

24 hr

35.31

28.28

1.73

2.98

3.06

1.63

0.33

1.21

standard deviation
Lower leve12

17 hr

0.33

0.26

0.02

0.04

0.02

0.01

0.04

Method

l8 hr

1.16

0.92

0.00

0.01

0.03

0.01

0.00

Hioher levell

19 hr

0.58

1.15

0.04

0.09

0.06

o.o2

0.02

0.03

Hiqher leve12

24 hr

0.98

0.86

0.01

0.03

0.05

0.03

0.00

0.01

23-FLU 2-FLU 3PHE
1-PHE
(no/mL)
averaqe concentration

4-PHE

Hydrolysis
temperature (oC)

2-NAP

1-NAP
28.84

1.68

3.07

37.94

31.71

1.74

36.39

28.79

1.73

Lower Level

320C

35.03

Method

370C

Hiqher Level

420C

PYR

3.11

1.64

0.49

1.22

2.98

3.00

1.57

0.42

1.20

2.99

3.15

1.60

o.45

1.14

standard deviation
Lower Level

320C

1.13

0.69

0.05

0.06

0.04

0.01

0.03

Method

370C

2.14

1.77

0.12

0.21

0.18

0.11

0.03

0.07

Hiqher Level

420C

0.73

0.65

0.04

0.07

0.04

0.02

0.03

127

AH in Urine

Page

DLS Method Gode: 6705.02

Results in Graphs
Ruggedness Testing - 2-NAP
45.0
40.0
35.0

30.0
J
E
o)

20.0

o
c
o

15.0

lLower level2

25.0

Lower levell

trMethod
oHigher levell

lHigher level2

10.0
5.0
0.0
Enzyme

amt

Buffer strength

De-conj

time

De-conj T

Ruggedness Testing - 1-NAP
40.0
35.0

300

1
E
È,
g
ej
c

25.0

lLower levell
tLower level2

20.0

trMethod
oHigher levell

15.0

¡H¡gher level2
1o.o
5.0
0.0
Enzyme

amt

Buffer strength

De-conj

time

De-conj T

Ruggedness Testing - 3-FLU
2.0
1.8
1.6

14
1.2

lLower level'1
tLower level2

1.0

tr Method

o
c
o

0.8

oHigher levell

0.6

J
E
o)

0.4
0.2
0.0
Enzyme

amt

Buffer strength

De-conj

time

De-conj T

Higher level2

128

NCEH/DLS/OATB

OH-PAH in Urine

Page 129

DLS Method Gode: 6705.02

Ruggedness Testing - 2-FLU
3.5
3.0

J
E
o)

c
o

ILower levell

Lower level2

oMethod

oHigher levell

1.0

Higher level2

0.5
0.0
Enzyme

amt

Buffer strength

De-conj

time

De-conj T

Ruggedness Testing - 2-3PHE
3.5
3.0

J
E

2.5

tLower levell

2.0

o
c
o

Lower level2

o Method

1.5

oHigher level'l

1.0

H¡gher level2

05
0.0
Enzyme

amt

Buffer strength

De-conj

time

De-conj T

Ruggedness Test¡ng - l-PHE
20

eq;
c

18
16
14
12
10

ILower levell

Lower level2

oMethod

08
06
04
02
00

trHigher levell

Enzyme

amt

Buffer strength

De-conj

time

De-conj T

Higher level2

Ruggedness Testing - 4-PHE
0.6
0.5

lLower levell

tLower level2

-J
E
(')

d
c
o

0.2

trMethod
oHigher levell

lHigher level2

0.0
Enzyme

amt

Buffer strength

De-conj

time

De-conj T

Ruggedness Testing - l-PYR
1.4
1.2
1.0

J
E
o)

o
c
o

lLower levell
¡Lower level2

0.8

oMethod
0.6

eHigher

0.4

rHigher level2

0.2
0.0
Enzyme

amt

Buffer strength

De-conj time

De-conj T

level'1

OH-PAH in Urine

NCEH'DLS/OATB

Page l3l

DLS Method Gode: 6705.02

Appendix C - Method Compar¡son Results
Method comparison was performed by analyzing samples using the previous method by
Agilent 7000 GC/MS/MS (Method #6703.04). The results are evaluated by both linear
regression plots between the previous ("GC") and current ('LC") results, as well as BlandAltman plot analysis (e.9., graphs of differences between the results from the two methods
against the mean of the two results). A total of 38 samples were included in the linear
regression analysis.

PLOTS
Representative Correlation plot and regression analysís
1-NAP

2-NAP
180

70
R'z=

É.

U
J

= L1,61,4x - 0.5896
R'z= 0.9986

160

y=1.1196x+0.1801

r40
! r20

0.9975

r.00

c.)

É.

U
J

3-FLU
y=L.I737x+0.0033...'

R'z=

=f
OJ

0.9978..,a'
!

200

GC-Result

1s0

L00

..o'

R'z: 0.999

ú.
(J 2
J

2
L)
J

7
1,

GC-Result

1.8

0.8

1.6

-y=1fl264x+O.O272

0.7

L,4

v
g

1.0

0.8
o.e

0.6

r.2

0.5

o.q

o.s

0.4

0.2

0,2

0.1

0,0

0.0

1.5

0.5

2.0

0.0

0.5

1.0

GC-Result

2,0

o.7
0,6

g
c)

ú.

,lJ

R2 =

0.s

0.9942

0.9973x +

1.5

P
5

0.4

o
É. 1,0

0.3

,lJ

0.2

0.5

0.1
0.0

0.0

0,0

0.4

0.2

0.6

0.0

0.8

2-NAP

160

120

y=-0,0023x+0.1943

R2 =

€zo

g
80
c

180

0.0729

Ë
ô

-ro
-40

5
80
c
Ë
i5

-oo
-80

-160

0.1501x:0;5571---R2

= 0.9426

-120

-60

1-NAP

b+o

2.O

and

Difference

1.5

1.0

GC-Result

0.5

20 40 60 80 100 L2A 740

160

AH in Urine

Page

DLS Method Gode: 6705.02

3-FLU

y=0,161x+0.0022

J¿

O3232

0.)

c)--1

'õ

80
c

L23456

0.,

õ
-4

-3

2.0

0.6716

.a
7

R'z=

-2

-4

y=-0,0644x+0.0159

ö¡r
(l,^
c

2-TLU

-6

Average (ng/mL)

2-3PHE

Average (nglmL)

1-PHE

1.5

110
E

b
5
I

y=0.029x+0.0252

0.5

y=-0.L427x-0.0193

E
b¡

os
00
20

9
-n(
@ ""

R':= 0.1157

5
I

0.0

td:É"

c)
c)

= 0.7183

eße,

1.0

õ -o.s

-10
-1.5
-2.O

1.0

-1.0

Average (nglmL)

4-PHE

Average (nelmL)

1-PYR

2.0
1.5

E
b!

5
Ic

R'z= 0.L213

y=-0.0054x+0.oI21'

1.0

R'?=

00
0.5

o)
o)

y=-0.0276x+0.0057

-os

1.0

b,0

0.5

(lJ

0.0

c(lJu
(lJ

-05

-L0

0.5

0.0046

1.0

-1-.5

-L.0

Average (nglmL)

-2.0

Average (nglmL)

1.5

2.0

133

Laboratory Procedure Manual
Analyte:

Polyfluoroalkyl Substances: 2-(N-methyl-perfluorooctane
sulfonamido) acetate, perfluorobutane sulfonate, perfluorohexane
sulfonate, n-perfluorooctane sulfonate, sum of
perfluoromethylheptane sulfonate isomers, sum of
perfluorodimethylhexane sulfonate isomers, perfluoroheptanoate, nperfluorooctanoate, sum of branched perfluorooctanoate isomers,
perfluorononanoate, perfluorodecanoate, perfluoroundecanoate, and
perfluorododecanoate
Matrix:
Method:

Method No:

Serum
Online Solid Phase Extraction-High Performance Liquid
Chromatography-Turbo Ion Spray-Tandem Mass Spectrometry
(online SPE-HPLC-TIS-MS/MS)
6304.06

As performed by:
Organic Analytical Toxicology Branch
Division of Laboratory Sciences
National Center for Environmental Health
Contact:
Xiaoyun (Sherry) Ye, M.S.
Phone: 770.488.7502
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.

Perfluoroalkyl and Polyfluoroalkyl Substances
NHANES 2013-2014

1 of 41

Public Release Data Set Information
This document details the Lab Protocol for testing the items listed in the following table:

File Name

Variable Name

SAS Label (and SI units)

LBXPFDE

Perfluorodecanoic acid (ng/mL)

LBXPFHS

Perfluorohexane sulfonic acid (ng/mL)

LBXMPAH

2-(N-methyl-PFOSA) acetic acid (ng/mL)

LBXPFBS

Perfluorobutane sulfonic acid (ng/mL)

LBXPFHP

Perfluoroheptanoic acid (ng/mL)

LBXPFNA

Perfluorononanoic acid (ng/mL)

LBXPFUA

Perfluoroundecanoic acid (ng/mL)

LBXPFDO

Perfluorododecanoic acid (ng/mL)

SSNPFOA

Linear perfluorooctanoate (ng/mL)

SSBPFOA

Branched isomers of perfluorooctanoate (ng/mL)

SSNPFOS

Linear perfluorooctane sulfonate (ng/mL)

SSMPFOS

Monomethyl branched isomers of PFOS (ng/mL)

PFAS_H

SSPFAS_H

Perfluoroalkyl and Polyfluoroalkyl Substances
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1. Clinical Relevance and Summary of Test Principle
a. Clinical Relevance
Some per- and polyfluoroalkyl substances (PFASs), including perfluorooctane
sulfonate (PFOS) and perfluorooctanoate (PFOA), persist in humans and the
environment and have been detected worldwide in wildlife 1. Exposure to PFOS
and PFOA in the general population is also widespread, although demographic,
geographic, and temporal differences exist 2-14. In animals, exposure to PFOS
and PFOA is associated with adverse health effects 15-17 albeit at serum
concentrations orders of magnitude higher than the concentrations observed in
the general population 18,19. PFOS was used in a wide variety of industrial and
consumer products including protective coatings for carpets and apparel, paper
coatings, insecticide formulations, and surfactants. In 2000, 3M, the sole
manufacturer of PFOS in the United States and the principal manufacturer
worldwide, announced that it was discontinuing its perfluorooctanyl chemistries,
including PFOS. Shortly after, EPA also identified possible related concerns with
respect to PFOA and fluorinated telomers. PFOA has been used primarily to
produce its salts which are used in the production of fluoropolymers and
fluoroelastomers. These polymers are used in many industrial and consumer
products, including soil, stain, grease, and water resistant coatings on textiles
and carpet; uses in the automotive, mechanical, aerospace, chemical, electrical,
medical, and building/construction industries; personal care products; and nonstick coatings on cookware.
The electrochemical fluorination (ECF) manufacturing method used from the
1950s until the early 2000s to produce PFASs including PFOA, and PFOS and
its precursors yielded branched and linear isomers. By contrast, another
method, telomerization, produces almost exclusively linear compounds 20. The
structural isomer patterns of PFOA and PFOS in humans may be useful for
understanding routes and sources of exposure 20.
b. Test Principle
Online solid phase extraction coupled to high performance liquid
chromatography-turboionspray ionization-tandem mass spectrometry (online
SPE-HPLC-TIS-MS/MS) is used for the quantitative detection of PFASs: 2-(Nmethyl-perfluorooctane
sulfonamido)
acetate
(Me-PFOSA-AcOH),
perfluorobutane sulfonate (PFBuS), perfluorohexane sulfonate (PFHxS), nperfluorooctane sulfonate (n-PFOS), sum of perfluoromethylheptane sulfonate
isomers (Sm-PFOS, monomethyl branched isomers of PFOS), sum of
perfluorodimethylhexane sulfonate isomers (Sm2-PFOS, dimethyl branched
isomers of PFOS), perfluoroheptanoate (PFHpA), n-perfluorooctanoate (nPFOA), sum of branched perfluorooctanoate isomers (Sb-PFOA, branched
PFOA isomers), perfluorononanoate (PFNA), perfluorodecanoate (PFDeA),
perfluoroundecanoate (PFUA), and perfluorododecanoate (PFDoA)21. Briefly,
after dilution with formic acid, one aliquot of 50 μL of serum is injected into a
commercial column switching system allowing for concentration of the analytes
on solid-phase extraction column. Separation of the analytes from each other
and from other serum components is achieved with high-performance liquid

Perfluoroalkyl and Polyfluoroalkyl Substances
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3 of 41

chromatography. Detection and quantification are done using negative-ion
TurboIonSpray ionization, a variant of electrospray ionization, tandem mass
spectrometry. This method allows for rapid detection of these PFASs in human
serum with limits of detection in the low parts per billion (ppb or ng/mL) range.
2. Safety Precautions
a. Reagent Toxicity or Carcinogenicity
Some of the reagents used are toxic. Special care should be taken to: 1) Avoid
contact with eyes and skin, 2) avoid use of the organic solvents in the vicinity of
an open flame, and 3) use solvents only in well-ventilated areas.
Note: Material Safety Data Sheets (MSDS) for the chemicals and solvents used
in this procedure can be found at www.ilpi.com/msds/index.html; some of them
may be found in a binder in the laboratory. Laboratory personnel are advised to
review the MSDS before using chemicals.
Care should be exercised in the handling of all chemical standards.
b. Radioactive Hazards
None.
c. Microbiological Hazards
The possibility of being exposed to various microbiological hazards exists.
Appropriate measures (i.e., universal precautions) should be taken to avoid any
direct contact with biological specimens (i.e., use gloves, laboratory coats, safety
glasses, chemical or biological hoods). Any residual biological material should be
appropriately discarded and prepared for autoclaving after analysis is completed.
All disposable laboratory supplies must also be placed in an autoclave bag for
disposal. The Hepatitis B vaccination series is recommended for health care and
laboratory workers who are exposed to human fluids and tissues. Laboratory
personnel who handles human fluids and tissues is required to take the
“Bloodborne Pathogens Training” course offered at CDC to insure proper
compliance with CDC safe work place requirements.
d. Mechanical Hazards
There are only minimal mechanical hazards when performing this procedure
using standard safety practices. Laboratorians should avoid any direct contact
with the electronics of the mass spectrometer, unless all power to the instrument
is off. Generally, only qualified technicians should perform the electronic
maintenance and repair of the mass spectrometer. Contact with the heated
surfaces of the mass spectrometer (e.g., interface) should be avoided.
e. Protective Equipment
Standard safety protective equipment should be utilized when performing this
procedure. This includes lab coat, safety glasses, durable gloves (e.g., nitrile or
vinyl), and/or a chemical fume hood or biological safety cabinet.

Perfluoroalkyl and Polyfluoroalkyl Substances
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f. Training
Training and experience in the use of a triple quadrupole mass spectrometer and
the on-line SPE extractor should be obtained by anyone using this procedure.
Operators are required to read the operation manuals or laboratory SOP. Formal
training is not necessary; however, an experienced user should train all of the
operators.
g. Personal Hygiene
Care should be taken in handling any biological specimen. Routine use of gloves
and proper hand washing should be practiced. No food or drink is allowed in
laboratory areas.
h. Disposal of Wastes
Solvents and reagents are disposed of in an appropriate container clearly
marked for waste products and temporarily stored in one of the chemical fume
hoods. Containers, glassware, etc., that come in direct contact with the specimen
are either autoclaved or decontaminated with 10% bleach. Contaminated
analytical glassware is treated with bleach, washed and reused; disposable
labware is autoclaved before disposal. To insure proper compliance with CDC
requirements, laboratory personnel are required to attend annual hazardous
waste disposal courses.
3. Computerization; Data-System Management
a. Software and Knowledge Requirements
All samples are queued for analysis in a database created using Microsoft
Access. Mass spectrometry data are collected and stored using the Analyst
Software of the ABI 5500 and ABI 6500 Qtrap mass spectrometers. During
sample preparation and analysis, samples are identified by their Sample Name
and Sample ID. The Sample Name is used to identify each specimen and links
the laboratory information with the demographic data recorded by the sample
takers. The Sample ID is used to identify each specimen and links the laboratory
information with the demographic data recorded by the sample takers. In case of
repeated measurements, one specimen in the database may have more than
one Sample Name, but only one Sample ID. All raw data files are processed
using the Analyst software and are archived for future reference. The Analyst
software selects the appropriate peak based on the precursor/product ion
combination and chromatographic retention time and subsequently integrates the
peak area. It also allows manual peak selection and area integration. The raw
data (peak area, peak height, retention time, analyte name, MRM transition
name) are exported to the Access database used for storage and retrieval of
data. The Access database is stored on a network drive; it may also be backed
up in additional archive locations. Statistical analysis of the data, programming,
and reporting are performed using the Statistical Analysis System (SAS) software
(SAS Institute, Cary, NC). Knowledge and experience with these software
packages (or their equivalent) are required to utilize and maintain the data
management structure.

Perfluoroalkyl and Polyfluoroalkyl Substances
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5 of 41

b. Sample Information
Sample names and Sample IDs are entered into the Access database before
sample preparation. If possible, for unknown samples, sample study IDs are
read in by a barcode reader directly from the vials labels. Sample names for
Standards, and Blanks (SBs, HSBs, QCBs) are entered manually. The Sample
Log Sheet, containing Sample Names, Sample IDs, and sample study IDs is
printed from the Access database and is used to record information during
sample preparation. Sample Names, Sample IDs, and sample study IDs are
exported as tab delimited text files from the Access database and imported into
the Acquisition Batch table (*.dab) of the Analyst program on the mass
spectrometer. After MS data collection and peak integration, data are saved as a
tab delimited file and imported into the Access database. Further manipulation of
the data, including QC evaluation and statistical analyses, are performed using
SAS statistical software. After any additional calculations or corrections by the
analyst are completed and the reviewing supervisor approves the final values for
release, a comma-delimited file (SAS output) is generated.
c. Data Maintenance
Raw files are regularly backed up onto an external hard drive. Sample and
analytical data are checked after being entered into the database for transcription
errors and overall validity. The database is routinely backed up onto a computer
hard drive and onto a network drive. Data from completed studies are saved on
an external hard drive and/or a network drive. Additionally, paper copies of
signed final report memos are scanned and saved as official government
records.
4. Procedures for Collecting, Storing, and Handling Specimens; Criteria for
Specimen Rejection
a. Sample Collection and Storage
Follow recommended phlebotomy practices for the collection of blood and
separation of blood serum. Preferably, a minimum of 0.5 mL of serum (plasma
may also be used) should be placed in standard collection containers,
refrigerated as soon as possible, and transferred to labeled containers for
storage. Sera should be stored frozen preferably in polypropylene or
polyethylene containers. Glass containers may be used if the specimens are to
be analyzed for other environmental chemicals for which storage in plastic may
be a problem. Teflon® coated materials should be avoided.
b. Sample Handling
In general, serum specimens should be shipped or transported cold (dry ice, ice
or blue ice can be used). Special care must be taken in packing to protect vials
from breakage during shipment.
Before analysis, samples are thawed, vortexed, aliquoted, and the residual
specimen is refrozen and stored. The integrity of samples thawed and refrozen
several times doesn’t appear to be compromised.

Perfluoroalkyl and Polyfluoroalkyl Substances
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c. Criteria for Specimen Rejection
Specimens can be rejected if tubes/vials leaked, are broken, appear
compromised or tampered with, or hold inadequate volume for analysis.
5. Procedures for Microscopic Examinations; Criteria for Rejecting Inadequately
Prepared Slides
Not applicable for this procedure.
6. Preparation of Reagents, Calibration (Standards), Controls, and All Other
Materials; Equipment and Instrumentation
a. Reagents and Sources
Methanol (MeOH), acetonitrile, and water were HPLC grade purchased from
Honeywell Burdick & Jackson (Muskegon, MI). Formic acid (99%) was
purchased from EM Science (Gibbstown, NJ). Acetic acid (glacial) was
purchased from J.T. Baker (Phillipsburg, NJ). The following PFASs were
purchased form Wellington Laboratories (Guelph, ON, Canada): Nmethylperfluoro-1-octanesulfonamidoacetic acid (Me-PFOSA-AcOH), sodium
perfluoro-1-hexanesulfonate (PFHxS), potassium perfluoro 1-butanesulfonate
(PFBuS), sodium perfluoro 1-octanesulfonate (n-PFOS), mixture of sodium
perfluoro-5-methylheptane sulfonate (P5MHpS) and perfluoro-5-methylheptanoic
acid (P5MHpA), mixture of sodium perfluoro-5,5-dimethylhexane sulfonate
(P55DMHxS)
and
perfluoro-5,5-dimethylhexanoic
acid
(P55DMHxA),
perfluoroheptanoic acid (PFHpA), ammonium perfluorooctanoate (n-PFOA),
perfluorononanoic
acid
(PFNA),
perfluorodecanoic
acid
(PFDeA),
perfluoroundecanoic acid (PFUA), and perfluorododecanoic acid (PFDoA).
Perfluoro-n-[1,2,3,4,5-13C]-heptanoic acid (13C 5 -PFHpA), perfluoro-n-[1,2,3,413C]-octanoic acid (13C -PFOA), perfluoro-n-[1,2,3,4,5-13C]-nonanoic acid (13C 4
5
PFNA), 2-Perfluorooctyl [1,2-13C]-ethanoic acid (13C 2 -PFDeA), 2-perfluorooctyl
[1,2-13C]-undecanoic acid (13C 2 -PFUA), perfluoro-n-[1,2-13C]-dodecanoic acid
(13C 2 -PFDoA), N-methyl-d 3 -perfluoro-1-octanesulfonamide acetic acid (D 3 -MePFOSA-AcOH), and sodium perfluoro 1-hexane [18O 2 ]-sulfonate (18O 2 -PFHxS),
sodium perfluoro 1-[1,2,3,4-13C]-octanesulfonate (13C 4 -PFOS) were purchased
form Wellington Laboratories. All reagents were used without further purification.
Other standards and reagents with similar specifications may be used.
b. Working Solutions
(1) HPLC Mobile Phase, 20mM Ammonium Acetate Buffer/acetonitrile
(95:5), pH 4.
To prepare 20 mM Ammonium acetate buffer (pH4.0), dilute 1140 µL of
concentrated acetic acid with approximately 800 mL water in a beaker.
Adjust pH to 4±0.1 by adding drop-wise 1:10 ammonium
hydroxide:water mixture. Transfer into a 1 L volumetric flask and fill up
to volume with deionized water.

Perfluoroalkyl and Polyfluoroalkyl Substances
NHANES 2013-2014

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Mix 950 mL of ammonium acetate buffer with 50 mL of acetonitrile in a
glass bottle. Prepare as needed and store at room temperature.
(2) HPLC Organic Mobile Phase, 100% HPLC acetonitrile
Refill as needed and store at room temperature.
(3) Organic solvent for SPE column regeneration, 100% Acetonitrile
Refill as needed and store at room temperature.
(4) Solid phase extraction (SPE) Acid Wash Solution, 0.1M formic acid
Dilute 3810 µL of 99% concentrated formic acid with water to 1000 mL in
a volumetric cylinder. Prepare monthly and store at room temperature.
c. Standards Preparation
(1) Analytical Calibration Standards
The native standard stock solutions of all the analytes are prepared in
methanol from the commercial solutions. The concentrations of the
commercial solutions are: 50 µg/mL for Me-PFOSA-AcOH and n-PFOS; 2
µg/mL for PFHxS, PFBuS, n-PFOS, PFHpA, n-PFOA, PFNA, PFDeA,
PFUA, and PFDoA; 1 µg/mL for P5MHpS and P55DMHxS; 1.96 µg/mL for
P5MHpA and P55DMHxA. We used P5MHpS to quantify Sm-PFOS and
P55DMHxS to quantify Sm2-PFOS. We used the combined response of
the P5MHpA and P55DMHxA standards for the quantitation of Sb-PFOA.
The PFOA isomers known to be included in Sb-PFOA are perfluoro-3methylheptanoic acid, perfluoro-4-methyheptanoic acid, perfluoro-5methyheptanoic acid, perfluoro-6-methyheptanoic acid, perfluoro-4,4dimethylhexanoic acid, perfluoro-5,5-dimethylhexanoic acid, perfluoro-3,5dimethylhexanoic acid, and perfluoro-4,5-dimethylhexanoic acid. Similarly,
the PFOS isomers known to be included in Sm-PFOS are perfluoro-3methylheptane sulfonate, perfluoro-4-methylheptane sulfonate, perfluoro5-methylheptane sulfonate, perfluoro-6-methylheptane sulfonate. The
PFOS isomers known to be included in Sm2-PFOS are perfluoro-4,4dimethylhexane sulfonate, perfluoro-5,5-dimethylhexane sulfonate,
perfluoro-4,5-dimethylhexane sulfonate, and perfluoro-3,5-dimethylhexane
sulfonate.
The spiking standard solutions are prepared in MeOH from native
standard stock solutions such as a 50-µL spike into 50 µL serum provides
concentrations that cover the linear range of the method (Table 1). The
spiking solutions are stored frozen in 1.0 mL aliquots in polypropylene
cryogenic vials until use.

Perfluoroalkyl and Polyfluoroalkyl Substances
NHANES 2013-2014

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Table 1. Concentrations of standards #1-9 (in µg/mL)
Standard
No

n-PFOS

Sm-PFOS
(P5MHpS)

Sm2-PFOS
(P55DMHxS)

PFHxS

PFBuS

Sb-PFOA
(P5MHpA+
P55DMHxA)

All other
analytes

Standard 1
Standard 2
Standard 3
Standard 4
Standard 5
Standard 6
Standard 7
Standard 8
Standard 9

0.015
0.06
0.15
0.60
1.5
6.0
15.0
60.0
115.

0.01
0.05
0.1
0.25
0.50
1.0
2.5
5.0
10.0

0.005
0.01
0.05
0.1
0.2
0.2
0.5
1.00
2.50

0.005
0.009
0.05
0.095
0.47
0.945
4.70
9.45
18.9

0.004
0.009
0.043
0.086
0.428
0.855
4.28
8.55
17.1

0.0294
0.118
0.294
0.686
1.37
2.45
5.88
11.8
24.5

0.005
0.01
0.05
0.1
0.5
1.0
5.0
10.0
20.0

(2) Internal Standard Spiking Solution
The internal standard spiking solution is prepared by dissolving
appropriate amounts of 13C 2 -PFHxA, 13C 5 -PFHpA, 13C 4 -PFOA, 13C 4 PFOS, 18O 2 -PFHxS, 13C 5 -PFNA, 13C 2 -PFDeA, 13C 2 -PFDoA, and D 3 -MePFOSA-AcOH (4-6 ng/mL) in water/methanol (50/50). A 50 µL spike of
this solution provides concentrations of 4-6 ng/mL in 50 μL serum.
Spiking solutions are stored frozen in 2.0 mL aliquots in polypropylene
cryogenic vials until use.
(3) Mass-Spec Operational Check Standard
The instrument test sample is prepared by spiking the reagent blank with
all analytes to final concentrations of 0.3-0.5 ng /mL.
(4) In-house Proficiency Testing (PT) Standards
Appropriate aliquots of each stock standard are added to calf serum
pools to produce 3 sets of in-house proficiency testing (PT) standards.
The PT standards are mixed, aliquoted into polypropylene vials and
frozen until needed. PT standards are characterized by at least 20
repeated analyses to determine the mean and standard deviation of the
measurements.
d. Materials
1) HySphere C8-SE (7µM) cartridge (i-Chrome solutions, Plainsboro, NJ)
2) Chromolith® HighResolution RP-18e column (4.6 × 100 mm) (Merck KGaA,
Germany).
3) Chromolith® HighResolution RP-18e Guard column (5 X 4.6 mm) (Merck
KGaA, Germany).
4) Chromolith® HighResolution RP-18e column (4.6 × 25 mm) (Merck KGaA,
Germany).
5) 750 µL polypropylene autosampler vials with polyethylene snap caps
(National Scientific Company, Rockwood, TN).
6) Tip ejector variable volume micropipettes (Wheaton, Millville, NJ) and pipette
tips (Rainin Instruments Co., Woburn, MA).

Perfluoroalkyl and Polyfluoroalkyl Substances
NHANES 2013-2014

9 of 41

7) 5.0 mL and 2.0 mL polypropylene cryovials (National Scientific Company,
Rockwood, TN).
8) Assorted glass and polypropylene labware.
e. Equipment
1)

2)
3)
4)
5)
6)
7)

Symbiosys extractor equipped with an Alias autosampler run by the
SparkLink software program (Spark Holland Inc. dba iChrom Solutions,
Plainsboro, NJ).
Agilent 1200 binary pump and degasser (Agilent Technologies).
Applied Biosystems ABI 5500 or ABI 6500 Qtrap mass spectrometer
(Applied Biosystems, Foster City, CA).
Sartorius Genius Series ME models Electronic Analytical & Semi –
microbalances (Sartorius AG, Goettingen, Germany).
Sartorius top – loading balance (Sartorius AG, Goettingen, Germany).
pH meter (AB 15 pH Meter, Fisher Scientific).
Vortex mixer (Type 16700, Barnstead International, Dubuque, Iowa).

f. Instrumentation
(1) Automated SPE
Tubing diagram for the Symbiosis column switching system used in
concurrent SPE/HPLC mode. (LCV: left clamp valve; DV-1: divert valve 1;
DV-2: divert valve 2; RCV: right clamp valve).
The method uses both left and right cartridge clamps, the four switching
valves, and the high pressure dispenser. The left clamp, the left clamp
valve (LCV), and left divert valve (DV-1) are used for SPE separation
while the right clamp, the right clamp valve (RCV) and right divert valve
(DV-2) are used for the HPLC elution. The SPE run of each sample starts
with the conditioning of a HySphere C8-SE (7µM) cartridge with HPLCgrade acetonitrile (2 mL) and 0.1 M formic acid (2 mL). Afterward, 500 µL
of the sample (containing 50 µL serum) injected into the 1 mL sample loop
is loaded onto the SPE column using 2 mL 0.1 M formic acid with 1
mL/min flow rate. Next, the SPE column is washed with 2 mL 90% 0.1 M
formic acid/10% Acetonitrile. The time of the SPE cleanup (including
injection time) is 10 min long. Before starting the clean up of the next
sample, the cartridge containing the extracted analytes is transferred by a
robotic gripper from the left clamp into the right clamp. Therefore, while
the right clamp is used for analyte elution and HPLC-MS/MS acquisition,
the left clamp could be used for the clean up of the next sample. Once,
the SPE column is in the right clamp, the right clamp valve remains in bypass (1-2) position until the HPLC-MS/MS system becomes ready to begin
acquisition.

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High Pressure
Dispenser

HPLC
pump

Filter column
HPLC
Precolumn

Autosampler
waste

3 LCV 6
4

2
3

waste

DV-1 6
4

Cont. delay col.

DV-2 6
4

RCV 6

3
4

waste

Left clamp

MS/MS

HPLC column

Right clamp

The Symbiosis system is used in concurrent SPE/HPLC mode controlled
by the SparkLink software (Table 2).

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Table 2. Valve configurations used for concurrent SPE clean up and
HPLC-MS/MS acquisition.

Stepsa

Method

LCV

DV-1

DV-2

RCV

Time
(min)

Move cartridge from left clamp to right
clamp

6-1

1-2

6-1

1-2

0.1

Load new cartridge into left clamp

6-1

1-2

6-1

1-2

0.2

Send contact closer signal to HPLC/MS/MS

6-1

1-2

6-1

1-2

0.1

Begin HPLC gradient elution by-pass
HPLC column and MS/MS

6-1

1-2

6-1

3.0b

Condition left cartridge
(2 mL acetonitrile, 2 mL/min)

1-2

6-1

1.2

Equilibrate left cartridge
(2 mL 0.1 M formic acid, 2 mL/min)

1-2

6-1

1.2

Load 500 µL sample on left cartridge
(2 mL, 0.1 M formic acid, 1 mL/min)

1-2

6-1

4.4

Forward wash left cartridge (2 mL
90% 0.1 M formic acid/10%
acetonitrile, 1 mL/min)

1-2

6-1

1.2

Return right cartridge to tray

6-1

1-2

6-1

1-2

0.1

a The

method used for the first sample included only steps 2 and 5-8. The method used for
the acquisition of the last sample included only steps 1, 3, 4, and 9.
b For the acquisition of the last sample duration of step 4 was 13 min.

(2) HPLC configuration
At the beginning of the HPLC-MS/MS acquisition, the right clamp valve
is turned into 6-1 position for the first 10 min of the HPLC gradient
program to transfer the analytes from the SPE column to the HPLC
column. At 10 min, the right clamp valve turns back to 1-2 position and
the SPE column is returned to the cartridge tray while the HPLC gradient
program continues. The HPLC pump is operated at a 1000 µL/min flow
rate with 95% of 20 mM ammonium acetate (pH 4) and 5% of acetonitrile
as mobile phase A and 100% acetonitrile as mobile phase B. The
analytes are separated from each other and other extracted components
on two Chromolith® HighResolution RP-18e columns (4.6 × 100 mm)
preceeded by a Chromolith® HighResolution RP-18e (5 X4.6 mm) guard
column and a Chromolith® HighResolution RP-18e (4.6 × 25 mm)
column. To delay the elution of the PFAS contaminants leaching out

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from Teflon parts of the HPLC pump, a 4.6 mm x 25 mm Chromolith®
HighResolution RP-18e column is inserted between the HPLC pump and
the right clamp valve. Because contaminants have to go through twice
the column length, their peaks elute 1 min after the main analytes bands
without interfering with the measured concentration.
Table 3. HPLC configuration
Parameters

Setting
95% 20 mM ammonium acetate, pH = 4/5%
acetonitrile
100% acetonitrile
1000 µL/min

Mobile Phase A
Mobile Phase B
Flow rate

Table 4. Mobile phase gradient
Time (min)

8.5

8.51

12.1

13.2

Mobile
phase B%
Flow rate
(mL/min)

1.0

Time (min)

13.3

13.5

13.6

15.5

15.6

16.5

16.6

18.1

1.5

1.8

2.0

1.5

1.0

Mobile
phase B%
Flow rate
(mL/min)

(3) Mass Spectrometer Configuration
Detection of the target analytes is conducted on the ABI 5500 or ABI
6500 Qtrap mass spectrometer in the negative ion Turbo Ion Spray (TIS)
mode. The TIS ionization source is a variant of the electrospray source
and is used to convert liquid phase ions into gas phase ions. We use
laboratory-grade air heated turbo ion spray gas (GS1=50 and GS2=50)
gas. The heated turbo ion spray gas temperature is set at 400 °C. The
curtain and collision gas (nitrogen) settings are as follows: collision
(medium), curtain gas (CUR=30 [ABI 5500], CUR=45 [ABI 6500]).
Ionization parameters and collision cell parameters are optimized
individually for each analyte (Table 5). Unit resolution is used for both
Q1 and Q3 quadrupoles. The dwell time is 50 msec for all compounds.

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Table 5. Mass spectrometric parameters for measuring PFASs
(M-H)- Precursor
ion (m/z)

Product
ion (m/z)

DP
(volts)

CE
(volts)

570
573
299
299
399
399
403
403
499
499
503
503
499
363
368
413
413
417
463
468
513
515
563
565
613
615

512
515
99
80
99
80
103
84
80
99
80
99
80
319
323
369
369
372
419
423
469
470
519
520
569
570

-45
-45
-70
-70
-70
-70
-70
-70
-70
-70
-70
-70
-70
-25
-25
-27
-27
-30-30
-30
--30
-30
-30
-30
-30
-45

-30
-30
-80
-85
-80
-85
-80
-85
-90
-80
-85
-85
-90
-13
-13
-14
-14
-15
-13
-13
-15
-15
-17
-17
-18
-15

Me-PFOSA-AcOH
D3-Me-PFOSA-AcOH(IS)
PFBuS-1
PFBuS-2a
PFHxS-1
PFHxS-2 a
PFHxS-18O2-1 (IS)b
PFHxS-18O2-2 (IS)a
n-PFOS-1 a
n-PFOS-2
PFOS-13C4-1 (IS)c
PFOS-13C4-2 (IS)
Sm-PFOS
PFHpA
PFHpA-13C5(IS)
n-PFOA
Sb-PFOA
PFOA-13C4 (IS)d
PFNA
PFNA-13C5 (IS))
PFDeA
PFDeA-13C2 (IS)
PFUA
PFUA -13C2 (IS)
PFDoA
PFDoA-13C2 (IS)
a

used only as confirmation ion
PFHxS-18O2-1 was used as IS for PFBuS.
c PFOS-13C was used as IS for n-PFOS, Sm-PFOS, and Sm2-PFOS.
4
d PFOA-13C was used as IS for both n-PFOA and Sb-PFOA.
4

1. Calibration and Calibration-Verification Procedures
a. Calibration Curve
Nine-point calibration curves are normally constructed with each quantitative
run from the analyte area ratios (i.e., analyte area/internal standard area)
obtained from extracted standards in calf serum. A linear regression analysis
(weighted by 1/x) of the area ratio versus standard concentration is
performed. The area of the Q1 ion for each analyte is used for quantification
(for PFOS we use Q2). Correlation coefficients are generally greater than
0.97. Samples with values exceeding the highest point in the calibration
curve are reanalyzed using less serum.
b. Mass Spectrometer Calibration

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The ABI 5500 or ABI 6500 Qtrap mass spectrometer is calibrated and tuned
at least once per year using a polypropylene glycol (PPG) solution according
to the instructions contained in the operator’s manual. The instrument
sensitivity is checked periodically by injecting the Instrument Test sample.
c. Calibration Verification
1) Calibration verification is not required by the manufacturer. However, it should
be performed after any substantive changes in the method or instrumentation
(e.g., new internal standard, change in instrumentation), which may lead to
changes in instrument response, have occurred.
2) Calibration verification must be performed at least once every 6 months.
3) All calibration verification runs and results shall be appropriately documented.
4) According
to
the
updated
CLIA
regulations
from
2003
(http://www.cms.gov/Regulations-andGuidance/Legislation/CLIA/downloads/6065bk.pdf), the requirement for
calibration verification is met if the test system’s calibration procedure
includes three or more levels of calibration material, and includes a low, mid,
and high value, and is performed at least once every six months.
5) All of the conditions above are met with the calibration procedures for this
method. Therefore, no additional calibration verification is required by CLIA.

d. Proficiency Testing (PT)
(1) Three pools of PT samples, which encompass the entire linear range of the
method, are prepared in-house as described in the standard preparation
section. Characterization of PT materials requires at least 20 separate
determinations.
Once the PT pools are characterized, the mean
concentration and standard deviation of the PT materials are forwarded to a
DLS representative (PT administrator) responsible for executing the PT
program. These PT samples are blind-coded by the PT administrator and
returned to the laboratory staff for storage.
(2) Proficiency testing should be performed a minimum of once per 6 months.
When proficiency testing is required, the laboratory supervisor or his/her
designee will notify the PT administrator, and the PT administrator will
randomly select five PT materials for analysis. Following analysis, the results
will be forwarded directly to the PT administrator for evaluation. A passing
score is obtained if at least four of the five samples fall within the prescribed
limits established by the PT administrator. The PT administrator will notify the
laboratory supervisor and/or his/her designee of the PT results (i.e., pass/fail).
(3) All proficiency results shall be appropriately documented.
(4) In addition to the in-house PT program, since 2005 we have successfully
participated in the international round-robin program organized by Intercal

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(Sweden) and RIVO (The Netherlands) when it is conducted for human
serum/plasma 22,23.
(5) Also, since 2006, at least once per year, we participate in the ongoing
German External Quality Assessment Scheme (G-EQUAS) for PFOS and
PFOA in serum, organized and managed by the Institute and Outpatient clinic
for Occupational, Social and Environmental Medicine of the University of
Erlangen-Nuremberg (Erlangen, Germany). The design, evaluation and
certification of G-EQUAS are based on the guidelines of the German Federal
Medical Council.
(6) Since 2011, three times a year we also participate in the ongoing Arctic
Monitoring and Assessment Program (AMAP) Ring Test for several PFASs in
human serum, conducted by the Institut National de Santé Publique du
Québec (INSP) in Canada.

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16 of 41

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8. Operating Procedures; Calculations; Interpretation of Results
a. Sample preparation
(1) Unknown, QC, Blank, and Standard Preparation
(a) Remove serum samples, working standard solutions and internal
standard solution from the freezer, and let them thaw. Label
polypropylene snap-cap autosampler vials with appropriate
Sample Names. Aliquot 0.1 M formic acid (500 μL for QCBs; 450
μL for UNKs, QCs, serum blanks (SBs), and human serum blanks
(HSBs); 400 μL for STDs) into appropriate vials.
(b) Dispense 50 µL of internal standard into each polypropylene
autosampler snap cap vials. In specific cases, the method can be
performed using a smaller volume of matrix; the applied dilution
factor must be noted appropriately.
(c) Add 50 µL of the appropriate native standard solution (S1-S9) into
the polypropylene vials designated for standards.
(d) Aliquot 50 μL of UNKs, QCs, SBs and HSBs into the designated
autosampler vials. For standards, aliquot 50 μL of blank serum.
Analysis may also be conducted with a smaller amount of serum;
in these circumstances, the volume used must be noted
appropriately throughout the analytical procedure.
(e) Vortex all vials for at least 10 seconds to make sure all the
internal standard and standard mixed into the sample.
(2) Automated SPE-HPLC-MS/MS Analysis Procedure
(a) Put the Alias into load position. Initialize the high pressure
dispenser (HPD) and the automated cartridge exchanger (ACE)
unit.
(b) Exchange the cartridge tray after every 500 samples.
(c) Purge the solvent lines on the HPLC binary pump and
equilibrate the HPLC column.
(d) In the SparkLink software, go to RunTables and open and set
up the batch table. For the first sample enter xx-method 1
which runs the injection and cleanup of the first sample. For the
second and consecutive samples use xx-method 2 which
initiates the HPLC/MS acquisition and runs the injection and
cleanup of the next sample. For the last sample enter xxmethod 3 which initiates the HPLC/MS acquisition of the last
sample. For injection volume, enter 500 μL. Make sure the

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right vial positions are entered and there is no sequential
duplication of cartridge numbers. The sample names and
sample IDs do not matter, since they will not be part of the
acquired data.
(e) Go to Excel, open the text file containing the batch table
created from Sample Login Table in the Microsoft Access
database. This file should not require any editing. Save the
table into the text file named import.txt into the Batch directory
(overwrite). Remember to CLOSE THE FILE IN EXCEL!!!!!
(f) Go to Analyst and import the import.txt file (Sample pull down,
go to gray header and click RMB, then Import From/File, select
Alias autosampler). Make sure that the proper Acquisition
Method and Quantitation Method are entered. Then, submit the
batch (highlight and/or click Submit, go to View Queue, and click
Start Sample). All samples on the Queue Manager should be in
“waiting”.
.

(g) Start the batch table in SparkLink. From this on everything
should run automatically.
b. Analysis
(1) Check out the LC/MS interface
(a) If the instrument is in ready mode, wait until the interface cools
down. When the interface is cool enough, take out the capillary
from the MS interface. Rinse the capillary with MeOH, sonicate in
MeOH for 20 min if necessary. Periodically, take off the interface
housing, and wipe out the skimmer plate.
(b) Open the rough pump cabinet, check for oil leaks and unusual
noise. Report anything unusual.
(2) Check out the LC system
After the column has been conditioned, click on the Equilibrate icon,
select the current method, and let the system equilibrate for
approximately 30 minutes. Run the Instrument Check sample by
opening the batch file named Instrument_test.dab. Change the date
in the Sample Name field. Make sure the proper Acquisition Method
and Vial Position are entered, and submit the batch. The file should
be saved into the Instrument_test.wiff file. Open the chromatogram
and compare the intensities and peak shape to those obtained a day
and a week before. If peaks appear distorted (tailing peaks, broad
peaks, etc.) change the column and submit the Instrument Check
sample again. If the absolute intensity is too low (peak intensity
should not be <70% less intense than before) check with the
laboratory supervisor or his/her designee.

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(3) Check out the SPE system
a) In SparkLink, put the Triathlon autosampler into load position.
Initialize the high pressure dispenser (HPD) and the automated
cartridge exchanger (ACE) unit.
b) Exchange the cartridge tray
approximately 500 samples).

needed

(generally

after

c) Make sure that the MS remote cable is connected ACE unit.
(4) Building batch files
(a) In the SparkLink software, go to RunTables and open and set up
the batch table. For the first sample enter xx-method 1 which
runs the injection and cleanup of the first sample. For the second
and consecutive samples use xx-method 2 which initiates the
HPLC/MS acquisition and runs the injection and cleanup of the
next sample. For the last sample enter xx-method 3 which
initiates the HPLC/MS acquisition of the last sample. For injection
volume, enter 500 μL. Make sure that the proper vial positions
are entered and there is no sequential duplication of cartridge
numbers. The sample names and sample IDs do not matter,
since they will not be part of the acquired data.
(b) In the Analyst software, open a new the subproject folder for each
new run. The subproject should have the same YYYY-MMDD
name as the unknowns it includes. Each subproject should have
separate Acquisition Methods, Quantitation Methods, Batch, Data,
and Results directories. Copy the latest Acquisition Method and
Quantitation Method from the previous subfolder.
(c) From Excel, open the text file containing the batch table created from the
Access database using Microsoft Access. This file should not require any
editing. Save the table into the text file named import.txt into the Batch
directory (overwrite). Remember to CLOSE THE FILE IN EXCEL!!!!! Go
to Analyst, open a new batch table and import the import.txt file (Sample
pull down, go to gray header and click RMB, then Import From/File, select
Alias autosampler).
(d) Make sure that the proper Acquisition Method and Quantitation
Method are entered. Although the vial positions entered in
Analyst will not be used they should agree with the vial positions
used on the Triathlon autosampler.
(5) Starting the SPE-HPLC-MS/MS run
a) Start the batch table in SparkLink.
b) Submit the batch table in Analyst (highlight and/or click Submit,
go to View Queue, and click Start Sample). From this on
everything should run automatically. After the SPE cleanup of the

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first sample, each N+1 sample in the SparkLink batch table will
correspond with sample N in the Analyst batch table.
c. Processing data
(1) Quantification
All raw data files are analyzed using the Quantitation Wizard application
in the Analyst software, which allows both automatic and manual peak
selection and area integration. The area values and retention times are
exported into a tab delimited text file and imported into the Access
database with the name YYYY-MMDD.txt.
(2) Importing Data into the Database
The tab-delimited file is read into the Access database. No prior editing
is required.
(3) Statistical Analysis and Interpretation of Data
Data are exported from the Access database to a fixed ASCII text file
and imported into SAS. SAS programs for standard curve generation,
QC analysis, blank analysis, limit of detection determination, unknown
calculations, and data distribution have been created and may be
executed in SAS when this information is needed.
d. Replacement and periodic maintenance of key components
(1) ABI 5500 or ABI 6500 Qtrap Mass Spectrometer
Preventative maintenance is done by a qualified engineer at least once a
year. In addition, to ensure proper performance of the system, a periodic
maintenance of the system may be required.
(a) When a partial blockage of the vacuum is suspected, the orifice is
probed with a syringe-cleaning wire.
(b) Cleaning of the spray shield and the entrance end of the heated
capillary is performed weekly as described in the Sciex ABI 5500
or ABI 6500 Qtrap Hardware Manual. First, wash with a solution
of water: methanol (1:1) and then, with 100% methanol. Wipe the
area using flake free paper wipes.
(c) The pump oil is changed approximately every six months as part
of the periodic maintenance of the system.
(2) Agilent 1200 HPLC
Preventative maintenance is done by a qualified engineer at least once a
year. Additional maintenance may be necessary if there is a general
decrease in instrument performance (see below). In general,
performance maintenance procedures are performed after detecting a
decrease in the system performance (sensitivity and/or S/N ratio) without
any other apparent technical reasons.

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(a) The HPLC column is replaced when analyte resolution decreases.
Once the analyte peaks start tailing, the HPLC column should be
replaced.
(b) If high pressure (>250 bar) error messages are observed, the
purge valve frit, the guard column, analytical column frit, HPLC
lines, needle seat, or injector components may need to be
replaced. See also section 8b.
(c) Reestablishment of performance and calibration. Every time the
system is disturbed for cleaning or maintenance, a mass spec
operational check standard is analyzed to assess the HPLC and
MS performance. For the mass spectrometer, a retune of the
system may or may not be necessary. If the instrument does not
pass this test, then the instrument is retuned using PPG as
described previously.
(3) Spark system
Preventative maintenance is done by a qualified engineer at least once a
year. Additional maintenance may be necessary if there is a general
decrease in instrument performance.
If the SparkLink error “HPD 1 high pressure problem” occurs, check the
SPE lines and HPD 6 port valve. The HPD valve stator and/or rotor may
need to be replaced.
The instrumentation used is serviced according to the manufacturer’s
guidance included in the instrument manuals or based on the
recommendation of experienced analysts/operators after following
appropriate procedures to determine that the instrument performs
adequately for the intended purposes of the method.
9. Reportable Range of Results
The linear range of the standard calibration curves and the method limit of detection
(LOD) determine the reportable range of results. The reportable range must be within
the range of the calibration curves. However, samples with concentrations exceeding
the highest reportable limit may be diluted, re-extracted, and reanalyzed so that the
measured value will be within the range of the calibration.
If a sample needs more than 100 times dilution (which would require using less than 1
μL of specimen) the dilution can be performed in at least two steps. For example, first,
at least 10 μL specimen is diluted up to 1 mL with water in a 2 mL Eppendorf tube (or
equivalent), then a second dilution is performed by aliquoting the appropriate fraction of
the dilute into an autosampler vial and adding 100 μL blank calf serum. With very
concentrated specimens it may be difficult to estimate the dilution that is necessary, and
the measured value may be higher than the highest calibration point even after the
dilution.
Formula to calculate the dilution factor to be entered into the Analyst batch file:

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D= (1000 / V 1st ) x (200 / V 2nd ).
Formula to calculate the volume of specimen to be entered into the Access database:
V= V 1st x (V 2nd / 1000)
Where V 1st is the volume of the aliquot taken from the original specimen and V 2nd is the
volume of the dilute measured into the autosampler vial.
1) Analytical Sensitivity
The limits of detection (LOD) for each analyte are listed in Table 6.
2) Analytical Specificity
This is a highly selective method that requires that the PFASs 1) elute at a
specific retention time; 2) have precursor ions with specific mass/charge ratios;
3) have specific product ions formed from the precursor ion with specific
mass/charge ratios.
3) Linearity Limits
The calibration curve is linear for all analytes (generally R2>0.95). The limit on
the linearity is determined by the highest standard analyzed in the method. Due
to the wide variation of PFASs levels in humans, we set our highest standard
near the high end of the linear range (Table 6). Unknown samples whose
concentrations exceed the highest standard concentration must be re-extracted
using a smaller aliquot. The low end of the linear range is limited by the method
LOD. Concentrations below the method LOD (or the concentration of the lowest
standard in the calibration curve) are reported as non-detectable.

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Table 6. Linear range (lowest – highest standard concentration) and LOD for
each PFAS measured in serum.
Analyte

Me-PFOSA-AcOH
PFBuS
PFHxS
n-PFOS
Sm-PFOS
PFHpA
n-PFOA
Sb-PFOA
PFNA
PFDeA
PFUA
PFDoA

LOD

0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1

Linear range
(ng/mL)

0.005-20
0.004-17.1
0.005-18.9
0.015-115
0.01-10
0.01-20
0.01-20
0.029-24.5
0.01-20
0.01-20
0.01-20
0.01-20

1) Accuracy
The accuracy of the method is determined by enriching serum samples with known
concentrations of PFASs and comparing the calculated and expected concentrations. To
examine their consistency over the range of levels encountered in serum, the
measurements are taken at 3 different concentrations, namely using standards near
3*LOD, middle level (~1.0 ng/mL, except n-PFOS (6 ng/mL), Sm-PFOS and Sm2-PFOS
(0.5 ng/mL), and Sb-PFOA (0.7 ng/mL)), and high level (~10.0 ng/mL, except n-PFOS
(60 ng/mL), Sm-PFOS and Sm2-PFOS (1.0 ng/mL), and Sb-PFOA (2.5 ng/mL)). The
accuracy is calculated from 5 independent measurements (Table 7).

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Table 7. Spiked recoveries of extracted standards in serum

Analyte
Me-PFOSA-AcOH
PFBuS
PFHxS
n-PFOS
Sm-PFOS
PFHpA
n-PFOA
Sb-PFOA
PFNA
PFDeA
PFUA
PFDoA

Accuracy (%)
at ~3*LOD/middle/high
105±20
93±5
101±3
113±25
105±19
110±12
106±12
98±5
98±6
110±14
99.7±6
97.0±2
105±23
92±4
90±4
115±12
110±11
103±10
92±14
105±5
101±2
112±15
106±8
102±5
95±13
103±7
102±3
103±13
96±6
98±4
92±17
102±3
101±3
110±22
96±10
101±3

1) Precision
The precision of this method is reflected in the variance of two quality control (QC)
pools over a period of three weeks. The coefficient of variation (CV) of repeated
measurements of these QC pools, which reflects both inter and intra-day
variations, is used to estimate precision (Table 8).
Table 8. Mean QC concentrations (ng/mL) and CV%
QCH

CV%

14.3

6.5

10.5

0.4

18.5

6.7

15.7

PFHxS

0.4

9.3

6.3

7.0

n-PFOS

1.0

10.6

15.8

9.8

Sm-PFOS

0.4

10.5

2.3

10.3

PFHpA

0.5

6.5

6.3

9.4

n-PFOA

0.4

13.6

7.4

12.0

Sb-PFOA

0.8

15.5

6.5

11.0

PFNA

0.4

10.6

8.1

9.4

PFDeA

0.5

10.9

6.3

9.7

PFUA

0.5

12.5

6.4

9.7

PFDoA

0.5

20.0

6.4

9.1

Analyte

VQC

CV%

Me-PFOSA-AcOH

0.4

PFBuS

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10. Quality Control (QC) Procedures
a. Individual samples (i.e., standards, unknown samples, serum blanks, and quality
control (QC) materials) QC procedures
1) For each analyte, the relative retention time (RT) (ratio of RT analyte and RT IS ) of
standards, unknowns, and QCs should be checked. If the relative RT falls
outside the range, check the integration to make sure the analyte or IS peak
was properly picked up.
2) For each analyte, the IS area counts should meet minimum area count
requirements. Low IS area counts suggest a) strong ion suppression from the
matrix, or b) missing of IS. Depending on the findings, either re-extract the
sample as usual or re-extract the sample after dilution.
3) For each analyte, the calculated concentration of the calf serum blanks (SB)
should be less than three times the LOD. Using the current method, all
standards, blanks and unknown samples are prepared following the same
procedure, thus background blank values (reflected in the intercept of the
calibration curve) are automatically subtracted from the concentrations of
unknown samples. If background levels are above the threshold above, the
reagents used for sample preparation and (or) mobile phases need to be
checked for potential contamination.
4) For each analyte, if the concentration in an unknown sample is above the
highest calibration standard, the sample needs to be re-extracted with a
smaller volume of serum.
b. Quality control of the QC materials
1) QC Materials
The QC materials were prepared in bulk from calf serum (Gibco, Grand Island,
NY). The target ranges for the pools were set to encompass the expected
concentration ranges in human populations.
2) Preparation of QC Pools
The calf serum purchased was pooled and the QC pools were mixed uniformly,
divided into four subpools and stored frozen. One subpool was used as a blank
QC and to prepare the calibration standards, and the other three were enriched
with PFASs as needed to afford very low concentration (VQC, ~0.3-1.0 ng/mL),
low concentration (QCL, ~2.0 ng/mL) and high concentration (QCH, ~0.8-15.8
ng/mL) subpools. The QC pools were characterized to define the mean and the
95% and 99% control limits of PFASs concentrations by a minimum of 30
repeated measurements in a three week period. QC materials reextracted and
analyzed after the initial characterization showed that the PFASs remained stable
frozen for at least 3 months 21.

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3) Characterization of QC Materials
For characterization, a minimum of 30 runs of QCL and QCH were measured
over 1 month. In each run, one pair of QCL and QCH materials were analyzed
and averaged. Using the pair average value from the 30 runs, the mean, and
upper and lower 99% and 95% control limits were established.
QC samples are analyzed along with unknown samples to monitor for accuracy
and precision throughout the analysis batch. Maximum 50 unknown samples are
run with randomly placed 2 QCL, 2 QCH, and 2 reagent blank samples. The
concentrations of the two QCL and two QCH in each batch are averaged to
obtain one average measurement of QCL and QCH.

4) Final evaluation of Quality Control Results
Standard criteria for run rejection based on statistical probabilities are used to
declare a run either in-control or out-of-control24.
QC rules for: Analytical run with 1 QC pool per run (must also include a blank
QC specimen):
One QC pool per run with one QC result per pool
1) If QC run result is within 2Si limits, then accept the run.
2) If QC run result is outside a 2Si limit - reject run if:
a) Extreme Outlier – Run result is beyond the characterization mean +/- 4Si
b) 1 3S Rule - Run result is outside a 3Si limit
c) 2 2S Rule - Current and previous run results are outside the same 2Si limit
d) 10 X-bar Rule – Current and previous 9 run results are on same side of the
characterization mean
e) R 4S Rule – The current and the previous run results differ by more than 4Si.
Note: Since runs have a single result per pool and only 1 pool, the R 4S rule is
applied across runs only.
One QC pool per run with two or more QC results per pool
1) If QC run mean is within 2Sm limits and individual results are within 2Si limits,
then accept the run.
2) If QC run mean is outside a 2Sm limit - reject run if:
a) Extreme Outlier – Run mean is beyond the characterization mean +/- 4Sm
b) 3S Rule - Run mean is outside a 3Sm limit
c) 2 2S Rule – Current and previous 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
3) If one of the two QC individual results is outside a 2Si limit - reject run if:

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a) R 4S Rule – Within-run range for the current run and the previous run exceeds
4Sw (i.e., 95% range limit)
Abbreviations:
S i = Standard deviation of individual results (the limits are not shown on the chart
unless run results are actually single measurements).
S m = Standard deviation of the run means (the limits are shown on the chart).
S w = Within-run standard deviation (the limits are not shown on the chart).
QC rules for: Analytical run with 2 QC pools per run:
Two QC pools per run with one QC result per pool
1) If both QC run results are within 2Si limits, then accept the run.
2) If 1 of the 2 QC run results is outside a 2Si limit - reject run if:
a) Extreme Outlier – Run result is beyond the characterization mean +/- 4Si
b) 3S Rule - Run result is outside a 3Si limit
c) 2S Rule - Both run results are outside the same 2Si limit
d) 10 X-bar Rule – Current and previous 9 run results are on same side of the
characterization mean
e) R 4S Rule – Two consecutive standardized run results differ by more than 4Si. Note:
Since runs have a single result per pool for 2 pools, comparison of results for the R 4S
rule will be with the previous result within run or the last result of the previous run.
Standardized results are used because different pools have different means.
Two QC pools per run with two or more QC results per pool
1) If both QC run means 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:
a) Extreme Outlier – Run mean is beyond the characterization mean +/- 4Sm
b) 3S Rule - Run mean is outside a 3Sm limit
c) 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
3) If one of the 4 QC individual results is outside a 2Si limit - reject run if:
a) 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.

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QC rules for: Analytical run with 3 QC pools per run:
Three QC pools per run with one QC result per pool
1) If all 3 QC run results are within 2Si limits, then accept the run.
2) If 1 of the 3 QC run results is outside a 2Si limit - reject run if:
a) Extreme Outlier – Run result is beyond the characterization mean +/- 4Si
b) 3S Rule - Run result is outside a 3Si limit
c) 2S Rule - 2 or more of the 3 run results are outside the same 2Si limit
d) 10 X-bar Rule – Current and previous 9 run results are on same side of the
characterization mean
e) R 4S Rule – Two consecutive standardized run results differ by more than 4Si. Note:
Since runs have a single result per pool for 3 pools, comparison of results for the R 4S
rule will be with the previous result within the current run or with the last result of the
previous run. Standardized results are used because different pools have different
means.
Three QC pools per run with two or more QC results per pool
1) If all 3 QC run means are within 2Sm limits and individual results are within 2Si limits,
then accept the run.
2) If 1 of the 3 QC run means is outside a 2Sm limit - reject run if:
a) Extreme Outlier – Run mean is beyond the characterization mean +/- 4Sm
b) 3S Rule - Run mean is outside a 3Sm limit
c) 2S Rule - 2 or more of the 3 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
3) If one of the QC individual results is outside a 2Si limit - reject run if:
a) R 4S Rule - 2 or more of the within-run ranges in the same run exceed 4Sw (i.e.,
95% range limit). Note: Since runs have multiple results per pool for 3 pools, the R 4S
rule is applied within runs only.
11. Remedial Action if Calibration or QC Systems Fail to Meet Acceptable Criteria
If the QC systems or the calibrations failed to meet acceptable criteria, operations are
suspended until the source or cause of failure is identified and corrected. If the source
of failure is easily identifiable (e.g., failure of the mass spectrometer or a pipetting error),
the problem is immediately corrected. Otherwise, fresh reagents are prepared and the
mass spectrometer is cleaned. Before beginning another analytical run, several QC
materials (in the case of QC failure) or calibration standards (in the case of calibration
failure) are reanalyzed. After calibration or quality control has been reestablished,
analytical runs may be resumed.
12. Limitations of Method; Interfering Substances and Conditions
Occasionally, the concentration of the PFASs in serum may be higher than the highest
standard in the calibration curves, and 0.1 mL of sample may be too much to use. This

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is evident by the low recovery of the isotope-labeled standard after the SPE extraction.
In this case, a smaller aliquot of serum can be used. Most likely, the LOD is not higher
in this case because of the concentrated nature of the specimen.
13. Reference Ranges (Normal Values)
Results (http://www.cdc.gov/exposurereport) from the National Health and Nutrition
Examination Survey (NHANES) can be used as reference ranges for the general US
population 25.
14. Critical-Call Results (“Panic” Values)
Critical call values have not been established for any PFAS concentrations.
15. Specimen Storage and Handling During Testing
Specimens are stored in the laboratory frozen prior to analysis. Frozen samples are
allowed to thaw completely at room temperature prior to the initiation of the analytical
procedure.

16. Alternate Methods for Performing Test and Storing Specimens if Test System
Fails
Alternate procedures do not exist in-house for the measurement of PFASs. If the
analytical system fails, storage of samples refrigerated is recommended until the system
is operational again.
17. Test-Result Reporting System; Protocol for Reporting Critical Calls (If Applicable)
a. The Quality Control officer reviews each analytical run, identifies the quality control
samples within each analytical run and determines whether the analytical run is
performed under acceptable quality control conditions.
b. The data from analytical runs of unknowns are initially reviewed by the laboratory
supervisor.
c. If the quality control data and results are acceptable the laboratory supervisor
generates a memorandum to the Branch Chief reporting the results.
d. These data are then sent to the person(s) that made the initial request.
e. Final hard copies of correspondence are maintained in the office of the Branch
Chief and with the quality control officer.

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18. Transfer or Referral of Specimens; Procedures for Specimen Accountability and
Tracking
Standard record keeping systems (e.g., notebooks, sample logs, data files) should be
employed to keep track of all specimens. One spreadsheet form with information for
receiving/transferring specimens is kept in the laboratory. In this form, the samples
received are logged in when received and when stored/transferred after analysis. For
NHANES samples, the person receiving the specimens signs and dates the shipping
manifests. The shipping manifests for NHANES and other samples are kept in a binder in
the Laboratory.

Use of trade names is for identification only and does not imply endorsement by the Public Health
Service or the U.S. Department of Health and Human Services.

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2013-2014 Summary Statistics and QC Chart for 2-(N-methyl-PFOSA) acetate (ng/mL)

Lot

Start
N Date

End
Date

Standard Coefficient of
Mean Deviation
Variation

HQC-122014 50 02JUL14 11MAR15 7.09100

0.48089

6.8

LQC-122014 50 02JUL14 11MAR15 1.69470

0.13635

8.0

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2013-2014 Summary Statistics and QC Chart for Perfluorobutane sulfonic acid (ng/mL)

Lot

Start
N Date

End
Date

Standard Coefficient of
Mean Deviation
Variation

HQC-122014 50 02JUL14 11MAR15 5.629

0.548

9.7

LQC-122014 50 02JUL14 11MAR15 2.088

0.202

9.7

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2013-2014 Summary Statistics and QC Chart for Perfluorodecanoic acid (ng/mL)

Lot

Start
N Date

End
Date

Standard Coefficient of
Mean Deviation
Variation

HQC-122014 50 02JUL14 11MAR15 6.484

0.272

4.2

LQC-122014 50 02JUL14 11MAR15 2.461

0.129

5.3

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2013-2014 Summary Statistics and QC Chart for Perfluorododecanoic acid (ng/mL)

Lot

Start
N Date

End
Date

Standard Coefficient of
Mean Deviation
Variation

HQC-122014 50 02JUL14 11MAR15 6.324

0.406

6.4

LQC-122014 50 02JUL14 11MAR15 2.390

0.263

11.0

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2013-2014 Summary Statistics and QC Chart for Perfluoroheptanoic acid (ng/mL)

Lot

Start
N Date

End
Date

Standard Coefficient of
Mean Deviation
Variation

HQC-122014 50 02JUL14 11MAR15 6.458

0.328

5.1

LQC-122014 50 02JUL14 11MAR15 2.429

0.144

5.9

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2013-2014 Summary Statistics and QC Chart for Perfluorohexane sulfonic acid (ng/mL)

Lot

Start
N Date

End
Date

Standard Coefficient of
Mean Deviation
Variation

HQC-122014 50 02JUL14 11MAR15 6.105

0.243

4.0

LQC-122014 50 02JUL14 11MAR15 2.291

0.114

5.0

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2013-2014 Summary Statistics and QC Chart for Perfluorononanoic acid (ng/mL)

Lot

Start
N Date

End
Date

Standard Coefficient of
Mean Deviation
Variation

HQC-122014 50 02JUL14 11MAR15 6.3701

0.3081

4.8

LQC-122014 50 02JUL14 11MAR15 2.3770

0.1517

6.4

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2013-2014 Summary Statistics and QC Chart for Perfluoroundecanoic acid (ng/mL)

Lot

Start
N Date

End
Date

Standard Coefficient of
Mean Deviation
Variation

HQC-122014 50 02JUL14 11MAR15 6.223

0.297

4.8

LQC-122014 50 02JUL14 11MAR15 2.364

0.145

6.1

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