GC/MS Determination of PAH in Urine

Att9e4_PAHs.pdf

Biomonitoring of Great Lakes Populations Program II

GC/MS Determination of PAH in Urine

OMB: 0923-0052

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2015-06-23 - No material or nonsubstantive change to a currently approved collection

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Laboratory of Organic Analytical Chemistry
Wadsworth Center
New York State Department of Health

STANDARD OPERATING PROCEDURE

Analyte: Monohydroxy-Polycyclic Aromatic Hydrocarbons (OH-PAHs)
Matrix: Urine
Method: Isotope Dilution Gas Chromatography/High-Resolution Mass
Spectrometry *
*This Method was Modified Based on CDC Method No. 09-0D
Revised: July 27, 2006

As performed by:
Division of Environmental Health Sciences
Wadsworth Center
Department Health of New York State
Contact:
Dr. Shijun Lu
Phone: (518) 474-3979
E-mail: [email protected]
Dr. Kenneth Aldous,
Director, Division of Environmental Health Sciences
Phone: (518) 473-0030
E-mail: [email protected]

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

Table of Contents
1. Clinical Relevance and Summary of Test Principle
2. Safety Precautions
a. Reagent Toxicity or Carcinogenicity
b. Radioactive Hazards
c. Microbiological Hazards
d. Mechanical Hazards
e. Protective Equipment
f. Training
g. Personal Hygiene
h. Disposal of Wastes
3. Computerization, Data-System Management
a. Software and Knowledge Requirements
b. Sample Information
c. Data Maintenance
4. Procedures for Collecting, Storing, and Handling Specimens; Criteria for Specimen
Rejection
a. Sample Collection
b. Sample Handling
c. Criteria for Specimen Rejection
5. Preparation of Reagents, Calibration (Standards), Controls, and All Other Materials;
Equipment and Instrumentation
a. Reagents and Sources
(1) Reagent Preparation
(2) Standards Preparation
b. Manual Equipment (indicated equipment or equivalent)
c. Other Materials 7
d. Instrumentation (indicated equipment or equivalent) 7
(1) High Resolution Mass Spectrometer (HRMS) Configuration
(2) Mass Spectrometer Instrument Control Language (ICL) Programs
(3) Gas Chromatography (GC) Configuration and Temperature Program

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

6. Calibration and Calibration-Verification Procedures
a.

Mass Spectrometer

Calibration Curve

Calibration Verification

Proficiency Testing (PT)

7. Operating Procedures; Calculations; Interpretation of Results
a.

Sample Preparation

Hydrolysis

Extraction

Concentration

Derivatization Procedure

Analysis

Processing of Data

Replacement and Periodic Maintenance of Key Components

8. Reportable Range of Results
a.

Linearity Limits

Analytical Sensitivity

Accuracy

Precision

9. Quality Control (QC) Procedures
a.

QC Material

Urine Enrichment

Dispensing

Characterization of QC Materials

Use of QC Samples

Final Evaluation of QC Results

10. Recoveries of Method
11. Remedial Action if Calibration or QC Systems Fail to Meet Acceptable Criteria.
12. Limitations of Method; Interfering Substances and Conditions
13. Reference Ranges (Normal Values)
14. Critical-Call Results ("Panic" Values)

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

15. Specimen Storage and Handling During Testing
16. Alternate Methods for Performing Test and Storing Specimens if Test System Fails
17. Transfer or Referral of Specimens; Procedures for Specimen Accountability and
Tracking References

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

1. Clinical Relevance and Summary of Test Principle
This method is used to assess human exposure to selected polycyclic aromatic
hydrocarbons (PAHs), a class of potential human carcinogens (1, 2), by determining the
concentrations of their mono-hydroxy metabolites in urine. Common routes of
occupational exposure to PAHs may include work involving diesel fuels and coal tars
through paving and roofing as well as possible environmental exposures including
smoking, diet, smog, and forest fires.Threshold levels for carcinogenicity have not been
determined for most PAHs.
The specific analytes measured in this method are monohydroxy-polycyclic
aromatic hydrocarbons (OH-PAHs). The procedure involves enzymatic hydrolysis of urine
(to hydrolyze PAH conjugates), liquid-liquid extraction, derivatization, and analysis using
capillary gas chromatography coupled to high-resolution mass spectrometry (GC/HRMS).
This method uses isotope dilution with carbon C-13-labeled internal standards. Ions from
each analyte and each C-13-labeled internal standard are monitored, and the abundances of
each ion are measured. The ratios of these ions are used as criteria for evaluating the data.
(See Table 1 for the analytes measured in this procedure.) By evaluating the
concentrations of these analytes in urine, laboratorians can assess human exposure to the
particular PAH analyzed.

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

Table 1. Analytes Measured and Their Abbreviations
Parent PAH

Metabolite/Analyte

Abbreviation

Naphthalene

1-hydroxynaphthalene

1-nap

Naphthalene

2-hydroxynaphthalene

2-nap

Fluorene

2-hydroxyfluorene

2-fluo

Fluorene

3-hydroxyfluorene

3-fluo

Fluorene

9-hydroxyfluorene

9-fluo

Phenanthrene

1-hydroxyphenanthrene

1-phen

Phenanthrene

2-hydroxyphenanthrene

2-phen

Phenanthrene

3-hydroxyphenanthrene

3-phen

Phenanthrene

4-hydroxyphenanthrene

4-phen

Phenanthrene

9-hydroxyphenanthrene

9-phen

Fluoranthene

3-hydroxyfluoranthene

3-flran

Pyrene

1-hydroxypyrene

1-pyr

Benzo(c)phenanthrene

1-hydroxybenzo(c)phenanthrene

1-bcp

Benzo(c)phenanthrene

2-hydroxybenzo(c)phenanthrene

2-bcp

Benzo(c)phenanthrene

3-hydroxybenzo(c)phenanthrene

3-bcp

Chrysene

1-hydroxychrysene

1-chry

Chrysene

2-hydroxychrysene

2-chry

Chrysene

3-hydroxychrysene

3-chry

Chrysene

4-hydroxychrysene

4-chry

Chrysene

6-hydroxychrysene

6-chry

Benzo(a)anthracene

1-hydroxybenzo(a)anthracene

1-baa

Benzo(a)anthracene

3-hydroxybenzo(a)anthracene

3-baa

Benzo(a)anthracene

9-hydroxybenzo(a)anthracene

9-baa

Benzo(a)pyrene

3-hydroxybenzo(a)pyrene

3-bap

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

2. Safety Precautions
a. Reagent Toxicity or Carcinogenicity
Some of the reagents necessary to perform this procedure are toxic. Exercise caution
to avoid inhalation of or dermal exposure to these reagents by wearing a lab coat and
gloves.
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
a laboratorian being exposed to various microbiological hazards exists. Take
appropriate measures to avoid any direct contact with the specimen (see Section
2.e.). A Hepatitis B vaccination series and a baseline test for health care and
laboratory workers who may be exposed to human fluids and tissues are
recommends. Observe universal precautions.
d. Mechanical Hazards
Following standard safety practices while performing this procedure minimizes the
risk for mechanical hazards. Avoid any direct contact with the electronic
components of the mass spectrometer unless all power to the instrument has been
shut off. Only qualified technicians should perform electronic maintenance and
repair.
e. Protective Equipment
Use standard personal protective equipment when performing this procedure. This
includes wearing a lab coat, safety glasses, durable gloves (nitrile medical
examination gloves or equivalent gloves), and appropriate footwear. Process
specimens and prepare samples using a chemical fume hood.
f. Training
Anyone performing this procedure must be trained and experienced in the use of a
high-resolution mass spectrometer. Formal training by an experienced operator of
the instrument is necessary. Personnel must also read the operation manuals from
the manufacturer. Anyone involved in sample preparation must be trained in
liquid-liquid extraction and have basic chemistry laboratory skills.

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

g. Personal Hygiene
Follow universal precautions. Exercise special care when handling any biological
specimen. Use personal protective equipment and wash hands properly after
completing lab work.
h. Disposal of Wastes
According to Wadsworth Hazardous Chemical Waste Management, collect solvents
and reagents in appropriate containers clearly marked for waste products, and
temporarily store them in a flame-resistant cabinet. Autoclave or decontaminate
with 10 percent bleach containers, glassware, etc., that come in direct contact with
the specimens. Wash, recycle, or dispose of the glassware in an appropriate manner
according to the Wadsworth Center Safety Guidelines. Dispose of all biological
samples and diluted specimens in a biohazard autoclave bag at the end of the
analysis according to Wadsworth Center Safety Guidelines for disposal of
hazardous waste.

3. Computerization, Data-System Management
a. Software and Knowledge Requirements
Raw data are processed for qualitative and quantitative analysis using
QualBrowser®, QuanBrowser®/QuanDesk® (Thermo Electron Corporation),
respectively. Perform statistical data analyses using Statistical Analysis System
(SAS)® software (SAS Institute, Cary, NC), or Excel® (Microsoft ). Knowledge and
experience with these software packages (or their equivalent) are required to use and
maintain the data-management structure.
b. Sample Information
Electronically transfer or manually enter information pertaining to particular
specimens into the database. Data that are manually entered should include the
sample identification number, sample type, standard number, and other information
not associated with the mass spectral analysis.
c. Data Maintenance

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

After inputting sample and analytical data into the database, backup the database
onto a computer hard drive and/or writable compact disk (CD-R) weekly. Backup
raw data onto the CD-RW, and/or DVD-RW monthly.

4. Procedures for Collecting, Storing, and Handling Specimens; Criteria for
Specimen Rejection
a. Sample Collection
Use standard urine collection cups to collect urine specimens from donors.
Refrigerate samples as soon as possible and transfer them within 4 hours of
collection. Collect a minimum of 20 ml of urine and pour it into sterile, 30-ml
Qorpak vials with screw-cap tops. Label specimens, immediately freeze them to
-20°C, and store them in dry ice for shipping. Carefully pack vials to avoid breakage
during shipment. If long-term storage is anticipated, then store all samples at -80 °C.
b. Sample Handling
Samples and residual specimens are stored at -80 °C until needed to be thawed,
aliquoted, and analyzed.
c. Criteria for Specimen Rejection
Specimens must be frozen when delivered to the lab. The minimum volume required
is 1.0 ml. If either of these criteria is violated or contamination due to improper
collection procedures or collection devices is suspected, the specimen should be
rejected.

5. Preparation of Reagents, Calibration (Standards), Controls, and All Other
Materials; Equipment and Instrumentation
a. Reagents and Sources (see Table 2)

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

Table 2. Reagents and the Respective Manufacturers
Reagent
1-nap; 2-nap, 2-fluo, 3-fluo, 9- fluo,
9-phen, 1-pyr
1-phen, 2-phen, 4-phen
3-phen, 3-flran, 1-bcp, 2-bcp, 3-bcp,
1-chry, 2-chry, 3-chry, 4-chry, 1-baa,
3-baa, 3-bap

Manufactures*
Sigma Chemicals, St Louis, MO
RCT, Laramie, WY
Midwest Research Institute, National Cancer
Institute, Kansas City, MO

6-chry, PCB-61

AccuStandard,

C6 1-nap

CDC, synthesized in house, Atlanta, GA

C6 1-baa, 13C6 3-bcp, 13C6 3-chry, 13C6 Midwest Research Institute, NCI Kansas

3- flran, 13C6 1-pyr

City, MO

Cambridge Isotope Laboratories, Andover,

C6 3-phen, 13C6 6-chry

β-Glucuronidase / arylsulfatase (Type
H-3, 98,000 Unit/ml, from Helix

Sigma Chemicals, St Louis, MO

pomatia)
N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA)

Sigma Chemicals, St Louis, MO

Toluene (Nanograde)

Mallinckrodt Baker, Inc., Paris, KY

Pentane (HPLC grad)

J.T. Baker, Philipsburg, NJ

Organic Pure Water (o.p. H2O)

Prepared in house, (Milli Q water)

n-Dedocane

ChemService, West Chester, PA 19381

Buffer Solution, (pH 5.00)

Anachemia, Rouses Point, NY

Argon

BOC Group Inc., Murray Hill, NJ

* Use an equivalent manufacturer when the manufacture noted is not available.
All OH-PAH compounds are stored at -70°C and protected with Argon.

(1). Reagent Preparation

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

MSTFA. Open the sealed vial containing N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) from the vendor and place the MSTFA solution in
a clean GC sample vial. Displace the air over the MSTFA with a gentle stream
of argon. Immediately screw the cap on the vial. Store the MSTFA in the vial at
4oC could last more than 1 month.

ii.

Enzymatic-buffer Combine 250mL of buffer solution (pH=5.0, Anachemia)
and 0.6mL of β-glucuronidase / sulfatase enzyme (derived from Helix Pomatia,
98900units/ml, Sigma) in a sterilized 250ml Wheaton bottle to generate
Enzymatic-buffer (β-glucuronidase / sulfatase enzyme 300 units/ml). Store at
4oC could last more than 1 month.

(2) Standards Preparation
Due to the potential carcinogenicity of these analytes, weigh each analyte on a
microbalance inside a ventilated plexiglass box. Because all OH-PAHs are
light- sensitive, take precautions to minimize their exposure to light.
i. Stock solutions of Individual Analytes (100ng/µL). Weigh approximately 1mg
of neat OH-PAH in a screw-cap vial. Add exactly 1 mL of acetonitrile and allow
the OH-PAH material to dissolve by gently swirling it or placing it in an
ultrasonic bath. Calculate and perform the dilution to100 ng/µL for a final volume
of ~10 mL of acetonitrile. Ampoulize the individual standard solution in clean
amber ampoules, use argon to displace any air in the ampoule and seal it. Stored
at -80°C (for up to 1.5 years).
ii. Working Standard Solution of 23 OH-PAH Mix. Combine 500 µL of each
OH-PAH standard stock solutions (100 ng/µL) and 1mL acetonitrile in a 20mL
scintillant tube to generate a mixture of 23 OH-PAHs working standard solution
(each OH-PAH concentration is 4 ng/µL). Solutions of other concentrations may
also be prepared, if needed. Use argon to displace any air in the vials. Then cap
the vials tightly, place the vials in a sealed double layer plastic bag filled with
argon, and store them at -80 oC until needed (for up to 6 months).
iii. Calibration Curve Standards. Concentrations for the six points that establish
the calibration curve are 10, 50, 100, 500, 2000, 5000 and 10000 ng/L. These

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

standards are made from the dilution of 23 OH-PAH working standard solution
[see Section 6.c.(2)].
iv. Working Solution of C-13-labeled OH-PAH. For each C-13-labeled OH- PAH,
make 100 ng/µL of the stock solution, combine and dilute aliquots of the
individual C-13-labeled OH-PAH solutions with acetonitrile to produce the
working solution with 100 pg/µL of each C-13- labeled OH-PAH. Use argon to
displace any air in the vials. Then cap the vials and store them at -80 °C until
needed (for up to12 months).
v. Mass Spectrometric Check Solution. Use this solution at least weekly to
monitor the operating performance of the GC column and the mass spectrometer.
Use a solution of 10 fg/µL of a tetra-chlorinated dioxin (2378 TCDD) as the
check solution. For a 1-µL injection, a minimum signal-to-noise ratio (using 4
standard deviations of baseline noise) of 4 is required. Use the ion intensity of
mass 321.894 for the signal-to-noise ratio measurement.

b. Manual Equipment (indicated manufacture or its equivalent)
(1) Water bath/sonicator (Cole Parmer, Vernon Hills, IL).
(2) Incubator (Fisher Scientific, Pittsburgh, PA).
(3) Oven for derivatization (Cole Parmer, Vernon Hills, IL).
(4) Microbalance (Sartorius Ultramicro, Westbury, NY).
(5) TurboVap® LV evaporator (Zymark Corp., Hopkinton, MA).
(6) Centrifuge 5804 (Eppendorf Corp., Westbury, NY)
(7) Repeater Plus (Eppendorf Corp., Westbury, NY)
(8) Vortex Genie vortex mixer (Scientific Industries Inc., Springfield, MA).

c. Other Materials (indicated manufacture or its equivalent)
(1) Miscellaneous glassware (Pyrex or Kimax, Scientific Services).
(2) 1.5ml amber autosampler vials (National Scientific, Inc.).
(3) 1.1ml 12x32mm, Pulled PT Vial (Sun Sri, Rockwood, TN)
(4) 4-mL, amber screw-top vials (Supelco, Inc.).
(5) 10-ml screw-top culture tubes

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

(6) 15-mL conical centrifuge tubes (Kimble, Vineland, NJ).
(7) 2-micron filter (Nalgene, Rochester, NY)
(8) Pipetman 10µL, 25µL, 100µL, 250µL,1mL, 5mL (Gilson Co., Middletown,
WI)

d. Instrumentation (indicated manufacture or its equivalent)
Perform the analyses on a Finnigan MAT -95XL high-resolution mass spectrometer
that is equipped with an electron impact ionization source and is interfaced to a
Thermo Finnigan Trace Ultra 2000 gas chromatograph system (Thermo Finnigan,
San Jose, CA). A PAL injection system (CTC Analytics, Switzerland) handles the
GC injections automatically. All instruments are connected and controlled by a Dell®
computer with Microsoft® Windows XP® operating system

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

(1) High Resolution Mass Spectrometer (HRMS) Configuration (see Table 4)

Table 4. Thermo Finnigan MAT 95 XL HRMS Configuration
MS Parameter

Setting

Scan mode

Multiple ion detection

Ionization type

Electron impact

Ion polarity mode

Positive

Electron energy

40 eV

Resolution

10,000

Ion source

260 °C

Transfer line

290 °C

Conversion dynode voltage

Positive

Continuous dynode electron

1.45 -2.25 kV (106 gain)

multiplier voltage

(2) Masses and retention times of targeted OH-PAHs on MAT 95 XP HRMS are
shown in the Table 5.

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

Table 5. Masses and Retention Time of Targeted OH-PAHs
Analyte-O-Si (CH3)3

Mole. Mass

Ion Mass

R.T. min

1-nap
C6 1-nap
2-nap
9-fluo
13
C6 9-fluo
3-fluo
13
C6 3-fluo
2-fluo
13
C6 2-fluo
4-phen
9-phen
3-phen
13
C6 3-phen
1-phen
2-phen
13
C6 2-phen
1-bcp
3-flran
13
C6 3-flran
1-pyr
13
C6 1-pyr
2-bcp
1-baa
13
C6 1-baa
4-chry
6-chry
13
C6 6-chry
3-bcp
13
C6 3-bcp
3-chry
13
C6 3-chry
1-chry
3-/9-baa
2-chry
3-bap
13
C6 3-bap
7-bap

216.0970
222.1172
216.0970
254.1127
260.1328
254.1127
260.1328
254.1128
260.1328
266.1127
266.1127
266.1127
272.1328
266.1127
266.1127
272.1328
316.1283
290.1127
296.1328
290.1127
296.1328
316.1283
316.1283
322.1485
316.1283
316.1283
322.1485
316.1283
322.1485
316.1283
322.1485
316.1283
316.1283
316.1283
340.1282
346.1484
340.1282

201.0735
207.0937
201.0735
239.0892
245.1093
239.0892
245.1093
239.0892
245.1093
251.0890
251.0890
251.0890
257.1093
251.0890
251.0890
257.1093
301.1048
275.0892
281.1093
275.0892
281.1093
301.1048
301.1048
307.1250
301.1048
301.1048
307.125
301.1048
307.1250
301.1048
307.1250
301.1048
301.1048
301.1048
325.1042
331.1249
325.1042

8.44

I-Std.

x
8.70
11.13
12.65
12.85
13.73
14.10
14.37
x
14.43
14.73
16.79
17.03
x
17.40
x
19.07
19.13
x
19.22
19.30
x
19.71
x
20.03
20.10
20.35
20.43
23.63

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

(3) Gas Chromatography (GC) Configuration and Temperature Program
Chromatographic separation is performed on a Finnigan Thermo Trace GC
Ultra 2000 gas chromatograph fitted with a J&W DB-5MS, 25-m, fused silica
capillary column. The column ID is 0.25 mm and the film thickness is 0.25
micron (J&W, # 22-5022 or its equivalent). The GC purge flow is 10 mL/min,
the saver time is 2 min, and the saver flow is 20 mL/min. The temperature
program lasts a total of 33.3 min. (See Table 6 for GC Configuration and Table
7 for the GC Temperature Program.)

Table 6. GC Configuration
GC Parameter

Setting

Carrier gas

Helium

Constant flow rate

1.0 ml/minute

Injection mode

Splitless

Injector purge delay

2.0 minutes

Injector temperature

270 °C

Mode

Constant pressure

Table 7. GC Oven Temperature Program for Hydroxy PAHs
Time (minutes)

Temperature ('C)

100

160

19.5

295

25.5

295

300

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

6. Calibration and Calibration-Verification Procedures
a. Mass Spectrometer
Calibrate and tune the Thermo Finnigan MAT 95 XP mass spectrometer using
FC43 (perfluorotributylamine) according to the instructions in the operator's
manual. After calibrating the instrument with 10,000 resolution and maximum
sensitivity, prepare the instrument to analyze the PAH metabolites (see Section
8).
b. Calibration Curve
(a). Construct a 6-point calibration curve by performing a weighted regression
(i.e., weighted to 1/concentration) analysis of relative response factor (i.e.,
area native/area internal label) versus standard concentration. Perform a
minimum of seven repeat determinations for each point on the standard
curve.
(b). The lowest point (10 ng/L) on the calibration curve is near the measurable
detection limits; the highest point (10,000 ng/L) is above the expected range
of normal results.
(c). Measure and calculate blank levels; subtract the blank amounts from the
calibration points.
(d). Determine the slope and intercept of the calibration curve by linear least
squares fit with weighted 1/concentration using QuanBrowser®,
QuanDesk® , or SAS® software.
(e). R-squared values for the curve must be greater than 0.90. Linearity of
standard curves should extend over the entire standard range. Intercepts
(calculated from the least squares fit of the data) should not be significantly
different from 0; if they are, then identify the source of bias, make necessary
corrections, and run again.
(f). Periodically recalculate the standard curve to incorporate the newest data
points. Reestablish the standard curve when new working standard
solutions or new isotope solutions are prepared.
c. Calibration Verification

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

(a). Analyze the calibration-verification materials after any substantive change
in the method or instrumentation occurs to verify the integrity of the
calibration curve, slope, linearity, and dynamic range (e.g., the instrument
has used in another method; installed a new column; performed
preventative maintenance; or cleaned the ion source of the mass
spectrometer).
(b). Perform calibration verification at least once every 6 months while the
method is in use.
(c). If there is a significant difference from the established
calibration-verification pool limits, stop doing analyses using this method
until you can correct the problem and ensure that calibration-verification
materials are consistent with the calibration curve.
(d). Document all calibration-verification runs and results.
d. Proficiency Testing (PT)
Because no standard reference materials are available for urinary analysis of
hydroxy-PAH levels within an analytical study, duplicate measurements of
hydroxy-PAH levels were performed on a series of selected individual samples
at designated intervals during sample analysis. The analytical reproducibility of
a set of individual samples where levels of duplicate samples were determined
after every calculated sample increment in the study was considered an
acceptable measure of the proficiency of the method. The calculated increment
is a value that ranges from N/0.05N to N/0.02N (N= number of samples in the
study). PT criteria are under evaluation.

7. Operating Procedures; Calculations; Interpretation of Results
a. Sample Preparation
Allow unknown urine samples, QC samples, and a blank (2-mL o.p. water) to
thaw slowly in a refrigerator overnight to avoid cracking the glass containers.
b. Hydrolysis
In this stage, carefully pre-washed culture tubs with screw-cap tops, caps with
PTFE liner, glass pipets, and conical centrifugal tubes are used. Aliquot 2 mL of

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

urine into 10mL culture tube. Add 3mL enzymatic-buffer (pH=5.0, with
β-glucuronidase / sulfatase enzyme 300 units/ml, derived from Helix Pomatia),
and 10µL of combined C-13-labeled standards (100 pg/µL of each labeled
compound). Prepare QC samples, the same way. Aliquots of calibration
standards and QCs are usually made earlier in batches and stored in a freezer.
Place the caps loosely on the tubes and use a vortex mixer to mix the resulting
solution well. Incubate the samples at 37 °C over night. Remove samples from
the incubator and cool down them for a brief period. Urine samples can be
hydrolyzed and then place them in a refrigerator for extraction in the following
morning. (Note: This is an acceptable stopping point if you immediately
refrigerate the hydrolysates.) For the blank, and the calibration standard the solid
phase extraction should be done same day of the preparation.
c. Extraction
Add 5mL pentane to hydrolyzed samples tube. Screw on cap, mix and vent
pressure twice. Screw on cap tightly and mix samples by shaking rocker for 5
min. Evenly place the culture tubes on rocker of Eppendorf centrifuger and
centrifuge for 15min at 3000rpm, check the separation and set another 15min
run. Upper layer solvent transferred to a cleaned conical centrifugal tubes using
glass pipet. Due to the extracts are directly concentrated without further dry-up
in next stage, the transferring the solvent needs special care, no aqueous is
allowed to touch. Extraction repeated with another 5mL of pentane. Add 5µL
dodecane (keeper) to each tube prior to further concentrate.
d. Concentration
Concentrate the extracts using a TurboVap® (Caliper Life Sciences), set at 40
°C and with 5 psi of nitrogen, the final volume is about 10µL. The evaporation
takes approximately 10 minutes (Do not let the nitrogen stop until remove the
tubes from TurboVap®). Reconstitute the residual with 20µL high purity
toluene. Add 10µL PCB-61 (50pg/µL) as the external recovery standard (ERS),
use vortex mixer 15 seconds and transfer solution into a cleaned pulled 1.1 ml
GC vial use 100 µL pipettor.
e. Derivatization Procedure

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

The derivatization step converts the OH functional group to an O-trimethylsilyl
group. This step produces better limits of detection (LOD) and chromatography.
To derivatize the analytes after evaporation, add 20 µL of MSTFA in each vial
and then displace the air in the vial with a gentle stream of argon. Quickly screw
the vial. Place the vials in an incubator set at 60 °C for 1hour. Cover the samples
with aluminum foil. The derivatized samples are then ready for analysis on the
mass spectrometer. The samples can also be stored in a refrigerator (-4 °C) for
up to 1 week.
f. Analysis
Preliminary MAT 95 XL System Setup and Performance Check
(a) In the TUNE window, adjust the resolution between 9950 and 10050 using
2 µL of FC43.
(b) Run a GC/HRMS analysis of 10 fg tetra-chlorinated dioxin, check the
solution, and verify chromatographic resolution and peak intensity.
(c) In the logbook, record the signal-to-noise ratio of the check compound.
g. Processing of Data
(1) Quantification
All raw data files are quantified using the quantitation software of the MAT 95
XP in the QUAN window. This allows manual peak selection and baseline
determination for each ion. The entire quantification procedure is accomplished
by using the user buttons defined in the QUAN application.
(2) Transfer of Data
Use the USB jump drive or CD-R/RW copy the data files to other computer.
(3) Backup Data
Backup data to CD-R or other net hard drive weekly.
(4) Statistical Analysis and Interpretation of Data
Export information in the cleanup and mass spectrometry database tables from
the database to a fixed text (ASCII) file, and then import the file into SAS® .
Use SAS® programs for standard curve generation, QC analysis, blank analysis,
limit-of-detection determination, unknown calculations, data distribution, etc.
as needed.

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

h. Replacement and Periodic Maintenance of Key Components
(1) Routine Maintenance
(a) MAT 95 XL Mass Spectrometer
(i) Check cooling water level and temperature monthly.
(ii) Clean the ion volume or replace it monthly.
(iii) Clean the ion source or replace it annually.
(iv) Replace the calibration gas septum every two weeks.
(v) Trained Thermo Finnigan technicians will perform all other
maintenance based on an annual schedule or as requested.
(b) Trace Ultra 2000 GC gas chromatograph
(i) Change the injection port liner and septum daily.
(ii) Clean the injection port, clean or replace the gold seal monthly. (iii)
Replace the GC column at 1000 analyses or sooner.
(iv) Replace Helium tank when the pressure is lower than 500 psi.
(Note: The above maintenance schedules may be accelerated if necessary.)
(2) Performance Maintenance
Perform maintenance procedures if you detect a decrease in system
performance (i.e., sensitivity and/or S/N ratio) without other apparent technical
reasons.

8. 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. The calibration verification of the
method encompasses this reportable range. However, you can dilute and analyze a urine
sample with an analytical data value exceeding the highest reportable limit so that the
result will be within the reportable range.
a. Linearity Limits
Analytical standards were linear for all analytes through the range of concentrations
evaluated. The linear range for all analytes was 10 ng/L to 5,000 ng/L. Urine samples
whose concentrations exceed these ranges must be analyzed again with a diluted
solution or using a smaller aliquot.

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

b. Analytical Sensitivity
The method detection limit (MDL) is defined as 3 times the standard deviation (SD)
at zero concentration (3SD). It is determined by weighted regression
(1/concentration) analysis of the absolute SD versus the concentration. For analytes
with detectable blank signals, the MDL is calculated by adding the average blank
level to 3SD (see Table 8 for the MDL determined for the analytes). Generally, MDL
range from 2ng/L to 10 ng/L, depending upon blank levels.

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

Table 8. Method Detection Limits (MDLs)
Analyte

MDL (ng/L)

1-nap

5.7

2-nap

4.7

2-fluo

4.4

3-fluo

7.0

9-fluo

2.0

1-phen

8.0

2-phen

4.4

3-phen

1.2

4-phen

1.3

9-phen

1.9

3-flran

5.8

1-pyr

4.9

1-bcp

4.5

2-bcp

2.2

3-bcp

6.9

1-chry

4.8

2-chry

3.8

3-chry

1.3

4-chry

9.5

6-chry

5.3

1-baa

8.6

3-baa

8.7

3-bap

72.1

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

c. Accuracy
Enriching urine samples with known concentrations of the OH-PAH and comparing
the calculated and expected concentrations determine the accuracy of the method.
The accuracy can be expressed as the slope and the intercept of a weighted regression
(1/concentration) analysis of the expected values versus the calculated values. A
slope of 1.0 and an intercept of 0 indicate that the results are identical. The method
accuracy is shown in Table 9.

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

Table 9. Accuracy of the Method
Weighting
Analyte

Slope

Intercept

R-Squared

1-nap

0.0015489

0.0142736

0.9982

1/ Conc.

2-nap

0.0016431

0.00441331

0.9982

1/ Conc.

2-fluo

0.0029785

0.00425533

0.9968

1/ Conc.

3-fluo

0.0019075

0.0050077

0.9975

1/ Conc.

9-fluo

0.0014534

0.000940282

0.9971

1/ Conc.

1-phen

0.0029675

0.00299927

0.9969

1/ Conc.

2-phen

0.0026923

0.005438

0.9979

1/ Conc.

3-phen

0.0028350

0.00563754

0.9959

1/ Conc.

4-phen

0.0029070

0.00333788

0.9972

1/ Conc.

9-phen

0.0021514

0.00336125

0.9983

1/ Conc.

3-flran

0.0015743

0.00224427

0.9975

1/ Conc.

1-pyr

0.0025646

0.00356015

0.9958

1/ Conc.

1-bcp

0.0013202

0.00498609

0.9939

1/ Conc.

2-bcp

0.0016979

0.00632489

0.9973

1/ Conc.

3-bcp

0.0019940

0.00548789

0.9961

1/ Conc.

1-chry

0.0034276

0.0211208

0.9962

1/ Conc.

2-chry

0.0017045

0.00689675

0.9962

1/ Conc.

3-chry

0.0028450

0.0210635

0.9977

1/ Conc.

4-chry

0.0035895

0.00712345

0.9987

1/ Conc.

6-chry

0.0021580

0.00605751

0.9950

1/ Conc.

1-baa

0.0020282

0.00339295

0.9917

1/ Conc.

3-/9-baa

0.0007313

0.00185813

0.9968

1/ Conc.

3-bap

0.0009369

-0.00932439

0.9940

1/ Conc.

index

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

d. Precision
The precision of this method is reflected in the variance of QC samples over time.
The major contributor to the overall coefficient of variation (CV) is the variation
between runs. (See Table 10 and Table 12 for CV values.)

9. Quality Control (QC) Procedures
a. QC Material
Use urine pools enriched with known amounts of OH-PAH as the control materials
for each analytical run.
b. Urine Enrichment
Split the urine pool into four smaller urine pools. Reserve the first pool for blank
check. Enrich the second pool with the 23 OH-PAH working solution to yield a low
pool with an added concentration of 100 ng/L. Enrich the third pool with the 23 OHPAH working solution to yield a middle pool with an added concentration of 300
ng/L. Enrich the fourth pool with the 23 OH- PAH working solution to yield a High
pool with an added concentration of 900 ng/L.
c. Dispensing
Dispense 2-mL aliquots of urine into screw-capped vials. Mark the vials
appropriately with identification stickers and store the QC materials at -70 °C until
time of analysis. Buffer and internal standards could be added at the day of analysis
when unknown urines are prepared.
d. Characterization of QC Materials
Characterize the QC pools (including the unspiked pool) by 20 consecutive runs of
each QC material. Use the data from these runs to establish the mean and upper- and
lower- 99th and -95th confidence intervals. Determine the confidence intervals and
adjust them according to the number of each QC material analyzed in each run.
e. Use of QC Samples
During each analytical run, analyze at least one QC material from each pool.
f. Final Evaluation of QC Results

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

After the completion of a run, consult the QC limits to determine if the run is in
control. The following QC rules apply to the average of the beginning and
ending analyses of each of the bench QC pools:
(1) If both the low and the high QC results are within the 2s limits, then accept the
run.
(2) If one of two QC results is outside the 2s limits, then apply the rules below and
reject the run if any condition is met.
(Note: each one is given a code [0- 4] to be entered in the database.)

Code
0 -Extreme outlier: the result is outside the characterization mean by more than 4SD.
1 -13s -The average of both low QC results QB the average of both high QC results is
outside a 3s limit.
2 –22s -The average of both low QC results AND the average of both high QC results is
outside a 2s limit on the same side of the mean.
3- R4s 5equential- The average of both low QC results AND the average of both high
QC results is outside a 2s limit on opposite sides of the mean.
4 -1Ox sequential -The previous nine average QC results (for the previous nine runs)
were on the same side of the mean for either the low QB high QC results.
If the run is declared out of control, the analysis results for all patient samples analyzed
during that run are invalid for reporting. If more than ten consecutive QCs are on the
same side of the mean of the characterized QC material, suspend all operations until
you determine whether a bias is present in the method. (See Tables 10- 12 for the
characterization of both QC pools.)

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

Table 10. Mean Value, SD, and CV of Low QC Pool
(Replicates =20)
Analyte

Mean (ng/L)

1-nap

231

2-nap

590

2-fluo

166

3-fluo

114

9-fluo

108

1-phen

168

2-phen

146

3-phen

171

4-phen

166

9-phen

100

3-flran

116

1-pyr

135

1-bcp

2-bcp

3-bcp

1-chry

2-chry

3-chry

4-chry

6-chry

1-baa

3-baa

104

3-bap

137

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

Table 11. Mean Value, SD, and CV of Middle QC Pool
(Replicates =20)
Analyte

Mean (ng/L)

SD (%)

1-nap

369

2-nap

781

2-fluo

357

3-fluo

317

9-fluo

326

1-phen

429

2-phen

361

3-phen

291

4-phen

357

9-phen

214

3-flran

238

1-pyr

248

1-bcp

173

2-bcp

222

3-bcp

222

1-chry

215

2-chry

246

3-chry

234

4-chry

195

6-chry

221

1-baa

219

3-baa

248

3-bap

288

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

Table 12. Mean Value, SD, and CV of High QC Pool
(Replicates =20)
Analyte

Mean (ng/L)

1-nap

930

2-nap

1189

127

2-fluo

806

3-fluo

766

9-fluo

656

1-phen

868

2-phen

813

3-phen

862

4-phen

801

9-phen

656

3-flran

752

1-pyr

789

1-bcp

628

2-bcp

790

3-bcp

765

1-chry

759

2-chry

859

3-chry

812

4-chry

698

6-chry

813

107

1-baa

762

3-baa

861

105

3-bap

1148

161

116

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

10. Recovery of Method
The recoveries of OH-PAHs use this method are calculated based on the amount of
spiked isotope labeled internal standards vs measured amount with the external
recovery standard correction. The recovery values reflect the performance of sample
preparation.

Table 13 Internal Standard Acceptable Recovery Ranges
Spike

Recovery Range, %

1-OH-Naphthol C13

35-95

3-OH-Phenanthrene C13

35-95

3-OH-Fluoranthene C13

35-95

1-OH-Pyrene C13

50-120

1-OH-Benzo(a)anthracene C13

50-120

6-OH-Chrysene C13

50-120

3-OH-Benzo(c)phenanthrene C13

50-120

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

11. Remedial Action if Calibration or QC Systems Fail to Meet Acceptable Criteria
If the calibration or QC systems fail to meet acceptable criteria, suspend all operations
until you identify the source or cause of failure. If you can easily identify the source of
failure (e.g., failure of the mass spectrometer or an error in pipeting), then correct the
problem. Otherwise, prepare fresh reagents and clean the mass spectrometer system.
Before beginning another analytical run, reanalyze several QC materials (in the case of
QC failure) or calibration-verification materials (in the case of calibration failure).
After reestablishing calibration or QC, resume analytical runs.

12. Limitations of Method; Interfering Substances and Conditions
This method is an isotope-dilution mass-spectrometry method, which is widely
regarded as the definitive method for the measurement of organic toxicants in human
body fluids. By using high-resolution mass spectrometry, it eliminates most
interferants. Occasional unknown interferants have been encountered because of the
matrix used in this procedure. Chromatographic interferences with the internal
standards will result in the rejection of analysis. If repeat analysis results in an
interference with the internal standard, do not report the results for that analyte.

13. Reference Ranges (Normal Values)
The reference ranges for OH-PAH in the U.S. population are based on the NHANES
2005 study. (See Table 14 for reference range values from NHANES 2005.)

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

Table 14. Reference Range of OH-PAHs in Urine

Analyte
1-nap

Without Creatine Correction
Mean
Low 50th High 95th
95% C.I.
(ng/L)
(ng/L)
(ng/L)
2050
1490
26600

Creatine Corrected
Mean
Low 50th High 95th
95% C.I.
(ng/L)
(ng/L)
(ng/L)
1910
1370
20800

2-nap

2470

1920

29700

2310

1670

19200

2-fluo

441

422

9470

408

382

6860

3-fluo

170

86.3

4680

157

79.9

4060

9-fluo

219

185

1300

205

181

1120

1-phen

154

105

1390

142

102

1270

2-phen

98.3

79.7

937

90.3

67.8

748

3-phen

127

113

721

116

101

574

4-phen

41.9

46.0

339

39.2

56.5

411

9-phen

33.9

36.0

396

31.7

36.3

290

3-flran

13.4

17.5

147

12.3

14.7

127

1-pyr

79.8

76.9

926

74.2

63.1

607

1-bcp

< LOD

54.0

< LOD

70.8

2-bcp

< LOD

28.1

< LOD

30.9

3-bcp

< LOD

16.0

< LOD

16.7

1-chry

< LOD

138

< LOD

104

2-chry

< LOD

45.0

< LOD

35.0

3-chry

< LOD

46.0

< LOD

62.5

4-chry

< LOD

6-chry

< LOD

10.1

< LOD

76.4

1-baa

< LOD

32.4

3-/9-baa

< LOD

31.0

< LOD

35.5

3-bap

< LOD

251

< LOD

248

< LOD means less than the limit of detection
* Not calculated. Portion of results below limit of
From CDC Third Report on Human Exposure to Environmental Chemicals

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

14. Critical-Call Results ("Panic" Values)
There are no established critical-call values for this method. It is unlikely that any result
would be a critical-call, which would only occur with poisonings.

15. Specimen Storage and Handling During Testing
Refrigerate urine samples overnight at -4 °C to expedite thawing prior to aliquoting the
samples. Store the urine extracts in autosampler vials at -70 °C after analysis. Current
studies indicate (CDC data) that the extracts are stable for up to 3 weeks.

16. Alternate Methods for Performing Test and Storing Specimens if Test System
Fails
The method is designed to run on a GC/HRMS instrument and is not generally
transferable to other instrumentation.
There are no acceptable alternative methods for analysis or backup systems. If the
analytical system fails, store specimens in a refrigerator overnight. If long-term
interruption is anticipated, store at -80 °C.

17. Transfer or Referral of Specimens; Procedures for Specimen Accountability and
Tracking
Use standard record-keeping systems (i.e., notebooks, sample logs, data files,
creatinine logs, demographic logs) to keep track of all specimens.

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

References
1. International Agency for Research on Cancer. IARC Monographs on the Evaluation of
Carcinogenic Risk of Chemicals to Man: Polycyclic Aromatic Compounds. Part 1.
Chemical and environmental data 1983; Vol 32. (IARC, Lyon).
2. International Agency for Research on Cancer. IARC Monographs on the Evaluation of
Carcinogenic Risk of Chemicals to Man: Tobacco Smoking 1985; Vol. 38. (IARC,
Lyon).
3. Angerer J, Mannschreck C, GOndel J. Biological monitoring and biochemical effect
monitoring in the case of PAH exposure. Int Arch Environ Health 1997; 70: 365.
4. Chaung JC, Callahan PJ, Lyu CW, Wilson NK. Polycyclic aromatic hydrocarbon
exposures of children in low-income families. J Expos Anal Environ Epidemiol 1999;
2: 85.
5. Dor.F;Haguenoer J-M, Zmirou D, Empereur-Bissonnet P, Jongeneelen FJ, Nedellec V,
Person A, Ferguson CC. Urinary 1-hydroxypyrene as a biomarker of polycyclic
aromatic hydrocarbons exposure of workers on a contaminated site: Influence of
exposure conditions. J Occup Environ Medicine 2000; 42: 391.
6. Gmeiner G, Krassnig C, Schmid E, Tausch H. Fast screening method for the profile
analysis of polycyclic aromatic hydrocarbon metabolites in urine using derivatisationsolid phase microextraction. J Chromatog B 1998; 705: 132.
7. Grimmer G, Jacob J, Dettbarn G, Naujack K-W, Heinrich U. Urinary metabolite profile
of PAH as a potential mirror of the genetic disposition for cancer. Exp Toxic
Patho11995; 47: 421.
8. Grimmer G, Jacob J, Dettbarn G, Naujack K-W. Determination of urinary metabolites of
polycyclic aromatic hydrocarbons for the risk assessment of PAH-exposed workers. Int
Arch Occup Environ Health 1997; 69: 231.
9. GOndel J, Angerer J. High performance liquid chromatographic method with
fluorescence detection for the determination of 3-hydroxybenzo(a)pyrene and 3hydroxybenzo(a)anthracene in the urine of polycyclic aromatic hydrocarbon exposed
workers. J Chromatog B 2000; 728: 47.
10. GOndel J, Schaller KH, Angerer J. Occupational exposure to polycyclic aromatic
hydrocarbons in a fireproof stone producing plant: biological monitoring of 1hydroxypyrene, 1-,2-,3- and 4- hydroxybenz(a)anthracene and 3hydroxybenzo(a)pyrene. Int Arch Occup Environ Health 2000; 73: 270.
11. Hansen AM, Christensen JM, Sherson D. Estimation of reference values for urinary
1-hydroxypyrene and alpha-naphthol in Danish workers. Sci Total Environ 1995; 163:
211.

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

12. CDC, Third National Report on Human Exposure to Environmental Chemicals. NCEH
Pub. 2005; No. 05-0570, 77-131
13. Zheng Li, Lovisa C. Romanoff, Debra A. Trinidad, Measurement of Urinary
Monohydroxy Polycyclic Aromatic Hydrocarbons Using Automated Liquid-Liquid
Extraction and Gas Chromatography/Isotope Dilution High-Resolution Mass
Spectrometry, Anal. Chem. 2006;

Standard Operating Procedure - Hydroxy PAHs in Urine

July 2006

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