NOAA Technical Memorandum NMFS-NWFSC-125

Northwest Fisheries Science 

Center’s Analyses of Tissue, 

Sediment, and Water Samples 

for Organic Contaminants 

by Gas Chromatography/Mass 

Spectrometry and Analyses 

of Tissue for Lipid Classes 

by Thin Layer Chromatography/

Flame Ionization Detection

January 2014

U.S. DEPARTMENT OF COMMERCE

National Oceanic and Atmospheric Administration

National Marine Fisheries Service

Northwest Fisheries Science Center
NOAA Technical Memorandum

NMFS-NWFSC Series

The Northwest Fisheries Science Center of NOAA’s

National Marine Fisheries Service uses the NOAA Technical 

Memorandum NMFS-NWFSC series to issue scientific and 

technical publications. Manuscripts have been peer reviewed 

and edited. Documents published in this series can be cited 

in the scientific and technical literature.

The Northwest Fisheries Science Center’s NOAA Technical 

Memorandum NMFS-NWFSC series continues the NMFSF/NWC series established in 1970 by the Northwest and

Alaska Fisheries Science Center, which subsequently was 

divided into the Northwest Fisheries Science Center and the 

Alaska Fisheries Science Center. The latter center uses the 

NOAA Technical Memorandum NMFS-AFSC series.

Reference throughout this document to trade names does not 

imply endorsement by the National Marine Fisheries Service.

Reference this document as follows:

Sloan, C.A., B.F. Anulacion, K.A. Baugh, J.L. Bolton, 

D. Boyd, R.H. Boyer, D.G. Burrows, D.P. Herman, 

R.W. Pearce, and G.M. Ylitalo. 2014. Northwest Fisheries 

Science Center’s analyses of tissue, sediment, and water 

samples for organic contaminants by gas chromatography/ 

mass spectrometry and analyses of tissue for lipid classes by 

thin layer chromatography/flame ionization detection. U.S. 

Dept. Commer., NOAA Tech. Memo. NMFS-NWFSC-125.
NOAA Technical Memorandum NMFS-NWFSC-125

Northwest Fisheries Science 

Center’s Analyses of Tissue, 

Sediment, and Water Samples 

for Organic Contaminants 

by Gas Chromatography/Mass 

Spectrometry and Analyses 

of Tissue for Lipid Classes 

by Thin Layer Chromatography/

Flame Ionization Detection

Catherine A. Sloan, Bernadita F. Anulacion, Keri A. Baugh, 

Jennie L. Bolton, Daryle Boyd, Richard H. Boyer, 

Douglas G. Burrows, David P. Herman, Ronald W. Pearce, 

and Gina M. Ylitalo

Northwest Fisheries Science Center

2725 Montlake Boulevard East

Seattle, Washington 98112

January 2014

U.S. DEPARTMENT OF COMMERCE

National Oceanic and Atmospheric Administration

National Marine Fisheries Service
Most NOAA Technical Memorandums

NMFS-NWFSC are available at the 

Northwest Fisheries Science Center

Web site, http://www.nwfsc.noaa.gov

Copies are also available from the

National Technical Information Service

5285 Port Royal Road

Springfield, VA 22161

Phone orders: 1-800-553-6847

E-mail orders: orders@ntis.fedworld.gov

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Contents

List of Figures.............................................................................................................................................vii

List of Tables...............................................................................................................................................ix

Executive Summary.....................................................................................................................................xi

Acknowledgments......................................................................................................................................xiii

List of Abbreviations ..................................................................................................................................xv

1. Introduction and Summary of Analytical Procedures..............................................................................1

2. Materials ..................................................................................................................................................8

2.1. Instruments and Accessories.............................................................................................................8

2.1.1. GC/MS System..........................................................................................................................8

2.1.2. GC Gas and Accessories...........................................................................................................8

2.1.3. GC Column and Accessories.....................................................................................................8

2.1.4. HPLC System and Accessories.................................................................................................9

2.1.5. Sample Extractor and Accessories............................................................................................9

2.1.6. TLC/FID and Accessories.........................................................................................................9

2.2. Solvents and Reagents....................................................................................................................10

2.3. Silica/Alumina Column Packing Material......................................................................................10

2.4. Labware ..........................................................................................................................................10

2.5. Disposable Items.............................................................................................................................11

2.6. Standard Solutions and GC Conditioning Solutions ......................................................................11

2.6.1. Surrogate Standard and Internal Standard Solutions...............................................................11

2.6.2. Calibration Standards..............................................................................................................12

2.6.3. Spike Solutions........................................................................................................................13

2.6.4. GC Conditioning Solutions.....................................................................................................13

2.7. Standard Reference Materials.........................................................................................................13

2.8. Preparation of Silica/Alumina Column Packing Material and Other Reagents..............................14

2.8.1. Silica........................................................................................................................................14

2.8.2. Alumina...................................................................................................................................14

2.8.3. Glass Wool and Glass Beads...................................................................................................14

2.8.4. Copper.....................................................................................................................................14
iv

2.8.5. Sodium Sulfate ........................................................................................................................14

2.8.6. Magnesium Sulfate..................................................................................................................14

2.9. Purity Testing .................................................................................................................................14

2.9.1. Isooctane Purity Test...............................................................................................................15

2.9.2. Dichloromethane Purity Test...................................................................................................15

2.9.3. Sodium Sulfate Purity Test .....................................................................................................15

2.9.4. Magnesium Sulfate Purity Test...............................................................................................16

2.9.5. Silica or Alumina Purity Test..................................................................................................16

3. Sample Extraction..................................................................................................................................17

3.1. Accelerated Solvent Extraction of Sediment and Tissue Samples.................................................17

3.1.1. Sample Preparation .................................................................................................................17

3.1.2. Surrogate Standards and Standard Check Solutions ...............................................................18

3.1.3. ASE Operation ........................................................................................................................19

3.1.4. Preparation of Extracts............................................................................................................20

3.2. Liquid-liquid Extraction of Water Samples....................................................................................23

4. Sample Cleanup .....................................................................................................................................26

4.1. Silica/Alumina Column Chromatography ......................................................................................26

4.1.1. Calibration of Silica/Alumina Column....................................................................................26

4.1.2. Sample Cleanup by Silica/Alumina Column Chromatography...............................................27

4.1.3. Preparation of Concentrated Extracts......................................................................................28

4.2. Size-exclusion High-performance Liquid Chromatography...........................................................29

4.2.1. Size-exclusion HPLC Start-up ................................................................................................29

4.2.2. Size-exclusion HPLC System Calibration ..............................................................................30

4.2.3. Daily Calibration of the Size-exclusion HPLC System ..........................................................33

4.2.4. Sample Cleanup by Size-exclusion HPLC..............................................................................33

4.2.5. Preparation of Extracts and Standard Check Solutions...........................................................34

5. Gas Chromatography/Mass Spectrometry .............................................................................................36

5.1. GC/MS Operating Conditions........................................................................................................36

5.2. GC/MS for Quantitating PAHs, Alkyl PAH Isomers, and Alkanes...............................................37

5.2.1. GC/MS SIM Windows for PAHs and Alkyl PAH Isomers ....................................................37

5.2.2. GC/MS SIM Windows for PAHs, Alkyl PAH Isomers, and Alkanes ....................................41

5.2.3. Verification of GC/MS Performance for PAHs and Alkyl PAH Isomers...............................41

5.2.4. GC/MS Analysis of Sediment Samples for PAHs ..................................................................41

5.2.5. GC/MS Analysis of Tissue Samples for PAHs and Alkyl PAH Isomers................................41

5.2.6. GC/MS Analysis of Water Samples for PAHs, Alkyl PAH Isomers, and Alkanes................42
v

5.3. GC/MS for Quantitating POPs .......................................................................................................42

5.3.1. GC/MS SIM Windows for POPs.............................................................................................42

5.3.2. Verification of GC/MS Performance for POPs.......................................................................42

5.3.3. GC/MS Analysis of Sediment Samples for POPs...................................................................42

5.3.4. GC/MS Analysis of Low-lipid Tissue Samples or Water Samples for POPs.........................45

5.3.5. GC/MS Analysis of High-lipid Tissue Samples for POPs......................................................45

6. Calculations of GC/MS Results.............................................................................................................46

6.1. Calculating Analyte Concentrations...............................................................................................46

6.2. Calculating Internal Standard Recoveries ......................................................................................50

6.3. Calculating and Reporting Lower Limits of Quantitation..............................................................51

6.4. Calculating and Reporting Estimated Concentrations for Analytes Exceeding the 

Calibration Range...................................................................................................................................51

7. Quality Assurance for PAHs, POPs, and Alkanes.................................................................................52

7.1. GC/MS Stability .............................................................................................................................52

7.2. Surrogate and Internal Standard Recoveries...................................................................................52

7.3. Analyte Concentrations in Reference Materials.............................................................................52

7.4. Precision of Replicate Samples ......................................................................................................53

7.5. Method Blank Contamination.........................................................................................................53

7.6. Spiked Blank Recoveries................................................................................................................53

8. Gravimetric Determinations...................................................................................................................54

8.1. Dry Weight Determination .............................................................................................................54

8.2. Total Extractables Determination...................................................................................................55

8.2.1. Batches of Tissue Samples Requiring Macro Total Extractables Determination ...................55

8.2.2. Batches of Tissue Samples Requiring Micro Total Extractables Determination ....................55

9. Lipid Classes..........................................................................................................................................56

9.1. Separation of Lipid Classes............................................................................................................56

9.2. TLC/FID Analysis..........................................................................................................................57

9.3. Calculations of Lipid Class Concentrations ...................................................................................57

9.4. Quality Assurance for Lipid Classes..............................................................................................59

9.4.1. TLC/FID Stability ...................................................................................................................59

9.4.2. Percent Summed Lipid Value for Reference Materials...........................................................59

9.4.3. Precision of Replicate Samples...............................................................................................59

9.4.4. Method Blank Contamination .................................................................................................59

10. Conclusions..........................................................................................................................................60

References...................................................................................................................................................61
vi
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Figures

Figure 1. Flow diagram of tissue or sediment sample preparation and analysis..........................................5

Figure 2. Flow diagram of water sample preparation and analysis..............................................................7

Figure 3. Size-exclusion high-performance liquid chromatograph calibration..........................................32

Figure 4. GC/MS SIM chromatograms of C3-naphthalenes in an oil sample, a brown shrimp sample, 

and a sample of NIST SRM 1974b .............................................................................................................47

Figure 5. GC/MS SIM chromatograms of C2-fluorenes in an oil sample, a brown shrimp sample, and 

a sample of NIST SRM 1974b....................................................................................................................48
viii
ix

Tables

Table 1. PAH analytes .................................................................................................................................1

Table 2. PAH analytes, including sums of alkyl PAH isomers ...................................................................2

Table 3. Persistent organic pollutant analytes, including PCB congeners and PBDE congeners................4

Table 4. Size-exclusion HPLC events........................................................................................................29

Table 5. GC/MS operating conditions.......................................................................................................36

Table 6. SIM windows for quantitating selected PAHs in tissue and water samples................................38

Table 7. SIM windows for quantitating PAHs, including sums of alkyl isomers in tissue and 

water samples..............................................................................................................................................39

Table 8. SIM windows for quantitating POPs in tissue, sediment, and water samples .............................43

Table 9. Compounds used for response factors to quantitate isomer groups of alkyl PAH isomers.........49
x
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Executive Summary

This document describes the analytical methods developed and currently used by the 

Environmental Chemistry Program in the Environmental Conservation Division of the Northwest 

Fisheries Science Center for the analysis of sediment, tissue, and water samples for part-perbillion concentrations (nanograms per gram or nanograms per milliliter) of selected polycyclic 

aromatic hydrocarbons and persistent organic pollutants, plus selected alkanes in water samples 

as needed. Detailed descriptions are presented for sample extraction, cleanup by silica/alumina 

columns and by size-exclusion high-performance liquid chromatography, and analysis by gas 

chromatography/mass spectrometry, as well as the calculation of analyte concentrations and 

important quality assurance measurements. Detailed descriptions are also given for measuring 

lipid classes in tissues, determining the percent total extractable content of tissues, and 

determining the percent dry weight of tissues and sediments. Information pertaining to the 

necessary laboratory supplies and instruments is provided. The step-wise procedures are 

presented in a descriptive manner, which facilitates the use of this document by other 

laboratories.
xii
xiii

Acknowledgments

We thank the past Environmental Chemistry Program chemists for their expert 

contributions, in particular, Donald Brown and Margaret Krahn.
xiv
xv

Abbreviations

ASE Accelerated Solvent Extractor

CS calibration standard

DOB dibromooctafluorobiphenyl

GC gas chromatograph

GC/MS gas chromatography/mass spectrometry

HMB hexamethylbenzene

HPLC high-performance liquid chromatography

id inside diameter

IS internal standard

LOQ lower limit of quantitation

MS mass spectrometer

NIST National Institute of Standards and Technology

PAH polycyclic aromatic hydrocarbon

PBDE polybrominated diphenyl ether

PCB polychlorinated biphenyl

PER perylene

POP persistent organic pollutant

QA quality assurance

RF response factor

RRF relative response factor

RSD relative standard deviation

RT retention time

SIM selected-ion monitoring

SRM Standard Reference Material

SS surrogate standard

TCMX tetrachloro-m-xylene

TCOX tetrachloro-o-xylene

TLC/FID thin layer chromatography/flame ionization detection

UV ultraviolet
xvi
1. Introduction and Summary of 

Analytical Procedures

A wide range of environmental studies has included the analyses of sediments, tissues of 

aquatic organisms, and water for toxic contaminants such as chlorinated pesticides, 

polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), collectively 

referred to as persistent organic pollutants (POPs) hereafter, and polycyclic aromatic 

hydrocarbons and sulfur-containing heterocycles, collectively referred to as polycyclic aromatic 

hydrocarbons (PAHs) hereafter. High-quality data with documented quality assurance (QA) 

measures are needed for such analyses in order to draw valid conclusions and reach appropriate 

decisions, for example, in damage assessment cases, for policy making, and to facilitate 

comparisons of the results with those of other studies. New techniques are continually sought 

and evaluated to further optimize the analytical accuracy, precision, sensitivity, robustness, 

efficiency, and safety. The Environmental Chemistry Program of the Northwest Fisheries 

Science Center (NWFSC) Environmental Conservation Division develops, validates, and uses 

advanced methods for analyzing marine samples for organic contaminants at part-per-billion 

concentrations (ng per g or ng per mL). As improvements to procedures are validated, they are 

incorporated into the analytical protocols used for a multitude of projects in which selected 

PAHs (Table 1 and Table 2) and POPs (Table 3) are quantitated in sediment, tissue, and water.

Table 1. PAH analytes.

Naphthalene Retene

1-Methylnaphthalene Fluoranthene

2-Methylnaphthalene Pyrene

2,6-Dimethylnaphthalene Benz[a]anthracene

Acenaphthylene Sum of chrysene and triphenylene*

Acenaphthene Benzo[b]fluoranthene

2,3,5-Trimethylnaphthalene Sum of benzo[j]fluoranthene 

Fluorene and benzo[k]fluoranthene*

Dibenzothiophene Benzo[e]pyrene

Phenanthrene Benzo[a]pyrene

Anthracene Perylene

1-Methylphenanthrene Indeno[1,2,3-cd]pyrene

3-Methylphenanthrene Sum of dibenz[a,h]anthracene 

9-Methylphenanthrene and dibenz[a,c]anthracene*

1,7-Dimethylphenanthrene Benzo[ghi]perylene

* These analytes are quantitated and reported as the sum 

of their concentrations because they coelute during gas 

chromatography/mass spectrometry (GC/MS) analysis; 

the first analyte is present in the calibration standard,

whereas the additional analyte is not.
2

Table 2. PAH analytes, including sums of alkyl PAH isomers.

Naphthalene

C1-Naphthalenes: sum of 1-methylnaphthalene and 2-methylnaphthalene

C2-Naphthalenes: sum of dimethyl- and ethyl-naphthalenes, including 2,6-dimethylnaphthalenea

C3-Naphthalenes: sum of trimethyl-, methylethyl- and propyl-naphthalenes, including 2,3,5-

trimethylnaphthalenea

C4-Naphthalenes: sum of tetramethyl-, dimethylethyl-, diethyl-, methylpropyl- and butyl-naphthalenes

Acenaphthylene

Acenaphthene

Fluorene

C1-Fluorenes: sum of methyl-fluorenes

C2-Fluorenes: sum of dimethyl- and ethyl-fluorenes

C3-Fluorenes: sum of trimethyl-, methylethyl- and propyl-fluorenes

Dibenzothiophene

C1-Dibenzothiophenes: sum of methyl-dibenzothiophenes

C2-Dibenzothiophenes: sum of dimethyl- and ethyl-dibenzothiophenes

C3-Dibenzothiophenes: sum of trimethyl-, methylethyl-, and propyl-dibenzothiophenes

C4-Dibenzothiophenes: sum of tetramethyl-, dimethylethyl-, diethyl-, methylpropyl- and butyldibenzothiophenes

Phenanthrene

Anthracene

C1-Phenanthrenes and anthracenesb: sum of methyl-phenanthrenes and anthracenes, including 

1-methylphenanthrene a, 3-methylphenanthrene and 9-methylphenanthrene

C2-Phenanthrenes and anthracenesb: sum of dimethyl- and ethyl-phenanthrenes and anthracenes, 

including 1,7-dimethylphenanthrenea

C3-Phenanthrenes and anthracenesb: sum of trimethyl-, methylethyl-, and propyl-phenanthrenes and 

anthracenes

C4-Phenanthrenes and anthracenesb: sum of tetramethyl-, dimethylethyl-, diethyl-, methylpropyl- and 

butyl-phenanthrenes and anthracenes

Retene

Fluoranthene

Pyrene

C1-Fluoranthenes and pyrenesb: sum of methyl-fluoranthenes and pyrenes

C2-Fluoranthenes and pyrenesb: sum of dimethyl- and ethyl-fluoranthenes and pyrenes

C3-Fluoranthenes and pyrenesb: sum of trimethyl-, methylethyl-, and propyl-fluoranthenes and pyrenes

C4-Fluoranthenes and pyrenesb: sum of tetramethyl-, dimethylethyl-, diethyl-, methylpropyl- and butylfluoranthenes and pyrenes

Benz[a]anthracene

Sum of chrysene and triphenylenec

C1-Benz[a]anthracenes and chrysenesb: sum of methyl-benzanthracenes and chrysenes

C2-Benz[a]anthracenes and chrysenesb: sum of dimethyl- and ethyl-benzanthracenes and chrysenes

C3-Benz[a]anthracenes and chrysenesb: sum of trimethyl-, methylethyl-, and propyl-benzanthracenes 

and chrysenes

C4-Benz[a]anthracenes and chrysenesb: sum of tetramethyl-, dimethylethyl-, diethyl-, methylpropyland butyl-benzanthracenes and chrysenes

Benzo[b]fluoranthene

Sum of benzo[k]fluoranthene and benzo[j]fluoranthenec
3

Table 2 continued. PAH analytes, including sums of alkyl PAH isomers.

Benzo[e]pyrene

Benzo[a]pyrene

Perylene

Indeno[1,2,3-cd]pyrene

Sum of dibenz[a,h]anthracene and dibenz[a,c]anthracenec

Benzo[ghi]perylene

a This alkyl PAH is used to calculate the GC/MS response factor for the quantitation of its group of isomers (also see 

Table 9). b These analytes are quantitated and reported as the sum of their concentrations because these isomers co-occur, and 

in some cases coelute, in the same region of the GC/MS chromatogram.

c These analytes are quantitated and reported as the sum of their concentrations because they coelute during GC/MS 

analysis; the first analyte is present in the calibration standard, whereas the additional analyte is not.

This document incorporates the latest revisions to the methods described in Sloan et al. 

(2004, 2005) and Ylitalo et al. (2005), including the addition of quantitating PBDE analytes by 

gas chromatography/mass spectrometry (GC/MS). Among the additions is the method for 

quantitating the following lipid classes by thin layer chromatography/flame ionization detection 

(TLC/FID):

Wax esters and sterol esters,

Triglycerides,

Free fatty acids,

Cholesterol, and

Phospholipids and other polar lipids.

Also included is the method for analysis of water samples for PAHs and POPs plus the following 

selected alkanes as needed:

Pristane,

Phytane,

n-Heptadecane, and

n-Octadecane.

These detailed descriptions of our current procedures are provided for complete documentation 

as well as for ease of use by other laboratories.

Preparation and analyses of tissue and sediment samples are summarized in Figure 1. 

Weighed portions of sediment or tissue samples are mixed with sodium sulfate and magnesium 

sulfate, then extracted with dichloromethane using an Accelerated Solvent Extractor (ASE) 

(Dionex, Salt Lake City, UT). Extracts of tissues, such as marine mammal tissues, may be split 

into portions: one for isolating and quantitating the organic contaminants, one for quantitating 

lipid classes as needed, and one for determining the percent total extractables (i.e., mostly lipids) 

as needed. The extract or portion of extract to be analyzed for contaminants is filtered through a 

gravity-flow silica/alumina column. The filtered sample extracts are then concentrated, and a 

portion of each filtered extract is chromatographed on a size-exclusion high-performance liquid 

chromatography (HPLC) column, using dichloromethane as the mobile phase, to collect a 

fraction containing the PAHs and POPs. The dichloromethane is replaced with isooctane while 
4

Table 3. Persistent organic pollutant analytes, including PCB congenersa and PBDE congeners.

b

PCB 17 PCB 206

PCB 18 PCB 208

PCB 28 PCB 209

PCB 31 2,4'-DDD (o,p’-DDD)

PCB 33 4,4'-DDD (p,p’-DDD)

PCB 44 2,4'-DDE (o,p’-DDE)

PCB 49 4,4'-DDE (p,p’-DDE)

PCB 52 2,4'-DDT (o,p’-DDT)

PCB 66 4,4'-DDT (p,p’-DDT)

PCB 70 Aldrin

PCB 74 cis-Chlordane

PCB 82 trans-Chlordane

PCB 87 Dieldrin

PCB 95 Endosulfan I

PCB 99 Heptachlor

Sum of PCB 101 and PCB 90c Heptachlor epoxide

PCB 105 Hexachlorobenzene

PCB 110 alpha-Hexachlorocyclohexane

PCB 118 beta-Hexachlorocyclohexane

PCB 128 gamma-Hexachlorocyclohexane (lindane)

Sum of PCB 138, PCB 163, and PCB 164c Mirex

PCB 149 cis-Nonachlor

PCB 151 trans-Nonachlor

Sum of PCB 153 and PCB 132c Nonachlor III

PCB 156 Oxychlordane

PCB 158 PBDE 28

PCB 170 PBDE 47

PCB 171 PBDE 49

PCB 177 PBDE 66

PCB 180 PBDE 85

PCB 183 PBDE 99

Sum of PCB 187, PCB 159, and PCB 182c PBDE 100

PCB 191 PBDE 153

PCB 194 PBDE 154

PCB 195 PBDE 155

PCB 199 PBDE 183

PCB 205 a PCB congeners are numbered according to 

Ballschmiter et al. 1992.

b PBDE congeners are numbered as is done for PCBs 

according to Ballschmiter et al. 1992.

c These analytes are quantitated and reported as the 

sum of their concentrations because they coelute 

during GC/MS analysis; the first congener is present 

in the calibration standard, whereas the additional 

congeners are not.
5

 Figure 1. Flow diagram of tissue or sediment sample preparation and analysis.

Mix a portion of sample with sodium 

sulfate and magnesium sulfate, add 

surrogate standard(s), then extract 

with dichloromethane using 

Accelerated Solvent Extraction. 

Add HPLC internal standard(s).

Tissue or sediment sample

Dichloromethane extract of sample

For tissues as needed, take a portion 

of the extract and evaporate the 

solvent; gravimetrically determine 

the percent total extractables.

For tissues as needed, take a portion 

of the extract and analyze it for lipid 

classes using TLC/FID.

Concentrated extract

in dichloromethane

Concentrate extract to 5 mL, 

filter extract through a 

silica/alumina column, then 

concentrate it to 1 mL.

Chromatograph 1⁄2 of extract using size-exclusion HPLC; collect the 

PAH/POP fraction, add GC/MS internal standard(s), and concentrate 

the fraction to 100 μL in isooctane.

Concentrated extract in isooctane

Analyze the extract using GC/MS 

for PAH quantitation, as needed.

As needed, dry a portion of the 

sample; gravimetrically determine 

the percent dry weight.

Analyze the extract using GC/MS 

for POP quantitation, as needed.
6

the extracts are reduced to a final volume of approximately 100 μL each. The concentrated 

extracts are then analyzed by GC/MS to measure PAHs or POPs or both. Analytes are 

quantitated relative to surrogate standards (SSs), also known as extraction internal standards 

(ISs), using multiple concentration levels of GC/MS calibration standards (CSs). The 

concentrations of analytes in sediment and tissue samples are reported on a nanogram per gram 

(ng/g) wet weight or dry weight basis. The percent dry weight of tissues or sediments is 

determined gravimetrically using a separate portion of the unprocessed sample. The percent total 

extractables in tissues is determined gravimetrically using a portion of the extract taken prior to 

cleanup. Lipid classes are quantitated by TLC/FID using another portion of the tissue extract 

taken prior to cleanup.

Preparation and analyses of water samples are summarized in Figure 2. Measured 

volumes of water samples are extracted with dichloromethane using separatory funnels. The 

extract is cleaned up, prepared for GC/MS, and analyzed as is done for sediments and tissues, 

except that if the sample is to be analyzed for alkanes, the extract is not chromatographed on 

HPLC prior to being prepared for GC/MS and the alkanes are quantitated concurrently with the 

PAHs. The concentrations in water samples are reported in nanograms per milliliter (ng/mL).

Samples are typically analyzed in batches of 10 to 14 field samples. QA measures are 

incorporated into each batch according to the Environmental Chemistry Program’s Quality 

Assurance Plan (Sloan et al. 2006). Each batch of samples includes a method blank, a sample of 

an appropriate National Institute of Standards and Technology (NIST) Standard Reference 

Material (SRM) for tissues or sediment that has certified concentrations for many analytes, a 

spiked blank for water, and as needed, replicate samples. SSs are added to the samples before 

extraction to monitor and account for any losses during sample preparation. HPLC ISs are added 

after extraction, before any aliquots of the extract are taken (e.g., for percent total extractables 

determination), in order to account for the amount of total extract that is chromatographed by 

HPLC, and GC ISs are added before GC/MS analysis to measure the recovery of the HPLC ISs. 

Typical goals for QA measurements are given in Section 7.

Samples are prepared in fume hoods to minimize exposure of the analysts to solvent 

vapors, and personal protective equipment (e.g., nitrile gloves, safety glasses) is worn by the 

analysts whenever organic solvents are used. In some instances (e.g., for tissues and fluids of 

stranded marine mammals), samples may need to be weighed and prepared for the ASE in a 

biological safety cabinet to minimize exposure of the analysts to potential pathogens. Additional 

laboratory safety practices are followed according to the NWFSC Chemical Hygiene Plan and 

the NWFSC Chemical Waste Management Guide.
7

 Figure 2. Flow diagram of water sample preparation and analysis.

Add surrogate standard(s). Extract the sample with dichloromethane 

using a separatory funnel. Add HPLC internal standard(s).

Water sample

Dichloromethane extract of sample

Concentrated extract in dichloromethane

Dry the extract with sodium sulfate. Concentrate extract to 5 mL, filter 

extract through a silica/alumina column, then concentrate it to 1 mL.

For analyses not including alkanes 

quantitation, chromatograph 1⁄2 of 

the extract using size-exclusion 

HPLC; collect the PAH/POP 

fraction, add GC/MS internal 

standard(s), and concentrate the 

fraction to 100 μL in isooctane.

Concentrated extract in isooctane

Analyze the extract using GC/MS 

for PAH quantitation or PAH plus 

alkane quantitation, as needed.

Analyze the extract using GC/MS 

for POP quantitation, as needed.

For analyses including alkanes 

quantitation, add GC/MS internal 

standard(s) and concentrate the 

extract to 100 μL in isooctane.
8

2. Materials

2.1. Instruments and Accessories

2.1.1. GC/MS System

• Autosampler, Agilent 7693A, with on-column injection syringe and needle, and needle guide 

for 0.53-mm columns (Agilent Technologies, Wilmington, DE).

• Data system software, ChemStation, Version E (Agilent Technologies, Wilmington, DE).

• Gas chromatograph (GC) with cool on-column inlet fitted with inset and septum cap for 

0.53-mm columns and syringe needles, Agilent 7890A (Agilent Technologies, Wilmington, 

DE).

• Mass spectrometer (MS), Agilent 5975C Mass Selective Detector (Agilent Technologies, 

Wilmington, DE).

• Sample vial tray, Agilent 7693 (Agilent Technologies, Wilmington, DE).

• Septum, GC inlet, bleed/temperature-optimized, 5 mm, 5183-4758 (Agilent Technologies, 

Wilmington, DE).

2.1.2. GC Gas and Accessories

• Helium, grade 5.0, ultra-high purity, 99.999% (Central Welding Supply, Marysville, WA).

• Alltech indicating oxygen trap, 4004 (Grace Davison Discovery Sciences, Deerfield, IL).

• Combination indicating oxygen/moisture/hydrocarbon trap, helium-specific, 21982 (Restek 

Corp., Bellefonte, PA).

• Regulator, two-stage, 400 series.

• Regulator, one-stage, 400 series.

2.1.3. GC Column and Accessories

• Capillary column cleaving tool, 23740-u (Supelco Inc., Bellefonte, PA).

• Column nut, GC inlet, 05921-21170 (Agilent Technologies, Wilmington, DE).

• Column nut, MS interface, 05988-20066 (Agilent Technologies, Wilmington, DE).

• Column union, Ultimate Union with UltiMetal Plus ferrules (Agilent Technologies, 

Wilmington, DE).

• Ferrule for installing column in GC inlet, UltiMetal Plus, 0.53 mm, G3188-27503 (Agilent 

Technologies, Wilmington, DE).

• Ferrule for installing columns in MS interface, 15% graphite: 85% Vespel, 1/16′′, 5181-3308, 

no-hole, drilled in-house (Agilent Technologies, Wilmington, DE).
9

• Guard column, deactivated fused-silica, 10 m × 0.53 mm, 160-2535-10 (Agilent 

Technologies, Wilmington, DE).

• GC column, fused-silica, J & W Scientific DB-5, 60 m × 0.25 mm, 0.25-μm film thickness, 

122-5062 (Agilent Technologies, Wilmington, DE).

• Electronic Leak Detector, 22839 (Restek Corp., Bellefonte, PA).

2.1.4. HPLC System and Accessories

• Autosampler, Waters 717 plus (Waters Corp., Milford, MA).

• Data system software, Empower (Waters Corp., Milford, MA).

• Filter, 2-μm particle size, 39-04-03524, in-line after autosampler (removed from inside 

autosampler) (Optimize Technologies Inc., Oregon City, OR).

• Fraction collector, Gilson FC 204 (Gilson Co., Middleton, WI).

• Helium for solvent degassing, grade 5.0, 99.999% (Central Welding Supply, Marysville, 

WA).

• Helium sparger for solvent reservoir, 10 μm, 25312 (Restek Corp., Bellefonte, PA).

• Regulator for helium tank, two-stage with stainless steel diaphragm.

• HPLC pump, 515 (Waters Corp., Milford, MA).

• Size-exclusion column, Envirosep ABC, 350 mm × 21.2 mm, 00W-3035-P0 (Phenomenex

Inc., Torrance, CA).

• Size-exclusion guard column, Envirosep ABC, 60 mm × 21.2 mm, 03R-3035-P0

(Phenomenex Inc., Torrance, CA).

• Solvent inlet filter for solvent reservoir, 10 μm, 59277 (Supelco Inc., Bellefonte, PA).

• UV/VIS detector, Spectra 100 (Spectra-Physics, San Jose, CA).

• Valve, six-port, 7010 (Rheodyne Inc., Cotati, CA).

2.1.5. Sample Extractor and Accessories

• Accelerated Solvent Extractor, ASE 200 (Dionex, Salt Lake City, UT).

• Extraction cells, 33 cc, 048764 (Dionex, Salt Lake City, UT).

• Nitrogen, grade 4.8 (Central Welding Supply, Marysville, WA).

2.1.6. TLC/FID and Accessories

• Thin layer chromatography/flame ionization detector, Iatroscan MK-6 (Bioscan, Washington, 

DC).

• Data system software, Empower (Waters Corp., Milford, MA).

• TLC rods, Chromarod, silica gel, type S-4 (Shell-USA, Fredericksburg, VA).

• TLC rod holder, SD-5 (Shell-USA, Fredericksburg, VA).

• TLC rod spotting guide (Shell-USA, Fredericksburg, VA).

• TLC development tank, 6′′ wide × 7.7′′ high × 1.25′′ deep (Shell-USA, Fredericksburg, VA).
10

• Filter paper, Whatman grade no. 1, 32′′ diameter cut to 5.75′′ wide × 7.35′′ high (VWR 

Scientific, West Chester, PA).

• Syringe, Hamilton, 2 μL (Fisher Scientific, Pittsburgh, PA).

• Ultra-high-purity hydrogen, 5.0 grade (Central Welding Supply, Marysville, WA).

2.2. Solvents and Reagents

• Acetone (C3H6O), HPLC-UV grade (Pharmco-APER, Brookfield, CT).

• Copper, reagent grade, granular 20–30 mesh, 1720-05 (JT Baker, Phillipsburg, NJ).

• Dichloromethane (CH2Cl2), high purity, 300-4 (Burdick & Jackson, Muskegon, MI).

• Diethyl ether, 31685 (Sigma-Aldrich, Saint Louis, MO).

• Formic acid (CH2O2), 88%, A118P-100 (Fisher Scientific, Fair Lawn, NJ).

• Hexane (C6H14), capillary GC-GC/MS grade, GC215-4 (Burdick & Jackson, Muskegon, MI).

• Hydrochloric acid (HCl), concentrated, A144-212 (Fisher Scientific, Fair Lawn, NJ).

• Isooctane (C8H18), Optima, 0301-4 (Fisher Scientific, Fair Lawn, NJ).

• Magnesium sulfate (MgSO4), anhydrous, M65-3 (Fisher Scientific, Fair Lawn, NJ).

• Methanol (CH3OH), HPLC grade, MX0475-1 (EM Science, Darmstadd, Germany).

• Sodium sulfate (Na2SO4), reagent grade, anhydrous, 10–60 mesh, S1461 (Spectrum, 

Gardena, CA).

2.3. Silica/Alumina Column Packing Material

• Alumina, 80–200 mesh, 0537-01 (JT Baker, Phillipsburg, NJ).

• Glass beads, borosilicate, 3-mm diameter, 26396-630 (VWR, Brisbane, CA).

• Glass wool, Pyrex, 3950 (Corning Inc., Corning, NY).

• Silica, 100–200 mesh, S679-212 (Fisher Scientific, Fair Lawn, NJ).

2.4. Labware

Labware that contact samples or reagents are rinsed with acetone before use.

• Balances, analytical, readable to 0.001 g, 0.0001 g, and 0.001 mg.

• Boiling chips, Teflon, AW0919120 (All-World Scientific, Monroe, WA).

• Desiccator with desiccant.

• Glass rod, 1.6-cm diameter, 45-cm length.

• Graduated cylinders, 25 mL and 100 mL, Pyrex glass.

• Microdispensers and micropipettors, variable volume, Teflon tip, disposable glass capillary 

bores.

• Muffle furnace.

• Ovens, drying, 50°C, 60°C, and 120°C.
11

• Separatory funnels with stopcocks, 500 mL.

• Spatulas, stainless steel.

• Vial heater, with a wire holder to accommodate ASE vials and an aluminum vial rack.

2.5. Disposable Items

Items are one-use only. Glass items are heated in a muffle furnace at 400°C for 18 hours,

then stored in glass containers at room temperature before use. Weighing pans are dried in an 

oven at 120°C overnight, then cooled in a desiccator for 30 minutes before use.

• ASE glass fiber filters, 047017 (Dionex, Salt Lake City, UT).

• ASE vials, 60 mL, with caps and septa (Dionex, Salt Lake City, UT).

• Chromatography column, plain, custom made, 22-mm id × 25-cm length (DJ’s Glass,

Morgan Hill, CA).

• GC vials, 1.5-mL, with caps and septa (SUN-SRI, Rockwood, TN).

• GC vials with limited volume inserts (approximately 200 μL), with caps and septa (Wheaton, 

Millville, NJ).

• HPLC vials, 4 mL, with caps, septa, and 1-mL inserts (SUN-SRI, Rockwood, TN).

• Jars, 10 ounce, 2.5-inch id mouth (Aaron Packaging, Kent, WA).

• Pipettes, transfer, Pasteur-style, 9′′ and 53⁄4′′.

• Weighing pans, aluminum, 28-mm id, 12577-081 (VWR, Brisbane, CA).

• Weighing pans, aluminum, 43-mm id, 180-5537 (All-World Scientific, Monroe, WA).

• Weighing pans, aluminum, 75-mm id, 25433-020 (VWR, Brisbane, CA).

2.6. Standard Solutions and GC Conditioning Solutions

Solutions are prepared commercially or in-house from commercial stock solutions using 

isooctane as the solvent, except the PAH/POP HPLC retention time (RT) standard and the lipid 

classes TLC/FID calibration standards, which are prepared using dichloromethane. These 

solutions are stored at −20°C. PAH and POP standards do not expire unless a problem occurs 

(e.g., evaporation). Concentrations given are approximate; actual concentrations will vary 

slightly from batch to batch of the solution. The GC CSs, SSs, and ISs are prepared such that the 

concentrations of the SSs and ISs in final sample extracts analyzed by GC/MS are approximately 

equal to the concentrations of the SSs and ISs in the GC CSs. GC conditioning solutions are 

used to simulate tissue or sediment samples during GC/MS performance verification.

2.6.1. Surrogate Standard and Internal Standard Solutions

• PAH SS (surrogate standard for quantitating PAH analytes)—contains naphthalene-d8, 

acenaphthene-d10, and benzo[a]pyrene-d12 at 1.7 ng/μL each compound.

• PAH HPLC IS (HPLC internal standard for quantitating the PAH SS)—contains 

phenanthrene-d10 at 1.6 ng/μL.
12

• PAH GC IS (GC internal standard for quantitating the PAH HPLC IS)—contains 

hexamethylbenzene (HMB) at 10 ng/μL.

• POP SS (surrogate standard for quantitating POP analytes)—contains PCB 103 and 

dibromooctafluorobiphenyl (DOB) at 1 ng/μL.

• POP HPLC IS (HPLC internal standard for quantitating the POP SS)—contains tetrachlorom-xylene (TCMX) at 1 ng/μL.

• POP GC IS (GC internal standard for quantitating the POP HPLC IS)—contains tetrachloroo-xylene (TCOX) at 1 ng/μL.

2.6.2. Calibration Standards

• PAH/POP HPLC RT standard (retention time standard for daily HPLC calibration)—

contains DOB and perylene (PER) at 0.075 ng/μL each compound in dichloromethane.

• PAH GC/MS calibration standards—contain the PAHs in Table 1, except 9-

methylphenanthrene, retene, triphenylene, benzo[j]fluoranthene and dibenz[a,c]anthracene,

at the following concentrations in isooctane: Level 1 at 0.001 ng/μL each compound, Level 2 

at 0.003 ng/μL each compound, Level 3 at 0.01 ng/μL each compound, Level 4 at 0.03 ng/μL 

each compound, Level 5 at 0.1 ng/μL each compound, Level 6 at 0.3 ng/μL each compound, 

Level 7 at 1 ng/μL each compound, Level 8 at 3 ng/μL each compound, and Level 9 at 10 

ng/μL each compound. All levels contain naphthalene-d8, acenaphthene-d10, 

benzo[a]pyrene-d12, and phenanthrene-d10 at 0.5 ng/μL each compound, and HMB at 3 

ng/μL in all levels.

• PAH plus alkanes GC/MS calibration standards—contain the PAHs in Table 1, except 

9-methylphenanthrene, retene, triphenylene, benzo[j]fluoranthene and dibenz[a,c]anthracene,

plus contain the alkanes listed in Section 1 (pristane, phytane, n-heptadecane, and 

n-octadecane) at the following concentrations in isooctane: Level 1 at 0.003 ng/μL each 

compound, Level 2 at 0.01 ng/μL each compound, Level 3 at 0.03 ng/μL each compound,

Level 4 at 0.1 ng/μL each compound, Level 5 at 0.3 ng/μL each compound, and Level 6 at 

1 ng/μL each compound. All levels contain naphthalene-d8, acenaphthene-d10, 

benzo[a]pyrene-d12, and phenanthrene-d10 at 0.5 ng/μL each compound, and HMB at 

3 ng/μL in all levels.

• POP GC/MS calibration standards—contain the POPs in Table 3, except nonachlor III, 

PBDE 85, and as noted for coeluting PCBs, at the following concentrations in isooctane: 

Level 1 at 0.0003 ng/μL each compound, Level 2 at 0.001 ng/μL each compound, Level 3 at 

0.003 ng/μL each compound, Level 4 at 0.01 ng/μL each compound, Level 5 at 0.03 ng/μL 

each compound, Level 6 at 0.1 ng/μL each compound, Level 7 at 0.3 ng/μL each compound, 

Level 8 at 1 ng/μL each compound, Level 9 at 3 ng/μL each compound, Level 10 at 10 ng/μL 

each trans-nonachlor and 2,4'-DDE only, and Level 11 at 50 ng/μL each trans-nonachlor and 

2,4'-DDE only. All levels contain PCB 103, DOB, TCMX, and TCOX at 0.3 ng/μL each 

compound.

• Lipid classes TLC/FID calibration standards—contain lauryl stearate, triolein, oleic acid, 

cholesterol and L-α-phosphatidylcholine at the following concentrations in dichloromethane: 

Level 1 at 6.67 mg/mL each lauryl stearate, triolein, and oleic acid and 1.33 mg/mL each 

cholesterol and L-α-phosphatidylcholine; Level 2 at 5.0 mg/mL each lauryl stearate, triolein,

and oleic acid and 1.0 mg/mL each cholesterol and L-α-phosphatidylcholine; Level 3 at 3.75 
13

mg/mL each lauryl stearate, triolein, and oleic acid and 0.75 mg/mL each cholesterol and 

L-α-phosphatidylcholine; Level 4 at 2.5 mg/mL each lauryl stearate, triolein, and oleic acid 

and 0.50 mg/mL each cholesterol and L-α-phosphatidylcholine; Level 5 at 1.25 mg/mL each 

lauryl stearate, triolein, and oleic acid and 0.25 mg/mL each cholesterol and L-αphosphatidylcholine; Level 6 at 0.63 mg/mL each lauryl stearate, triolein, and oleic acid and 

0.13 mg/mL each cholesterol and L-α-phosphatidylcholine. Calibration standards are 

prepared from primary standards every 4-6 months to ensure that the standards have not 

decomposed or oxidized. Primary standards are prepared every 1–1.5 years at the following 

concentrations in dichloromethane: 10 mg/mL each lauryl stearate, triolein, and oleic acid 

and 2 mg/mL each cholesterol and L-α-phosphatidylcholine.

2.6.3. Spike Solutions

• POP spike solution—contains the POPs in Table 3, except nonachlor III, PBDE 85, and as 

noted otherwise for coeluting PCBs, at 0.3 ng/μL each compound in isooctane.

• PAH spike solution—contains the PAHs in Table 1, except 9-methylphenanthrene, retene, 

triphenylene, and dibenz[a,c]anthracene, at 0.2 ng/μL each compound in isooctane.

• Alkane spike solution—contains the alkanes listed in Section 1 (pristane, phytane,

n-heptadecane, and n-octadecane) at 1 ng/μL each compound in isooctane.

2.6.4. GC Conditioning Solutions

• Sediment GC Conditioning solution—a composite of moderately contaminated sediment 

extracts that have been prepared for GC/MS analysis, as described in Section 3 and Section 

4, in isooctane.

• Low-lipid Tissue GC Conditioning solution—a composite of moderately contaminated, lowlipid tissue extracts that have been prepared for GC/MS analysis, as described in Section 3 

and Section 4, in isooctane.

• High-lipid Tissue GC Conditioning solution—a composite of moderately contaminated, 

high-lipid tissue extracts that have been prepared for GC/MS analysis, as described in 

Section 3 and Section 4, in isooctane.

2.7. Standard Reference Materials

SRMs are purchased from NIST, Gaithersburg, MD.

• SRM 1974c Organics in Mussel Tissue (Mytilus edulis)—analyzed for QA measurements in 

batches of low-lipid tissue samples.

• SRM 1941b Organics in Marine Sediment—analyzed for QA measurements in batches of 

sediment samples.

• SRM 1945 Organics in Whale Blubber—analyzed for QA measurements in batches of highlipid tissue samples.

• SRM 1947 Lake Michigan Fish Tissue—analyzed for QA measurements in batches of fish 

tissue samples.
14

• SRM 1958 Organic Contaminants in Fortified Human Serum—analyzed for QA

measurements in batches of marine mammal blood/serum/plasma samples.

2.8. Preparation of Silica/Alumina Column Packing Material 

and Other Reagents

2.8.1. Silica

The silica is heated at 700°C for 18 hours then stored at 120°C. It is allowed to cool to 

room temperature in a desiccator just prior to use.

2.8.2. Alumina

The alumina is heated at 120°C for 2 hours then allowed to cool to room temperature in a 

desiccator just prior to use.

2.8.3. Glass Wool and Glass Beads

The glass wool and glass beads are prepared for use (separately) by heating them at 

400°C for 18 hours. They are stored in respective covered glass containers at room temperature 

until use.

2.8.4. Copper

Less than 1 hour before use, the copper is activated in a fume hood by placing it in a glass 

beaker, covering it with concentrated hydrochloric acid, stirring it with a glass rod, and allowing 

it to stand for 5 minutes. The copper is then washed three times with water, followed by three 

times with methanol then three times with dichloromethane. The copper is stored covered with 

dichloromethane until use to avoid contact with air.

2.8.5. Sodium Sulfate

The sodium sulfate is heated at 700°C for 18 hours then stored at 120°C. It is allowed to 

cool to room temperature in a desiccator just prior to use.

2.8.6. Magnesium Sulfate

The magnesium sulfate is heated at 400°C for 18 hours then stored at 120°C. It is 

allowed to cool to room temperature in a desiccator just prior to use.

2.9. Purity Testing

New brands of reagents are tested for purity before their initial use in sample analyses. 

Triplicate samples of the new brand of reagent are analyzed along with triplicate samples of the 

brand of reagent currently in use, for comparison. For solid reagents, triplicate solvent blanks 

are also analyzed to demonstrate whether any impurities found are from the reagent being tested 

or are from the solvent or laboratory. New glassware is used during purity testing. All 
15

glassware and equipment contacting the sample are heated in a muffle furnace or rinsed with 

acetone before use.

2.9.1. Isooctane Purity Test

PAH GC IS (30 μL), POP GC IS (30 μL), and isooctane (100 μL) are added to a labeled 

GC vial with a limited volume insert, and the sample is mixed thoroughly.

The sample and GC/MS calibration standards are analyzed for PAHs and POPs using 

GC/MS selected-ion monitoring (SIM) as described in Section 5 for low-lipid tissues. The 

GC/MS responses of any impurities that are detected at the same retention times as analytes 

should be smaller than the corresponding analyte responses in the lowest level of GC/MS 

calibration standard.

2.9.2. Dichloromethane Purity Test

Dichloromethane (60 mL) is added to an ASE vial. Five boiling chips are added to the 

ASE vial containing the sample, and the sample volume is reduced to approximately 2 mL using 

a vial heater.

PAH GC IS (30 μL), POP GC IS (30 μL), and isooctane (150 μL) are added to the 

sample, and the sample is mixed thoroughly.

The sample is transferred to a labeled 4-mL HPLC vial, a boiling chip is added, and the 

sample volume is reduced to 100 μL using the vial heater. All dichloromethane should be 

evaporated from the sample, indicated by the remaining isooctane ceasing to boil.

The sample is transferred to a labeled GC vial with a limited volume insert.

The sample and GC/MS calibration standards are analyzed for PAHs and POPs using

GC/MS SIM, as described in Section 5 for low-lipid tissues. The GC/MS responses of any 

impurities that are detected at the same retention times as analytes should be smaller than the 

corresponding analyte responses in the lowest level of GC/MS calibration standard.

2.9.3. Sodium Sulfate Purity Test

Two glass-fiber filters are placed at the bottom of an ASE cell. Sodium sulfate (20 cc) is 

added to the labeled ASE cell. One glass-fiber filter is placed on top of the sodium sulfate in the 

cell.

The sample is extracted using the ASE as described in Section 3.

Five boiling chips are added to the ASE vial containing the sample, and the sample 

volume is reduced to approximately 2 mL using a vial heater.

PAH GC IS (30 μL), POP GC IS (30 μL), and isooctane (150 μL) are added to the 

sample, and the sample is mixed thoroughly.
16

The sample is transferred to a labeled 4-mL HPLC vial, a boiling chip is added, and the 

sample volume is reduced to 100 μL using the vial heater. All dichloromethane should be 

evaporated from the sample, indicated by the remaining isooctane ceasing to boil.

The sample is transferred to a labeled GC vial with a limited volume insert.

The sample and GC/MS calibration standards are analyzed for PAHs and POPs using 

GC/MS SIM, as described in Section 5 for low-lipid tissues. The GC/MS responses of any 

impurities that are detected at the same retention times as analytes should be smaller than the 

corresponding analyte responses in the lowest level of GC/MS calibration standard.

2.9.4. Magnesium Sulfate Purity Test

The Magnesium Sulfate Purity Test is performed following the same procedure as for the 

Sodium Sulfate Purity Test in Subsection 2.9.3, except that magnesium sulfate (15 cc) is added 

to the labeled ASE cell instead of sodium sulfate.

2.9.5. Silica or Alumina Purity Test

A silica/alumina column is prepared by adding a 10- to 15-mm plug of glass wool to a 

chromatography column and tamping it with a glass rod, then adding alumina (10 cc), followed 

by silica (20 cc) and glass beads (5 cc).

A solution of methanol (10%) in dichloromethane (35 mL total) is slowly added to the 

column and allowed to drain into a waste container. Then to flush the methanol from the 

column, dichloromethane (35 mL) is slowly added to the column and allowed to drain into a 

waste container. Next the tip of the column is rinsed with dichloromethane.

A labeled ASE vial is placed under the column.

Dichloromethane (55 mL) is added to the column and allowed to drain into the vial.

Five boiling chips are added to the ASE vial containing the sample, and the sample 

volume is reduced to approximately 2 mL using a vial heater.

PAH GC IS (30 μL), POP GC IS (30 μL), and isooctane (150 μL) are added to the 

sample, and the sample is mixed thoroughly.

The sample is transferred to a labeled 4-mL HPLC vial, a boiling chip is added, and the 

sample volume is reduced to 100 μL using the vial heater. All dichloromethane should be 

evaporated from the sample, indicated by the remaining isooctane ceasing to boil.

The sample is transferred to a labeled GC vial with a limited volume insert.

The sample and GC/MS calibration standards are analyzed for PAHs and POPs using 

GC/MS SIM, as described in Section 5 for low-lipid tissues. The GC/MS responses of any 

impurities that are detected at the same retention times as analytes should be smaller than the 

corresponding analyte responses in the lowest level of GC/MS calibration standard.
17

3. Sample Extraction

3.1. Accelerated Solvent Extraction of Sediment and Tissue Samples

Sediments or tissues (e.g., muscle, liver, blubber, plasma, gastroenteric contents) are 

customarily analyzed in batches of 12 to 14 field samples. In addition to the sediment or tissue 

samples in each batch, there are typically one method blank sample, one sample of an 

appropriate SRM, and as needed, replicates of a field sample. Standard Check Solutions are 

prepared as described below for each class of analytes. If either POPs or PAHs are not to be 

quantitated in the samples, all the internal standards in the samples and the Standard Check 

Solution for that class of analytes in the batch are omitted.

The typical amount of sediment or low-lipid tissue analyzed is ≤2 g to avoid overloading 

the ASE with water. The amount of high-lipid tissue analyzed is ≤0.5 g to avoid overloading the 

size-exclusion HPLC with lipids.

3.1.1. Sample Preparation

A 10-ounce jar, an ASE cell, and an ASE collection vial are labeled with the sample 

number.

For sediment samples, standing water is decanted from the original sample container, and 

the sample is stirred to homogenize it. All pebbles, shells, biota, and other extraneous material 

are discarded.

For tissue samples, the sample is homogenized in a glass jar (the original sample 

container is used if appropriate).

For a batch of tissue samples requiring Total Extractables Determination (Macro or 

Micro, Subsection 8.2) or Lipid Classes (Section 9) or both, the ASE collection vial is weighed 

to the nearest 0.001 g, and the weight is recorded as the “ASE Vial Weight.”

The sample is transferred to the labeled 10-ounce jar and weighed to the nearest 0.001 g. 

The weight is recorded as the “Sample Weight.”

Sodium sulfate (15 cc) is added to the sample in the jar and mixed thoroughly with the 

sample to absorb the water in the sample, which allows greater extraction efficiency of the 

organic compounds. To avoid clumping and hardening of the sodium sulfate, the sample is 

mixed immediately after adding the sodium sulfate to the jar. The sample is mixed until it 

appears dry to avoid generating excessive heat in the next step, which could volatilize analytes 

such as the naphthalenes.
18

For increased absorption of water from the sample, magnesium sulfate (15 cc) is then 

added to the sample in the jar and mixed thoroughly with the sample.

Two glass-fiber filters are placed at the bottom of the labeled ASE cell.

The mixture of sample and drying agent is transferred to the ASE cell using the ASE 

funnel. The bottom of the cell is tapped firmly but carefully on the countertop to completely 

settle the cell contents.

The remaining cell volume is filled with sodium sulfate. Again, the bottom of the cell is 

tapped firmly but carefully on the countertop to completely settle the cell contents, leaving 

approximately 3 mm of void volume at the top of the cell.

One glass-fiber filter is placed on top of the sodium sulfate in the cell and pressed in 

without overlapping the rim of the cell.

3.1.2. Surrogate Standards and Standard Check Solutions

The volumes of standards used depends on the amount of extract injected on GC/MS, 

which depends on the sample type and, in the case of tissues, the method of gravimetrically 

determining the percent mass of nonvolatile extractable material (Total Extractables 

Determination). Macro Total Extractables Determination uses one-third of the extract, whereas 

Micro Total Extractables Determination uses 1.5 mL of the extract in order to reduce the lower 

limit of quantitation (LOQ) for PAHs and POPs.

3.1.2.1. Batches of tissue samples requiring Macro Total Extractables Determination

PAH SS (150 μL) and POP SS (150 μL) are added onto the top filter in the cell, and then

dichloromethane (approximately 1 mL) is added onto the top filter in the cell to rinse the internal 

standards into the cell.

The cell threads are cleared of sulfates, if present, and the cell is capped firmly but not 

forcefully.

A PAH Standard Check Solution is prepared by adding isooctane (50 μL) and PAH SS 

(150 μL) to a labeled GC vial.

A POP Standard Check Solution is prepared by adding isooctane (50 μL) and POP SS 

(150 μL) to a labeled GC vial.

3.1.2.2. Batches of sediment samples or tissue samples (except plasma) not requiring 

Macro Total Extractables Determination

PAH SS (75 μL) and POP SS (75 μL) are added onto the top filter in the cell, and then 

dichloromethane (approximately 1 mL) is added onto the top filter in the cell to rinse the internal 

standards into the cell.
19

The cell threads are cleared of sulfates, if present, and the cell is capped firmly but not 

forcefully.

A PAH Standard Check Solution is prepared by adding isooctane (25 μL) and PAH SS 

(75 μL) to a labeled GC vial.

A POP Standard Check Solution is prepared by adding isooctane (25 μL) and POP SS 

(75 μL) to a labeled GC vial.

3.1.2.3. Batches of plasma samples

PAH SS (40 μL) and POP SS (40 μL) are added onto the top filter in the cell, and then 

dichloromethane (approximately 1 mL) is added onto the top filter in the cell to rinse the internal 

standards into the cell.

The cell threads are cleared of sulfates, if present, and the cell is capped firmly but not 

forcefully.

A PAH Standard Check Solution is prepared by adding isooctane (25 μL) and PAH SS 

(40 μL) to a labeled GC vial.

A POP Standard Check Solution is prepared by adding isooctane (25 μL) and POP SS 

(40 μL) to a labeled GC vial.

3.1.3. ASE Operation

The ASE cell and the ASE collection vial are loaded in the corresponding positions in the 

ASE carousels. Collection vials for system rinses are loaded in the rinse vial positions.

The ASE system-rinse function is activated three times to flush the solvent lines.

The sample is extracted using an ASE schedule with the Rinse parameter set to [ON], and 

using a method with the parameter values set as follows.

PREHEAT [0] min PRESSURE [2,000] psi

HEAT [5] min TEMPERATURE [100]°C

STATIC [5] min SOL A [MeCl2] [100]%

FLUSH % [115] vol SOL B [Other] [0]%

PURGE [180] sec SOL C [Other] [0]%

CYCLES [2] SOL D [Other] [0]%

After the extraction is completed, the ASE cell and the ASE collection vial are removed 

from the ASE carousel. The contents of the ASE cell are discarded appropriately.
20

3.1.4. Preparation of Extracts

3.1.4.1. Batches of tissue samples requiring Macro Total Extractables Determination

PAH HPLC IS (150 μL) and POP HPLC IS (150 μL) are added to the ASE collection 

vial, and the contents of the vial are mixed thoroughly.

PAH HPLC IS (150 μL) is added to the PAH Standard Check Solution, and the solution 

is mixed thoroughly.

POP HPLC IS (150 μL) is added to the POP Standard Check Solution, and the solution is 

mixed thoroughly.

The ASE collection vial is weighed to the nearest 0.001 g, and the weight is recorded as 

the “ASE Vial w/Extract Weight.”

The sample number is etched on the tab of a 75-mm id aluminum weighing pan.

The weighing pan is weighed to the nearest 0.001 g, and the weight is recorded as the 

“Pan Weight.”

Approximately one-third of the sample in the ASE collection vial is transferred to the 

pan. The ASE vial is capped immediately afterward.

The ASE collection vial is weighed to the nearest 0.001 g, and the weight is recorded as 

the “ASE Vial w/out TE Extract Weight.”

The pan containing one-third of the sample proceeds to Total Extractables Determination 

as described in Subsection 8.2.

The sample in the ASE collection vial proceeds to Sample Cleanup by Silica/Alumina 

Column Chromatography as described in Section 4.

3.1.4.2. Batches of tissue (except plasma) samples requiring Micro Total Extractables 

Determination

PAH HPLC IS (75 μL) and POP HPLC IS (75 μL) are added to the ASE collection vial, 

and the contents of the vial are mixed thoroughly.

PAH HPLC IS (75 μL) is added to the PAH Standard Check Solution, and the solution is 

mixed thoroughly.

POP HPLC IS (75 μL) is added to the POP Standard Check Solution, and the solution is 

mixed thoroughly.

The ASE collection vial is weighed to the nearest 0.001 g, and the weight is recorded as 

the “ASE Vial w/Extract Weight.”
21

A 1.5-mL GC vial with label and cap is weighed to the nearest 0.0001 g, and the weight 

is recorded in the logbook as the “GC Vial Weight.”

Approximately 1.5 ml of the sample in the ASE vial is transferred to the 1.5-mL vial

using a Pasteur pipet. The ASE vial and the 1.5-mL vial are capped immediately afterward.

The 1.5-mL GC vial with the 1.5 mL of sample is weighed to the nearest 0.0001 g, and 

the weight is recorded as the “GC Vial w/TE Aliquot Weight” in the logbook.

The sample number is marked on a 28-mm id aluminum weighing pan.

The weighing pan is weighed to the nearest 0.001 mg using a 6-place balance, and the 

weight is recorded as the “Pan Weight.”

The 1.5 mL aliquot of sample in the GC vial is poured into the weighing pan. The GC 

vial is rinsed with 0.2 mL of dichloromethane, and then this dichloromethane is added to the pan. 

This GC vial rinsing is repeated twice, with the dichloromethane added to the pan.

The pan containing 1.5 mL of the sample plus rinses proceeds to Total Extractables 

Determination as described in Subsection 8.2.

The sample in the ASE collection vial proceeds to Sample Cleanup by Silica/Alumina 

Column Chromatography as described in Section 4.

3.1.4.3. Batches of plasma samples requiring Micro Total Extractables Determination

PAH HPLC IS (40 μL) and POP HPLC IS (40 μL) are added to the ASE collection vial,

and the contents of the vial are mixed thoroughly.

PAH HPLC IS (40 μL) is added to the PAH Standard Check Solution, and the solution is 

mixed thoroughly.

POP HPLC IS (40 μL) is added to the POP Standard Check Solution, and the solution is 

mixed thoroughly.

The ASE collection vial is weighed to the nearest 0.001 g, and the weight is recorded as 

the “ASE Vial w/Extract Weight.”

A 1.5-mL GC vial with label and cap is weighed to the nearest 0.0001 g, and the weight 

is recorded in the logbook as the “GC Vial Weight.”

Approximately 1.5 ml of the sample in the ASE vial is transferred to the 1.5-mL vial 

using a Pasteur pipet. The ASE vial and the 1.5-mL vial are capped immediately afterward.

The 1.5-mL GC vial with the 1.5 mL of sample is weighed to the nearest 0.0001 g, and 

the weight is recorded as the “GC Vial w/TE Aliquot Weight” in the logbook.

The sample number is marked on a 28-mm id aluminum weighing pan.
22

The weighing pan is weighed to the nearest 0.001 mg using a 6-place balance, and the 

weight is recorded as the “Pan Weight.”

The 1.5 mL aliquot of sample in the GC vial is poured into the weighing pan. The GC 

vial is rinsed with 0.2 mL of dichloromethane, and then this dichloromethane is added to the pan. 

This GC vial rinsing is repeated twice, with the dichloromethane added to the pan.

The pan containing 1.5 mL of the sample plus rinses proceeds to Total Extractables 

Determination as described in Subsection 8.2.

The sample in the ASE collection vial proceeds to Sample Cleanup by Silica/Alumina 

Column Chromatography as described in Section 4.

3.1.4.4. Batches of sediment samples or tissue (except plasma) samples not requiring 

Macro or Micro Total Extractables Determination

PAH HPLC IS (75 μL) and POP HPLC IS (75 μL) are added to the ASE collection vial, 

and the contents of the vial are mixed thoroughly.

PAH HPLC IS (75 μL) is added to the PAH Standard Check Solution, and the solution is 

mixed thoroughly.

POP HPLC IS (75 μL) is added to the POP Standard Check Solution, and the solution is 

mixed thoroughly.

The sample in the ASE collection vial proceeds to Sample Cleanup by Silica/Alumina 

Column Chromatography as described in Section 4.

3.1.4.5. Batches of plasma samples not requiring Macro or Micro Total Extractables 

Determination

PAH HPLC IS (40 μL) and POP HPLC IS (40 μL) are added to the ASE collection vial,

and the contents of the vial are mixed thoroughly.

PAH HPLC IS (40 μL) is added to the PAH Standard Check Solution, and the solution is 

mixed thoroughly.

POP HPLC IS (40 μL) is added to the POP Standard Check Solution, and the solution is 

mixed thoroughly.

The sample in the ASE collection vial proceeds to Sample Cleanup by Silica/Alumina 

Column Chromatography as described in Section 4.

3.1.4.6. Batches of tissue samples requiring Lipid Classes Determination

Lipid Class Determination may be done in addition to Macro or Micro Total Extractables 

Determination after the corresponding steps in Subsection 3.1.4.1, Subsection 3.1.4.2, or 

Subsection 3.1.4.3.
23 

The ASE collection vial is weighed to the nearest 0.001 g, and the weight is recorded as 

the “ASE Vial w/Extract Weight,” unless this has been done previously for Macro or Micro 

Total Extractables Determination.

A 1.5-mL GC vial with label and cap is weighed to the nearest 0.001 g, and the weight is 

recorded in the logbook as the “Iatro Vial Weight.”

Approximately 1 ml of the sample in the ASE vial is transferred to the 1.5-mL vial using 

a micropippetor. The ASE vial and the 1.5-mL vial are capped immediately afterward.

The 1.5-mL GC vial with the 1 mL of sample is weighed to the nearest 0.001 g, and the 

weight is recorded as the “Iatro Vial w/Extract Weight” in the logbook. 

The sample in the 1.5-mL vial proceeds to Lipid Classes determination as described in 

Section 9. 

The sample in the ASE collection vial proceeds to Sample Cleanup by Silica/Alumina 

Column Chromatography as described in Section 4. 

3.2. Liquid-liquid Extraction of Water Samples

Water samples are customarily analyzed in batches of 10 samples. In addition to the 

water samples in each batch, there are typically one method blank sample and one spiked blank 

sample (using project-specific control water for both), one laboratory solvent blank sample (no 

water), and as needed, one or more replicate water samples. Standard Check Solutions are 

prepared as described below for each class of analytes. If either POPs or PAHs are not to be 

quantitated in the samples, all the internal standards and the Standard Check Solution for that 

class of analytes are omitted. Alkanes are quantitated concurrently with PAHs and therefore 

require the PAH internal standards and the PAH Standard Check Solution. 

Typically, the amount of water analyzed is 200 mL, depending on the expected level of 

contamination. Water samples are contained in 250-mL amber bottles. Dichloromethane is 

added (10% of volume of water, e.g., 20 mL of dichloromethane for 200 mL of water) as soon as 

possible after collection to inhibit biological activity.

PAH SS (200 μL) and POP SS (200 μL) are added to each water sample in its bottle. 

For the spiked water sample, PAH Spike (200 μL) and POP Spike (200 μL) are also 

added to the sample in its bottle. If the alkanes are to be quantitated, Alkane Spike (200 μL) is 

also added. 

The laboratory solvent blank is prepared by adding dichloromethane (40 mL), PAH SS 

(200 μL), and POP SS (200 μL) to a labeled ASE vial. This sample is set aside until the other 

samples have been extracted as described below. 

A PAH Standard Check Solution is prepared by adding PAH SS (200 μL) and PAH 

Spike (200 μL) to a labeled GC vial. 
24

A POP Standard Check Solution is prepared by adding POP SS (200 μL) and POP Spike 

(200 μL) to a labeled GC vial.

If the alkanes are to be quantitated, an Alkane Standard Check Solution is prepared by 

adding PAH SS (200 μL) and Alkane Spike (200 μL) to a labeled GC vial.

The water sample bottle is inverted several times to mix the sample, and then (with the 

bottle right-side up) the cap is slowly opened to vent the sample. The sample is poured into a 

separatory funnel (stopcock closed) on a ring stand, and the funnel is capped.

Dichloromethane (20 mL) is added to the sample bottle, the bottle is capped, and the 

dichloromethane is swirled in the bottle to rinse the bottle. The bottle is set aside until after the 

first extraction of the sample as described below.

While keeping the cap securely on the separatory funnel, the funnel is shaken a few times 

to mix the contents. With the funnel upside-down, the stopcock is slowly opened to vent the 

sample, and the stopcock is closed. This shaking and venting of the sample is repeated for 2

minutes to thoroughly mix and extract the sample.

The separatory funnel is set on a ring stand, and the water and dichloromethane phases

are allowed to separate.

After the phases have separated in the separatory funnel, the lower (dichloromethane) 

phase is drained into a labeled ASE vial, leaving behind any emulsion.

The dichloromethane in the sample bottle is added to the water in the separatory funnel.

The extraction and phase separation are repeated as above, and the lower 

(dichloromethane) phase is drained into the labeled ASE vial, leaving behind any emulsion.

The water remaining in the separatory funnel is disposed of appropriately.

The laboratory solvent blank sample is treated the same as all other samples from this 

point on.

Sodium sulfate (15 cc) is added to the sample in the ASE vial to absorb any water 

present, the vial is capped, and the sample is mixed thoroughly. The sample is allowed to sit 

overnight.

The extract in the ASE vial is decanted slowly into a second labeled ASE vial, leaving 

the sodium sulfate in the first vial. Any extract remaining in the sodium sulfate is transferred to 

the vial using a pipette.

Dichloromethane (approximately 3 mL) is added to the first ASE vial (that had contained 

the extract) and swirled in the vial to rinse the vial, and then it is slowly decanted into the second 

ASE vial, leaving the sodium sulfate in the first vial. Any solvent remaining in the sodium 

sulfate is transferred to the second vial using a pipette.
25

The rinsing of the first ASE vial and the decanting into the second ASE vial are repeated 

as above.

PAH HPLC IS (200 μL) and POP HPLC IS (200 μL) are added to the second ASE 

collection vial, and the contents of the vial are mixed thoroughly.

PAH HPLC IS (200 μL) is added to the PAH Standard Check Solution and the Alkane 

Standard Check Solution, and each solution is mixed thoroughly.

POP HPLC IS (200 μL) is added to the POP Standard Check Solution, and the solution is 

mixed thoroughly.

The sample in the second ASE collection vial proceeds to Sample Cleanup by 

Silica/Alumina Column Chromatography as described in Section 4.
26

4. Sample Cleanup

4.1. Silica/Alumina Column Chromatography

A gravity-flow silica/alumina column is used for removing extraneous polar compounds 

from the sample.

4.1.1. Calibration of Silica/Alumina Column

Calibration is performed once per lot of silica and once per lot of alumina before their use 

in sample analyses.

A spiked blank is prepared by adding dichloromethane (5 mL), POP SS (60 μL), PAH SS 

(60 μL), POP Spike (80 μL), and PAH Spike (80 μL) to a labeled ASE vial.

A POP Standard Check Solution is prepared by adding POP SS (60 μL) and POP Spike 

(80 μL) to a labeled GC vial.

A PAH Standard Check Solution is prepared by adding PAH SS (60 μL) and PAH spike 

(80 μL) to a labeled GC vial.

A silica/alumina column is prepared by adding a 10- to 15-mm plug of glass wool to a 

chromatography column and tamping it down with a glass rod, then adding alumina (10 cc), 

followed by silica (20 cc) and glass beads (5 cc).

The column packing is slightly deactivated for better PAH recovery by slowly adding a 

solution of methanol (10% by volume) in dichloromethane (35 mL total solution) to the column 

and allowing the solvent to drain into a waste container. Then to flush the methanol from the 

column, dichloromethane (35 mL) is slowly added to the column and allowed to drain into a 

waste container. The tip of the column is then rinsed with dichloromethane.

A second ASE vial labeled “F1” is placed under the column. The spiked blank is slowly 

decanted into the column, and the eluant is collected in the second ASE vial.

The first ASE vial, containing the spiked blank, is washed with dichloromethane 

(approximately 5 mL), the washings are slowly decanted into the column, and the eluant is 

collected in the second ASE vial. This step is repeated twice more.

Dichloromethane (35 mL) is added to the column, and the eluant is collected in the 

second ASE vial. The ASE vial containing the F1 fraction is then set aside.

A third ASE vial labeled “F2” is placed under the column. Dichloromethane (10 mL) is 

added to the column, and the eluant is collected in the third ASE vial.
27

Five boiling chip are added to each of the ASE vials containing the F1 and F2 fractions, 

and each fraction is concentrated to approximately 2 mL using a vial heater.

PAH GC IS (60 μL), POP GC IS (60 μL), and isooctane (200 μL) are added to each 

fraction, and each fraction is mixed thoroughly.

POP GC IS (60 μL) is added to the POP Standard Check Solution, and the solution is 

mixed thoroughly.

PAH GC IS (60 μL) is added to the PAH Standard Check Solution, and the solution is 

mixed thoroughly.

Each fraction is transferred to a labeled 4-mL HPLC vial, a boiling chip is added, and the 

fraction is concentrated to 200 μL using the vial heater. All dichloromethane should be 

evaporated from the sample, indicated by the remaining isooctane ceasing to boil.

Each fraction is transferred to a labeled GC vial with a limited volume insert.

Each fraction and Standard Check Solution is analyzed for PAHs and POPs using 

GC/MS SIM as described in Section 5 to compare the recoveries of the analytes and internal 

standards in the F1 and F2 fractions to those in the Standard Check Solutions. The analytes and 

internal standards should completely elute in F1 and none in F2. If any analytes elute in F2, then 

the volume of dichloromethane for eluting the PAH/POP fraction in Subsection 4.1.2 must be 

increased from 35 mL to 45 mL.

4.1.2. Sample Cleanup by Silica/Alumina Column Chromatography

Five boiling chips are added to the ASE collection vial containing the sample extract 

from Section 3, referred to as the first vial in this subsection, and the extract is concentrated to 

5 mL using a vial heater.

A silica/alumina column is prepared by adding a 10- to 15-mm plug of glass wool to a 

chromatography column and tamping it down with a glass rod, then adding alumina (10 cc), 

followed by silica (20 cc) and glass beads (5 cc).

The column packing is slightly deactivated for better PAH recovery by slowly adding a

solution of methanol (10% by volume) in dichloromethane (35 mL total solution) to the column 

and allowing the solvent to drain into a waste container. Then to flush the methanol from the 

column, dichloromethane (35 mL) is slowly added to the column and allowed to drain into a 

waste container. The tip of the column is then rinsed with dichloromethane.

A second labeled ASE vial is placed under the column. The sample extract in the first 

ASE vial is slowly decanted into the column, and the eluant is collected in the second ASE vial.

The inside of the first vial is washed with dichloromethane (approximately 5 mL), the 

washings are decanted into the column, and the eluant is collected in the second ASE vial. This 

step is repeated twice more.
28

Dichloromethane (35 mL) is added to the column, and the eluant is collected in the 

second ASE vial.

Five boiling chips are added to the ASE vial containing the sample eluted from the 

silica/alumina column, and the sample volume is reduced to approximately 2 mL using a vial 

heater.

The sample is transferred to a labeled 4-mL HPLC vial, a boiling chip is added, and the 

sample volume is reduced to approximately 1 mL using the vial heater.

For a batch of sediment samples, activated copper is added to the HPLC vial a few grains 

at a time until no further discoloring of the copper occurs. The HPLC vial is capped and stored 

overnight.

4.1.3. Preparation of Concentrated Extracts

4.1.3.1. Batches of sediment or tissue (except plasma) samples or water samples not 

analyzed for alkanes

The sample is transferred to an HPLC vial with a 1-mL insert (leaving the copper in the 

4-mL HPLC vial in the case of sediment samples), and the volume brought to 1 mL either by 

adding dichloromethane or by evaporating the excess under a gentle stream of nitrogen gas.

The sample in the vial proceeds to HPLC as described in Subsection 4.2.

4.1.3.2. Batches of plasma samples

The sample is transferred to a GC vial and the volume brought to 0.35 mL under a gentle 

stream of nitrogen gas.

The sample is transferred to an HPLC vial with a 0.35-mL insert.

The sample in the vial proceeds to HPLC as described in Subsection 4.2, injecting 

350 μL of sample (the entire sample).

4.1.3.3. Batches of water samples analyzed for alkanes

Approximately one-half of the sample is transferred to a GC vial, capped, and set aside in 

case additional cleanup by size-exclusion HPLC is needed for PAH or POP analyses (prior to 

size-exclusion HPLC, this aliquot is prepared as is done for plasma samples in Subsection 

4.1.3.2). Note that the alkanes cannot be measured in samples cleaned up by size-exclusion 

HPLC.

PAH GC IS (100 μL), POP GC IS (100 μL), and isooctane (100 μL) are added to the 

remaining sample in the 4-mL vial, and the sample is mixed thoroughly.

PAH GC IS (200 μL) is added to the PAH Standard Check Solution and to the Alkane 

Standard Check Solution, and each solution is mixed thoroughly.
29

POP GC IS (200 μL) is added to the POP Standard Check Solution, and the solution is 

mixed thoroughly.

A boiling chip is added to the remaining sample in the 4-mL vial, and the sample volume 

is reduced to 100 μL using the vial heater. All dichloromethane should be evaporated from the 

sample, indicated by the remaining isooctane ceasing to boil. Samples known to have high 

concentrations of analytes may need to be diluted with additional isooctane.

The sample is transferred to a labeled GC vial with a limited volume insert.

The sample in the GC vial insert and the PAH Standard Check Solution are analyzed by 

GC/MS for PAHs as described in Section 5. Samples and an Alkane Standard Check Solution 

may be analyzed for alkanes concurrently with PAHs, as described in Subsection 5.2.2.

The sample in the GC vial insert and the POP Standard Check Solution are analyzed by 

GC/MS for POPs as described in Section 5.

4.2. Size-exclusion High-performance Liquid Chromatography

Size-exclusion HPLC is used for removing extraneous high-molecular-weight 

compounds from the sample. The HPLC system is programmed according to Table 4. The 

fraction-collection time is determined immediately before chromatographing a batch of samples, 

as in Subsection 4.2.3, using the results from the system calibration performed in Subsection 

4.2.2.

4.2.1. Size-exclusion HPLC Start-up

A 4-L bottle of dichloromethane is installed in the solvent reservoir holder, and the 

solvent inlet filter and helium diffuser are placed at the bottom of the bottle. The 

dichloromethane is degassed for a minimum of 10 minutes prior to HPLC system operation by 

diffusing helium into the dichloromethane. The helium flows through a hydrocarbon trap and an 

oxygen trap installed in-line between the helium tank and the diffuser.

The purge valve on the pump is opened, and the pump is started in purge mode to purge 

the pump with the degassed solvent for 5 minutes, with the solvent flowing to the waste 

collection bottle. The purge valve is closed after the pump purge has finished.

Table 4. Size-exclusion HPLC events.

Approx. time (min) HPLC module Activity

<0 Autosampler Sample is loop loaded with 500 μL of sample or 

calibration standard.

0 Data system Signal is sent to autosampler to inject sample and to 

fraction collector to start timer.

Continuous Pump Isocratic mobile phase flows at 5 mL/min.

1 Autosampler Injection port is rinsed; needle is rinsed.

14–23 Fraction collector Fraction is collected.

25 Computer Data system ends integration.
30

The pump is started with the flow rate of the solvent set at 1 mL/min for purging the 

autosampler, and the autosampler purge mode is started. If the autosampler compression test 

fails, the autosampler purge is repeated.

The pump flow is set to 5 mL/min, and the system pressure is monitored for stability.

The wavelength on the detector is set at 254 nm, and the baseline of the ultraviolet (UV) 

signal is zeroed.

The data system is programmed and initiated so that the PAH/POP HPLC RT standard is 

injected and chromatographed according to Table 4, except that the fraction collector is set for no 

collection.

A 4-mL vial is filled with the PAH/POP HPLC RT standard and placed in the 

autosampler carousel.

The PAH/POP HPLC RT standard is chromatographed three or more times, as necessary, 

to obtain three consecutive analyses having retention time stability to within +0.05 minutes of 

each other for 4,4'-dibromooctafluorobiphenyl (DOB, first peak) and for perylene (PER, last 

peak). It may be necessary to re-zero the baseline of the UV signal at approximately 10 minutes 

into the first analysis of the PAH/POP HPLC RT standard.

For each analysis of the RT standard, the system pressure and the retention times of DOB 

and PER are recorded.

When the retention times of the DOB and PER peaks in the RT standard have stabilized 

for three consecutive analyses, the system is ready to proceed with the System Calibration

described in Subsection 4.2.2 or the Daily Calibration described in Subsection 4.2.3, using the 

retention times of the DOB and PER peaks to determine the fraction collection times.

4.2.2. Size-exclusion HPLC System Calibration

The HPLC system calibration is performed only when any component of the HPLC 

system (e.g., pump, column, length of tubing, etc.) is changed.

The HPLC is started, and the PAH/POP HPLC RT standard is chromatographed three or 

more times, as necessary, as described in Subsection 4.2.1.

Using the retention times in the last chromatogram of the PAH/POP HPLC RT standard, 

the fraction collector is programmed to collect 10 consecutive 0.1-minute fractions starting 1 

minute before the retention time of DOB, and to collect 15 consecutive 0.1-minute fractions 

starting at the retention time of PER.

The fraction collector is configured with a tray for 60-mL ASE vials. Twenty-five 

60-mL ASE vials labeled F1 through F25 are positioned in the fraction collector tray.

An HPLC system calibration solution is prepared by adding dichloromethane (900 μL) 

and PAH/POP HPLC RT standard (100 μL) to a 1.5-mL GC vial, then thoroughly mixing the 
31

contents of the vial. The contents are transferred to a 4-mL HPLC vial with a 1-mL insert. This 

vial is positioned in the autosampler tray.

The autosampler is programmed and initiated so that the HPLC system calibration 

solution is injected and chromatographed according to the times in Table 4, except that the 

fractions are collected as programmed above. The retention times of DOB and PER are 

recorded.

When the analysis is finished, the pump, autosampler, and fraction collector are stopped

and the detector is turned off.

The ASE vials containing fractions F1 through F25 are removed from the collector tray,

and the remaining HPLC system calibration solution is removed from the autosampler.

POP GC IS (30 μL) and isooctane (150 μL) are added to each of the vials containing 

fractions F1 through F10, and the contents of each vial are mixed thoroughly and transferred to a 

labeled 1.5-mL GC vial.

PAH GC IS (30 μL) and isooctane (150 μL) are added to each vial containing fractions 

F11 through F25, and the contents of each vial are mixed thoroughly and transferred to a labeled 

1.5-mL GC vial.

POP GC IS (30 μL), PAH GC IS (30 μL), and isooctane (120 μL) are added to the 

remaining HPLC system calibration solution, and the solution is mixed thoroughly and

transferred to a labeled 1.5-mL GC vial.

A small boiling chip is added to each GC vial containing the fractions and the remaining 

HPLC system calibration solution, and the contents are reduced to 100 μL each using a vial 

heater. All dichloromethane should be evaporated from the sample, indicated by the remaining 

isooctane ceasing to boil.

The remaining HPLC system calibration solution and each fraction are transferred to a 

labeled GC vial with a limited volume insert.

The fractions F1 through F10, the remaining HPLC system calibration solution, and the 

Level 6 POP GC/MS calibration solution are analyzed by GC/MS for POPs as described in 

Section 5. The Level 6 POP GC/MS calibration solution is used to check the reproducibility of 

the GC/MS for DOB relative to TCOX. The POP GC IS in the fractions is used to show that 

each fraction is injected and analyzed properly by the GC/MS.

The GC/MS chromatograms for fractions F1 through F10 are inspected to find the first 

fraction in which DOB appears with an area ≥1% of the DOB area in the remaining HPLC 

system calibration solution. The starting time for the collection of this fraction from the HPLC 

system is designated as the starting time (t1) of the DOB elution from the HPLC system (see 

Figure 3).
32

To allow for possible retention time drift, 0.2 minute is subtracted from t1 to obtain t2. 

Then t2 is subtracted from the DOB HPLC retention time (DOB RT) in the analysis of the HPLC 

system calibration standard to obtain t3 (see Figure 3).

The value for t3 is recorded for future use in the daily calibration of the HPLC 

(Subsection 4.2.3).

The fractions F11 through F25, the remaining HPLC system calibration solution, and the 

Level 5 PAH GC calibration standard are analyzed by GC/MS SIM for PAHs as described in 

Section 5. The Level 5 PAH GC calibration standard is used to check the reproducibility of the 

GC/MS for PER relative to HMB. The PAH GC IS in the fractions is used to show that each 

fraction is injected and analyzed properly by the GC/MS.

The GC/MS chromatograms for fractions F11 through F25 are inspected to find the last 

fraction in which PER appears with an area that is ≥1% of the PER peak area in the remaining 

HPLC system calibration solution. The ending time for the collection of this fraction is 

designated as the ending time (t5) of the PER elution from the HPLC system (see Figure 3).

To allow for possible retention time drift, 0.5 minute is added to t5 to obtain t6. Then the 

PER HPLC retention time (PER RT) in the analysis of the HPLC system calibration standard is 

subtracted from t6 to obtain t7 (see Figure 3).

The value for t7 is recorded for future use in the daily calibration of the HPLC 

(Subsection 4.2.3).

Figure 3. Size-exclusion high-performance liquid chromatograph calibration.
33

4.2.3. Daily Calibration of the Size-exclusion HPLC System

The daily calibration of the HPLC system is performed immediately before 

chromatographing a batch of samples to determine the collection time of the PAH/POP fraction.

The HPLC is started, and the PAH/POP HPLC RT standard is chromatographed three or 

more times, as necessary, as described in Subsection 4.2.1.

Collection start and end times for the PAH/POP fraction are determined using the 

retention times of DOB and PER in the most recent analysis of the PAH/POP HPLC RT 

standard, and the values t3 and t7 are derived from the HPLC system calibration described in 

Subsection 4.2.2.

For the daily calibration, the formula for the starting time t4 for collecting a PAH/POP 

fraction is

t4 = DOB RT – t3 (1)

where DOB RT is the DOB HPLC retention time in the most recent analysis of the PAH/POP 

RT standard and t3 is the value determined in Subsection 4.2.2.

For the daily calibration, the formula for the ending time t8 for collecting a PAH/POP 

fraction is 

t8 = PER RT + t7 (2)

where PER RT is the PER HPLC retention time in the most recent analysis of the PAH/POP RT 

standard during the daily calibration and t7 is the value determined in Subsection 4.2.2.

4.2.4. Sample Cleanup by Size-exclusion HPLC

A batch of samples is chromatographed immediately following the daily calibration of 

the HPLC system described in Subsection 4.2.3, except for plasma samples the injection volume 

is 350 μL.

The fraction collector is programmed to collect the PAH/POP fraction for each sample in 

the batch, using the start and end times (t4 and t8, respectively) determined during the daily 

calibration of the HPLC system described in Subsection 4.2.3. The start and end times of the 

collection are recorded in the HPLC logbook.

Labeled ASE vials are placed in the rack of the fraction collector.

An HPLC Blank sample is prepared by filling a labeled 4-mL vial with dichloromethane. 

This sample is monitored for contamination from the HPLC system.

The batch of samples from Subsection 4.1.3, the HPLC Blank, and the PAH/POP HPLC 

RT standard are positioned in the autosampler tray such that the HPLC Blank is 

chromatographed at the beginning of the sequence and the PAH/POP HPLC RT standard is 

analyzed in the middle and at the end of the batch.
34

The data system is programmed to sequentially chromatograph the samples in the batch 

plus the PAH/POP HPLC RT standard in the middle and end of the batch.

For each analysis of the RT standard, the system pressure and the retention times of DOB 

and PER are recorded. If the retention time for DOB in the middle analysis differs from the first 

analysis by ±0.05 minute or more, then the starting time for collection of the fraction is changed 

accordingly for the remaining samples in the batch. If the retention time for PER in the middle 

analysis changed by ±0.05 minute or more with respect to the retention time for DOB in the 

middle analysis, then the collection time for the fraction is changed accordingly for the 

remaining samples in the batch.

After the sequence of samples and PAH/POP HPLC RT standard has finished, the guard 

column is backflushed for 15 minutes. Then the pump, autosampler, and fraction collector are 

stopped, and the detector is turned off.

The remaining sample in the HPLC vial insert is transferred to a GC vial and stored in a 

freezer as a reserve if needed.

4.2.5. Preparation of Extracts and Standard Check Solutions

Five boiling chips are added to the ASE vial containing the cleaned up extract, and the 

extract volume is reduced to approximately 2 mL using a vial heater.

For a batch of tissue, sediment, or water samples to be analyzed for both PAHs and 

POPs, PAH GC IS (30 μL), POP GC IS (30 μL), and isooctane (150 μL) are added to the ASE

vial, and the contents are mixed thoroughly.

For a batch of tissue, sediment, or water samples to be analyzed for PAHs only, PAH GC 

IS (30 μL) and isooctane (175 μL) are added to the ASE vial, and the contents are mixed 

thoroughly.

For a batch of tissue, sediment, or water samples to be analyzed for POPs only, POP GC 

IS (30 μL) and isooctane (175 μL) are added to the ASE vial, and the contents are mixed 

thoroughly.

For the check solutions in a batch of tissue samples requiring Macro Total Extractables 

Determination: PAH GC IS (150 μL) is added to the PAH Standard Check Solution, and the 

solution is mixed thoroughly; POP GC IS (150 μL) is added to the POP Standard Check 

Solution, and the solution is mixed thoroughly.

For the check solutions in a batch of sediment or water samples or tissue samples not 

requiring Macro Total Extractables Determination: PAH GC IS (75 μL) is added to the PAH 

Standard Check Solution, and the solution is mixed thoroughly; POP GC IS (75 μL) is added to 

the POP Standard Check Solution, and the solution is mixed thoroughly.

For all batches of sediment, tissue, or water, the sample is transferred from the ASE vial 

to a labeled 4-mL HPLC vial, a boiling chip is added, and the sample volume is reduced to 100 
35

μL using the vial heater. All dichloromethane should be evaporated from the sample, indicated 

by the remaining isooctane ceasing to boil.

The sample is transferred to a labeled GC vial with a limited volume insert.

The sample and the appropriate Standard Check Solution are analyzed by GC/MS for 

PAHs or POPs as described in Section 5.
36

5. Gas Chromatography/Mass Spectrometry

GC/MS is used to measure the amounts of the analytes and the internal standards in the 

samples. The compounds are separated by GC and detected by MS, producing a chromatogram 

for each analysis, where the detector’s response to the compounds produces peaks. The area 

under each peak is proportional to the concentration of the compound in the sample or standard 

analyzed.

5.1. GC/MS Operating Conditions

The operating conditions for the GC/MS are listed in Table 5.

Table 5. GC/MS operating conditions.

Injection volume: 2 μL

Injection technique: cool on-column

Inlet temperature program: oven temperature plus 3°C

Transfer line temperature: 320°C

Carrier gas: helium

Carrier gas flow: 1.3 mL/min, constant flow

Column

Material: fused-silica capillary tubing

Length: 60 m

Internal diameter: 0.25 mm

Stationary phase: DB-5

Phase composition: 5% phenyl, 95% methylpolysiloxane

Film thickness: 0.25 μm

Guard column

Material: deactivated fused-silica capillary tubing

Length: 10 m

Internal diameter: 0.53 mm

Quadrupole temperature: 150°C

Ion-source pressure: <5 × 10-5 Torr

Ion-source temperature: 300°C

Ionization mode: electron ionization

Filament: chemical-ionization type (accommodates 300°C)

Tune mode: Standard Tune; then the electron multiplier 

voltage is increased to achieve the desired 

sensitivity

Emission current maximum: 35 μA
37

The helium flows through a combination oxygen/moisture/hydrogen-indicating trap 

installed in-line between the helium regulators and the GC. In addition, an indicating oxygen 

trap is installed in-line at the rear of the GC.

The GC/MS is checked for atmospheric leaks, and the MS is tuned or checked for 

stability prior to analyzing a sequence of samples and standards.

5.2. GC/MS for Quantitating PAHs, Alkyl PAH Isomers, 

and Alkanes

The GC oven temperature is programmed for PAHs as follows.

Initial temperature: 80°C

Initial time: 1 min

First rate: 10°C/min to 200°C

Isothermal hold: 0 min

Second rate: 4°C/min to 300°C

Isothermal hold: 12 min at 300°C

Third rate: 10°C/min to 315°C

Isothermal hold: 15 min at 315°C

Total run time: 66.5 min

Sediment samples are analyzed using scan mode, scanning from 60 to 300 daltons. 

Tissue samples and water samples are analyzed using SIM mode, scanning only the quantitation 

and confirmation ions during the specified time window as shown in Table 6 for the analytes in 

Table 1 or as shown in Table 7 for the analytes in Table 2. See Subsection 5.2.2 for the alkane 

analytes. Approximate time windows are shown; see below for determining actual start and end 

times. Scan mode provides greater ability to confirm analyte identification in the complex 

sediment samples, whereas SIM mode generally provides greater sensitivity for tissue or water 

samples, which typically contain lower concentrations of contaminants.

5.2.1. GC/MS SIM Windows for PAHs and Alkyl PAH Isomers

For SIM analyses using Table 6, the actual time for ending a given window and starting 

the next window is determined as approximately the midpoint between the retention time of the 

last compound in the given window and the retention time of the first compound in the next 

window. Initially, the retention times of the compounds are determined from a scan analysis of a 

mid- or high-level GC/MS calibration standard. The time windows are adjusted as necessary 

when the GC column or the guard column are changed or trimmed.

Dwell times for the SIM windows in Table 6 are set to achieve 10–20 scans per peak.

For SIM analyses using Table 7, the start times for windows are determined using 

formulae such as those shown. These formulae are determined from the analysis of an 

appropriate NIST SRM or a project-specific sample of oil that has been diluted with 

dichloromethane then cleaned up by a silica/alumina column and size-exclusion HPLC as 

described above for tissue samples. The SRM or oil sample is analyzed by GC/MS using scan 
38

Table 6. SIM windows for quantitating selected PAHs in tissue and water samples.

Approximate time 

window (minutes) Compounds

Quantitation ion,*

confirmation ion* (daltons)

1) 10 to 12 Naphthalene-d8 136

Naphthalene 128, 127

2-methylnaphthalene 142, 141

1-methylnaphthalene 142, 141

2) 12 to 13 2,6-dimethylnaphthalene 156, 141

3) 13 to 14 Hexamethylbenzene 147

Acenaphthylene 152, 151

Acenaphthene-d10 164

Acenaphthene 154, 153

4) 14 to 17 2,3,5-trimethylnaphthalene 170, 155

Fluorene 166, 165

5) 17 to 20 Dibenzothiophene 184, 139

Phenanthrene-d10 188

Phenanthrene 178, 176

Anthracene 178, 176

6) 20 to 29 3-methylphenanthrene 192, 191

1-methylphenanthrene 192, 191

1,7-dimethylphenanthrene 192, 191

Fluoranthene 202, 101

Pyrene 202, 101

7) 29 to 42 Benz[a]anthracene 228, 226

Chrysene + triphenylene 228, 226

Benzo[b]fluoranthene 252, 250

Benzo[j]fluoranthene + benzo[k]fluoranthene 252, 250

Benzo[e]pyrene 252, 250

Benzo[a]pyrene-d12 264

Benzo[a]pyrene 252, 250

Perylene 252, 250

8) 42 to 53 Indeno[1,2,3-cd]pyrene 276, 138

Dibenz[a,h]anthracene + dibenz[a,c]anthracene 278, 139

Benzo[ghi]perylene 276, 138

* Nominal mass.
39

Table 7. SIM windows for quantitating PAHs, including sums of alkyl isomers in tissue and 

water samples.

Approximate time 

window (minutes) Compounds

Quantitation ion,*

confirmation ion*

(daltons)

1) 10 to 13.5 Naphthalene-d8 136

Naphthalene 128, 127

C1-naphthalenes 142, 141

C2-naphthalenes 156, 141

2) 13.5 to 14.8

Start = 2,6-

dimethylnaphthalene 

retention time (RT) plus 

0.2 min.

C2-naphthalenes 156, 141

Hexamethylbenzene 147

Acenaphthylene 152, 151

Acenaphthene-d10 164

Acenaphthene 154, 153

C3-naphthalenes 170, 155

3) 14.8 to 17

Start = 2,3,5-

trimethylnaphthalene RT 

minus 0.75 min

C3-naphthalenes 170, 155

C4-naphthalenes 184, 169

Fluorene 166, 165

4) 17 to 20

Start = Dibenzothiophene 

RT minus 1.7 min

C4-naphthalenes 184, 169

C1-fluorenes 180, 179

C2-fluorenes 194, 193

C3-fluorenes 208, 193

Dibenzothiophene 184, 139

Phenanthrene-d10 188

Phenanthrene 178, 176

Anthracene 178, 176

5) 20 to 23

Start = Phenanthrene RT 

plus 0.9 min

C2-fluorenes 194, 193

C3-fluorenes 208, 193

C1-dibenzothiophenes 198, 197

C1-phenanthrenes/anthracenes 192, 191

C2-dibenzothiophenes 212, 211

C2-phenanthrenes/anthracenes 206, 191

6) 23 to 25

Start = Fluoranthene RT 

minus 1.1 min

Fluoranthene 202, 101

C3-fluorenes 208, 193

C2-phenanthrenes/anthracenes 206, 191

C3-phenanthrenes/anthracenes 220, 205

C2-dibenzothiophenes 212, 211

C3-dibenzothiophenes 226, 211

C4-dibenzothiophenes 240, 225
40

Table 7 continued. SIM windows for quantitating PAHs, including sums of alkyl isomers in tissue 

and water samples.

Approximate time 

window (minutes) Compounds

Quantitation ion,*

confirmation ion*

(daltons)

7) 25 to 28

Start = Pyrene RT minus 

0.2 min

Pyrene 202, 101

Retene 234, 219

C3-dibenzothiophenes 226, 211

C4-dibenzothiophenes 240, 225

C3-phenanthrenes/anthracenes 220, 205

C4-phenanthrenes/anthracenes 234, 219

C1-fluoranthenes/pyrenes 216, 215

8) 28 to 32

Start = Pyrene RT plus 

2.85 min

Benz[a]anthracene 228, 226

Chrysene + triphenylene 228, 226

C4-dibenzothiophenes 240, 225

C4-phenanthrenes/anthracenes 234, 219

C1-fluoranthenes/pyrenes 216, 215

C2-fluoranthenes/pyrenes 230, 215

C3-fluoranthenes/pyrenes 244, 229

9) 32 to 36

Start = Chrysene RT plus 

0.3 min

C3-fluoranthenes/pyrenes 244, 229

C4-fluoranthenes/pyrenes 258, 243

C1-benz[a]anthracenes/chrysenes 242, 241

C2-benz[a]anthracenes/chrysenes 256, 241

10) 36 to 44

Start = 

Benzo[b]fluoranthene RT 

minus 0.6 min

C2-benz[a]anthracenes/chrysenes 256, 241

C3-benz[a]anthracenes/chrysenes 270, 255

C4-benz[a]anthracenes/chrysenes 284, 269

Benzo[b]fluoranthene 252, 250

Benzo[j]fluoranthene + benzo[k]fluoranthene 252, 250

Benzo[e]pyrene 252, 250

Benzo[a]pyrene-d12 264

Benzo[a]pyrene 252, 250

Perylene 252, 250

11) 44 to 53

Start = Indeno[1,2,3-cd]-

pyrene RT minus 0.8 min

Indeno[1,2,3-cd]pyrene 276, 138

Dibenz[a,h]anthracene + dibenz[a,c]anthracene 278, 139

Benzo[ghi]perylene 276, 138

* Nominal mass.
41

mode, scanning from 60 to 300 daltons, to identify the groups of alkyl PAH isomers (Table 2) 

and their retention times in the sample, as well as the retention times of the individual PAH 

analytes that are not present in the calibration standards. The windows are set such that multiple

groups of isomers are monitored in more than one SIM window. This is because, in many cases, 

the elution of one or more groups of isomers overlap (e.g., the elution of C2-phenanthrenes/

anthracenes partially overlaps with that of C3-fluorenes and C3-phenanthrenes/anthracenes), and 

the signals are optimized by minimizing and balancing the number of ions per window. The 

formulae shown in Table 7 are derived for the GC column in use to relate the start times of SIM 

windows to retention times of PAHs that are in the calibration standard. The resulting formulae 

can be used to update the SIM-window start times when retention times shift, for example, after 

the GC column or the guard column are changed or trimmed.

Dwell times for the SIM windows in Table 7 are set at 65 for all ions in windows 1 

through 10 for consistent response, and 100 for all ions in window 11 for smoother signals.

The quantitation ions for PAHs in tissues and water shown in Table 6 and Table 7 are 

also the ions used for quantitating PAHs in sediments. 

5.2.2. GC/MS SIM Windows for PAHs, Alkyl PAH Isomers, and Alkanes

If water samples are to be analyzed for the alkanes (pristane, phytane, n-heptadecane, and 

n-octadecane), the alkanes are quantitated concurrently with the PAHs using the SIM windows in 

Table 7, modified to include the quantitation ion 71 and the confirmation ion 57 in window 5 for 

pristane and n-heptadecane and in window 6 for phytane and n-octadecane, as needed.

5.2.3. Verification of GC/MS Performance for PAHs and Alkyl PAH Isomers

The sensitivity of the GC/MS is checked by analyzing the lowest level of PAH GC 

calibration standard that will be used to quantitate PAHs in the samples (Level 1 for tissues and 

water, Level 3 for sediments).

5.2.4. GC/MS Analysis of Sediment Samples for PAHs

One or more batches of sediment samples are analyzed in a sequence that includes one 

each of PAH GC calibration standard Levels 3, 4, 5, 6, 8, and 9, alternating with the samples, 

plus one Level 7 before the first sample and replicate Level 7 analyses at the middle and end of 

each batch. Included in this sequence is a representative sediment sample at the beginning of the 

sequence, which serves as a GC/MS system conditioner.

5.2.5. GC/MS Analysis of Tissue Samples for PAHs and Alkyl PAH Isomers

One or more batches of tissues samples are analyzed in a sequence that includes one each 

of PAH GC calibration standard Levels 1, 2, 3, 5, and 6, alternating with the samples in the 

sequence, plus one Level 4 before the first sample and replicate Level 4 analyses at the middle 

and end of each batch. Included in this sequence is one injection of a representative tissue 

sample at the beginning of the sequence, which serves as a GC/MS system conditioner.
42

5.2.6. GC/MS Analysis of Water Samples for PAHs, Alkyl PAH Isomers, and Alkanes

One or more batches of water samples are analyzed in a sequence that includes one each 

of PAH plus alkanes GC calibration standard Levels 1, 2, 4, 5, and 6, alternating with the 

samples in the sequence, plus one Level 3 before the first sample and replicate Level 3 analyses 

at the middle and end of each batch. Included in this sequence is one injection of a 

representative water sample at the beginning of the sequence, which serves as a GC/MS system 

conditioner.

5.3. GC/MS for Quantitating POPs

The GC oven temperature is programmed for POPs as follows.

Initial temperature: 1 min at 80°C

First rate: 10°C/min to 150°C

Isothermal hold: 0 min

Second rate: 0.5°C/min to 195°C

Isothermal hold: 0 min

Third rate: 3°C/min to 315°C

Isothermal hold: 15 min at 315°C

Total run time: 153 min

5.3.1. GC/MS SIM Windows for POPs

Sediment, tissue, and water samples are analyzed using SIM mode, scanning only the 

quantitation ions during the specified time window as shown in Table 8. For SIM analyses using 

Table 8, the actual time for ending a given window and starting the next window is determined 

as approximately the midpoint between the retention time of the last compound in the given 

window and the retention time of the first compound in the next window. Initially, the retention 

times of the compounds are determined from a scan analysis of a mid-level or high-level GC/MS 

calibration standard. The time windows are adjusted as necessary when the GC column is 

changed or the guard column is trimmed.

Dwell times for the SIM windows in Table 8 are set to achieve 10–20 scans per peak.

5.3.2. Verification of GC/MS Performance for POPs

The sensitivity of the GC/MS is checked by analyzing the lowest level of POP GC 

calibration standard that will be used to quantitate POPs in the samples.

5.3.3. GC/MS Analysis of Sediment Samples for POPs

One or more batches of sediment samples are analyzed in a sequence that includes one 

each of POP GC calibration standard Levels 2 through 5 and Level 7 alternating with the 

samples, plus one Level 6 before the first sample and replicate Level 6 analyses at the middle 

and end of each batch. Included in this sequence is one injection of a representative sediment 

sample at the beginning of the sequence, which serves as a GC/MS system conditioner.
43

Table 8. SIM windows for quantitating POPs in tissue, sediment, and water samples.

Approximate time 

window (minutes) Compounds

Quantitation 

ionsa, b

1) 20 to 28 Tetrachloro-o-xylene 207

Tetrachloro-m-xylene 207

2) 28 to 35 Dibromooctafluorobiphenyl 456

alpha-Hexachlorocyclohexane 181

Hexachlorobenzene 284

3) 35 to 39 beta-Hexachlorocyclohexane 219

gamma-Hexachlorocyclohexane 181

4) 39 to 44 PCB 18 256

PCB 17 256

5) 44 to 54 PCB 31 256

PCB 28 256

PCB 33 256

Heptachlor 272

6) 54 to 64 PCB 52 292

PCB 49 292

Aldrin 263

PCB 44 292

7) 64 to 68 PCB 103 326

8) 68 to 74 Heptachlor epoxide 353

Oxychlordane 115

PCB 74 292

PCB 70 292

PCB 66 292

PCB 95 326

9) 74 to 77 trans-Chlordane 373

10) 77 to 84 2,4'-DDE 246

Endosulfan I 241

PCB 101 + PCB 90 326

Nonachlor III 409

cis-Chlordane 373

PCB 99 326

trans-Nonachlor 409
44

Table 8 continued. SIM windows for quantitating POPs in tissue, sediment, and water samples.

Approximate time 

window (minutes) Compounds

Quantitation 

ionsa, b

11) 84 to 91 Dieldrin 263

PCB 87 326

4,4'-DDE 246

PCB 110 326

2,4'-DDD 235

12) 91 to 96 PCB 82 326

PCB 151 360

13) 96 to 99 PCB 149 360

PBDE 28 405.8, 407.8

PCB 118 326

14) 99 to 102 cis-Nonachlor 409

4,4'-DDD 235

2,4'-DDT 235

15) 102 to 106 PCB 153 + PCB 132 360

PCB 105 326

16) 106 to 109 4,4'-DDT 235

PCB 138 + PCB 163 + PCB 164 360

PCB 158 360

17) 109 to 112 PCB 187 + PCB 159 + PCB 182 394

PCB 183 394

PCB 128 360

18) 112 to 115 PCB 177 394

PCB 171 394

PBDE 49 483.7, 485.7

PCB 156 360

19) 115 to 120 PCB 180 394

PBDE 47 483.7, 485.7

PCB 191 394

PBDE 66 483.7, 485.7

Mirex 272

PCB 170 394

PCB 199 430
45

Table 8 continued. SIM windows for quantitating POPs in tissue, sediment, and water samples.

Approximate time 

window (minutes) Compounds

Quantitation 

ionsa, b

20) 120 to 125 PCB 208 464

PCB 195 430

PBDE 100 563.6, 565.6

PCB 194 430

PCB 205 430

PBDE 99 563.6, 565.6

21) 125 to 134 PCB 206 464

PCB 209 498

PBDE 85 563.6, 565.6

PBDE 155 641.5, 643.5

PBDE 154 641.5, 643.5

PBDE 153 641.5, 643.5

22) 134 to 142 PBDE 183 561.6, 563.6

a Nominal mass, except the exact mass to one significant figure for PBDEs.

b For quantitating PBDEs, the signals of two ions are summed.

5.3.4. GC/MS Analysis of Low-lipid Tissue Samples or Water Samples for POPs

One or more batches of low-lipid tissue samples or water samples are analyzed in a 

sequence that includes one each of POP GC calibration standard Levels 1 through 5 and Level 7 

alternating with the samples (Level 1 is optional, depending on the project), plus one Level 6 

before the first sample and replicate Level 6 analyses at the middle and end of each batch. 

Included in this sequence is one injection of a representative tissue or water sample at the 

beginning of the sequence, which serves as a GC/MS system conditioner.

5.3.5. GC/MS Analysis of High-lipid Tissue Samples for POPs

One or more batches of high-lipid tissue samples are analyzed in a sequence that includes 

one each of POP GC calibration standard Levels 1 through 5 and Levels 7 through 11 alternating 

with the samples (Level 1 is optional, depending on the project), plus one Level 6 before the first 

sample and replicate Level 6 analyses at the middle and end of each batch. Included in this 

sequence is one injection of a representative tissue sample at the beginning of the sequence, 

which serves as a GC/MS system conditioner.
46

6. Calculations of GC/MS Results

6.1. Calculating Analyte Concentrations

The acquired GC/MS data for the samples and calibration standards are processed using 

the Agilent ChemStation software to determine areas for the analytes’ peaks and the internal 

standards’ peaks in the chromatograms. The identifications and integrations of the peaks are 

performed by the ChemStation software to generate the peak areas, and then they are checked for 

appropriateness and manually reintegrated as needed.

For quantitating the groups of alkyl PAH isomers (Table 2), the responses for all isomers 

within a group are summed by integrating the area for all peaks in the entire group to get one 

response area, and the isomer group is treated as a single analyte. The retention times and 

relative peak abundances of the isomers determined from a SIM analysis of an appropriate SRM 

or a project-specific oil sample can be used for guidance. For example, panel A of Figure 4

shows the group of C3-naphthalenes in an oil sample (between arrows) and panel B and panel C 

show the correspondingly integrated C3-naphthalenes in a brown shrimp sample and a sample of 

NIST SRM 1974b, respectively. Similarly, the panels of Figure 5 show the chromatograms for 

C2-fluorenes.

The analyte areas produced by the ChemStation software are then used by a BASIC 

program written in-house to compute the analyte amounts in the samples (ng analyte per sample) 

based on point-to-point calibration (equations provided below). The point-to-point calibration 

method is equivalent to plotting the relative response factors (RRFs) of the calibration standards 

versus their analyte areas to achieve two-dimensional points. Linear equations generated 

between each pair of consecutive points are used for the calibration in order to gain a better fit 

over the entire range of calibration standards than would be provided by a single regression 

equation, such as linear, weighted linear, or quadratic regression.

For each level of calibration standard (CS), the RRF is computed for each analyte relative 

to its surrogate standard (SS) as

CS RRF = CS Analyte Concentration × CS SS Area (3)

CS Analyte Area CS SS Concentration

For the PAHs listed in Table 1, the surrogate standards (SSs) are naphthalene-d8 for the 

analytes naphthalene through 1-methylnaphthalene, acenaphthene-d10 for analytes acenaphthene 

through pyrene, and benzo[a]pyrene-d12 for analytes benz[a]anthracene through 

benzo[ghi]perylene. For the PAHs including alkyl PAH isomers listed in Table 2, the SSs are 

naphthalene-d8 for the analytes naphthalene and C1-naphthalenes, acenaphthene-d10 for 

analytes C2-naphthalenes through C4-fluoranthenes and pyrenes, and benzo[a]pyrene-d12 for 

analytes benz[a]anthracene through benzo[ghi]perylene. The POP SS is PCB 103 for all 
47

Figure 4. GC/MS SIM chromatograms of C3-naphthalenes in an oil sample (panel A), a brown shrimp 

sample (panel B), and a sample of NIST SRM 1974b (panel C). Arrows point to the start and end 

of the integration baseline.
48

Figure 5. GC/MS SIM chromatograms of C2-fluorenes in an oil sample (panel A), a brown shrimp 

sample (panel B), and a sample of NIST SRM 1974b (panel C). Arrows point to the start and end 

of the integration baseline.
49

analytes in Table 3, except DOB is used as the POP SS for all analytes in cases when 

endogenous PCB 103 is evident, such as in highly contaminated marine mammal blubber. For 

the alkanes (pristane, phytane, n-heptadecane, and n-octadecane), the SS is acenaphthene-d10.

For the analytes not contained in the CSs, the response factor of a similar analyte is used, 

that is, 1-methylphenanthrene for retene, chrysene for chrysene + triphenylene,

benzo[k]fluoranthene for benzo[k]fluoranthene + benzo[j]fluoranthene, dibenz[a,h]anthracene 

for dibenz[a,h]anthracene + dibenz[a,c]anthracene, PCB 101 for PCB 101 + PCB 90, PCB 138 

for PCB 138 + PCB 163 + PCB 164, PCB 153 for PCB 153 + PCB 132, PCB 187 for PCB 187 + 

PCB 159 + PCB 182, trans-nonachlor for nonachlor-III, and as shown in Table 9 for the groups 

of alkyl PAH isomers.

Next, specific for each sample, a sample RRF for each analyte relative to its surrogate 

standard is computed by interpolating between the RRFs of the two consecutive calibration 

standards whose areas for that analyte bracket the area of the analyte in the sample, as

Sample RRF = Lower CS RRF

+ Sample Analyte Area – Lower CS Analyte Area × (Upper CS RRF – Lower CS RRF) (4)

 Upper CS Analyte Area – Lower CS Analyte Area

Table 9. Compounds used for response factors to quantitate isomer groups of alkyl PAH isomers.

Analyte Compound used for response factor

C2-Naphthalenes 2,6-Dimethylnaphthalene

C3-Naphthalenes 2,3,5-Trimethylnaphthalene

C4-Naphthalenes 2,3,5-Trimethylnaphthalene

C1-Fluorenes Fluorene

C2-Fluorenes Fluorene

C3-Fluorenes Fluorene

C1-Dibenzothiophenes Dibenzothiophene

C2-Dibenzothiophenes Dibenzothiophene

C3-Dibenzothiophenes Dibenzothiophene

C4-Dibenzothiophenes Dibenzothiophene

C1-Phenanthrenes and anthracenes 1-Methylphenanthrene

C2-Phenanthrenes and anthracenes 1,7-Dimethylphenanthrene

C3-Phenanthrenes and anthracenes 1,7-Dimethylphenanthrene

C4-Phenanthrenes and anthracenes 1,7-Dimethylphenanthrene

C1-Fluoranthenes and pyrenes Fluoranthene

C2-Fluoranthenes and pyrenes Fluoranthene

C3-Fluoranthenes and pyrenes Fluoranthene

C4-Fluoranthenes and pyrenes Fluoranthene

C1-Benz[a]anthracenes and chrysenes Chrysene

C2-Benz[a]anthracenes and chrysenes Chrysene

C3-Benz[a]anthracenes and chrysenes Chrysene

C4-Benz[a]anthracenes and chrysenes Chrysene
50

where Lower CS is the calibration standard whose analyte area is closest to and smaller than that 

of the sample’s analyte area and Upper CS is the calibration standard whose analyte area is 

closest to and larger than that of the sample’s analyte area.

The amount of each analyte in each sample (ng Analyte per Sample) is then calculated as

ng Analyte/Sample = Sample RRF × Sample Analyte Area × Sample SS ng (5)

 Sample SS Area

where Sample SS ng is the ng amount of surrogate standard added to the sample prior to 

extraction.

These analyte amounts for the samples are then used by a Filemaker Pro database to 

compute the analyte concentrations in the samples. Concentrations on a wet weight basis or a 

dry weight basis are calculated as

ng/g wet weight = ng Analyte per Sample (6)

 Sample Weight (g)

and

ng/g dry weight = ng Analyte per Sample × 100% (7)

 Sample Weight (g) × % Dry Weight

where the % Dry Weight is determined as described in Subsection 8.1.

6.2. Calculating Internal Standard Recoveries

The percent recoveries of the PAH SSs (naphthalene-d8, acenaphthene-d10, and 

benzo[a]pyrene-d12) are computed for each sample, including the PAH Standard Check 

Solution, based on the HPLC IS (phenanthrene-d10). Furthermore, the PAH HPLC IS recovery 

is computed based on the PAH GC IS (HMB). For these calculations, a single analysis of the

Level 3 GC CS in the case of SIM analyses, or the Level 6 GC CS in the case of scan analyses, is

used for the RRFs of the SSs.

The percent recovery of the POP SS (PCB 103) is computed for each sample, including 

the POP Standard Check Solution, based on the POP HPLC IS (TCMX). Furthermore, the POP 

HPLC IS recovery is computed based on the POP GC IS (TCOX). For these calculations, a 

single analysis of the Level 6 GC CS is used for the RRF of the SS.

The percent recovery of each surrogate standard (SS % Recovery) is calculated as

SS % Recovery = CS SS RRF × Sample SS Area × Sample HPLC IS ng × 100% (8)

 Sample SS ng Sample HPLC IS Area

where

CS SS RRF = CS SS Concentration × CS HPLC IS Area (9)

 CS SS Area CS HPLC IS Concentration

and Sample HPLC IS ng is the ng amount of HPLC internal standard added to the sample.
51

The percent recovery of the HPLC internal standard (HPLC IS % Recovery) is monitored 

for in-house QA information and is calculated as

HPLC IS % Recovery

= CS HPLC IS RRF × Sample HPLC IS area × Sample GC IS ng × 100% (10)

Sample HPLC IS ng Sample GC IS Area

where

CS HPLC IS RRF = CS HPLC IS Concentration × CS GC IS Area (11)

 CS HPLC IS Area CS GC IS Concentration

and Sample GC IS ng is the ng amount of GC internal standard added to the sample.

6.3. Calculating and Reporting Lower Limits of Quantitation 

The LOQ for a given analyte in a given sample is the concentration that would be 

calculated if the analyte had a GC/MS response area equivalent to that analyte’s area in the 

lowest level CS used in the calibration for that analyte (not all levels are used for some analytes). 

When an analyte is not detected in a sample or has an area that is smaller than its area in the 

lowest level CS used, the concentration of the analyte in that sample is reported to be less than 

the value of its LOQ.

6.4. Calculating and Reporting Estimated Concentrations for 

Analytes Exceeding the Calibration Range

When an analyte in a given sample has a GC/MS area that is larger than its area in the 

highest level CS used in the calibration, that sample is diluted as needed (up to fivefold) to get 

the analyte area within the calibration range then reanalyzed by GC/MS along with its 

corresponding QA samples. Alternatively, the analyte amount is calculated using the RRF of 

that analyte in the highest level CS used for that analyte (not all levels are used for some 

analytes), and the concentration is footnoted as exceeding the calibration range and is therefore 

an estimate.
52

7. Quality Assurance for PAHs, POPs, 

and Alkanes

QA measures are incorporated into the analyses of each batch of samples according to the 

Environmental Chemistry Program Quality Assurance Plan. Specific QA goals for batches and 

individual samples, as well as the percent of the total results that must meet the goals, depend on 

the project for which the samples are analyzed and the purpose of the data. Typical goals are 

presented here. When QA goals are not met, the data may be qualified or the sample(s) 

reanalyzed.

7.1. GC/MS Stability

The stability of the GC/MS is evaluated using the repetitions of the mid-level GC CS

analyzed intermittently in the sequence of samples and other GC calibration standards. The 

GC/MS is considered stable if, for each analyte, the percent relative standard deviation (RSD) of 

the analyte responses relative to its SS responses is ≤15%.

7.2. Surrogate and Internal Standard Recoveries

The percent recoveries of SSs in all samples are considered acceptable if they are 

between 60% and 130%. The percent recoveries of these standards in the Standard Check 

Solutions are monitored for problems with the SS and IS solutions. The percent recoveries of 

HPLC ISs are monitored in-house and do not have QA criteria.

7.3. Analyte Concentrations in Reference Materials

At least one sample of an appropriate NIST SRM is analyzed in each batch of tissue or 

sediment samples to indicate the accuracy of the data for the entire batch. An appropriate NIST 

SRM for water analyses is not available at this time. NIST provides certified concentrations and 

uncertainty values for many of the individual compounds in Table 1, Table 2, and Table 3. For 

the analytes having NIST certified concentrations, upper and lower control limits are set as is 

done by NIST in the Intercomparison Exercise Program for Organics in the Marine Environment, 

and are defined as

Upper Control Limit = 1.3 × (Certified Concentration + Uncertainty Value) (12)

and

Lower Control Limit = 0.7 × (Certified Concentration − Uncertainty Value) (13)

The accuracy of the SRM result is considered acceptable if the measured concentration 

falls within the Upper and Lower Control Limits. This goal does not apply to analytes with 

concentrations below their LOQ when their LOQ is greater than their Lower Control Limit. The 
53

analysis of the SRM is acceptable if at least 70% of the analytes having NIST certified 

concentrations are within their control limits. Analytes which do not have NIST certified 

concentrations do not have QA goals for accuracy; however, their SRM results can be monitored 

and compared to NIST reference concentrations, where available, or to previous in-house results.

7.4. Precision of Replicate Samples

When three or more replicate samples are analyzed, the precision of the results can be 

evaluated based on the RSD of the concentrations of each analyte. The precision is considered 

acceptable if the RSD is ≤15% for at least 90% of the analytes with concentrations greater than 

1 ng/g. For duplicate samples, this translates to a relative percent difference of ≤30% for at least 

90% of the analytes.

7.5. Method Blank Contamination

A method blank is analyzed with each batch of samples to monitor for contamination 

from laboratory sources. Ideally, analytes would not be detected in the method blank above their 

LOQ. However, if analytes are measured in the method blank, their concentrations need to be 

compared to the concentrations in the sediment, tissue, or water samples in the same batch. For 

each project, criteria need to be determined for contaminants in the method blank; for example, 

the method blank contamination might be considered acceptable if no more than 10% of the 

analytes have concentrations exceeding two times the LOQ. Also, analytes in tissue, sediment,

or water samples having concentrations between the LOQ and four times the concentration in the 

method blank for the batch might be footnoted or qualified as such. Analyte concentrations in 

method blanks are not subtracted from analyte concentrations in field or SRM samples.

The HPLC blank is monitored in-house and does not have QA criteria.

7.6. Spiked Blank Recoveries

At least one spiked blank water sample is analyzed in each batch of water samples to 

indicate the accuracy of the data for the entire batch, because an appropriate NIST SRM for 

water analyses is not available at this time. The percent recoveries of spikes in spiked blank 

samples are considered acceptable if they are between 60% and 130%. The percent recoveries of 

spikes in the Standard Check Solutions are monitored for problems with the spike solutions.
54

8. Gravimetric Determinations

8.1. Dry Weight Determination

The sample number is etched on the tab of a 43-mm id aluminum weighing pan.

The pan is placed in a drying oven at 120°C overnight then cooled in a desiccator for 30 

minutes.

The pan is weighed to the nearest 0.001 g, and the weight is recorded as the “Pan 

Weight.”

For sediment samples, using a spatula, the sediment is stirred in its container until 

thoroughly homogenized. Standing water was decanted from the sediment sample container and 

all pebbles, shells, biota, and other extraneous material were discarded previously in Section 3; 

no further decanting of excess water is done here so that the water content is the same as when 

the portion of sediment was taken for PAH and POP analysis.

The sediment (2 g, ±0.5) is placed into the pan, and the pan is weighed to the nearest 

0.001 g. The weight is recorded as the “Pan w/Wet Sample Weight.”

For tissue samples, using a spatula, the tissue is stirred in its container to ensure that it is 

homogeneous. The tissue was previously homogenized in Section 3.

The tissue (1 g, ±0.5) is placed into the pan, and the pan is weighed to the nearest 0.001 

g. The weight is recorded as the “Pan w/Wet Sample Weight.”

For sediment or tissue samples, the pan is placed in a drying oven at 120°C for 24 hours

then cooled in a desiccator for 30 minutes.

The pan is weighed to the nearest 0.001 g, and the weight is recorded as the “Pan w/Dry 

Sample Weight.”

The percent dry weight of the sample is determined as

% Dry Weight = (Pan w/Dry Sample Weight – Pan Weight) × 100% (14)

 Pan w/Wet Sample Weight – Pan Weight

The % Dry Weight values for QA samples are monitored in-house and do not have QA

criteria.
55

8.2. Total Extractables Determination

The pan containing the sample for total extractables from Section 3 is placed on a 

covered rack in the hood, and the solvent is allowed to completely evaporate (approximately 1–2 

hours).

The pan is dried in a 50°C oven for 2 hours then cooled in a desiccator overnight.

8.2.1. Batches of Tissue Samples Requiring Macro Total Extractables Determination

The pan is weighed to the nearest 0.001g, and the weight is recorded as the “Pan w/TE 

Weight.”

The percent total extractables (% TE) content of the sample is calculated as

% TE = (Pan w/TE – Pan Weight) × (ASE Vial w/Extract Weight – ASE Vial Weight) × 100% (15)

(ASE Vial w/Extract Weight – ASE Vial w/out TE Extract Weight) × Sample Weight

8.2.2. Batches of Tissue Samples Requiring Micro Total Extractables Determination

The pan is weighed to the nearest 0.001mg, and the weight is recorded as the “Pan w/TE 

Weight.”

The percent total extractables (% TE) content of the sample is calculated as

% TE = (Pan w/TE – Pan Weight) × (ASE Vial w/Extract Weight – ASE Vial Weight) × 100% (16)

(GC Vial w/TE Extract Weight – GC Vial Weight) × Sample Weight

The % TE values for QA samples are monitored in-house and do not have QA criteria.
56

9. Lipid Classes

Aliquots of sample extracts are analyzed for percentages of lipid classes by TLC/FID. 

Lipid classes are separated by TLC and detected by FID, producing a chromatogram for each 

analysis, where the detector’s response to the compounds produces peaks. The area under each 

peak is proportional to the concentration of the lipid class in the sample or standard analyzed.

The portion of sample extract in the 1.5-mL vial for lipid classes from Section 3 is stored 

at between 4 and −20°C until analyzed.

For low-lipid tissues (<10% lipid), the lipid classes extract portion is reduced in volume 

to between 10 and 100 μL using a gentle stream of ultra-pure nitrogen, and the cap is 

immediately replaced. The weight of the concentrated extract in the capped vial is weighed to 

the nearest 0.001 g, and the weight is recorded as “Iatro Vial w/Conc Extract Weight.”

9.1. Separation of Lipid Classes

A TLC development tank containing 70 mL of hexane/diethyl ether/formic acid 

[60:10:0.02 (v:v:v)] and a 5.75′′ × 7.35′′ piece of filter paper is set in a fume hood (temperature 

range 20–25°C). To ensure that the development tank is completely saturated, it is then allowed 

to sit at least 30 minutes prior to placing the first set of Chromarods in the tank.

Ten Chromarods are placed into a SD-5 Chromarod holder, and each rod is blank 

scanned (from top to bottom at a speed of 60 seconds per scan) one to three times by the 

Iatroscan to remove any organic compounds that are present and “activate” the Chromarod.

The rod holder containing the Chromarods is removed from the Iatroscan and placed onto 

a spotting guide.

The Chromarods are spotted with one sample extract or one calibration standard per rod 

as follows. A 1-μL aliquot is carefully spotted onto a Chromarod near the base of the 

Chromarod using a 2-μL syringe, keeping the spot as small as possible in order to provide good 

separation of the lipid classes and reproducible analyses. Each sample extract is analyzed in 

duplicate. Also, within each holder of 10 Chromarods, one Chromarod is spotted with a 

continuing calibration check standard (one of CSs Levels 1–5) to be analyzed concurrently with 

sample extracts.

The rod holder containing the Chromarods is placed in the TLC development tank for 

exactly 24 minutes to separate the various lipid classes listed in Section 1 (wax esters/sterol 

esters, triglycerides, free fatty acids, cholesterol, and phospholipids/other polar lipids). Then the 

rod holder is removed immediately from the tank, and the remaining solvent is allowed to 

evaporate for 2 minutes in a 60°C oven.
57

The developed Chromarods in the holder proceed to TLC/FID analysis.

9.2. TLC/FID Analysis

The operating conditions for the Iatroscan FID are as follows.

Hydrogen flow rate: 160 mL/min

Air flow rate: 2,000 mL/min

Scan rate: 30 s/scan

The Chromarod holder containing the 10 Chromarods is placed in the Iatroscan FID.

Each Chromarod is sequentially passed through the hydrogen flame to ionize the sample 

extract or standard, and the detected changes in current are recorded by the Empower data 

acquisition system to generate the chromatogram.

The TLC/FID calibration standards are analyzed at least every 4 weeks to generate the 

calibration equations for each lipid class as described below. As noted above, a continuing 

calibration check standard (one of CSs Levels 1–5) is analyzed concurrently with the sample 

extracts within each holder of 10 Chromarods.

9.3. Calculations of Lipid Class Concentrations

The acquired TLC/FID data for the samples and calibration standards are processed using 

the Empower software to determine areas for the lipid classes’ peaks in the samples’ 

chromatograms and for individual compounds’ peaks in the CSs’ chromatograms. The 

identification and integration of the peaks are performed by the Empower software to generate 

the peak areas, and then they are checked for appropriateness and manually reintegrated as 

needed.

The individual compounds used to calibrate or quantitate the corresponding lipid classes

are as follows.

Lipid class Lipid compound used for response factor

Wax esters and sterol esters Lauryl stearate

Triglycerides Triolein

Free fatty acids Oleic acid

Cholesterol Cholesterol

Phospholipids and other polar lipids L-α-phosphatidylcholine

The lipid areas produced by the Empower software are then used by a FileMaker Pro 

database to compute the amounts of each lipid class in the samples (mg lipid) based on point-topoint calibration (equations provided below). The point-to-point calibration method is 

equivalent to plotting the response factors (RFs) of the CSs versus their analyte areas to achieve 

two-dimensional points. Linear equations generated between each pair of consecutive points are 

used for the calibration in order to gain a better fit over the entire range of CSs than would be 

provided by a single regression equation, such as linear, weighted linear, or quadratic regression.
58

For each level of calibration standard (CS), a RF is computed for each lipid as

CS RF = CS Lipid Concentration (mg/mL) (17)

 CS Lipid Area

Next, specific for each sample, a sample RF for each lipid class is computed by 

interpolating between the RFs of the two consecutive calibration standards whose areas for that 

lipid bracket the area of the lipid class in the sample, as

Sample RF = Lower CS RF

+ (Sample Lipid Class Area – Lower CS Lipid Area) × (Upper CS RF – Lower CS RF) (18)

 Upper CS Lipid Area – Lower CS Lipid Area

where Lower CS is the calibration standard whose lipid area is closest to and smaller than that of 

the sample’s lipid class area, and Upper CS is the calibration standard whose lipid area is closest 

to and larger than that of the sample’s lipid class area. If the Sample Lipid Class Area is larger 

than the Level 1 lipid area, then the sample is diluted to get the area within the calibration range 

and the sample is reanalyzed; if the Sample Lipid Class Area is smaller than the Level 6 lipid 

area, then the Sample Lipid Class Area is set to zero.

The concentration of each lipid class in each sample is then calculated in terms of mg 

lipid per g sample wet weight as

mg Lipid Class

g Sample

= Sample RF × Sample Lipid Class Area × (ASE Vial w/Extract Weight − ASE Vial Weight) (19)

 1.33 g/mL × Iatro Extract Concentration Factor × Sample Weight

where 1.33 g/mL is the density of dichloromethane, and if the extract in the Iatro vial is 

concentrated prior to analysis,

Iatro Extract Concentration Factor = Iatro Vial w/Extract Weight − Iatro Vial Weight (20)

 Iatro Vial w/Conc Extract Weight − Iatro Vial Weight

or if the extract in the Iatro vial is not concentrated prior to analysis,

Iatro Extract Concentration Factor = 1 (21)

The percent of each lipid class per sum of all lipid classes is calculated as

Percent Lipid Class = Lipid Class Concentration × 100% (22)

Sum of All Lipid Classes’ Concentrations

Duplicate TLC/FID analyses are performed for each lipid extract, and the mean value is

reported for each lipid class’s Percent Lipid Class.

The limits of quantitation for each of the five lipids contained in the calibration standards 

are the following: 0.63 mg/mL lauryl stearate, 0.63 mg/mL triolein, 0.63 mg/mL oleic acid, 0.13 

mg/mL cholesterol, and 0.13 mg/mL L-α-phosphatidylcholine.
59

9.4. Quality Assurance for Lipid Classes

QA measures are incorporated into the analyses of each batch of samples according to the 

Environmental Chemistry Program Quality Assurance Plan. Specific QA goals for batches and 

individual samples, as well as the percent of the total results that must meet the goals, depend on 

the project for which the samples are analyzed and the purpose of the data. Typical goals are 

presented here. When QA goals are not met, the data may be qualified or the sample(s) 

reanalyzed.

9.4.1. TLC/FID Stability

External standard curves for each lipid class are created at least every 4 weeks. To 

determine whether the Iatroscan TLC/FID is operating properly, a calibration standard will be 

analyzed with each Chromarod rack that consists of 10 Chromarods. If the concentration of a 

lipid class in the calibration standard is not within ±20% of the known concentration of that 

compound, the field samples will be reanalyzed (unless the lipid class is not measured in the 

associated field samples).

9.4.2. Percent Summed Lipid Value for Reference Materials

To monitor the accuracy of the lipid extraction and analyses, the Percent Summed Lipid

value of the SRM determined by the TLC/FID method is compared to the NIST percent 

nonvolatile extractable material value. The accuracy of the NIST SRM result is considered 

acceptable if the Percent Summed Lipid value is within 35% of either end of NIST’s “95% 

confidence interval for the mean” for the NIST percent nonvolatile extractable material value. 

However, for low-lipid containing SRMs (<2%), the TLC/FID value is considered acceptable if 

the Percent Summed Lipid value is within 50% of either end of NIST’s “95% confidence interval 

for the mean” for the NIST percent nonvolatile extractable material value. There are no NIST 

certified values for lipid classes.

9.4.3. Precision of Replicate Samples

When three or more replicate samples are extracted and analyzed, the precision of the 

results can be evaluated based on the RSD of the percentages of each lipid class and the Percent 

Summed Lipid value. The precision is considered acceptable if the RSD is ≤25% for analytes 

with concentrations greater than their LOQ. For duplicate samples, this translates to a relative 

percent difference of ≤50%. This does not apply to the duplicate TLC/FID analyses of the same 

extract.

9.4.4. Method Blank Contamination

The method blank is analyzed to determine whether the extraction solvents, glassware, or 

other supplies were free of lipid contaminants. Our acceptance criterion is that no peak is to be 

detected above the noise level in a method blank.
60

10. Conclusions

The analytical methods presented here resulted from research, development, and 

application by chemists in the Environmental Chemistry Program. These methods will continue 

to be evaluated and upgraded as feasible for improved accuracy, precision, sensitivity, 

robustness, safety, and cost-effectiveness, as well as for minimizing contamination from 

laboratory materials and from sample-to-sample carryover. QA measures are included, which 

are monitored and reported for every batch of samples. Our methods are provided here in detail 

for complete documentation as well as for ease of use by other laboratories.
61

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Recent NOAA Technical Memorandums

published by the

Northwest Fisheries Science Center

NOAA Technical Memorandum NMFS-NWFSC124 Anderson, L.E., and S.T. Lee. 2013. Washington and Oregon saltwater sportfishing surveys: 

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122 Frame, E., and K. Lefebvre. 2013. ELISA methods for domoic acid quantification in multiple marine 

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121 Lian, C.E. 2012. West Coast limited entry groundfish cost earnings survey: Protocol and results for 

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120 Pollock, M.M., J.M. Wheaton, N. Bouwes, C. Volk, N. Weber, and C.E. Jordan. 2012. Working 

with beaver to restore salmon habitat in the Bridge Creek intensively monitored watershed: Design 

rationale and hypotheses. U.S. Dept. Commer., NOAA Tech. Memo. NMFS-NWFSC-120, 47 p. NTIS 

number PB2013-101722.

119 Waples, R.S., K. Hindar, and J.J. Hard. 2012. Genetic risks associated with marine aquaculture. 

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118 Stout, H.A., P.W. Lawson, D.L. Bottom, T.D. Cooney, M.J. Ford, C.E. Jordan, R.G. Kope, 

L.M. Kruzic, G.R. Pess, G.H. Reeves, M.D. Scheuerell, T.C. Wainwright, R.S. Waples, E. Ward, 

L.A. Weitkamp, J.G. Williams, and T.H. Williams. 2012. Scientific conclusions of the status review 

for Oregon coast coho salmon (Oncorhynchus kisutch). U.S. Dept. Commer., NOAA Tech. Memo. 

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Most NOAA Technical Memorandums NMFS-NWFSC are available at the

Northwest Fisheries Science Center Web site, http://www.nwfsc.noaa.gov