U.S. DEPARTMENT OF COMMERCE

National Oceanic and Atmospheric Administration

National Marine Fisheries Service

NOAA Technical Memorandum NMFS-NWFSC-59

Extraction, Cleanup, and

Gas Chromatography/Mass

Spectrometry Analysis

of

Sediments and Tissues for

Organic Contaminants

March 2004
NOAA Technical Memorandum

NMFS Series

The Northwest Fisheries Science Center of the National

Marine Fisheries Service, NOAA, uses the NOAA

Technical Memorandum NMFS series to issue informal scientific and technical publications when complete formal review and editorial processing are not

appropriate or feasible due to time constraints. Documents published in this series may be referenced in the

scientific and technical literature.

The NMFS-NWFSC Technical Memorandum series of

the Northwest Fisheries Science Center continues the

NMFS-F/NWC series established in 1970 by the

Northwest & Alaska Fisheries Science Center, which

has since been split into the Northwest Fisheries

Science Center and the Alaska Fisheries Science

Center. The NMFS-AFSC Technical Memorandum

series is now being used by the Alaska Fisheries

Science Center.

Reference throughout this document to trade names

does not imply endorsement by the National Marine

Fisheries Service, NOAA.

This document should be cited as follows:

Sloan, C.A., D.W. Brown, R.W. Pearce, R.H. Boyer,

J.L. Bolton, D.G. Burrows, D.P. Herman, and M.M.

Krahn. 2004. Extraction, cleanup, and gas chromatography/mass spectrometry analysis of sediments and

tissues for organic contaminants. U.S. Dept.

Commer., NOAA Tech. Memo. NMFS-NWFSC-59,

47 p.
NOAA Technical Memorandum NMFS-NWFSC-59

Extraction, Cleanup, and

Gas Chromatography/Mass

Spectrometry Analysis

of

Sediments and Tissues for

Organic Contaminants

Catherine A. Sloan, Donald W. Brown, Ronald W. Pearce,

Richard H. Boyer, Jennie L. Bolton, Douglas G. Burrows,

David P. Herman, and Margaret M. Krahn

Northwest Fisheries Science Center

Environmental Conservation Division

2725 Montlake Boulevard East

Seattle, Washington 98112

March 2004

U.S. DEPARTMENT OF COMMERCE

Donald L. Evans, Secretary

National Oceanic and Atmospheric Administration

Vice Admiral Conrad C. Lautenbacher, Jr. USN (Ret), Administrator

National Marine Fisheries Service

William T. Hogarth, Assistant Administrator for Fisheries
Most NOAA Technical Memorandums NMFS-NWFSC

are available online at the Northwest Fisheries Science

Center web site (http://www.nwfsc.noaa.gov)

Copies are also available from:

National Technical Information Service

5285 Port Royal Road

Springfield, VA 22161

phone orders (1-800-553-6847)

e-mail orders (orders@ntis.fedworld.gov)

ii
TABLE OF CONTENTS

List of Figures...............................................................................................................................................v

List of Tables ..............................................................................................................................................vii

Executive Summary .....................................................................................................................................ix

Acknowledgments........................................................................................................................................xi

1. Introduction..............................................................................................................................................1

2. Materials ..................................................................................................................................................6

2.1. Instruments and Accessories ............................................................................................................6 

2.1.1. GC/MS System..........................................................................................................................6 

2.1.2. GC Gas and Accessories...........................................................................................................6 

2.1.3. GC Column and Accessories.....................................................................................................6 

2.1.4. HPLC System and Accessories.................................................................................................7 

2.1.5. Sample Extractor and Accessories............................................................................................7 

2.2 Solvents and Reagents.......................................................................................................................7 

2.3 Silica/Alumina Column Packings......................................................................................................8 

2.4 Labware .............................................................................................................................................8 

2.5 Disposable Glassware........................................................................................................................9 

2.6. Standard Solutions and GC Conditioning Solutions ........................................................................9 

2.6.1. Internal Standard Solutions.......................................................................................................9 

2.6.2. Calibration Standards..............................................................................................................10 

2.6.3. Spike Solutions........................................................................................................................10 

2.6.4. GC Conditioning Solutions.....................................................................................................10 

2.7 Standard Reference Materials..........................................................................................................11 

2.8. Preparation of Silica/Alumina Column Packings...........................................................................11 

2.8.1. Silica Preparation ....................................................................................................................11 

2.8.2. Alumina Preparation ...............................................................................................................11 

2.8.3. Copper Preparation..................................................................................................................11 

2.8.4. Sand Preparation .....................................................................................................................11 

2.8.5. Glass Wool Preparation...........................................................................................................12 

2.9. Purity Testing .................................................................................................................................12 

2.9.1. Isooctane Purity Test...............................................................................................................12 

2.9.2. Dichloromethane Purity Test...................................................................................................12 

2.9.3. Sodium Sulfate Purity Test .....................................................................................................13 

2.9.4. Magnesium Sulfate Purity Test...............................................................................................13 

2.9.5. Silica or Alumina Purity Test..................................................................................................13 

3. Sample Extraction..................................................................................................................................15

3.1 Extraction ........................................................................................................................................15 

3.2 Extract Preparation ..........................................................................................................................17 

4. Sample Cleanup .....................................................................................................................................19

4.1. Silica/Alumina Column Chromatography ......................................................................................19 

4.1.1. Calibration of Silica/Alumina Column....................................................................................19 

4.1.2. Sample Cleanup by Silica/Alumina Column Chromatography...............................................20 

4.2. Size-Exclusion High-Performance Liquid Chromatography..........................................................21 

iii
4.2.1. SEC HPLC Start Up................................................................................................................21 

4.2.2. SEC HPLC System Calibration...............................................................................................23 

4.2.3. Daily Calibration of the SEC HPLC System...........................................................................26 

4.2.4. Sample Cleanup by SEC HPLC..............................................................................................27 

5. Gas Chromatography/Mass Spectrometry .............................................................................................29

5.1. GC/MS Operating Conditions........................................................................................................29 

5.2. GC/MS for Quantitating AHs.........................................................................................................29 

5.2.1 Verification of GC/MS Performance for AHs .........................................................................29 

5.2.2. GC/MS Analysis of Sediment Samples for AHs.....................................................................33 

5.2.3. GC/MS Analysis of Tissue Samples for AHs.........................................................................33 

5.3. GC/MS for Quantitating CHs.........................................................................................................33 

5.3.1. Verification of GC/MS Performance for CHs.........................................................................33 

5.3.2. GC/MS Analysis of Sediment Samples for CHs......................................................................37 

5.3.3. GC/MS Analysis of Low-Lipid Tissue Samples for CHs.......................................................37 

5.3.4. GC/MS Analysis of High-Lipid Tissue Samples for CHs.......................................................37 

6. Calculations of Results ..........................................................................................................................38

6.1. Calculation of Analyte Concentrations...........................................................................................38 

6.2. Calculation of Internal Standard Recoveries..................................................................................39 

6.3. Calculating and Reporting Lower Limits of Quantitation..............................................................40 

6.4. Calculating and Reporting Estimated Concentrations for Analytes Exceeding the 

Calibration Range...................................................................................................................................40 

7. Quality Assurance..................................................................................................................................41

7.1. GC/MS Stability .............................................................................................................................41

7.2. Internal Standard Recoveries..........................................................................................................41 

7.3. Reference Material Analyte Concentrations...................................................................................41 

7.4. Precision of Replicate Samples ......................................................................................................42 

7.5. Method Blank Contamination ........................................................................................................42 

8. Gravimetric Determinations...................................................................................................................43

8.1. Dry Weight Determination .............................................................................................................43 

8.2. Total Extractables Determination...................................................................................................43 

9. Conclusions............................................................................................................................................45

10. Citations...............................................................................................................................................47

iv
LIST OF FIGURES 

Figure 1. Flow diagram of sample preparation and analysis. ......................................................................5

Figure 2. Size-exclusion high-performance liquid chromatograph calibration..........................................25

v
vi
LIST OF TABLES 

Table 1. Aromatic hydrocarbon analytes. ....................................................................................................2

Table 2. Chlorinated hydrocarbon analytes including polychlorinated biphenyl (PCB) congeners (by 

IUPAC number).............................................................................................................................3

Table 3. Size-exclusion high-performance liquid chromatography events................................................22

Table 4. Gas chromatograph/mass spectrometer operating conditions......................................................30

Table 5. Gas chromatograph oven temperature program for aromatic hydrocarbons................................31

Table 6. Selected-ion monitoring for quantitating aromatic hydrocarbons in tissue samples. ..................32

Table 7. Gas chromatograph oven temperature program for chlorinated hydrocarbons............................34

Table 8. Selected-ion monitoring for quantitating chlorinated hydrocarbons in tissue and sediment 

samples. .......................................................................................................................................35

vii
viii
EXECUTIVE SUMMARY 

This document describes the analytical methods developed and used by the 

Environmental Conservation Division of the National Marine Fisheries Service’s Northwest 

Fisheries Science Center for the analysis of sediment and tissue samples for nanograms per gram

concentrations of selected aromatic hydrocarbons and chlorinated hydrocarbons. Detailed 

descriptions are presented for sample extraction, cleanup by silica/alumina columns and sizeexclusion 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 determining the percent dry 

weight of tissues and sediments, and for determining the percent total extractable content of

tissues. Information pertaining to the necessary laboratory supplies and instruments is provided. 

In addition, to facilitate use of this document by other laboratories, the step-wise procedures are 

presented in a descriptive manner. 

ix
x
ACKNOWLEDGMENTS 

The authors are pleased to acknowledge the support from John Stein, Northwest Fisheries 

Science Center, and we thank the past and present Environmental Assessment Program chemists 

for their expert contributions to the development and validation of the methods described in this 

document. We also thank Barbara French and Jon Buzitus from the Environmental Conservation 

Division, Northwest Fisheries Science Center, for manuscript review. 

xi
xii
1. INTRODUCTION 

Various environmental studies have involved the analyses of marine sediments and 

tissues for toxic contaminants such as chlorinated pesticides, polychlorinated biphenyls, and 

aromatic hydrocarbons. High quality data with documented quality assurance is needed for such 

analyses in order to draw valid conclusions and reach appropriate decisions; for example, in 

damage assessment situations, 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 Assessment Program of the Environmental Conservation Division of the National 

Marine Fisheries Service’s Northwest Fisheries Science Center (NWFSC) develops and employs 

advanced methods for analyzing marine environmental samples for organic contaminants at partper-billion concentrations. As improvements to procedures are validated, they are incorporated 

into the analytical protocols that are used for a multitude of projects in which selected aromatic 

hydrocarbons (AHs, Table 1) and chlorinated hydrocarbons (CHs, Table 2) are quantitated in 

tissues and sediment. This document includes the latest revisions to the methods described in 

Sloan et al. (1993) and Krahn et al. (1988), which updated the methods of MacLeod et al. (1985). 

Detailed descriptions of our current procedures are provided for complete documentation as well 

as for ease of use by other laboratories. 

Several changes have been made to the earlier methods to improve the analyses of 

samples, which involve three steps: 1) extraction, 2) cleanup by silica/alumina columns and sizeexclusion high-performance liquid chromatography (SEC HPLC), and 3) quantitation of CHs 

and AHs by gas chromatography/mass spectrometry (GC/MS). One of the major procedural 

modifications is the extraction of samples using an Accelerated Solvent Extractor (ASE), which 

reduces time, labor, solvent use, hazardous waste, and potential exposure of analysts to 

extraction solvent (in our case, dichloromethane) compared to our previous method. This ASE 

method provides an exhaustive extraction of organic compounds from various matrices while 

excluding water; thus both AHs and CHs are quantitatively recovered in a single extract, and the 

percent total extractables (lipids) in tissues can be gravimetrically determined from a portion of 

the ASE extract. Another major modification is the use of GC/MS selected-ion monitoring 

(SIM) (Burrows et al. 1990) in place of GC/electron-capture detection for CH analyses; thus the 

concentrated, cleaned-up extracts are analyzed for both AHs and CHs (separately) using the 

same GC system and configuration, as well as similar operating conditions. As MS 

instrumentation has improved, the sensitivity of GC/MS SIM for CH detection has approached 

that of electron-capture detection while being more selective, thus reducing interferences. 

Alterations made to the GC/MS system that allow greater instrument stability and accuracy 

include replacing the electron ionization filaments with chemical ionization filaments in order to 

use a higher source temperature, using a cool on-column injection system in the GC in place of a 

splitless injection system, and adding a guard column before the analytical column. Quantitation 

has also been optimized by using point-to-point calibration, which provides a better data fit over 

the entire range of GC/MS calibration standards than would be given by a single equation, such 

as linear, weighted linear, or quadratic regression. 
Table 1. Aromatic hydrocarbon analytes. 

Naphthalene 

1-Methylnaphthalene 

2-Methylnaphthalene 

Biphenyl 

2,6-Dimethylnaphthalene 

Acenaphthylene 

Acenaphthene 

2,3,5-Trimethylnaphthalene

Fluorene 

Dibenzothiophene 

Phenanthrene 

Anthracene 

1-Methylphenanthrene 

Fluoranthene

Pyrene 

Retene 

Benz[a]anthracene 

Chrysene + Triphenylene *

Benzo[b]fluoranthene 

Benzo[j]fluoranthene + Benzo[k]fluoranthene *

Benzo[e]pyrene 

Benzo[a]pyrene 

Perylene 

Indeno[1,2,3-cd]pyrene 

Dibenz[a,h]anthracene + Dibenz[a,c]anthracene *

Benzo[ghi]perylene 

*

 These analytes are quantitated and reported as the sum of their concentrations 

because they co-elute during GC/MS analysis. 

2
Table 2. Chlorinated hydrocarbon analytes including polychlorinated biphenyl (PCB) congeners 

(by IUPAC number). 

PCBs Other chlorinated hydrocarbons

PCB 17 2,4'-DDD 

PCB 18 4,4'-DDD 

PCB 28 2,4'-DDE 

PCB 31 4,4'-DDE 

PCB 33 2,4'-DDT 

PCB 44 4,4'-DDT 

PCB 49 Hexachlorobenzene 

PCB 52 alpha-Hexachlorocyclohexane 

PCB 66 beta-Hexachlorocyclohexane 

PCB 70 gamma-Hexachlorocyclohexane 

PCB 74 Aldrin 

PCB 82 Dieldrin 

PCB 87 Endosulfan I

PCB 95 Endosulfan II

PCB 99 Endosulfan sulfate 

PCB 101 + PCB 90 * Mirex 

PCB 105 Heptachlor 

PCB 110 Heptachlor epoxide 

PCB 118 Oxychlordane

PCB 128 trans-Chlordane 

PCB 138 + PCB 163 + PCB 164 * cis-Chlordane 

PCB 149 trans-Nonachlor 

PCB 151 cis-Nonachlor 

PCB 153 + PCB 132 * Nonachlor III 

PCB 156 

PCB 158 

PCB 170 + PCB 190 *

PCB 171 

PCB 177 

PCB 180 

PCB 183 

PCB 187 + PCB 159 + PCB 182 *

PCB 191 

PCB 194 

PCB 195 

PCB 199 

PCB 205 

PCB 206 

PCB 208 

PCB 209 

*

 These analytes are quantitated and reported as the sum of their concentrations because they co-elute during

GC/MS analysis. 

3
The sample preparation and analyses are summarized in Figure 1. Sediment and tissue 

samples are mixed with sodium sulfate and magnesium sulfate, then extracted using an ASE. 

Extracts of tissues, such as marine mammal tissues, may be split into two portions: one for 

isolation and quantitation of the analytes, and one for determining the percent total extractables

(i.e., mostly lipids). The portions of extracts to be analyzed for AHs, CHs, or both are filtered 

through gravity-flow silica/alumina columns. The filtered sample extracts are then concentrated, 

and a portion of each filtered extract is chromatographed on a SEC HPLC column, using 

dichloromethane as the mobile phase, to collect a fraction containing AHs and CHs. The solvent 

is “exchanged” to isooctane while the fraction is reduced to a final volume of approximately 

100 μL. The concentrated AH/CH fraction is then analyzed by GC/MS SIM to measure AHs, 

CHs, or both. Analytes are quantitated relative to extraction internal standards (also known as 

surrogate standards) using multiple concentration levels of GC/MS calibration standards. The 

concentrations of analytes in the samples are reported on a nanogram (ng) per gram (g) wet 

weight or dry weight basis. 

Samples are typically analyzed in batches of 12 to 14 samples. Quality assurance (QA) 

measures are incorporated into each batch according to the Environmental Assessment Program

Quality Assurance Plan (NMFS unpubl. data). Each batch of samples includes a method blank, a 

sample of a Standard Reference Material that has certified concentrations for many analytes, and 

as needed, replicate samples. Internal standards are added to the samples before extraction to 

monitor and account for any losses during sample preparation. HPLC internal standards are 

added before sample cleanup to account for the amount of total extract that is chromatographed 

by HPLC, and GC/MS internal standards are added before GC/MS to measure the recovery of 

the extraction and the HPLC internal standards. Typical goals for QA measurements are 

provided in Section 7. 

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

vapors, and personal protective equipment (e.g., rubber gloves, face shield, rubber apron) is worn

by the analyst whenever concentrated acids are used. Additional laboratory safety practices are 

followed according to the NWFSC Chemical Hygiene Plan and the NWFSC Chemical Waste 

Management Guide. Analysts at other institutions would follow safety practices according to 

their in-house chemical hygiene and chemical waste management guidelines. 

4
Mix a portion of sample with sodium 

sulfate and magnesium sulfate, then 

extract with dichloromethane using 

Accelerated Solvent Extraction 

Dichloromethane extract of sediment or tissue

For tissue extracts as needed, take 

1/2 of extract and evaporate the 

solvent; gravimetrically determine 

the percent total extractables

Chromatograph 1/2 of the concentrated extract using size-exclusion HPLC; 

collect the AH/CH fraction, then concentrate it to 100 μL

Concentrated AH/CH fraction

Analyze fraction using GC/MS for 

AH quantitation, as needed

Analyze fraction using GC/MS for 

CH quantitation, as needed

Filter extract through a silica/alumina 

column, then concentrate it to 1 mL

Concentrated extract

As needed, dry a portion of sample; 

gravimetrically determine the 

percent dry weight

Sediment or tissue sample

 Figure 1. Flow diagram of sample preparation and analysis. 

5
2. MATERIALS 

As a convenience, equipment lists in Subsection 2.1 through Subsection 2.5 include 

names and locations of suppliers. These are not necessarily the preferred or sole suppliers. 

When no supplier is listed, the item is considered widely available. 

2.1. Instruments and Accessories 

2.1.1. GC/MS System 

• Autosampler, Agilent 7683, with on-column injection syringe and needle guide. Agilent 

Technologies, Wilmington, DE 

• ChemStation software, Version DA. Agilent Technologies, Wilmington, DE 

• Gas chromatograph with cool on-column injection, Agilent 6890N. Agilent Technologies, 

Wilmington, DE 

• Mass Selective Detector, Agilent 5973N. Agilent Technologies, Wilmington, DE 

2.1.2. GC Gas and Accessories 

• Helium, grade 5, ultra-high purity, 99.999% 

• Indicating moisture trap, GMT-4-HP. Agilent Technologies, Wilmington, DE 

• Indicating oxygen trap, 4004. Alltech Associates, Inc., Deerfield, IL 

• Oxygen trap, disposable. 803088. Supelco, Inc., Bellefonte, PA 

• Regulator, two-stage, 400 series 

• Regulator, one-stage, 400 series 

2.1.3. GC Column and Accessories 

• Capillary column cleaving tool, 2-3740m. Supelco Inc., Bellefonte, PA 

• Column nut, GC inlet, 5181-8830. Agilent Technologies, Wilmington, DE 

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

• Column union, glass, universal, 705-0825. Agilent Technologies, Wilmington, DE 

• Ferrule for inlet end of guard column, 5062-3538. Agilent Technologies, Wilmington, DE 

• Ferrule for MS end of GC column, 15% graphite:85% Vespel, 1/16", 100/0-VG1, no-hole, 

drilled in-house. Alltech Associates, Inc., Deerfield, IL 

• Guard column, deactivated fused-silica, Agilent, 10-m x 0.53-mm, 460-2535-10. Agilent 

Technologies, Wilmington, DE 

6
• GC column, fused-silica, J & W Scientific DB-5, 60-m x 0.25-mm, 0.25-μm film thickness, 

122-5062. Agilent Technologies, Wilmington, DE 

• Gas leak detector, Leak Detective II, 20413. Restek Corporation, Bellefonte, PA 

• Sealing resin for column union, polyimide, 500-1200. Agilent Technologies, 

Wilmington, DE 

2.1.4. HPLC System and Accessories 

• Autosampler, Waters 717 plus. Waters, Milford, MA 

• Data system, Dynamax DA, Version 1.2B4. Rainin Instrument Co., Woburn, MA 

• Filter, in-line after autosampler, 2-μm particle size, 7302. Rheodyne, Inc., Cotati, CA

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

• Helium for solvent degassing, Grade 5.0, 99.999%. PraxAir, Danbury, CT 

• Helium sparger for solvent reservoir, 10-μm, 25312. Restek, Bellefonte, PA 

• Hydrocarbon trap for helium line, Big Supelco Carb HC, 24564. Supelco, Bellefonte, PA 

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

• Oxygen trap for helium line, Supelco Pure O, 2-2449. Supelco, Bellefonte, PA 

• HPLC pump, 515. Waters, Milford, MA 

• Size-exclusion column, Envirosep ABC, 350 mm x 21.2 mm. Phenomenex, Inc., 

Torrance, CA

• Size-exclusion guard column, Envirosep ABC, 60 mm x 21.2 mm. Phenomenex, Inc., 

Torrance, CA

• Solvent inlet filter for solvent reservoir, 10-μm, 59277. Supelco, Bellefonte, PA 

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

• Valve, six-port, 7030. 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. PraxAir, Danbury, CT 

2.2 Solvents and Reagents

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

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

• Dichloromethane (CH2Cl2), High Purity, 300-4. Burdick & Jackson, Muskegon, MI 

7
• Hydrochloric acid (HCl), concentrated, reagent grade, A144-212. Fischer Scientific, Fair 

Lawn, NJ 

• Isooctane (C8H16), Optima, 0301-4. Fischer Scientific, Fair Lawn, NJ 

• Magnesium Sulfate (MgSO4), anhydrous, M65-3. Fischer Scientific, Fair Lawn, NJ 

• Methanol (CH3OH), Optima, A454-4. Fischer Scientific, Fair Lawn, NJ 

• Nitric acid (HNO3), concentrated, reagent grade, A200-500. Fischer Scientific, Fair 

Lawn, NJ 

• Sodium sulfate (Na2S04), reagent grade, anhydrous, granular, S1461. Spectrum, 

Gardena, CA 

2.3 Silica/Alumina Column Packings 

• Alumina, 80-200 mesh, O537-01. J.T. Baker, Phillipsburg, NJ

• Glass wool, Pyrex 3950, 11-388. Fischer Scientific, Fair Lawn, NJ 

• Sand, Ottawa, kiln-dried, 30-40 mesh, SX0075-3. EMD Chemicals, Gibbstown, NJ 

• Silica, 100-200 mesh, Davisil Grade 634, S734-1. Fischer Scientific, Fair Lawn, NJ 

2.4 Labware 

All glassware that contacts samples or reagents is heated in a muffle furnace at 700°C for 

18 hours or rinsed with acetone before use. Other labware that contacts samples or reagents is 

rinsed with acetone before use. 

• ASE glass fiber filters, 047-017. Dionex, Salt Lake City, UT 

• Balances, analytical 

• Boiling chips, Teflon, AW0919120. All-World Scientific, Lynnwood, WA 

• Desiccator with desiccant 

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

• Muffle furnace 

• Ovens, drying, 50°C, 120°C, and 170°C 

• Steam table 

• Teflon sheeting, 2-mil thickness, for lining caps and lids 

• Tube heater, 720000-0000, with glass-cylinder shroud and wire tube holder, aluminum 

inserts bored out to accepted 50-mL conical tubes. Kontes, Vineland, NJ 

• Vial heater, column heater SP 8792 modified to hold 2-mL GC vials with a rack that allows 

the solvent level in the vials to be seen. Spectra-Physics, San Jose, CA 

• Volume static eliminator, VSE 3000. Chapman, Hatfield, PA 

8
• Weighing pans, aluminum, disposable, 43-mm i.d., 180-5537. All-World Scientific, 

Lynnwood, WA 

• Weighing pans, aluminum, disposable, 75-mm i.d., 25433-020. VWR, Brisbane, CA 

2.5 Disposable Glassware 

Items are one-use only. 

• ASE collection vials, 60-mL with cap and septa. Dionex, Salt Lake City, UT 

• Bottles, amber, 250-mL, Boston round, 28-mm mouth, with Teflon-lined plastic caps 

• Chromatography column, plain, custom made, 22-mm i.d. x 25-cm length. DJ’s Glass 

Factory, San Jose, CA 

• Jars, 10-ounce, 2.5-inch i.d. mouth. Aaron Packaging, Kent, WA

• Pipettes, transfer, Pasteur-style 

• Tubes, centrifuge, 50-mL, 18-mm i.d. mouth, conical with Teflon-lined plastic caps 

• Vials, GC, 2-mL, C4000-1W, with caps and septa, C4000-54R, and 200 μL inserts, C4010-

627L. National Scientific Co., Duluth, GA 

• Vials with caps and septa, HPLC, 4-mL, 99300-A, and 750-μL inserts, 200756. Sun 

International, Wilmington, NC 

2.6. Standard Solutions and GC Conditioning Solutions 

All solutions are prepared in-house using isooctane as the solvent, except the HPLC 

retention-time standard, which is prepared using dichloromethane. Concentrations given in 

parentheses are approximate; actual concentrations will vary slightly from batch to batch of the 

solution. The GC calibration standards and the internal standards, including GC and HPLC 

internal standards, are prepared such that the concentrations of the internal standards in final 

sample extracts analyzed by GC/MS are approximately equal to the concentrations of the internal 

standards in the GC calibration standards. GC conditioning solutions are used to simulate tissue 

or sediment samples during GC/MS performance verification and as GC/MS “warm up” samples 

at the beginning of a sequence of analyses. 

2.6.1. Internal Standard Solutions 

• CH I-Std (internal standard for quantitating CH analytes)—contains PCB 103 at 1 ng/μL

• CH HPLC I-Std (HPLC internal standard for CHs)—contains tetrachloro-m-xylene (TCMX) 

at 1 ng/μL 

• CH GC I-Std (GC internal standard for CHs)—contains tetrachloro-o-xylene (TCOX) at 

1 ng/μL 

9
• AH I-Std (internal standard for quantitating AH analytes)—contains naphthalene-d8, 

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

• AH HPLC I-Std (HPLC internal standard for AHs)—contains phenanthrene-d10 at 1.6 ng/μL 

• AH GC I-Std (GC internal standard for AHs)—contains hexamethylbenzene (HMB) at 

10 ng/μL 

2.6.2. Calibration Standards 

• AH/CH HPLC retention-time standard—contains 4,4'-dibromooctafluorobiphenyl and 

perylene at 1.5 ng/μL each compound 

• CH GC calibration standards—contain the CHs in Table 2 (except Nonachlor III); not all 

compounds are contained in all levels; 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 1.0 ng/μL each compound, Level 8 at 4.0 ng/μL each compound, Level 9 at 10 

ng/μL each compound, Level 10 at 20 ng/μL each compound, Level 11 at 100 ng/μL each 

compound, plus all levels contain PCB 103, TCMX, and TCOX at 0.3 ng/μL each compound 

• AH GC calibration standards—contain the AHs in Table 1 (except Retene); Level 1 at 

0.0011 ng/μL each compound, Level 2 at 0.0044 ng/μL each compound, Level 3 at 0.015 

ng/μL each compound, Level 4 at 0.044 ng/μL each compound, Level 5 at 0.11 ng/μL each 

compound, Level 6 at 0.33 ng/μL each compound, Level 7 at 1.13 ng/μL each compound, 

Level 8 at 3.3 ng/μL each compound, and Level 9 at 10.5 ng/μL each compound, plus all 

levels contain naphthalene-d8, acenaphthene-d10, and benzo[a]pyrene-d12, phenanthrened10 at 0.5 ng/μL each compound and HMB at 3.2 ng/μL in all levels 

2.6.3. Spike Solutions 

• CH Spike solution—contains the CHs in Table 2 (except Nonachlor III); 0.3 ng/μL each 

compound 

• AH Spike solution—contains the AHs in Table 1 (except Retene); 7 ng/μL each compound 

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 Sections 3 and 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 Sections 3 

and 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 

Sections 3 and 4, in isooctane 

10
2.7 Standard Reference Materials 

Standard Reference Materials (SRM) are obtained from the National Institute of

Standards and Technology (NIST), Gaithersburg, Maryland. 

• SRM 1974b Organics in Mussel Tissue (Mytilus edulis)—analyzed for quality assurance 

measurements in batches of low-lipid tissue samples 

• SRM 1941b Organics in Marine Sediment—analyzed for quality assurance measurements in 

batches of sediment samples 

• SRM 1945 Organics in Whale Blubber—analyzed for quality assurance measurements in 

batches of high-lipid tissue samples 

2.8. Preparation of Silica/Alumina Column Packings 

2.8.1. Silica Preparation 

The silica is activated by heating it at 700°C for 18 hours, then stored at 170°C. It is allowed to 

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

2.8.2. Alumina Preparation 

The alumina is activated by heating it at 120°C for 2 hours. It is allowed to cool to room 

temperature in a desiccator just prior to use. 

2.8.3. Copper Preparation 

Less than one hour before use, the copper is activated by 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 each with water, methanol, and dichloromethane, in that order. The 

copper is stored covered with dichloromethane until use to avoid contact with air. 

2.8.4. Sand Preparation 

The sand is soaked overnight in a 1:3 v:v mixture of concentrated nitric acid:concentrated 

hydrochloric acid. The sand is washed three times each with water, methanol and 

dichloromethane, in that order, then dried, and stored at 120°C. It is allowed to cool to room

temperature in a desiccator just prior to use. 

11
2.8.5. Glass Wool Preparation 

The glass wool is prepared for use by heating it at 400°C for 18 hours. It is stored in a covered 

glass container at room temperature until use. 

2.9. Purity Testing 

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

Duplicate samples of the new lot of reagent are analyzed along with duplicate samples of the lot 

of reagent currently in use, for comparison. For solid reagents, duplicate 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 glassware and 

equipment contacting the sample are heated in a muffle furnace or rinsed with acetone before 

use. 

2.9.1. Isooctane Purity Test 

AH GC I-Std (30 μL), CH GC I-Std (30 μL), and isooctane (40 μL) are added to a labeled GC 

vial with an insert, and the sample is mixed thoroughly. 

The sample and GC calibration solutions are analyzed for AHs and CHs 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. 

2.9.2. Dichloromethane Purity Test 

Dichloromethane (200 mL) is added to a 250-mL bottle. Five boiling chips are added to the 

bottle, and using the steam table, the sample volume is reduced to 15–20 mL. 

The sample is transferred to a 50-mL tube. No dichloromethane washes are used. A boiling 

chip is added to the tube, and using the tube heater, the sample is concentrated to approximately 

1 mL. 

AH GC I-Std (30 μL), CH GC I-Std (30 μL), and isooctane (40 μL) are added to the sample, and 

the sample is mixed thoroughly. 

The sample is transferred to a labeled GC vial, a small boiling chip is added, and the sample is 

concentrated to 100 μL using the vial heater. 

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

The sample and GC calibration solutions are analyzed for AHs and CHs using GC/MS SIM as 

described in Section 5 for low-lipid tissues. The GC/MS responses of any impurities that are 

12
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. 

A boiling chip is added to the vial containing the sample, and the sample is concentrated to 

approximately 1 mL using the tube heater. 

AH GC I-Std (30 μL), CH GC I-Std (30 μL), and isooctane (40 μL) are added to the sample, and 

the sample is mixed thoroughly. 

The sample is transferred to a labeled GC vial, a small boiling chip is added, and the sample is 

concentrated to 100 μL using the vial heater. 

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

The sample and GC calibration solutions are analyzed for AHs and CHs 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 down with a glass rod, then adding alumina (10 cc), 

followed by silica (20 cc), and sand (5 cc). 

A solution of methanol (10% by volume) in dichloromethane (35 mL) 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. The tip of the column is then rinsed with dichloromethane. 

A labeled 250-mL bottle is placed under the column. 

13
Dichloromethane (100 mL) is added to the column and allowed to drain into the bottle. 

The sample is concentrated to 15–20 mL using a steam table, then transferred to a labeled 50-mL

tube. 

A boiling chip is added to the tube and the sample is concentrated to approximately 1 mL using a 

tube heater. 

AH GC I-Std (30 μL), CH GC I-Std (30 μL), and isooctane (40 μL) are added to the sample, and 

the sample is mixed thoroughly. 

The sample is transferred to a labeled GC vial, a small boiling chip is added, and the sample is 

concentrated to 100 μL using the vial heater. 

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

The sample and GC calibration solutions are analyzed for AHs and CHs 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. 

14
3. SAMPLE EXTRACTION 

Sediments or tissues are customarily analyzed in batches of 12 to 14 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, one or more replicate sediment or tissue samples. 

Typically, the amount of sediment or low-lipid tissue analyzed is 1 to 2.5 g, depending on the 

expected level of contamination. The amount of high-lipid tissue analyzed is typically 1 g. If 

only one of the two classes of analytes (CHs or AHs) is to be quantitated in the samples, all the 

internal standards and the I-Std check solution for the other class of analytes should be omitted. 

The volume static eliminator is used on the balances whenever weighing items, in order to 

reduce weighing errors caused by static charge. 

3.1 Extraction 

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

Samples are prepared depending on the matrix type as follows: 

• 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 the original sample container. 

• For a batch of tissue samples requiring Total Extractables Determination (Subsection 8.2), 

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. 

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. 

15
The sample/drying agent mixture is transferred to the ASE cell using the ASE funnel. The 

bottom of the cell is tapped firmly but carefully on the counter top during the transfer 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 counter top 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 without overlapping the 

rim of the cell. 

The procedural steps are performed depending on the type of sample batch as follows: 

• For a batch of tissue samples requiring Total Extractables Determination

o AH I-Std (150 μL) and CH I-Std (150 μL) are added onto the top filter in the cell, then 

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

internal standards into the cell. 

o The cell threads are cleared of sample/drying agent as necessary, and the cell is capped 

firmly but not forcefully. 

o An AH I-Std check solution is prepared by adding isooctane (50 μL) and AH I-Std (150 

μL) to a labeled GC vial. 

o A CH I-Std check solution is prepared by adding isooctane (50 μL) and CH I-Std (150 

μL) to a labeled GC vial. 

• For a batch of tissue or sediment samples not requiring Total Extractables Determination 

o AH I-Std (75 μL) and CH I-Std (75 μL) are added onto the top filter in the cell, then 

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

internal standards into the cell. 

o The cell threads are cleared of sample/sulfate as necessary, and the cell is capped firmly 

but not forcefully. 

o An AH I-Std check solution is prepared by adding isooctane (25 μL) and AH I-Std (75 

μL) to a labeled GC vial. 

o A CH I-Std check solution is prepared by adding isooctane (25 μL) and CH I-Std (75 μL) 

to a labeled GC vial. 

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 below. The method parameters as set for the 

Accelerated Solvent Extractor would appear on screen as follows: 

16
PREHEAT [0] min PRESSURE [2000] 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 has finished, the ASE collection vial is removed from the ASE carousel. 

3.2 Extract Preparation 

The procedural steps are performed depending on the type of sample batch as follows: 

• For a batch of tissue samples requiring Total Extractables Determination

o AH HPLC I-Std (150 μL) and CH HPLC I-Std (150 μL) are added to the ASE collection 

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

o AH HPLC I-Std (150 μL) is added to the AH I-Std check solution, which is then mixed 

thoroughly. 

o CH HPLC I-Std (150 μL) is added to the CH I-Std check solution, which is then mixed 

thoroughly. 

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

the “ASE Vial w/Extract” weight. 

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

o The pan is placed in a drying oven at 120°C overnight, then cooled in a desiccator for 30 

minutes. 

o The weighing pan is weighed to the nearest 0.0001 g, and the weight is recorded as the 

“Pan” weight. 

o One half of the sample in the ASE collection vial is transferred to the pan by pressing a 

spatula against the rim of the ASE vial and pouring the sample down the spatula into the 

pan. The ASE vial is capped immediately afterward. 

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

the “ASE vial w/o TE Extract” weight. 

o The pan containing half of the sample proceeds to Total Extractables Determination as 

described in Subsection 8.2, and the remaining sample in the ASE collection vial 

proceeds to Sample Cleanup by Silica/Alumina Column Chromatography as described in 

Subsection 4.1.2. 

• For a batch of tissue or sediment samples not requiring Total Extractables Determination 

o AH HPLC I-Std (75 μL) and CH HPLC I-Std (75 μL) are added to the ASE collection 

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

o AH HPLC I-Std (75 μL) is added to the AH I-Std check solution, which is then mixed 

thoroughly. 

17
o CH HPLC I-Std (75 μL) is added to the CH I-Std check solution, which is then mixed 

thoroughly. 

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

Column Chromatography as described in Subsection 4.1.2. 

18
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 (55 mL), CH I-Std (60 μL), 

AH I-Std (60 μL), CH Spike (80 μL), and AH Spike (60 μL) to a labeled 250-mL bottle. 

A CH analyte check solution is prepared by adding CH Spike (80 μL) and CH I-Std (60 μL) to a 

labeled GC vial. 

An AH analyte check solution is prepared by adding AH spike (80 μL) and AH I-Std (60 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 sand (5 cc). 

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

column and allowed to drain into a waste container. 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 250-mL bottle labeled “F1” is placed under the column. The spiked blank is slowly 

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

The first bottle, 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 bottle. This step is repeated twice more. 

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

The bottle containing the F1 fraction is then set aside. 

A 50-mL tube labeled “F2” is placed under the column. Dichloromethane (10 mL) is added to 

the column and the eluant is collected in the tube. The tube containing the F2 fraction is then 

set aside. 

19
Five boiling chips are added to the bottle containing the F1 fraction and the F1 fraction is 

concentrated to 15–20 mL using a steam table. 

The F1 fraction is transferred to a labeled 50-mL tube. 

The bottle is washed with dichloromethane (approximately 5 mL) and the washings are added to 

the tube. This step is repeated twice more. 

A boiling chip is added to the tubes containing the F1 and F2 fractions and each fraction is 

concentrated to approximately 1 mL using a tube heater. 

AH GC I-Std (60 μL), CH GC I-Std (60 μL), and isooctane (80 μL) are added to each fraction, 

which is then mixed thoroughly. 

CH GC I-Std (60 μL) is added to the CH analyte check solution, which is then mixed thoroughly. 

AH GC I-Std (60 μL) is added to the AH analyte check solution, which is then mixed thoroughly. 

Each fraction is transferred to a labeled GC vial, a small boiling chip is added, and the fraction is 

concentrated to 200 μL using the vial heater. 

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

Each fraction and analyte check solution is analyzed for AHs and CHs 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 analyte check solutions. The analytes and internal standards 

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

volume of dichloromethane for eluting the AH/CH 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 

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 sand (5 cc). 

The column packing is slightly deactivated for better AH recovery by slowly adding a solution of 

methanol (10% by volume) in dichloromethane (35 mL) to the column and allowing the solvent 

to drain into a waste container. 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 labeled 250-mL bottle is placed under the column. The sample extract in the ASE collection 

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

20
The inside of the vial is washed with dichloromethane (approximately 5 mL), the washings 

are decanted into the column, and the eluant is collected in the bottle. This step is repeated 

twice more. 

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

Five boiling chips are added to the bottle containing the sample eluant and the sample volume is 

reduced to 15–20 mL using a steam table. 

The sample is transferred to a labeled 50-mL centrifuge tube. 

The inside of the bottle is washed with dichloromethane (approximately 5 mL) and the washings

are added to the tube. This step is repeated twice more. 

A boiling chip is added to the tube and the sample volume is reduced to approximately 1 mL

using a tube heater. 

• For a batch of sediment samples, activated copper is added to the tube a few grains at a time 

until no further discoloring of the copper occurs. The tube is capped and stored overnight. 

The sample is transferred to a GC vial (leaving the copper in the tube in the case of sediment 

samples), and the volume brought to 1.0 mL either by adding dichloromethane or by evaporating 

the excess under a gentle stream of nitrogen gas. 

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

4.2. Size-Exclusion High-Performance Liquid Chromatography 

SEC HPLC is used for removing extraneous high molecular weight compounds from the 

sample. The HPLC is programmed according to Table 3. 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. SEC 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 approximately 10 minutes prior to SEC 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, then 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. 

21
Table 3. Size-exclusion high-performance liquid chromatography events. 

Approximate time (min) HPLC module Activity 

<0 Autosampler Sample loop loaded with 500 μL of 

sample or calibration standard 

0 Autosampler Sample injected; signal sent to 

computer to start data system 

0–24 Pump Isocratic elution, 5 mL/min 

1 Autosampler Injection port rinsed; needle rinsed 

14–19 Fraction collector Fraction collected 

24 Computer Data system ends integration 

22
The pump is started with the flow rate of the solvent set at 0.75 mL/min for purging the 

autosampler, then the autosampler purge mode is started. If the compression test fails, the 

autosampler purge is repeated. 

The pump is stopped, then the flow is set to 5 mL/min and the pump is started. The pump is 

monitored for stable system pressure. 

The wavelength on the detector is set at 254 nm, and the baseline of the UV signal is zeroed. 

The fraction collector is set for no collection. 

A 4-mL vial is filled with AH/CH HPLC retention-time standard and placed in the autosampler 

carousel. 

The autosampler is programmed and initiated so that the AH/CH HPLC retention-time standard 

is injected and chromatographed according to Table 3, except that no fraction is collected. 

The AH/CH HPLC retention-time 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 in the first analysis of the AH/CH HPLC retention-time standard. 

For each analysis of the calibration 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 calibration standard have stabilized 

for three consecutive analyses, the system is ready to perform the system calibration described in 

Subsection 4.2.2, or the daily calibration described in Subsection 4.2.3. 

The autosampler program is stopped, and the vial of AH/CH HPLC retention-time standard is 

removed from the autosampler tray, recapped, and set aside until needed for the daily calibration 

of the SEC HPLC system as described in Subsection 4.2.3. 

4.2.2. SEC HPLC System Calibration 

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

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

The SEC HPLC is started and the AH/CH HPLC retention-time standard is chromatographed 

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

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 10 consecutive, 0.1-minute fractions 

starting at the retention time of PER. 

23
The fraction collector is configured with a tray for 2-mL GC vials. Twenty 2-mL GC vials 

labeled F1 through F20 are positioned in the fraction collector tray. 

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

AH/CH HPLC (100 μL) retention-time standard to a 4-mL HPLC vial and thoroughly mixing the 

contents of the vial. 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 3, 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 remaining HPLC system calibration solution is removed from the autosampler and discarded 

appropriately. The GC vials containing fractions F1 through F20 are removed from the fraction 

collector tray. 

CH GC I-Std (30 μL) and isooctane (70 μL) are added to each of the vials containing fraction F1 

through F10, and the contents of each vial are mixed thoroughly. 

AH GC I-Std (30 μL) and isooctane (70 μL) are added to each vial containing fractions F11 

through F20, and the contents of each vial are mixed thoroughly. 

A small boiling chip is added to each vial and each fraction is reduced to 100 μL using a 

vial heater. 

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

The fractions F1 through F10 and the Level 5 CH GC calibration solution are analyzed by 

GC/MS for CHs as described in Section 5. The CH GC I-Std 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, which indicates the start of the DOB elution from the SEC HPLC 

system. 

The starting time (t1) of the DOB elution from the SEC HPLC system is determined as the 

starting time for the collection of the first fraction in which DOB appears. 

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 2). 

24
Figure 2. Size-exclusion high-performance liquid chromatograph calibration. (DOB is 

4,4'-dibromooctafluorobiphenyl, PER is perylene, and RT is retention time.) 

25
The value for t3 is recorded for future use in the daily calibration of the SEC HPLC (Subsection 

4.2.3.). 

The fractions F11 through F20 and the Level 3 AH GC calibration solution are analyzed by 

GC/MS SIM for AHs as described in Section 5. The AH GC I-Std 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 F20 are inspected to find the last fraction 

in which PER appears, which indicates the end of the PER elution from the SEC HPLC system. 

The ending time (t5) of the PER elution from the SEC HPLC system is determined as the ending 

time for the collection of the last fraction in which PER appears. 

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. 

The value for t7 is recorded for future use in the daily calibration of the SEC HPLC 

(Subsection 4.2.3.). 

4.2.3. Daily Calibration of the SEC HPLC System 

The daily calibration of the SEC HPLC system is performed immediately before 

chromatographing a batch of samples to determine the collection time of the AH/CH fraction. 

The SEC HPLC is started and the AH/CH HPLC retention-time standard is chromatographed 

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

Collection start and end times for the AH/CH fraction are determined using the retention times of 

DOB and PER from the most recent analysis of the AH/CH HPLC retention-time standard and 

the values t3 and t7 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 an AH/CH fraction is 

t4 = DOB RT – t3 (1) 

where DOB RT is the DOB HPLC retention time in the most recent analysis of the AH/CH 

calibration 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 an AH/CH fraction is 

t8 = PER RT + t7 (2) 

where PER RT is the PER HPLC retention time in the most recent analysis of the AH/CH 

calibration standard during the daily calibration and t7 is the value determined in Subsection 

4.2.2. 

26
4.2.4. Sample Cleanup by SEC HPLC 

A batch of samples is chromatographed immediately following the daily calibration of the SEC 

HPLC system, as described in Subsection 4.2.3. 

The fraction collector is programmed to collect the AH/CH fraction for each sample in the batch, 

using the start and end times determined during the daily calibration of the SEC HPLC system

described in Subsection 4.2.3. The starting and ending times of the collection are recorded in the 

HPLC logbook. 

Labeled 50-mL tubes are placed in the rack of the fraction collector. 

The sample obtained from the silica/alumina column chromatography cleanup described in 

Subsection 4.1.2 is transferred to a labeled 4-mL vial with an insert. 

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 and the AH/CH HPLC retention-time standard are positioned in the 

autosampler tray such that the HPLC blank is chromatographed at the beginning of the 

sequence and the AH/CH HPLC retention-time standard is analyzed in the middle and at the end 

of the batch. 

The autosampler is programmed to sequentially chromatograph the samples in the batch plus the 

AH/CH HPLC retention-time standard in the middle and end of the batch. 

For each analysis of the calibration 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 minutes 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 minutes or more with respect to the retention time for DOB in the 

middle analysis, the collection time for the fraction is changed accordingly for the remaining 

samples in the batch. 

After the sequence of samples and AH/CH HPLC retention-time standard has finished, the SEC 

guard column is backflushed for 2–3 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 back to its 2-mL GC vial and stored 

in a freezer.

A boiling chip is added to the 50-mL tube containing the AH/CH fraction and the fraction 

volume is reduced to approximately 1 mL using a tube heater. 

27
The procedural steps are performed depending on the type of sample matrix as follows: 

• For a batch of tissue or sediment samples to be analyzed for both AHs and CHs, AH GC IStd (30 μL), CH GC I-Std (30 μL), and isooctane (40 μL) are added to the tube and the 

contents mixed thoroughly. 

• For a batch of tissue or sediment samples to be analyzed for AHs only, AH GC I-Std (30 μL) 

and isooctane (70 μL) are added to the tube and the contents mixed thoroughly. 

• For a batch of tissue or sediment samples to be analyzed for CHs only, CH GC I-Std (30 μL) 

and isooctane (70 μL) are added to the tube and the contents mixed thoroughly. 

The procedural steps are performed depending on the type of sample batch as follows: 

• For the check solutions in a batch of tissue samples requiring Total Extractables 

Determination 

o AH GC I-Std (150 μL) is added to the AH I-Std check solution, which is then mixed 

thoroughly. 

o CH GC I-Std (150 μL) is added to the CH I-Std check solution, which is then mixed 

thoroughly. 

• For the check solutions in a batch of tissue or sediment samples not requiring Total 

Extractables Determination 

o AH GC I-Std (75 μL) is added to the AH I-Std check solution, which is then mixed 

thoroughly. 

o CH GC I-Std (75 μL) is added to the CH I-Std check solution, which is then mixed 

thoroughly. 

The AH/CH fraction is transferred from the tube to a labeled GC vial. A small boiling chip is 

added to the vial and the fraction volume is reduced to 100 μL using a vial heater. 

The fraction is transferred to a labeled GC vial with an insert.

The AH/CH fraction and the AH I-Std check solution are analyzed by GC/MS for AHs as 

described in Section 5. In addition, the AH/CH fraction and the CH I-Std check solution are 

analyzed by GC/MS for CHs as described in Section 5. 

28
5. GAS CHROMATOGRAPHY/MASS SPECTROMETRY 

Gas chromatography/mass spectrometry (GC/MS) is used to measure the amounts of the 

analytes and the internal standards in the samples. The GC/MS response to each compound is 

measured in terms of the area of the response. 

5.1. GC/MS Operating Conditions 

The operating conditions for the GC/MS are listed in Table 4.

The helium flows through an indicating moisture trap, an oxygen trap, and an indicating oxygen 

trap installed in-line between the helium regulators and the GC. 

The GC/MS is checked for atmospheric leaks and the MS tune is performed or checked for 

stability prior to analyzing a sequence of samples and standards. 

5.2. GC/MS for Quantitating AHs

The GC oven temperature is programmed according to Table 5. 

Sediment samples are analyzed using scan mode, scanning all masses from 60 to 300 atomic 

mass units. Tissue samples are analyzed using SIM mode, scanning only the quantitation and 

confirmation ions during the specified time window as shown in Table 6. Scan mode provides 

greater ability to confirm analyte identification in the complex sediment samples, whereas SIM 

mode provides greater sensitivity for tissue samples that generally contain lower concentrations 

of contaminants. The quantitation ions for AHs in tissues shown in Table 6 are also the ions 

used for quantitating AHs in sediments. The time windows for SIM mode are adjusted as 

necessary when the GC column is changed or the guard column is trimmed. 

5.2.1 Verification of GC/MS Performance for AHs 

The sensitivity of the GC/MS is checked by analyzing the lowest level of AH GC calibration 

standard that will be used to quantitate AHs in the samples (Level 1 for tissues; Level 3 for 

sediments). 

The stability of the GC/MS is verified by analyzing a sequence of three or more repetitions of the 

mid-level AH GC calibration standard that will be used (Level 4 for tissues; Level 7 for 

sediments). Included in this sequence are repetitions of the appropriate conditioning solution 

(Low-Lipid Tissue Conditioning solution for tissues, or Sediment Conditioning solution for 

sediments), that is, one conditioning solution before each repetition of the mid-level AH GC 

calibration standard. The responses of the analytes relative to the responses of their internal 

standard are compared among the repetitions of the mid-level AH GC calibration standard to 

29
Table 4. Gas chromatograph/mass spectrometer operating conditions. 

Parameter Value

Injection volume 2 μL 

Injection technique cool on-column

Inlet temperature program oven temperature plus 3°C 

Transfer line temperature 300°C 

Carrier gas helium

Carrier gas linear velocity 30 cm/sec 

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 x 10-5 Torr

Ion-source temperature 300°C 

Ionization mode electron ionization 

Filament chemical ionization

Tune mode AHs: Autotune 

CHs: Autotune, then the multiplier voltage is 

increased to achieve the desired sensitivity

Emission current maximum 35 μA 

Scan rate Approximately 10 to 20 scans per peak 

30
Table 5. Gas chromatograph oven temperature program for aromatic hydrocarbons. 

Parameter Value

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 15 min at 300°C 

Total run time 53 min 

31
Table 6. Selected-ion monitoring for quantitating aromatic hydrocarbons in tissue samples. 

Approximate 

time window Compounds 

Quantitation ion, confirmation ion 

(atomic mass units) 

7–12 min Naphthalene-d8 136 

Naphthalene 128, 127

2-Methylnaphthalene 142, 141

1-Methylnaphthalene 142, 141

12–13 min Biphenyl 154, 153 

2,6-Dimethylnaphthalene 156, 141

13–14 min Hexamethylbenzene 147 

Acenaphthylene 152, 151

Acenaphthene-d10 164

Acenaphthene 154, 153

14–17 min 2,3,5-Trimethylnaphthalene 170, 155 

Fluorene 166, 165

17–20 min Dibenzothiophene 184, 139 

Phenanthrene-d10 188

Phenanthrene 178, 176

Anthracene 178, 176

20–29 min 1-Methylphenanthrene 192, 191 

Fluoranthene 202, 101

Pyrene 202, 101

Retene 234, 219

29–42 min Benz[a]anthracene 228, 226

Chrysene + Triphenylene 228, 226 

Benzo[b]fluoranthene 252, 250

Benzo[j+k]fluoranthenea 252, 250 

Benzo[e]pyrene 252, 250

Benzo[a]pyrene-d12 264

Benzo[a]pyrene 252, 250

Perylene 252, 250

42–53 min Indeno[1,2,3-cd]pyrene 276, 138

Benzo[ghi]perylene 276, 138

Dibenz[a,h+a,c]anthraceneb 278, 139 

a

Benzo[j]fluoranthene + Benzo[k]fluoranthene 

b

Dibenz[a,h]anthracene + Dibenz[a,c]-anthracene 

32
assess the GC/MS stability. The GC/MS is considered stable if the response of an analyte 

relative to the response of its internal standard in a given repetition is within +15% of the 

respective average for the repetitions. 

5.2.2. GC/MS Analysis of Sediment Samples for AHs 

One or more batches of sediment samples are analyzed in a sequence that includes one each of 

AH GC calibration standard Levels 3 through 6 and Levels 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 one injection of the Sediment Conditioning solution at 

the beginning of the sequence, which serves as a GC/MS system conditioner. 

5.2.3. GC/MS Analysis of Tissue Samples for AHs 

One or more batches of tissues samples are analyzed in a sequence that includes one each of AH 

GC calibration standard Levels 1, 2, 3, 5, 6, and 7, 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 the Low-Lipid Tissue Conditioning 

solution at the beginning of the sequence, which serves as a GC/MS system conditioner. 

5.3. GC/MS for Quantitating CHs

The GC oven temperature is programmed according to Table 7. 

Sediment and tissue samples are analyzed using SIM mode, scanning only the quantitation ion 

during the specified time window as shown in Table 8. The time windows for SIM mode are 

adjusted as necessary when the GC column is changed or the guard column is trimmed. 

5.3.1. Verification of GC/MS Performance for CHs 

The sensitivity of the GC/MS is checked by analyzing the lowest level of CH GC calibration 

standard that will be used to quantitate CHs in samples (Level 1 for sediments and low-lipid 

tissues, Level 2 for high-lipid tissues). 

The stability of the GC/MS is checked by analyzing a sequence of three or more repetitions of

the Level 5 CH GC calibration standard. Included in this sequence are repetitions of the 

appropriate conditioning solution (Low-Lipid Tissue Conditioning solution for low-lipid tissues, 

High-Lipid Tissue Conditioning solution for high-lipid tissues or Sediment Conditioning

solution for sediments), that is, one conditioning solution before each repetition of the Level 5 

CH GC calibration standard. The responses of the analytes relative to the responses of their 

internal standard are compared among the repetitions of the Level 5 CH GC calibration standard 

to assess the GC/MS stability. The GC/MS is considered stable if the response of an analyte 

33
Table 7. Gas chromatograph oven temperature program for chlorinated hydrocarbons. 

Parameter Value

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 315°C 

Total run time 153 min 

34
Table 8. Selected-ion monitoring for quantitating chlorinated hydrocarbons in tissue and sediment 

samples. 

Approximate 

time window Compounds 

Quantitation ion 

(atomic mass units) 

15–29 min Tetrachloro-o-xylene 207

Tetrachloro-m-xylene 207

29–35 min alpha-Hexachlorocyclohexane 181 

Hexachlorobenzene 284

35–39 min beta-Hexachlorocyclohexane 219

gamma-Hexachlorocyclohexane 181

39–45 min PCB 18 256 

PCB 17 256

45–55 min PCB 31 256 

PCB 28 256

PCB 33 256

heptachlor 272

55–65 min PCB 52 292 

PCB 49 292

aldrin 263

PCB 44 292

65–69 min PCB 103 326 

69–76 min Heptachlor epoxide 353 

Oxychlordane 115

PCB 74 292

PCB 70 292

PCB 66 292

PCB 95 326

76–79 min trans-Chlordane 373

79–86 min 2,4'-DDE 318 

Endosulfan I 241

PCB 101 + PCB 90 326 

Nonchlor III 409

cis-Chlordane 373

PCB 99 326

trans-Nonachlor 409

35
Table 8 continued. Selected-ion monitoring for quantitating chlorinated hydrocarbons in tissue and 

sediment samples. 

Approximate 

time window Compounds 

Quantitation ion 

(atomic mass units) 

86–93 min Dieldrin 79 

PCB 87 326

4,4'-DDE 165

PCB 110 326

2,4'-DDD 235

93–98 min PCB 82 326 

PCB 151 360

Endosulfan II 241

98–103 min PCB 149 360 

PCB 118 326

cis-Nonachlor 409

4,4'-DDD 165

2,4'-DDT 235

103–106 min PCB 153 + PCB 132 360 

PCB 105 326

106–110 min Endosulfan sulfate 272 

4,4'-DDT 235

PCB 138 + PCB 163 + PCB 164 360 

PCB 158 360

110–113 min PCB 187 + PCB 159 + PCB 182 394 

PCB 183 394

PCB 128 360

113–115 min PCB 177 394 

PCB 171 394

PCB 156 360

115–121 min PCB 180 394 

PCB 191 394

Mirex 272

PCB 170 + PCB 190 394 

PCB 198 430

PCB 199 430

121–153 min PCB 208 464 

PCB 195 430

PCB 194 430

PCB 205 430

PCB 206 464

PCB 209 498

36
relative to the response of its internal standard in a given repetition is within +15% of the 

respective average for the repetitions. 

5.3.2. GC/MS Analysis of Sediment Samples for CHs 

One or more batches of sediment samples are analyzed in a sequence that includes one each of 

CH GC calibration standard Levels 1 through 4 and Level 6 alternating with the samples, plus 

one Level 5 before the first sample and replicate Level 5 analyses at the middle and end of each 

batch. Also included in this sequence is one injection of the Sediment Conditioning solution at 

the beginning of the sequence which serves as a GC/MS system conditioner. 

5.3.3. GC/MS Analysis of Low-Lipid Tissue Samples for CHs 

One or more batches of low-lipid tissues samples are analyzed in a sequence that includes one 

each of CH GC calibration standard Levels 1 through 4 and Level 6 alternating with the samples, 

plus one Level 5 before the first sample and replicate Level 5 analyses at the middle and end of 

each batch. Included in this sequence is one injection of the Low-Lipid Tissue Conditioning 

solution at the beginning of the sequence which serves as a GC/MS system conditioner. 

5.3.4. GC/MS Analysis of High-Lipid Tissue Samples for CHs 

One or more batches of high-lipid tissues samples are analyzed in a sequence that includes one 

each of CH GC calibration standard Levels 2, 3, and 4 and Levels 6 through 11 alternating with 

the samples, plus one Level 5 before the first sample and replicate Level 5 analyses at the middle 

and end of each batch. Included in this sequence is one injection of the High-Lipid Tissue 

Conditioning solution at the beginning of the sequence which serves as a GC/MS system

conditioner.

37
6. CALCULATIONS OF RESULTS 

6.1. Calculation of Analyte Concentrations

The acquired GC/MS data for the samples and calibration standards are processed using the 

ChemStation software to determine response areas for the analytes and internal standards. These 

areas 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. The point-to-point 

calibration method plots the relative response factors of the calibration standards versus their 

analyte areas. The 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), a relative response factor (RRF) is computed for each 

analyte relative to its internal standard (IS) as follows: 

 CS RRF = (CS Analyte Concentration) x (CS IS Area) (3)

(CS Analyte Area) (CS IS Concentration)

The CH internal standard is PCB 103 for all analytes in Table 2; the AH internal standards are 

naphthalene-d8 for the analytes naphthalene through 2, 6-dimethylnaphthalene, acenaphthened10 for analytes acenaphthene through pyrene, and benzo[a]pyrene-d12 for analytes 

benz[a]anthracene through benzo[ghi]perylene as listed in Table 1. 

For the analytes not contained in the calibration standards (i.e., retene and nonachlor III), the 

response factor of a similar analyte is used (i.e., 1-methylphenanthrene and trans-nonachlor, 

respectively). 

Next, specific for each sample, a sample RRF for each analyte relative to its internal 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 follows: 

Sample RRF = Lower CS RRF + (Sample Analyte Area – Lower CS Analyte Area) x (Upper CS RRF – Lower CS RRF) (4)

(Upper CS Analyte Area – Lower CS Analyte Area) 

where “Lower CS” is the calibration standard whose analyte area is closest to and below that of 

the sample’s analyte area and “Upper CS” is the calibration standard whose analyte area is 

closest to and above that of the sample’s analyte area. 

 

38
The amount of each analyte in each sample (ng Analyte per Sample) is then calculated as 

follows: 

ng Analyte/Sample = Sample RRF x Sample Analyte Area x (Sample IS ng) (5) 

 (Sample IS Area) 

where “Sample IS ng” is the ng amount of internal 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 follows: 

ng/g Wet Weight = ng Analyte per Sample (6)

Sample Weight 

and 

ng/g Dry Weight = ng Analyte per Sample x 100 (7)

Sample Weight x % Dry Weight 

where the “% Dry Weight” is determined as described in Subsection 8.1. 

6.2. Calculation of Internal Standard Recoveries 

The FileMaker Pro database uses the internal standard areas for the samples and calibration 

standards to compute the internal standard recoveries, which are calculated depending on the 

type of analyses, as follows: 

• For CH analyses, for each sample, including the CH I-Std check solution, the percent 

recovery of the CH internal standard (PCB 103) is computed based on the CH HPLC internal 

standard (TCMX). Furthermore, the CH HPLC internal standard recovery is computed based 

on the CH GC internal standard (TCOX). For these calculations, the Level 5 GC calibration 

standard is used for the relative response factors of the internal standard. 

• For AH analyses, for each sample, including the AH I-Std check solution, the AH internal 

standards (naphthalene-d8, acenaphthene-d10, and benzo[a]pyrene-d12) are computed based 

on the HPLC internal standard (phenanthrene-d10). Furthermore, the AH HPLC internal 

standard recovery is computed based on the AH GC internal standard (HMB). For these 

calculations, the Level 3 GC calibration standard in the case of analyses using SIM mode, or 

the Level 6 GC calibration standard in the case of analyses using scan mode, is used for the 

relative response factors of the internal standards.

39
The percent recovery of each internal standard (IS % Recovery) is calculated as follows: 

IS % Recovery = CS IS RRF x Sample IS Area x (Sample HPLC IS ng) (8) 

 (Sample HPLC IS Area) 

where 

CS IS RRF = (CS IS Concentration) x (CS HPLC IS Area) (9)

 (CS IS Area) (CS HPLC IS Concentration) 

and “Sample HPLC IS ng” is the ng amount of HPLC internal standard added to the sample. 

Each HPLC internal standard percent recovery (HPLC IS % Recovery) is monitored for in-house 

quality assurance information and is calculated as follows: 

HPLC IS % Recovery = CS HPLC IS RRF x Sample HPLC IS Area x (Sample GC IS ng) (10)

 (Sample GC IS Area) 

where 

CS HPLC IS RRF = (CS HPLC IS Concentration) x (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 lower limit of quantitation 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 its area in the lowest 

level calibration standard used in the calibration. When an analyte is not detected in a sample or 

has an area that is smaller than its area in the lowest level calibration standard used, the 

concentration of the analyte in that sample is reported to be less than the value of its lower limit 

of quantitation. 

6.4. Calculating and Reporting Estimated Concentrations for 

Analytes Exceeding the Calibration Range 

When a given analyte in a given sample has a GC/MS area that is larger than its area in the 

highest level calibration standard used in the calibration, the analyte amount is calculated using 

the relative response factor of that analyte in the highest level calibration standard used. The 

concentration is footnoted as exceeding the calibration range and is therefore an estimate. 

40
7. QUALITY ASSURANCE 

The QA measures are incorporated into the analyses of each batch of samples according 

to the Environmental Assessment Program Quality Assurance Plan (NMFS unpubl. data). 

Specific quality assurance 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 quality assurance 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 calibration 

standard analyzed intermittently in the sequence of samples and other GC calibration standards. 

The GC/MS is considered stable if the response of an analyte relative to the response of its 

internal standard in a given repetition is within +15% of the respective average for the 

repetitions. 

7.2. Internal Standard Recoveries 

The percent recoveries of internal standards in samples are considered acceptable if they are 

between 60% and 130%. The percent recoveries of internal standards in the I-Std check 

solutions are monitored for problems with the internal standard solutions. 

7.3. Reference Material Analyte Concentrations 

At least one sample of an appropriate NIST SRM is analyzed in each batch of samples to 

indicate the accuracy of the data for the entire batch. NIST provides certified concentrations and 

uncertainty values (for approximately 95% confidence) for many of the analytes in Tables 1 and 

2. For the analytes having NIST certified concentrations, upper and lower control limits are set 

in accordance with those set by NIST in the Intercomparison Exercise Program for Organics in 

the Marine Environment. They are defined as follows: 

Upper Control Limit = 1.3 x (Certified Concentration + Uncertainty Value) (12) 

and 

Lower Control Limit = 0.7 x (Certified Concentration − Uncertainty Value) (13) 

The accuracy of the SRM result is considered acceptable if the determined concentration falls 

within the Upper and Lower Control Limits. This goal does not apply to analytes with 

concentrations below their limit of quantitation. The analysis of the SRM is acceptable if at least 

70% of the analytes having NIST certified concentrations are within their control limits. 

41
Analytes that do not have NIST certified concentrations, as a result, do not have QA goals for 

accuracy, but 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 percent relative standard deviation of the concentrations of each analyte. The 

precision is considered acceptable if the percent relative standard deviation is <15% for at least 

90% of the analytes. 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 set of samples to monitor for contamination from 

laboratory sources. Ideally, analytes would not be detected in the method blank above their limit 

of quantitation. However, if analytes are measured in the method blank, their concentrations

need to be compared to the concentrations in the sediment or tissue 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 five 

analytes are measured and the analyte concentrations in the method blank do not exceed two 

times the lower limit of quantitation. Also, analytes in tissue or sediment samples having 

concentrations between the lower limit of quantitation and four times the concentration in the 

method blank for the batch might be footnoted as such. 

42
8. GRAVIMETRIC DETERMINATIONS 

The volume static eliminator is used on the balances whenever weighing items, in order 

to reduce weighing errors caused by static charge. 

8.1. Dry Weight Determination 

The sample number is etched on the tab of a 43-mm i.d. 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. 

The procedural steps are performed depending on the type of sample matrix as follows: 

• 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 AH and CH analysis.) The sediment (2 + 0.5 g) 

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, the pre-homogenized tissue (1 + 0.5 g) 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. 

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 follows: 

% Dry Weight = (Pan w/Dry Sample – Pan) x 100% (14)

 (Pan w/Wet Sample – Pan) 

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). 

43
The pan is dried in a 50°C oven for 2 hours, then cooled in a desiccator overnight. 

The pan is weighed to the nearest 0.0001g and the weight is recorded as the “Pan w/TE” weight. 

The percent total extractables (% TE) content of the sample is calculated as follows: 

% TE = (Pan w/TE – Pan) x (ASE Vial w/Extract – ASE Vial) x 100% (15) 

 (ASE Vial w/Extract – ASE Vial w/o TE Extract) x Sample Weight 

44
9. CONCLUSIONS 

The analytical methods presented here result from research, development, and application 

by chemists in the Environmental Assessment Program. These methods are continuously 

upgraded for better accuracy, precision, sensitivity, robustness, safety, and cost effectiveness, as 

well as for minimizing contamination from laboratory materials and from sample-to-sample 

carry-over. Our productivity and data quality have increased due to the new and improved 

techniques and instrumentation described here. Quality assurance measures are also 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. 

45
46
10. CITATIONS 

Burrows, D. G., D. W. Brown, and W. D. MacLeod, Jr. 1990. A twenty-five fold increase in GC/MS 

sensitivity attained by switching through a sequence of ten MID descriptors during capillary GC

analysis. Unpubl. manuscr., 16 p. Presented at the 38th Annual ASMS Conference on Mass 

Spectrometry and Allied Topics, Tucson, AZ, June 3–8, 1990. (Available from Douglas G. 

Burrows, Environmental Conservation Division, Northwest Fisheries Science Center, 2725 

Montlake Blvd. East, Seattle, WA 98112-2097.) 

Krahn, M. M., C. A. Wigren, R. W. Pearce, L. K. Moore, R. G. Bogar, W. D. MacLeod, Jr., S-L. Chan, 

and D. W. Brown. 1988. Standard analytical procedures of the NOAA National Analytical 

Facility, 1988: New HPLC cleanup and revised extraction procedures for organic contaminants. 

U.S. Dept. Commerce, NOAA Tech. Memo. NMFS F/NWC-153, 52 p. 

MacLeod, W. D., D. W. Brown, A. J. Friedman, D. G. Burrows, O. Maynes, R. W. Pearce, C. A. Wigren, 

and R. G. Bogar. 1985. Standard analytical procedures of the NOAA National Analytical 

Facility, 1985–1986: Extractable toxic organic compounds. Second edition. U.S. Dept. 

Commerce, NOAA Tech. Memo. NMFS F/NWC-92, 121 p. 

NMFS (National Marine Fisheries Service). Unpubl. data. NWFSC Environmental Assessment Program

quality assurance plan, 2003, 30 p. (Available from Margaret Krahn, Northwest Fisheries 

Science Center, Environmental Conservation Division, 2725 Montlake Blvd. East, Seattle, WA

98112-2097.)

Sloan, C. A., N. G. Adams, R. W. Pearce, D. W. Brown, and S-L. Chan. 1993. Northwest Fisheries 

Science Center organic analytical procedures. In G. G. Lauenstein and A. Y. Cantillo (eds.), 

Sampling and analytical methods of the National Status and Trends Program, National Benthic 

Surveillance and Mussel Watch Projects 1984–1992. Volume IV. Comprehensive descriptions 

of trace organic analytical methods. U.S. Dept. Commerce, NOAA Tech. Memo. NOS ORCA 

71, p. 53–97.

47

Most NOAA Technical Memorandums NMFS-NWFSC are available online at the

Northwest Fisheries Science Center web site (http://www.nwfsc.noaa.gov).

Recent NOAA Technical Memorandums NMFS

published by the

Northwest Fisheries Science Center

NOAA Tech. Memo.

NMFS-NWFSC58 Beechie, T.J., E.A. Steel, P. Roni, and E. Quimby (editors). 2003. Ecosystem recovery planning for

listed salmon: an integrated assessment approach for salmon habitat. U.S. Dept. Commer., NOAA Tech.

Memo. NMFS-NWFSC-58, 183 p. NTIS number pending.

57 Rogers, J.B. 2003. Species allocation of Sebastes and Sebastolobus sp. caught by foreign

countries from 1965 through 1976 off Washington, Oregon, and California, USA. U.S. Dept. Commer.,

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