Organic Contaminant Analytical Methods of the 

National Status and Trends Program: 2000-2006 

 NOAA Technical Memorandum NOS NCCOS 30 
Mention of trade names or commercial products does not constitute 

endorsement or recommendation for their use by the United States 

government. 

Cover photograph of shrimping fleet, Palacios, TX 

Citation for this Report 

Kimbrough, K. L., G. G. Lauenstein and W. E. Johnson (Editors). 2006. Organic 

Contaminant Analytical Methods of the National Status and Trends Program: Update 

2000-2006. NOAA Technical Memorandum NOS NCCOS 30. 137 pp. 
Organic Contaminant Analytical Methods of the 

National Status and Trends Program: Update 

2000-2006 

K. L. Kimbrough, G. G. Lauenstein and W. E. Johnson 

(Editors)

Center for Coastal Monitoring and Assessment 

NOAA/NOS/NCCOS 

1305 East-West Highway 

Silver Spring, Maryland 20910 

NOAA Technical Memorandum NOS NCCOS 30 

June 2007 

United States Department of National Oceanic and National Ocean Service 

Commerce Atmospheric Administration 

Carlos M. Gutierrez Conrad C. Lautenbacher, Jr. John H. Dunnigan 

Secretary Administrator Assistant Administrator 

TABLE OF CONTENTS 

TABLE OF CONTENTS ..................................................................................................I

LIST OF TABLES ..........................................................................................................III

LIST OF FIGURES ........................................................................................................IV

CHAPTER 1. INTRODUCTION.................................................................................... 1

CHAPTER 2. EXTRACTION OF SEDIMENTS FOR AROMATIC AND 

CHLORINATED HYDROCARBONS AND POLYBROMINATED FLAME 

RETARDANTS 2000 – 2006............................................................................................ 3

ABSTRACT.................................................................................................................... 3 

1.0 INTRODUCTION .............................................................................................. 3 

2.0 APPARATUS AND MATERIALS.................................................................... 3 

3.0 PROCEDURE..................................................................................................... 5 

4.0 QUALITY CONTROL (QC).............................................................................. 6 

5.0 REFERENCES ................................................................................................... 8 

CHAPTER 3. EXTRACTION OF BIOLOGICAL TISSUES FOR AROMATIC 

AND CHLORINATED HYDROCARBONS AND POLYBROMINATED FLAME 

RETARDANTS 2000 – 2006............................................................................................ 9

ABSTRACT.................................................................................................................... 9 

1.0 INTRODUCTION .............................................................................................. 9 

2.0 APPARATUS AND MATERIALS.................................................................... 9 

3.0 PROCEDURE................................................................................................... 11 

4.0 QUALITY CONTROL (QC)............................................................................ 12 

5.0 REFERENCES ................................................................................................. 15 

CHAPTER 4. QUANTITATIVE DETERMINATION OF CHLORINATED 

HYDROCARBONS USING GAS CHROMATOGRAPHY/ELECTRON 

CAPTURE DETECTION 2000 – 2005 ......................................................................... 16

ABSTRACT.................................................................................................................. 16 

1.0 INTRODUCTION ............................................................................................ 16 

2.0 APPARATUS AND MATERIALS.................................................................. 16 

3.0 QUANTITATIVE DETERMINATION OF CHLORINATED 

 HYDROCARBONS BY GC/ECD .............................................................. 20 

4.0 QUANTITATION CALCULATIONS............................................................. 21 

5.0 QUALITY CONTROL (QC)............................................................................ 22 

6.0 REFERENCES ................................................................................................. 24 

CHAPTER 5. QUANTITATIVE DETERMINATION OF POLYCYCLIC 

AROMATIC HYDROCARBONS USING SELECTED ION MONITORING GAS 

CHROMATOGRAPHY/MASS SPECTROMETRY 2000 – 2006............................. 25

ABSTRACT.................................................................................................................. 25 

1.0 INTRODUCTION ............................................................................................ 25 

2.0 APPARATUS AND MATERIALS.................................................................. 25 

3.0 QUANTITATIVE DETERMINATION OF PAHS BY GC/MS-SIM............. 29 

i
4.0 QUANTITATION CALCULATIONS............................................................. 34 

5.0 QUALITY CONTROL (QC)............................................................................ 35 

6.0 REFERENCES ................................................................................................. 37 

CHAPTER 6. EXTRACTION OF SEDIMENTS FOR BUTYLTINS 2000 - 2006.. 38

ABSTRACT.................................................................................................................. 38 

1.0 INTRODUCTION ............................................................................................ 38 

2.0 APPARATUS AND MATERIALS.................................................................. 38 

3.0 PROCEDURE................................................................................................... 39 

4.0 QUALITY CONTROL (QC)............................................................................ 40 

CHAPTER 7. EXTRACTION OF BIOLOGICAL TISSUES FOR BUTYLTINS 

2000 - 2006 ....................................................................................................................... 43

ABSTRACT.................................................................................................................. 43 

1.0 INTRODUCTION ............................................................................................ 43 

2.0 APPARATUS AND MATERIALS.................................................................. 43 

3.0 PROCEDURE................................................................................................... 45 

4.0 QUALITY CONTROL (QC)............................................................................ 48 

CHAPTER 8. QUANTITATIVE DETERMINATION OF BUTYLTINS USING 

GAS CHROMATOGRAPHY/FLAME PHOTOMETRIC DETECTION 1999 -2006

........................................................................................................................................... 49

ABSTRACT.................................................................................................................. 49 

1.0 INTRODUCTION ............................................................................................ 49 

2.0 APPARATUS AND MATERIALS.................................................................. 49 

3.0 QUANTITATIVE DETERMINATION OF BUTYLTINS BY GC/FPD........ 51 

4.0 QUANTITATION CALCULATIONS............................................................. 53 

5.0 QUALITY CONTROL (QC)............................................................................ 54 

6.0 REFERENCES ................................................................................................. 57 

APPENDIX A. METHOD DETECTION LIMITS ..................................................... 58

ii
LIST OF TABLES 

TABLE 4-1. CHLORINATED HYDROCARBONS CONTAINED IN MATRIX SPIKE SOLUTION. ..... 18 

TABLE 4-2. CHLORINATED HYDROCARBONS CONTAINED IN CALIBRATION SOLUTIONS AND 

THEIR APPROXIMATE CONCENTRATIONS. ................................................................... 19 

TABLE 4-2 CONT’D. CHLORINATED HYDROCARBONS CONTAINED IN CALIBRATION 

SOLUTIONS AND THEIR APPROXIMATE CONCENTRATIONS. ......................................... 20 

TABLE 5-1. POLYCYCLIC AROMATIC HYDROCARBONS CONTAINED IN THE MATRIX-SPIKING 

SOLUTION................................................................................................................... 27 

TABLE 5-2. POLYCYCLIC AROMATIC HYDROCARBONS CONTAINED IN CALIBRATION 

SOLUTIONS AND THEIR APPROXIMATE CONCENTRATIONS. ......................................... 28 

TABLE 5-3. TARGET ANALYTE PARAMETERS. ................................................................... 32 

TABLE 5-3. TARGET ANALYTE PARAMETERS (CONT’D)..................................................... 33 

TABLE 8-1. BUTYLTINS CONTAINED IN THE MATRIX-SPIKING SOLUTION......................... 51 

TABLE 8-2. BUTYLTINS CONTAINED IN CALIBRATION SOLUTIONS AND THEIR 

APPROXIMATE CONCENTRATIONS. ............................................................................ 51 

TABLE A-1. REPORTING UNITS FOR ORGANIC CONTAMIANTS MEASURED BY THE MUSSEL 

WATCH PROJECT. ...................................................................................................... 58 

TABLE A-2. MUSSEL WATCH PROJECT TISSUE BUTYLTIN METHOD LIMITS OF DETECTION 

(NG SN/G DRY WEIGHT).............................................................................................. 58 

TABLE A-3. MUSSEL WATCH PROJECT SEDIMENT BUTYLTIN METHOD LIMITS OF DETECTION 

(NG SN/G DRY WEIGHT).............................................................................................. 58 

TABLE A-4. MUSSEL WATCH PROJECT TISSUE POLYCYCLIC AROMATIC HYDROCARBON 

METHOD LIMITS OF DETECTION (NG/G DRY WEIGHT). ................................................. 59 

TABLE A-4 (CONT’D). MUSSEL WATCH PROJECT TISSUE POLYCYCLIC AROMATIC 

HYDROCARBON METHOD LIMITS OF DETECTION (NG/G DRY WEIGHT)......................... 60 

TABLE A-5. MUSSEL WATCH PROJECT SEDIMENT POLYCYCLIC AROMATIC HYDROCARBON 

METHOD LIMITS OF DETECTION (NG/G DRY WEIGHT). ................................................. 61 

TABLE A-6. MUSSEL WATCH PROJECT PESTICIDE TISSUE METHOD LIMITS OF DETECTION 

(NG/G DRY WEIGHT). .................................................................................................. 62 

TABLE A-7. MUSSEL WATCH PROJECT PESTICIDE SEDIMENT METHOD LIMITS OF DETECTION 

(NG/G DRY WEIGHT). .................................................................................................. 63 

TABLE A-8. MUSSEL WATCH PROJECT POLYCHLORINATED BIPHENYL TISSUE METHOD 

LIMITS OF DETECTION (NG/G DRY WEIGHT). ............................................................... 64 

TABLE A-9. MUSSEL WATCH PROJECT POLYCHLORINATED BIPHENYL SEDIMENT METHOD 

LIMITS OF DETECTION (NG/G DRY WEIGHT). ............................................................... 65 

iii
iv

LIST OF FIGURES

FIGURE 2-1. METHODOLOGY FOR EXTRACTION, ISOLATION AND QUANTIFICATION OF 

SEDIMENT SAMPLES FOR POLYCYCLIC AROMATIC HYDROCARBONS (PAHS) AND 

ORGANIC CONTAMINANTS (OC)................................................................................... 7 

FIGURE 3-1. METHODOLOGY FOR EXTRACTION, ISOLATION, AND QUANTIFICATION OF 

TISSUE SAMPLES FOR POLYCYCLIC AROMATIC HYDROCARBONS (PAHS) AND ORGANIC 

CONTAMINANTS (OC). ............................................................................................... 14 

FIGURE 6-1. METHODOLOGY FOR EXTRACTION, ISOLATION, AND QUANTIFICATION OF 

SEDIMENT SAMPLES FOR BUTYLTINS. ......................................................................... 42 

FIGURE 7-1. METHODOLOGY FOR EXTRACTION, ISOLATION, AND QUANTIFICATION OF 

TISSUE SAMPLES FOR BUTYLTINS. .............................................................................. 47 
CHAPTER 1. INTRODUCTION 

K. L. Kimbrough, G. G. Lauenstein and W. E. Johnson 

National Status and Trends Program 

Center for Coastal Monitoring and Assessment 

National Oceanic and Atmospheric Administration 

Silver Spring, MD 

This document describes the analytical methods used to quantify core organic chemicals 

in tissue and sediment collected as part of NOAA’s National Status and Trends Program 

(NS&T) for the years 2000-2006. Organic contaminat analytical methods used during the 

early years of the program are described in NOAA Technical Memoranda NOS ORCA 

71 and 130 (Lauenstein and Cantillo, 1993; Lauenstein and Cantillo, 1998) for the years 

1984-1992 and 1993-1996, respectively. These reports are available from our website 

(http://www.ccma.nos.gov) 

The methods detailed in this document were utilized by the Mussel Watch Project and 

Bioeffects Project, which are both part of the NS&T program. The Mussel Watch Project 

has been monitoring contaminants in bivalves and sediments since 1986 and is the 

longest active national contaminant monitoring program operating in U.S. costal waters. 

Approximately 280 Mussel Watch sites are sampled on a biennial and decadal timescale 

for bivalve tissue and sediment respectively. Similarly, the Bioeffects Assessment Project 

began in 1986 to characterize estuaries and near coastal environs. Using the sediment 

quality triad approach that measures; (1) levels of contaminants in sediments, (2) 

incidence and severity of toxicity, and (3) benthic macrofaunal conmmunities, the 

Bioeffects Project describes the spatial extent of sediment toxicity. Contaminant 

assessment is a core function of both projects. These methods, while discussed here in 

the context of sediment and bivalve tissue, were also used with other matricies including: 

fish fillet, fish liver, nepheloid layer, and suspended particulate matter. 

The methods described herein are for the core organic contaminants monitored in the 

NS&T Program and include polycyclic aromatic hydrocarbons (PAHs), polychlorinated 

biphenyls (PCBs), butyltins, and organochlorines that have been analyzed consistently 

over the past 15-20 years. Organic contaminants such as dioxins, perfluoro compounds 

and polybrominated biphenyl ethers (PBDEs) were analyzed periodically in special 

studies of the NS&T Program and will be described in another document. 

All of the analytical techniques described in this document were used by B&B 

Laboratories, Inc, an affiliate of TDI-Brook International, Inc. in College Station, Texas 

under contract to NOAA. The NS&T Program uses a performance-based system 

approach to obtain the best possible data quality and comparability, and requires 

laboratories to demonstrate precision, accuracy, and sensitivity to ensure results-based 

performance goals and measures. 
REFERENCES 

Lauenstein, G. G. and A. Y. Cantillo (eds.) (1998) Sampling and analytical methods of 

the National Status and Trends Program Mussel Watch Project 1993-1996 Update: TERL 

Trace Element Quantification Techniques, Volume III. NOAA Technical Memorandum 

NOS ORCA 71, Silver Spring, MD. 219 pp. 

Lauenstein, G. G. and A. Y. Cantillo (eds.) (1993) Sampling and analytical methods of 

the National Status and Trends Program National Benthic Surveillance and Mussel 

Watch Projects 1984-1992: Comprehensive descriptions of elemental analytical methods, 

Volume III. NOAA Technical Memorandum NOS ORCA 71, Silver Spring, MD. 219 pp. 

2
CHAPTER 2. EXTRACTION OF SEDIMENTS FOR AROMATIC 

AND CHLORINATED HYDROCARBONS AND 

POLYBROMINATED FLAME RETARDANTS 2000 – 2006 

Juan. A. Ramirez, Bo Wang, Donell S. Frank, Thomas. J. McDonald, Rebecca Price, 

Susanne J. McDonald and James M. Brooks 

TDI-Brooks International/B&B Laboratories Inc. 

College Station, Texas 77845 

ABSTRACT 

Determining organic contaminant levels in sediments requires the extraction, isolation, 

and concentration of analytes from the matrix. Sediment samples are dried, pulverized, 

and homogenized prior to extraction. Sediments are extracted in dichloromethane using a 

Dionex® Accelerated Solvent Extractor. The extracts are purified using alumina/silica 

gel chromatography columns. The volume of the resultant eluent is reduced and analyzed 

for aromatic and chlorinated hydrocarbons and polybrominated flame retardants by gas 

chromatography/mass spectrometry and gas chromatography/electron capture detection. 

1.0 INTRODUCTION 

The procedure described is used to extract, isolate, purify, and concentrate aromatic and 

chlorinated hydrocarbons and polybrominated flame retardants from sediments. 

Contaminant concentrations in parts per billion or parts per trillion can be resolved in 

sediments. Sediment samples are homogenized, dried, and ground using a mortar and 

pestle. Approximately 15 g of dry sediment are extracted with 100% dichloromethane 

using a Dionex Accelerated Solvent Extractor (ASE200). The extracts are reduced in 

volume and then purified using alumina/silica gel column chromatography. Extracts are 

reduced to 1 mL and analyzed for PAHs, PCBs, pesticides, PBBs, and PBDEs by gas 

chromatography. 

2.0 APPARATUS AND MATERIALS 

2.1 EQUIPMENT 

Dionex, ASE200 Accelerated Solvent Extractor (ASE) with 33 mL extraction cells 

Water bath, capable of maintaining a temperature of 55-60 °C 

Balance, top loading, tare capacity to 300 g, capable of weighing to 1 mg 

Microbalance, capable of weighing to 1 μg 

Calibrated weights, certified 

3
Combustion furnace, electric, capable of combusting glassware at 400 °C for at least 4 

hours 

Oven, capable of maintaining 40 °C temperature maintenance 

Conditioning oven, electric, gravity convection, capable of maintaining a stable 

temperature of up to 200 °C 

Tumbler, Lortone rock tumbler or equivalent 

Collection vials, 60 mL certified pre-cleaned with open screw caps and Teflon lined septa 

Micropipettors, calibrated, 1% accuracy, disposable tips 

250 mL flat bottom, boiling flasks 

Borosilicate glass chromatography columns, 300 mm x 19 mm, with Teflon stopcock 

Kurderna-Danish (K-D tubes), 25 mL, slow dry concentrator tubes 

Synder columns, 3-ball 

Boiling chips, Teflon 

Glass wool 

2.2 REAGENTS 

Water (CAS 7732-18-5), gas chromatography/HPLC grade or equivalent purity 

Acetone (CAS 67-64-1), pesticide grade or equivalent purity 

Dichloromethane (CAS 75-09-2), pesticide grade or equivalent purity 

Hexane (110-54-3), pesticide grade or equivalent purity 

Pentane (109-66-0), pesticide grade or equivalent purity 

Copper (CAS 7440-50-8), granular, 20-30 mesh, ACS reagent grade, purified with 

hydrochloric acid 

Hydrochloric acid (7647-01-0), ACS reagent grade 

Sodium sulfate (CAS 7757-82-6), anhydrous granular powder, ACS reagent grade, 

purified by combusting at 400 °C for at least 4 hours and stored at 120°C. 

Alumina (CAS 1344-28-1), 80-325 mesh, basic or neutral, purified by combusting at 

400°C for at least 4 hours and stored at 120 °C 

4
Silica gel (CAS 1343-98-2), grade 923, 100-200 mesh, purified in an oven at 170 °C for 

at least 16 hours and stored at 170 °C 

Nitrogen (CAS 7727-37-9), 99.8% purity 

3.0 PROCEDURE 

Sediment samples are frozen upon receipt from the field at –20°C in the contract 

laboratory. Prior to extraction, sediment samples are thawed and homogenized using a 

stainless steel spatula. A subsample is removed for percent moisture determination 

(McDonald et al., 2006). At least 15 grams of sample are dried in an oven at 40 °C and 

then ground and homogenized using a mortar and pestle. Approximately 15 g of the 

dried sediment are extracted. 

Sediments are extracted with dichloromethane using an ASE200. Dried samples are 

loaded into 33 mL stainless steel ASE extraction cells. Appropriate surrogate and spikes 

are added to the top of samples. The ASE extractor tubes are sealed and placed in the 

ASE cell carousel. The ASE conditions are: 100% dichloromethane as the extraction 

solvent, 2,000-psi solvent pressure, 100°C cell temperature, and 2 static cycles for 2 

minutes each. Extracts are collected in 60 mL collection vials. The extracts are reduced 

to approximately 10 mL in the 60 mL collection vials in a 55-60 °C water bath. Extracts 

are then quantitatively transferred to Kurderna-Danish (K-D) tubes and the reduced to 1.0 

mL in a 55-60 °C water bath. Quality control samples (e.g., blanks, duplicates, matrix 

spikes and standard reference materials) are prepared and extracted in the same manner 

as samples. 

Extracts are purified using alumina/silica gel chromatography columns. Combusted and 

cooled alumina is deactivated by adding 1% (w/w) HPLC water and tumbled for at least 

1 hour using a Lortone rock tumbler. Combusted, cooled silica gel is deactivated by 

adding 5% (w/w) HPLC water and tumbled for at least 1 hour using a Lortone rock 

tumbler. Borosilicate glass columns (300 mm x 19 mm) are filled with dichloromethane 

and packed from the bottom with: glass wool, 1-2 g of sodium sulfate, 10 g of 

deactivated alumina, 20 g of deactivated silica gel, and another 1-2 g of sodium sulfate. 

The dichloromethane is drained to the top of the column followed by the addition of 50 

mL of pentane. The pentane is drained from the top of the upper sodium sulfate layer and 

discarded. The sample extract (approximately 1 mL) is added to the top of the column 

and eluted with 200 mL of a 50:50 mixture of pentane and dichloromethane at a flow rate 

of 1 mL/min. The eluent is collected in a 250 mL flat-bottom flask and reduced to 

approximately 10 mL in a 55-60 °C water bath. The extract is transferred to a 25 mL KD container and reduced to 1 mL. The dichloromethane is exchanged with hexane and 

reduced to a final volume of 1 mL. The concentrate is transferred to a 2 mL amber vial 

containing pre-cleaned copper granules (copper granules are carefully mixed with 

concentrated hydrochloric acid followed by thorough rinsing with HPLC-grade water) 

and stored at 20 °C until analysis. High sulfur-containing samples may require additional 

copper granules. Additional pre-cleaned copper granules are added if the initial batch of 

copper granules turns black, indicating the presence of excess sulfur in the extract. 

Figure 2-1 shows a flow chart of the extraction and purification procedure. 

5
4.0 QUALITY CONTROL (QC) 

Solvents are verified to be contaminant-free by lot tests prior to use. All equipment and 

glassware used to extract samples are thoroughly cleaned by solvent rinsing or 

combustion at 400 °C. The calibration and accuracy of balances, weights, pipettors and 

thermometers are checked daily using certified weights and thermometers with 

calibrations traced to the National Institute of Standards and Technology (NIST). The 

calibration and accuracy of balances, weight, pipettors, and thermometers are verified 

yearly by an independent source. A series of quality control samples are processed with 

each batch of 20 samples or less. The following quality controls are used to ensure the 

accuracy and precision of sediment data. 

• Surrogates. Solutions containing analytes that do not interfere with the analytes 

of interest are prepared at concentrations approximately 5 to 10 times the method 

detection limit (MDL). Specified surrogates are added to each sample extracted, 

including QC samples, at a specified volume (typically 100 μL) immediately prior 

to extraction. 

• Method Blank. Method blanks are extractions of all support material used for 

extraction of samples, with the exception of sediment. A method blank is 

analyzed with each extraction batch of 20 or fewer samples. The method blank is 

extracted and analyzed in a manner identical to samples. 

• Matrix Spike. Matrix spikes are extractions of sample matrix fortified with spikes 

of selected target analytes. Spikes are prepared at concentrations approximately 

10 times the MDL. A matrix spike and matrix spike duplicate are analyzed with 

each extraction batch of 20 or fewer samples. Matrix spikes are extracted and 

analyzed in a manner identical to samples. 

• Laboratory Duplicates. A sample is analyzed in duplicate with each extraction 

batch of 20 or fewer samples. 

• Standard Reference Material (SRM). A sediment standard reference material 

from NIST (SRM 1941b) is analyzed with each extraction batch of 20 or fewer 

samples for aromatic and chlorinated hydrocarbons only. 

6
SAMPLE RECEIPT

AND STORAGE

SAMPLE 

PREPARATION

SAMPLE 

EXTRACTION % MOISTURE

DETERMINATION

SAMPLE CLEANUP VIA 

ALUMINA/SILICA GEL 

COLUMN

EXCHANGE 

DICHLOROMETHANE WITH 

HEXANE

ANALYSIS BY 

GC/MS (PAHS) 

ANALYSIS BY

GC/ECD (OCS) 

Figure 2-1. Methodology for extraction, isolation and quantification of sediment samples 

for polycyclic aromatic hydrocarbons (PAHs) and organic contaminants (OC). 

7
5.0 REFERENCES 

McDonald, S. J., D. S. Frank, J. A. Ramirez, B. Wang, and J. M. Brooks. 2006. Ancillary 

Methods of the National Status and Trends Program: 2000-2006 Update. Silver Spring, 

MD. NOAA Technical Memorandum NOS NCCOS 28. 17 pp. 

8
CHAPTER 3. EXTRACTION OF BIOLOGICAL TISSUES FOR 

AROMATIC AND CHLORINATED HYDROCARBONS AND 

POLYBROMINATED FLAME RETARDANTS 2000 – 2006 

Donell. S. Frank, Bo Wang, Juan A. Ramirez, Susanne J. McDonald, Rebecca Price, and 

James M. Brooks 

TDI-Brooks International/B&B Laboratories Inc. 

College Station, Texas 77845 

ABSTRACT 

Determining organic contaminant levels in tissues require extraction, isolation, and 

concentration of analytes from the matrix. Tissue extracts require extensive purification 

procedures to remove lipids, which cause analytical interferences. Bivalves are shucked 

and homogenized. Aliquots of homogenized sample are chemically dried using 

Hydromatrix® and extracted in dichloromethane using a Dionex Accelerated Solvent 

Extractor. The extracts are purified using alumina/silica gel chromatography columns. 

The volume of the resultant eluent is further purified using a gel permeation column 

coupled to a high performance liquid chromatograph. The volume of the resultant eluant 

is reduced and analyzed for aromatic and chlorinated hydrocarbons and polybrominated 

flame retardants by gas chromatography. 

1.0 INTRODUCTION 

The procedure described is used to extract, isolate, purify, and concentrate aromatic, 

chlorinated hydrocarbons, and polybrominated flame retardant contaminants from tissues. 

Shell length and shell volume are determined for specimens collected at each location. 

Bivalves are shucked and multiple organisms are processed as one sample to ensure the 

sample is representative of a population at a given site and to have sufficient sample to 

complete the analyses. Tissue samples are homogenized using a stainless steel blender 

with titanium blades. Aliquots of approximately 15 g of wet tissue are chemically dried 

with Hydromatix®. The tissue/Hydromatix® mixtures are extracted with 100% 

dichloromethane using a Dionex Accelerated Solvent Extractor (ASE200) operated at 

100 °C and 2,000 psi. The extracts are reduced to 3 mL by evaporative solvent reduction. 

A 100 μL aliquot is removed and weighed to determine lipid weight (McDonald et al.,

2006). The remaining sample portion is purified using alumina/silica gel column 

chromatography and gel permeation column (GPC)/high performance liquid 

chromatography (HPLC). After HPLC purification, the eluents are reduced to 0.5 mL 

and analyzed for PAHs, PCBs, pesticides, PBBs, and PBDEs by gas chromatography. 

2.0 APPARATUS AND MATERIALS 

2.1 EQUIPMENT 

Dionex®, ASE200 Accelerated Solvent Extractor (ASE) with 33 mL extraction cells 

9
Water bath, capable of maintaining a temperature of 55-60 °C 

Balance, top loading, tare capacity to 300 g, capable of weighing to 1 mg 

Microbalance, capable of weight to 1 μg 

Calibrated weights, certified 

Combustion furnace, electric, capable of combusting glassware at 400 °C for at least 4 

hours 

Oven capable of maintaining 40°C temperature maintenance 

Conditioning oven, electric, gravity convection, capable of maintaining a stable 

temperature of up to 200 °C 

Tumbler, Lortone rock tumbler or equivalent 

HPLC system, Water Model 590 programmable solvent delivery module HPLC pump, 

Waters 717 plus autosampler, Waters UV absorbance detector, Waters 746 data module, 

Waters Fraction Collector, Phenogel 10μ GPC 100Å size exclusion columns and 

Phenogel 100Å guard column. 

Glass fiber filter circles, 2.4 cm diameter 

Collection vials, 60 mL certified pre-cleaned with open screw caps and Teflon lined septa 

Micropipettors, calibrated, 1% accuracy, disposable tips 

Zymark®, 50 mL concentration tubes 

250 mL flat bottom, boiling flasks 

Borosilicate glass chromatography columns, 300 mm x 19 mm, with Teflon stopcock 

Kurderna-Danish (K-D) tubes, 25 mL, slow dry concentrator tubes 

Synder columns, 3-ball 

Boiling chips, Teflon 

Glass wool 

2.2 REAGENTS 

Water (CAS 7732-18-5), gas chromatography/HPLC grade or equivalent purity 

Acetone (CAS 67-64-1), pesticide grade or equivalent purity 

10
Dichloromethane (CAS 75-09-2), pesticide grade or equivalent purity 

Hexane (CAS 110-54-3), pesticide grade or equivalent purity 

Pentane (CAS 109-66-0), pesticide grade or equivalent purity 

Hydromatrix® (CAS 68855-54-9/14464-46-1), conditioned by combustion at 400 °C for 

at least 4 hours and stored at 120°C 

Sodium sulfate (CAS 7757-82-6), anhydrous granular powder, ACS reagent grade, 

purified by combusting at 400°C for at least 4 hours and stored at 120°C. 

Alumina (CAS 1344-98-2), 80-325 mesh, basic, purified by combusting at 400 °C for at 

least 4 hours and stored at 120 °C 

Silica gel (CAS 1343-98-2), grade 923, 100-200 mesh, purified in an oven at 170 °C for 

at least 16 hours and store at 170°C 

Nitrogen (CAS 7727-37-9), 99.8% purity 

3.0 PROCEDURE 

Shell length and volume are determined for all bivalves collected at each sampling site. 

The bivalves are then shucked and the soft tissue homogenized using a stainless steel 

Waring® blender. Homogenized tissue samples are frozen at –20 oC until extraction. 

Prior to extraction, tissue samples are thawed and re-homogenized using a stainless steel 

spatula. A subsample is removed for percent moisture determination (McDonald et al., 

2006). 

Approximately 15 g of tissue are thoroughly mixed and ground with a sufficient quantity 

(approximately 40 g) of prepared (combusted) Hydromatrix® to “dry” the sample. The 

tissue samples must be thoroughly dry to optimize the extraction efficiency. 

Hydromatrix® chemically dries samples by binding moisture. The amount of 

Hydromatrix® necessary to dry a sample depends upon the amount of sample and the 

percent moisture in that sample. 

Tissues are extracted with dichloromethane using an ASE200. The tissue/Hydromatix®

mixture is loaded into 33 mL ASE extraction cells. Appropriate surrogates and spikes are 

added to the top of the samples. The ASE extractor tubes are sealed and placed in the 

ASE cell carousel. The ASE conditions are: 100% dichloromethane as the extraction 

solvent, 2,000-psi solvent pressure, 100 °C cell temperature, and 2 static cycles for 2 

minutes each. Extracts are collected in 60 mL collection vials. The extracts are reduced 

to approximately 10 mL in the 60 mL collection vials in a 55-60 °C water bath. Extracts 

are then quantitatively transferred to Kurderna-Danish (K-D) tubes and the volume 

reduced to 3 mL in a 55-60 °C water bath. A 100 μL aliquot is removed and weighed to 

determine lipid content (McDonald et al., 2006). Quality control samples (e.g., blanks, 

11
duplicates, matrix spikes and standard reference materials) are prepared and extracted in 

the same manner as samples. 

Extracts are initially purified using alumina/silica gel chromatography columns. 

Combusted and cooled alumina is deactivated by adding 1% (w/w) HPLC water and 

tumbled for at least 1 hour using a Lortone rock tumbler. Combusted and cooled silica 

gel is deactivated by adding 5% (w/w) HPLC water and tumbling for at least 1 hour using 

a Lortone rock tumbler. Borosilicate glass columns (300 mm x 19 mm) are filled with 

dichloromethane and packed from the bottom with: glass wool, 1-2 g of sodium sulfate, 

10 g of deactivated alumina, 20 g of deactivated silica gel, and another 1-2 g of sodium 

sulfate. The dichloromethane is drained to the top of the column followed by the addition 

of 50 mL of pentane. The pentane is drained from the top of the upper sodium sulfate 

layer and discarded. The sample extract (approximately 3 mL) is added to the top of the 

column and eluted with 200 mL of a 50:50 mixture of pentane and dichloromethane at a 

flow rate of 1 mL/min. The eluent is collected in a 250 mL flat-bottom flask. The eluent 

is reduced to approximately 10 mL in a 55-60°C water bath. The extract is transferred to 

25 mL K-D tubes and reduced to1-2 mL. The concentrate is transferred to 4 mL amber 

HPLC vials and brought up to 4 mL with dichloromethane. 

The extract is further purified using HPLC. The extract is injected using a Waters, Model 

717 Plus autosampler and eluted through one Phenogel 100Å guard column and two 

Phenogel 10μ GPC 100Å size exclusion columns with 100% dichloromethane at a flow 

rate of 7 mL per minute. Elution times for compounds of interest are monitored using 

standards and a UV absorbance detector (254 nm). The appropriate fraction is collected 

using a Waters Fraction Collector. The sample is collected in 50 mL Zymark tubes and 

reduced to 10 mL in a 50-60 oC water bath. The extract is transferred to K-D tubes and 

reduced to 1.0 mL. The dichloromethane is exchanged with hexane and reduced to a 

final volume of 0.5 mL. The concentrate is transferred to 2 mL amber vials and stored at 

20 °C until analysis. Figure 3-1 shows a flow chart of the extraction and purification 

procedure. 

4.0 QUALITY CONTROL (QC) 

Solvents are verified to be contaminant-free by lot tests prior to use. All equipment and 

glassware used to extract samples are thoroughly cleaned by solvent rinsing or 

combustion at 400 °C. The calibration and accuracy of balances, weights, pipettors and 

thermometers are checked daily using certified weights and thermometers with 

calibrations traced to the National Institute of Standards and Technology (NIST). The 

calibration and accuracy of balances, weight, pipettors and thermometers are verified 

yearly by an independent source. A series of quality control samples are processed with 

each batch of 20 samples or less. The following quality controls are used to ensure the 

accuracy and precision of tissue data. 

• Surrogates. Solutions containing analytes that do not interfere with the analytes of 

interest are prepared at concentrations approximately 5 to 10 times the method 

detection limit (MDL). Specified surrogates are added to each sample extracted, 

12
• Method Blank. Method blanks are extractions of all support material used for 

extraction of samples, with the exception of tissue. A method blank is analyzed with 

each extraction batch of 20 or fewer samples. The method blank is extracted and 

analyzed in a manner identical to samples. 

• Matrix Spike. Matrix spikes are extractions of sample matrix fortified with spikes of 

selected target analytes. Spikes are prepared at concentrations approximately 10 

times the MDL. A matrix spike and matrix spike duplicate are analyzed with each 

extraction batch of 20 or fewer samples. Matrix spikes are extracted and analyzed in 

a manner identical to samples. 

• Laboratory Duplicates. A sample is analyzed in duplicate with each extraction batch 

of 20 or fewer samples. 

• Standard Reference Material (SRM). A standard reference material from the NIST 

(SRM 1974a) is analyzed with each extraction batch of 20 or fewer samples for 

aromatic and chlorinated hydrocarbons. 

13
SAMPLE RECEIPT

AND STORAGE

SAMPLE 

PREPARATION

SAMPLE 

EXTRACTION % MOISTURE

DETERMINATION

EXCHANGE 

DICHLOROMETHANE WITH 

HEXANE

SAMPLE CLEANUP VIA 

ALUMINA/SILICA GEL 

COLUMN

ANALYSIS BY 

GC/MS (PAHS) 

% LIPID

DETERMINATION

ANALYSIS BY

GC/ECD (OCS) 

Figure 3-1. Methodology for extraction, isolation, and quantification of tissue samples for 

polycyclic aromatic hydrocarbons (PAHs) and organic contaminants (OC). 

14
5.0 REFERENCES

McDonald, S. J., D. S. Frank, J. A. Ramirez, B. Wang, and J. M. Brooks. 2006. Ancillary 

Methods of the National Status and Trends Program: 2000-2006 Update. Silver Springs, 

MD. NOAA Technical Memorandums NOS NCCOS 28. 17 pp. 

15
CHAPTER 4. QUANTITATIVE DETERMINATION OF 

CHLORINATED HYDROCARBONS USING GAS 

CHROMATOGRAPHY/ELECTRON CAPTURE DETECTION 2000 – 

2005 

Juan A. Ramirez, Donell S. Frank, Susanne J. McDonald and James M. Brooks 

TDI-Brooks International/B&B Laboratories Inc. 

College Station, Texas 77845 

ABSTRACT 

Selected chlorinated hydrocarbons, including polychlorinated biphenyls and pesticides, 

are detected using gas chromatograph/electron capture detector. This method is capable 

of detecting low concentration of chlorinated hydrocarbons in complex matrices such as 

tissues and sediments. 

1.0 INTRODUCTION 

A gas chromatograph/electron capture detector (GC/ECD), coupled to two capillary 

columns, is used to resolve and detect chlorinated hydrocarbons (polychlorinated 

biphenyls and pesticides) in tissues and sediments. Samples are injected into a 

temperature-programmed GC/ECD, operated in splitless mode. The capillary columns 

are DB-5 (30 m x 0.25 mm ID and 25 μm film thickness) and DB-17HT (30 m x 0.25 

mm ID and 0.15 μm film thickness). The DB-17HT column is used for analyte 

confirmation. A data acquisition system continuously acquires and stores all data for 

quantitation. This method is capable of producing data at parts-per billion and parts-per 

trillion concentrations. 

2.0 APPARATUS AND MATERIALS 

2.1 EQUIPMENT 

Gas chromatograph, split/splitless injection port and electronic pressure control, dual 

electron capture detectors, Agilent Technologies 5890-II 

Data acquisition system, Agilent Technologies ChemStation, capable of continuous 

acquisition and storage of all data during analysis 

Autosampler, capable of making 1 to 5 μL injections 

Capillary columns, J&W DB-5®(30 m x 0.25 mm ID and 0.25 μm film thickness) or 

equivalent, and J&W DB-17HT®(30 m X 0.25 mm ID and 0.15 μm film thickness) 

Micropipetters, calibrated, 1% accuracy, disposable tips 

16
2.2 REAGENTS 

Hexane (CAS 110-54-3), pesticide grade or equivalent purity 

Helium (CAS 7440-59-7), 99.8% purity 

95% Argon/5% Methane, 99.8% purity 

2.3 STANDARDS 

2.3.1 SURROGATE SPIKING SOLUTION 

A surrogate spiking solution is prepared from a commercially available solution (Ultra 

Scientific) that is diluted with hexane to a concentration of 1,000 pg/μL. The surrogate 

spiking solution includes 4,4’-dibromooctaflurobiphenyl (DBOFB), 2,2’,4,5’,6 

pentachlorobiphenyl (PCB 103), and 2,2’,3,3’,4,5,5’6 octachlorobiphenyl (PCB 198). 

Surrogate solution (100 μL) is added to all samples and quality control samples prior to 

extraction. Surrogate compounds are resolved from, but elute in close proximity to, the 

analytes of interest. The recovery of PCB 103 is used to correct analyte concentrations. 

2.3.2 INTERNAL STANDARD SOLUTION 

The internal standard solution is prepared from a commercially available solution (Ultra 

Scientific) of tetrachloro-m-xylene (TCMX) diluted with hexane to a final concentration 

of 1,000 pg/μL. The internal standard compound is resolved from, but elutes in close 

proximity to, the analytes of interest. The internal standard solution (100 μL) is added to 

all samples and quality control samples just prior to analysis. Internal standards are used 

to calculate relative response factors and specific analyte concentrations based on 

retention time. 

2.3.3 MATRIX SPIKING SOLUTION 

To prepare the matrix spiking solution, a certified solution (Accustandard) containing 

analytes of interest is purchased from commercial vendors and diluted with hexane 

(Table 4-1). The matrix spike solution is diluted to a concentration approximately 10 

times the MDL and is added to all matrix spike samples. 

2.3.4 CALIBRATION SOLUTION 

Calibration solutions are prepared at 5 concentrations ranging from approximately 5 to 

200 pg/μL (Table 2) by diluting a commercially prepared solutions (Ultra Scientific and 

Accustandard) containing the analytes of interest. 

17
Table 4-1. Chlorinated hydrocarbons contained in matrix spike solution.

Compound Name CAS Spiking Solution 

Concentration (pg/μL) 

1,2,4,5-Tetrachlorobenzene 95-94-3 40 

1,2,3,4-Tetrachlorobenzene 634-66-22 40 

Pentachlorobenzene 608-93-5 40 

Pentachloroanisole 1825-21-4 40 

Chlorpyrifos 2921-88-2 40 

Hexachlorobenzene 118-74-1 40 

α-HCH 319-84-6 40 

β-HCH 319-85-7 40 

γ−HCH (Lindane) 55-89-9 40 

δ-HCH 319-86-8 40 

Heptachlor 76-44-8 40 

Heptachlor epoxide 1024-57-3 40 

α-Chlordane (cis-) 5103-71-9 40 

γ -Chlordane (trans-) 5103-74-2 40 

Trans-nonachlor 39765-80-5 40 

Cis-nonachlor 5103-73-1 40 

Aldrin 309-00-2 40 

Dieldrin 60-57-1 40 

Endrin 72-20-8 40 

Mirex 2385-85-5 40 

2,4’ DDE 3424-82-6 40 

4,4’ DDE 72-55-9 40 

2,4’ DDD 53-19-0 40 

4,4’ DDD 72-54-8 40 

2,4’ DDT 789-02-6 40 

4,4’ DDT 50-29-3 40 

Endosulfan II 33213-65-9 40 

Oxychlordane 27304-13-8 40 

Endosulfan sulfate 1031-07-8 40 

PCB 8 34883-43-7 40 

PCB 18 37680-65-2 40 

PCB 28 7012-37-5 40 

PCB 44 41464-39-5 40 

PCB 52 35693-99-3 40 

PCB 66 32598-10-0 40 

PCB 101 37680-73-2 40 

PCB 105 32598-14-4 40 

PCB 118 31508-00-6 40 

PCB 128 38380-07-3 40 

PCB 138 35065-28-2 40 

PCB 153 35065-27-1 40 

PCB 170 35065-30-6 40 

PCB 180 35065-29-3 40 

PCB 187 52663-68-0 40 

PCB 195 52663-78-2 40 

PCB 206 40186-72-9 40 

PCB 209 2051-24-3 40 

18
Table 4-2. Chlorinated hydrocarbons contained in calibration solutions and their 

approximate concentrations. 

Compounds Contained CAS Level 1 Level 2 Level 3 Level 4 Level 5

in Calibration Solutions (pg/μL) (pg/μL) (pg/μL) (pg/μL) (pg/μL)

Internal Standard

TCMX 877-9-8 100 100 100 100 100 

Surrogates

DBOFB 10386-84-2 5 20 40 80 200 

PCB 103 60145-21-3 5 20 40 80 200 

PCB 198 68194-17-2 5 20 40 80 200 

Analytes

1,2,4,5-Tetrachlorobenzene 95-94-3 5 20 40 80 200 

1,2,3,4-Tetrachlorobenzene 634-66-22 5 20 40 80 200 

Pentachlorobenzene 608-93-5 5 20 40 80 200 

Pentachloroanisole 1825-21-4 5 20 40 80 200 

Chlorpyrifos 2921-88-2 5 20 40 80 200 

Hexachlorobenzene 118-74-1 5 20 40 80 200 

α-HCH 319-84-6 5 20 40 80 200 

β-HCH 319-85-7 5 20 40 80 200 

γ−HCH (Lindane) 319-86-6 5 20 40 80 200 

δ-HCH 58-89-9 5 20 40 80 200 

Heptachlor 76-44-8 5 20 40 80 200 

Heptachlor epoxide 1024-57-3 5 20 40 80 200 

Oxychlordane 27304-13-8 5 20 40 80 200 

α-Chlordane (cis-) 5103-71-9 5 20 40 80 200 

γ-Chlordane (trans-) 5103-74-2 5 20 40 80 200 

Trans-nonachlor 39765-80-5 5 20 40 80 200 

Cis-nonachlor 5103-73-1 5 20 40 80 200 

Aldrin 309-00-2 5 20 40 80 200 

Dieldrin 60-57-1 5 20 40 80 200 

Endrin 72-20-8 5 20 40 80 200 

Mirex 2385-85-5 5 20 40 80 200 

2,4’-DDE 3424-82-6 5 20 40 80 200 

4,4’-DDE 75-55-9 5 20 40 80 200 

2,4’-DDD 53-19-0 5 20 40 80 200 

4,4’-DDD 72-54-8 5 20 40 80 200 

2,4’-DDT 789-02-6 5 20 40 80 200 

4,4’-DDT 50-29-3 5 20 40 80 200 

Endosulfan II 33213-65-9 5 20 40 80 200 

Endosulfan sulfate 1031-07-8 5 20 40 80 200 

19
Table 4-2 cont’d. Chlorinated hydrocarbons contained in calibration solutions and their 

approximate concentrations. 

Compounds Contained CAS Level 1 Level 2 Level 3 Level 4 Level 5

in Calibration Solutions (pg/μL) (pg/μL) (pg/μL) (pg/μL) (pg/μL)

PCB 8 34883-43-7 5 20 40 80 200 

PCB 18 37680-65-2 5 20 40 80 200 

PCB 28 7012-37-5 5 20 40 80 200 

PCB 44 41464-39-5 5 20 40 80 200 

PCB 52 35693-99-3 5 20 40 80 200 

PCB 66 32598-10-0 5 20 40 80 200 

PCB 101 37680-73-2 5 20 40 80 200 

PCB 105 32598-14-4 5 20 40 80 200 

PCB 118 31508-00-6 5 20 40 80 200 

PCB 128 38380-07-3 5 20 40 80 200 

PCB 138 35065-28-2 5 20 40 80 200 

PCB 153 35065-27-1 5 20 40 80 200 

PCB 170 35065-30-6 5 20 40 80 200 

PCB 180 35065-29-3 5 20 40 80 200 

PCB 187 52663-68-0 5 20 40 80 200 

PCB 195 52663-78-2 5 20 40 80 200 

PCB 206 40186-72-9 5 20 40 80 200 

PCB 209 2051-24-3 5 20 40 80 200 

3.0 QUANTITATIVE DETERMINATION OF CHLORINATED 

HYDROCARBONS BY GC/ECD 

3.1 CALIBRATION 

An ECD exhibits limited linearity, particularly for low concentrations. Consequently, a 

calibration must be established for each analytical run. An analytical run consists of 

samples and 5 calibration standards (approximately 5 to 200 pg/μL or 5 to 200 ng/mL) 

that are interspersed throughout the run. A calibration curve is established by analyzing 

the 5 interspersed calibration standards and fitting the data to the following quadratic 

equation. 

( )

2

02

2

11

2b

Yb4bbb

x

−−+− =

Where: 

x = the concentration of the analyte (ng/mL)

Y = the ratio of the area of the analyte to the area of the internal standard multiplied 

by the amount of the internal standard (ng) 

b2, b1, b0 = the coefficients for the quadratic equation 

The data generated for each analyte in the calibration standards are subjected to the 

method of least squares to determine the coefficients for the corresponding quadratic 

20
equation. Each analyte has different coefficients based on the relative response of the 

analyte compared to the internal standard, and as a function of the amount of the analyte. 

The injected concentration of the internal standard analyte is held constant for each set of 

calibration standards. In order for the calibration to be valid, each analyte must have a 

correlation coefficient greater than 0.997. 

3.2 GC/ECD ANALYSIS 

Sample analyses are completed only if the calibration meets previously described criteria. 

Samples are analyzed in analytical sets that consist of standards, samples, and quality 

control samples. Quality control (QC) samples are method blanks, laboratory duplicates, 

matrix spikes, and standard reference material (SRM). An autosampler is used to inject 1 

or 5 μL of all samples, standards, and QC samples into the capillary column of the GC 

using the following instrument conditions. Slight modifications may be necessary 

depending upon the analysis. 

Inlet: Splitless

Carrier gas: Helium, 1 mL/min 

Temperatures: 

Injection port: 275 °C 

Detector: 325 °C 

Oven program: 

Initial oven temp: 100 °C 

Initial hold time: 1 minute 

Ramp rate: 5 °C/min to 140 °C 

Hold time: 1 minute 

Ramp rate: 1.5 °C/min to 250 °C 

Hold time: 1 minute 

Final oven rate: 10 °C/min to 300 °C 

Final hold time: 5 minutes 

3.3 ANALYTE IDENTIFICATION 

The retention time of a sample analyte must fall within 15 seconds of the retention time 

for that analyte in a calibration standard or a retention index solution. 

Chromatographic interferences may limit the ability to quantify peaks correctly and these 

data are reported but qualified to indicate interference. 

4.0 QUANTITATION CALCULATIONS 

Sample analyte concentrations are calculated based on the concentration and response of 

the internal standard (Table 4-2). The concentration (C) of each target analyte in the 

sample (ng/g) is calculated using the following equation: 

21
( ) DFV

W

X C ⎟ e

⎠

⎞ ⎜

⎝

⎛ =

Where: 

Ve = the final volume of the extract (mL) 

X = the concentration of the analyte (ng/mL) as found from solving the quadratic 

equation 

W = the sample weight (g) 

DF = the dilution factor 

μL)(dilution make tousedextract of Volume

L)(Extract ofVolume DF μ =

Analyte concentrations are reported as corrected for surrogate recoveries. Percent 

surrogate recoveries (SURecovery) for each surrogate are calculated using the following 

equation: 

x100

C

C SU

SU

ESU

Recovery =

Where: 

CESU = calculated surrogate concentration in the extract 

CSU = known concentration of surrogate added to extract 

Analyte concentration corrections (CCorrected) for surrogate recovery are calculated 

using the following equation: 

100x 

SU

C C

Recovery

Corrected

5.0 QUALITY CONTROL (QC) 

Samples are analyzed in analytical batches consisting of 19 samples or fewer and QC 

samples. The QC samples are a method blank, laboratory duplicate, matrix spike, matrix 

spike duplicate, and SRM. A method blank is a reagent blank prepared in the laboratory. 

A duplicate is a sample for which a second aliquot is analyzed. Matrix spikes are 

samples that are spiked with known concentrations of known analytes. The SRM used 

depends upon availability, matrix, and analytes. All SRMs are certified and traceable to 

the National Institute of Standards and Testing (NIST). 

The validity of the data is monitored using defined QC criteria. The following QC 

criteria are used to evaluate analytical batches: 

1) Calibration 

22
• The calibration criteria (Section 3.1) must be met prior to data analyses. If the 

calibration criteria are not met, then the run is aborted and the instrument recalibrated before further sample analysis. 

2) Method Blank 

• No more than two target analytes may exceed 3 times the concentration of the 

MDL. 

• Exceptions are that if an analyte detected in the method blank exceeds 3 times the 

concentration of the MDL, but is not present in the associated samples, or if a 

sample analyte concentration is greater than 10 times that analyte concentration in 

the method blank, the result is qualified and reported. 

• If a method blank exceeds these criteria then the source of contamination is 

determined and corrective action is taken before further sample analysis. 

3) Matrix Spikes 

• Analytes spiked into a matrix are considered valid only if they are spiked at 

concentrations equivalent to levels found in the sample. 

• The average recovery for all valid spiked analytes in a matrix spike is between 

60% and 120%. No more than two individual spiked analyte (valid) recoveries 

may exceed 40% - 120%, with the exception of chlorpyrifos and endosulfan 

sulfate. 

• If the QC criteria are not met then the matrix spike sample failing the criteria will 

be re-analyzed and if the re-analyzed spike meets the criteria then the data are 

reported. 

• If an analyte exceeds the criteria and is not present in the associated samples 

analyzed with the analytical batch, the result is qualified and reported. 

• If upon re-analysis, QC criteria are still not met, the entire batch of samples is 

reanalyzed. If sufficient sample is unavailable to re-extract the matrix-spike, 

another sample may be selected or a blank-spike may be substituted. 

• The average relative percent difference (RPD) for a valid matrix spike/matrix 

spike duplicate or blank spike/blank spike duplicate pair is 30%. No more than 

two individual analyte RPDs may exceed 35%. 

4) Duplicate 

• The average RPD between the duplicate and original sample, for analytes greater 

than 10 times the concentration of the MDL is 30%. The RPD for no more than 

two individual analytes may exceed 35%. 

23
• If the QC criteria are not met then the sample pair failing the criteria will be reanalyzed and if the re-analyzed samples meet the criteria then the data are 

reported. 

• If an analyte exceeds the criteria and is not present in the associated samples 

analyzed with the analytical batch, the result is qualified and reported. 

• If upon re-analysis, QC criteria are still not met, the entire batch of samples is 

reanalyzed. If sufficient sample is unavailable to re-extract the duplicate pair, 

another sample may be selected. 

5). Standard Reference Material 

• The average recovery for target analytes in a SRM should not exceed 30% of the 

upper and lower bounds of the mean certified values. No more than two target 

analytes should deviate more than 35% from the upper or lower bounds of the 

mean certified values. 

• If the QC criteria are not met then the SRM failing the criteria will be re-analyzed 

and if the re-analyzed SRM meets the criteria then the data are reported. 

• If an analyte exceeds the criteria and is not present in the associated samples 

analyzed with the analytical batch, the result is qualified and reported. 

• If upon re-analysis, QC criteria are still not met, the entire batch of samples is 

reanalyzed. 

6). Surrogates 

• The average recovery of surrogate compounds is between 50% and 150%. 

• Exceptions are analytical interferences with the surrogates and diluted samples. 

• If the average recovery of surrogates exceeds the criteria, and calculation and 

analytical errors are eliminated, the sample is re-analyzed. If sufficient sample is 

unavailable for re-extraction, the data are qualified and reported. 

7). Method Detection Limit 

• The method detection limit (MDL) is determined following the procedures 

outlined in the Federal Register (1984). 

6.0 REFERENCES 

Federal Registry (1984) Vol. 49, No. 209:198-199. 

24
CHAPTER 5. QUANTITATIVE DETERMINATION OF 

POLYCYCLIC AROMATIC HYDROCARBONS USING SELECTED 

ION MONITORING GAS CHROMATOGRAPHY/MASS 

SPECTROMETRY 2000 – 2006 

Thomas J. McDonald, Bo Wang, Susanne J. McDonald and James M. Brooks 

TDI-Brooks International/B&B Laboratories Inc. 

College Station, Texas 77845 

ABSTRACT 

Selected polycyclic aromatic hydrocarbons (PAHs), including alkylated homologues, are 

detected using a gas chromatograph/mass spectrometer in selected ion monitoring mode. 

This method is capable of detecting low concentrations of PAHs in complex matrices 

such as tissues and sediments. 

1.0 INTRODUCTION 

A gas chromatograph/mass spectrometer (GC/MS) in selected ion mode (SIM), coupled 

to a capillary column, is used to resolve and detect polycyclic aromatic hydrocarbons 

(PAHs) in tissues and sediments. Samples are injected into a temperature-programmed 

GC/MS, operated in splitless mode. The capillary column is an HP-5MS (60 m x 0.25 

mm ID and 0.25 μm film thickness). The mass spectrometer is capable of scanning from 

35 to 500 AMU every second or less and uses 70 electron volts energy in electron impact 

ionization mode. A data acquisition system continuously acquires and stores all data for 

quantitation. This method is capable of producing data at parts-per-billion 

concentrations. 

2.0 APPARATUS AND MATERIALS 

2.1 EQUIPMENT 

Gas chromatograph, split/splitless injection port and electronic pressure control, Agilent 

Technologies 5890-II 

Mass spectrometer, capable of scanning from 35 to 500 AMU, utilizing 70 electron volts 

of energy in impact ionization mode, Agilent Technologies 5972-MSD 

Data acquisition system, Agilent Technologies ChemStation, capable of continuous 

acquisition and storage of all data during analysis 

Autosampler, capable of making 1 to 5 μL injections 

Capillary column, Agilent Technologies HP-5MS (60 m x 0.25 mm ID and 0.25 μm film 

thickness) 

Micropipetters, calibrated, 1% accuracy, disposable tips 

25
2.2 REAGENTS 

Dichloromethane (CAS 75-09-02), pesticide grade or equivalent purity 

Helium (CAS 7440-59-7), 99.8% purity 

2.3 STANDARDS 

2.3.1 SURROGATE SPIKING SOLUTION 

The surrogate spiking solution is prepared from aliquots of pure compounds (Absolute 

Standards, Inc.) that are diluted with dichloromethane to a final concentration of 0.5 

μg/mL. The surrogate spiking solution includes naphthalene-d8, acenaphthalene-d10, 

phenanthrene-d10, chrysene-d12 and perylene-d12. Surrogate solution (100 μL) is added to 

all samples and quality control samples prior to extraction. Surrogate compounds are 

resolved from, but elute in close proximity to, the analytes of interest. Individual 

surrogate recoveries are used to correct specific analyte concentrations based on retention 

time. 

2.3.2 INTERNAL STANDARD SOLUTION 

The internal standard solution is made from aliquots of pure compounds (Cambridge 

Isotope Laboratories, Inc.) and diluted with dichloromethane to a final concentration of 

0.5 μg/mL. The internal standard solution includes fluorine-d10, pyrene-d10, and 

benzo(a)pyrene-d12. The internal standard compounds are resolved from, but elute in 

close proximity to, the analytes of interest. The internal standard solution (100 μL) is 

added to all samples and quality control samples just prior to instrument analysis. 

Internal standards are used to calculate relative response factors and specific analyte 

concentrations based on retention time. 

2.3.3 MATRIX SPIKING SOLUTION 

Certified solutions containing 2- to 5-ring PAH compounds are purchased from 

commercial vendors (Chiron, Aldrich and Absolute Standards) and diluted with 

dichloromethane to prepare the matrix spiking solution (Table 5-1). The matrix spiking 

solution is diluted to a concentration approximately 10 times the MDL and is added to all 

matrix spike samples. 

2.3.4 CALIBRATION SOLUTION 

Calibration solutions are prepared at 5 concentrations ranging from approximately 0.02 to 

1 μg/mL (Table 5-2) by diluting commercially available certified solutions containing 

analytes of interest (Absolute Standards, Inc., Cambridge Isotope Laboratories, Inc., 

Chiron, Aldrich, and Absolute Standards). 

26
2.3.5 RETENTION INDEX SOLUTIONS 

The mid-level calibration standard, containing analytes at approximately 0.25 μg/mL, is 

used as a retention index solution to determine the retention times of unsubstituted 

compounds and certain substituted compounds. A crude oil/coal oil standard material is 

used as a retention index solution for the determination of retention times for the 

remaining alkyl homologues. The retention index solutions are also used to evaluate 

instrument retention time drift over time. 

Table 5-1. Polycyclic aromatic hydrocarbons contained in the matrix-spiking solution. 

Analyte CAS Spiking Solution Concentration

(ng/mL)

Decalin 97-17-8 1.00 

Naphthalene 91-20-3 1.06 

2-Methylnaphthalene 91-57-6 1.05 

1-Methylnaphthalene 90-12-0 1.06 

Benzo[b]thiophene 95-15-8 1.86 

Biphenyl 92-52-4 1.06 

2,6-Dimethylnaphthalene 581-42-0 1.06 

Acenaphthylene 208-96-8 1.02 

Acenaphthene 83-32-9 1.10 

1,6,7-Trimethylnaphthalene 2245-38-7 0.940 

Dibenzofuran 132-64-9 1.00 

Fluorene 86-73-7 1.05 

Pentachlorophenol 87-86-5 4.00 

Carbazole 86-74-8 1.00 

Anthracene 120-12-7 0.801 

1-Methylphenanthrene 832-69-9 1.05 

Phenanthrene 85-07-8 1.06 

Dibenzothiophene 132-65-01 1.00 

Fluoranthene 206-44-0 1.06 

Pyrene 129-00-0 1.06 

Naphthobenzothiophene 239-35-0 1.00 

Benz[a]anthracene 56-55-3 0.919 

Chrysene 218-01-9 1.07 

C30-Hopane 13849-96-2 1.00 

Benzo[b]fluoranthene 200-99-2 1.06 

Benzo[k]fluoranthene 207-08-9 1.05 

Benzo[e]pyrene 192-97-2 1.06 

Benzo[a]pyrene 50-32-8 0.955 

Perylene 198-55-0 0.800 

Indeno[1,2,3-c,d]pyrene 193-39-5 0.938 

Dibenzo[a,h]anthracene 53-70-3 0.794 

Benzo[g,h,i]perylene 191-24-2 0.945 

27
Table 5-2. Polycyclic aromatic hydrocarbons contained in calibration solutions and their 

approximate concentrations. 

Compounds Contained CAS Level 1 Level 2 Level 3 Level 4 Level 5 

in Calibration Solutions (μg/mL) (μg/mL) (μg/mL) (μg/mL) (μg/mL) 

Internal Standards

Fluorene-d10 NA 0.05 0.05 0.05 0.05 0.05 

Pyrene-d10 NA 0.05 0.05 0.05 0.05 0.05 

Benzo[a]pyrene-d12 NA 0.05 0.05 0.05 0.05 0.05 

Surrogates

Naphthalene-d8 NA 0.02 0.10 0.25 0.50 1.00 

Acenaphthene-d10 NA 0.02 0.10 0.25 0.50 1.00 

Phenanthrene-d10 NA 0.02 0.10 0.25 0.50 1.00 

Chrysene-d12 NA 0.02 0.10 0.25 0.50 1.00 

Perylene-d12 NA 0.02 0.10 0.25 0.50 1.00 

5β(H)-Cholane 80373-86-0 0.02 0.10 0.25 0.50 1.00 

Analytes

Naphthalene 91-20-3 0.021 0.106 0.267 0.531 1.06 

2-Methylnaphthalene 91-57-6 0.021 0.104 0.263 0.524 1.05 

1-Methylnaphthalene 90-12-0 0.021 0.105 0.265 0.527 1.05 

Benzo[b]thiophene 95-15-8 0.037 0.184 0.464 0.928 1.86 

Biphenyl 92-52-4 0.021 0.105 0.266 0.529 1.06 

2,6-Dimethylnaphthalene 581-42-0 0.021 0.105 0.265 0.527 1.06 

Acenaphthylene 208-96-8 0.020 0.101 0.255 0.508 1.02 

Acenaphthene 83-32-9 0.022 0.109 0.276 0.548 1.10 

1,6,7-Trimethylnaphthalene 2245-38-7 0.019 0.093 0.236 0.470 0.939 

Dibenzofuran 132-64-9 0.020 0.100 0.250 0.500 1.00 

Fluorene 86-73-7 0.021 0.105 0.264 0.526 1.05 

Pentachlorophenol 87-86-5 0.250 0.500 1.000 1.500 2.00 

Carbazole 86-74-8 0.020 0.100 0.250 0.500 1.00 

Dibenzothiophene 132-65-01 0.020 0.100 0.250 0.500 1.00 

Phenanthrene 85-01-8 0.021 0.105 0.266 0.528 1.06 

Anthracene 120-12-7 0.016 0.080 0.201 0.400 0.800 

1-Methylphenanthrene 832-69-9 0.021 0.104 0.263 0.523 1.05 

Fluoranthene 206-44-0 0.021 0.106 0.267 0.530 1.06 

Pyrene 129-00-0 0.021 0.105 0.266 0.530 1.06 

Naphthobenzothiophene 239-35-0 0.020 0.099 0.250 0.500 1.00 

Benz[a]anthracene 56-55-3 0.018 0.091 0.231 0.459 0.918 

Chrysene 218-01-9 0.021 0.106 0.267 0.532 1.06 

C30-Hopane 13849-96-2 0.020 0.100 0.250 0.500 1.00 

Benzo[b]fluoranthene 200-99-2 0.021 0.105 0.265 0.528 1.06 

Benzo[k]fluoranthene 207-08-9 0.021 0.105 0.264 0.526 1.05 

Benzo[e]pyrene 192-97-2 0.021 0.105 0.265 0.528 1.06 

Benzo[a]pyrene 50-32-8 0.019 0.095 0.238 0.477 0.954 

Perylene 198-55-0 0.016 0.080 0.201 0.400 0.799 

Indeno[1,2,3-cd]pyrene 193-39-5 0.019 0.093 0.236 0.469 0.937 

Dibenzo[a,h]anthracene 53-70-3 0.016 0.079 0.199 0.396 0.793 

Benzo[g,h,i]perylene 191-24-2 0.019 0.094 0.237 0.472 0.944 

28
3.0 QUANTITATIVE DETERMINATION OF PAHS BY GC/MS-SIM 

3.1 MASS SPECTROMETER TUNING 

Prior to calibration, the MS is autotuned using perfluorotributylamine (PFTBA) to criteria 

established by the instrument manufacturer. 

3.2 INITIAL CALIBRATION 

A 5-point relative response factor (RRF) calibration curve is established for analytes of 

interest prior to the analysis of samples and quality control (QC) samples (Table 5-2). A 

RRF is determined for each analyte for each calibration level using the following 

equation: 

( )( )

( )( AIS )

ISA

CA

CA = RRF

Where: 

AA = the area of the characteristic ion for the analyte to be measured 

AIS = the area of the characteristic ion for the specific internal standard 

CA = the known concentration of the analyte in the calibration solution (μg/mL) 

CIS = the known concentration of the internal standard in the calibration solution 

(μg/mg) 

The response factors determined for each calibration level are averaged to produce a 

mean relative response factor (RRFi) for each analyte. The percent relative standard 

deviation (%RSD) for the 5 response factors must be less than or equal to 15%, for each 

analyte. 

100

RRFs the of Average

RRFs the of Deviation Standard %RSD = ×

Where: 

( )

1) - (n

2 n

1i

x - x

 Deviation Standard

∑ i

= =

Where: 

xi = each RRF value used to calculate the mean RRF 

x = the mean of n values 

n = total number of values (5) 

3.3 CONTINUING CALIBRATION 

A mid-level calibration standard is analyzed at the beginning and end of each analytical 

set or every 10 samples (whichever is more frequent). The daily relative response factor 

29
for each compound is compared to the mean relative response factor from the initial 

calibration curve and the average relative percent difference (RPD) of all analytes must 

be less than 25%. If the calibration check does not meet this criterion then the initial 

five-point calibration is repeated. 

100x 

RRF

RRF RRF RPD

i

c − i

=

Where: 

RRFi = mean relative response factor from the most recent initial calibration (meeting 

technical acceptance criteria) 

RRFc = relative response factor from the continuing calibration standard 

3.4 GC/MS-SIM ANALYSIS 

The initial calibration of the GC/MS must meet the previously described criteria prior to 

sample analysis. Samples are analyzed in analytical sets that consist of standards, 

samples, and QC samples. Quality control samples are method blanks, laboratory 

duplicates, matrix spikes, and standard reference materials. An autosampler is used to 

inject 1 or 2 μL of all samples, standards, and QC samples into the capillary column of 

the GC using the following instrument conditions. Slight modifications may be 

necessary depending upon the analysis. 

Inlet: Splitless

Carrier gas: Helium, 1 mL/min 

Temperatures: 

Injection port: 300 °C 

Transfer line: 290 °C 

Oven program: 

Initial oven temp: 60 °C 

Initial hold time: 0 minutes 

Ramp rate: 7 °C/min 

Final oven temp: 315 °C 

Final hold time: 22 minutes 

Total run time: 56 minutes 

The effluent from the GC capillary column is routed directly into the ion source of the 

MS. The MS is operated in the selected ion monitoring mode (SIM) and includes the 

quantitation and confirmation masses for the PAHs listed in Table 5-3. For all 

compounds detected at a concentration above the MDL, the confirmation ion is checked 

to confirm the analyte’s presence. 

30
3.5 ANALYTE IDENTIFICATION 

The extracted ion current profiles of the primary m/z and the confirmatory ion for each 

analyte must meet the following criteria: 

The characteristic masses of each analyte of interest must be in the same scan or within 

one scan of each other. The retention time must fall within ±5 seconds of the retention 

time of the authentic compound or alkyl homologue grouping determined by the analysis 

of the daily calibration check or reference oil standard. 

The alkylated PAH homologue groupings (e.g. C4-naphthalene) appear as a group of 

isomers. The pattern of each group and the retention time window for the group is 

established by the analysis of a reference oil standard. Each group of alkylated 

homologues is integrated in its entirety and the total area response is used to determine 

the concentration of the entire group. 

The relative peak heights of the primary mass ion, compared to the confirmation or 

secondary mass ion, must fall within ±30 percent of the relative intensities of these 

masses in a reference mass spectrum (Table 5-3). The reference mass spectrum is 

obtained from the continuing calibration solution or the reference oil standard for the 

parent compounds and alkylated homologues, respectively. In some instances, a 

compound that does not meet secondary ion confirmation criteria may still be determined 

to be present in a sample after close inspection of the data by a qualified mass 

spectrometrist. Supportive data includes the presence of the confirmation ion, but at a 

ratio different then that indicated in Table 5-3. 

Data not meeting the criteria established in this section are appropriately qualified or reanalyzed. 

31
Table 5-3. Target analyte parameters. 

Analyte CAS 

Reference to 

Internal Standard 

and Surrogate 

Quantitation 

Ion 

Confirmation 

Ion 

% Relative 

Abundance of 

Confirmation Ion 

Fluorene-d10 (I -1) NA 176 174 85 

Naphthalene-d8 (S-1) NA I-1 136 134 11 

Decalin 91-17-8 I-1, S-1 138 96 90 

C1-Decalins NA I-1, S-1 152 ND ND 

C2-Decalins NA I-1, S-1 166 ND ND 

C3-Decalins NA I-1, S-1 180 ND ND 

C4-Decalins NA I-1, S-1 180 ND ND 

Naphthalene 91-20-30 I-1, S-1 128 127 13 

2-Methylnaphthalene 91-57-6 I-1, S-1 142 141 80 

1-Methylnaphthalene 90-12-0 I-1, S-1 142 141 80 

C1-Naphthalenes NA I-1, S-1 142 141 ND 

Acenaphthene-d10 (S-2) NA I-1 164 162 89 

2,6-Dimethylnaphthalene 581-42-0 I-1, S-2 156 141 90 

1,6,7-Trimethylnaphthalene 2245-38-7 I-1, S-2 170 155 102 

C2-Naphthalenes NA I-1, S-2 156 141 ND 

C3-Naphthalenes NA I-1, S-2 170 155 ND 

C4-Naphthalenes NA I-1, S-2 184 169 ND 

Benzothiophene 11095-43-5 I-1, S-2 134 89 10 

C1-Benzothiophenes NA I-1, S-2 148 ND ND 

C2-Benzothiophenes NA I-1, S-2 162 ND ND 

C3-Benzothiophenes NA I-1, S-2 176 ND ND 

Biphenyl 92-52-4 I-1, S-2 154 152 30 

Acenaphthylene 208-96-8 I-1, S-2 152 153 15 

Acenaphthene 83-32-9 I-1, S-2 154 153 98 

Dibenzofuran 132-64-9 I-1, S-2 168 139 25 

Fluorene 86-73-7 I-1, S-2 166 165 95 

C1-Fluorenes NA I-1, S-2 180 165 ND 

C2-Fluorenes NA I-1, S-2 194 179 ND 

C3-Fluorenes NA I-1, S-2 208 193 ND 

Pyrene-d10 (I -2) NA 212 210 15 

Phenanthrene-d10 (S-3) NA I-2 188 184 15 

Pentachlorophenol 87-86-5 I-2, S-3 266 268 70 

Carbazole 86-74-8 I-2, S-3 167 139 10 

Dibenzothiophene 132-65-01 I-2, S-3 184 152 18 

C1-Dibenzothiophenes NA I-2, S-3 198 184 ND 

C2-Dibenzothiophenes NA I-2, S-3 212 197 ND 

C3-Dibenzothiophenes NA I-2, S-3 226 211 ND 

C4-Dibenzothiophenes NA I-2, S-3 240 ND ND 

Phenanthrene 85-01-8 I-2, S-3 178 176 20 

Anthracene 120-12-7 I-2, S-3 178 176 20 

C1-Phenanthrene/anthracenes NA I-2, S-3 192 191 ND 

C2-Phenanthrene/anthracenes NA I-2, S-3 206 191 ND 

C3-Phenanthrene/anthracenes NA I-2, S-3 220 205 ND 

C4-Phenanthrene/anthracenes NA I-2, S-3 234 219 ND 

Naphthobenzothiophene NA I-2, S-3 234 ND ND 

C1-Naphthobenzothiophenes NA I-2, S-3 248 ND ND 

C2-Naphthobenzothiophenes NA I-2, S-3 262 ND ND 

C3-Naphthobenzothiophenes NA I-2, S-3 276 ND ND 

Fluoranthene 206-44-0 I-2, S-3 202 101 15 

Pyrene 129-00-0 I-2, S-3 202 101 15 

C1-Fluoranthene/pyrenes NA I-2, S-3 216 215 ND 

C2-Fluoranthene/pyrenes NA I-2, S-3 230 ND ND 

32
Table 5-3. Target analyte parameters (cont’d). 

Analyte CAS 

Reference to 

Internal Standard 

and Surrogate 

Quantitation 

Ion 

Confirmation 

Ion 

% Relative 

Abundance of 

Confirmation Ion 

C3-Fluoranthene/pyrenes NA I-2, S-3 244 ND ND 

Chrysene-d12 (S-4) NA I-2 240 236 20 

Benz[a]anthracene 56-55-3 I-2, S-4 228 226 30 

Chrysene 218-01-9 I-2, S-4 228 226 30 

C1-Chrysenes/Benzo[a]anthracenes NA I-2, S-4 242 241 ND 

C2-Chrysenes/Benzo[a]anthracenes NA I-2, S-4 256 241 ND 

C3-Chrysenes/Benzo[a]anthracenes NA I-2, S-4 270 255 ND 

C4-Chrysenes/Benzo[a]anthracenes NA I-2, S-4 284 269 ND 

Benzo(a)pyrene-d12 (I-3) NA 264 260 20 

5β(H)-Cholane(S-6) 80373-86-0 I-3 217 ND ND 

C29-Hopane 53584-60-4 I-3, S-3 191 398 5 

18α−Oleanane 30759-92-3 I-3, S-3 191 412 5 

C30-Hopane 13849-96-2 I-3, S-3 191 412 5 

Benzo[b]fluoranthene 200-99-2 I-3, S-4 252 253 30, 

Benzo[k]fluoranthene 207-08-9 I-3, S-4 252 253 30, 

Benzo[e]pyrene 192-97-2 I-3, S-4 252 253 30 

Benzo[a]pyrene 50-32-8 I-3, S-4 252 253 30 

Indeno[1,2,3-cd]pyrene 193-39-5 I-3, S-4 276 277 25, 

Dibenzo[a,h]anthracene 53-70-3 I-3, S-4 278 279 25, 

C1-Dibenzo[a,h]anthracenes NA I-3, S-4 292 ND ND 

C2-Dibenzo[a,h]anthracenes NA I-3, S-4 306 ND ND 

C3-Dibenzo[a,h]anthracenes NA I-3, S-4 320 ND ND 

Benzo[g,h,i]perylene 191-24-2 I-3, S-4 276 277 25, 

Perylene-d12 (S-5) NA I-3 264 260 20 

Perylene 198-55-0 I-3, S-5 252 253 20 

ND = Not determined 

(I-#) = Internal standard reference number 

(S-#) = Surrogate reference number 

33
4.0 QUANTITATION CALCULATIONS 

Sample analyte concentrations are calculated based on the concentration and response of 

the internal standard compounds (Table 5-2). The equations in Section 3.2 are used to 

calculate the RRF of each analyte relative to the concentration and area of the internal 

standard in the initial calibration. Response factors for alkyl homologues are presumed 

equal to the response factor of the respective unsubstituted (parent) compound. 

The mass (MA) of each target analyte (ng), including alkyl homologues, is calculated 

using the following equation: 

( )

( iIS )

ISA

A

RRFA

MA M =

Where: 

AA = the area of the characteristic ion for the analyte measured 

AIS = the area of the characteristic ion for the specific internal standard 

MIS = mass of internal standard added to the extract (ng) 

RRFi = the average relative response factor for the analyte from the current 

calibration 

The concentration (C) of each target analyte in a sample (ng/g) is calculated using the 

following equation: 

( )

( ) W

DFM C A

=

 

Where: 

DF = the dilution factor applied to the extract 

μL)(dilution make tousedextract of Volume

L)(Extract ofVolume DF μ =

W = the sample weight (g) 

Analyte concentrations are reported as corrected for individual surrogate recoveries. The 

corrections for each compound are based on the surrogates referenced in Table 5-3. 

Percent surrogate recoveries (SURecovery) for each surrogate are calculated using the 

following equation: 

x100

C

C SU

SU

ESU

Recovery =

Where: 

CESU = calculated surrogate concentration in the extract 

34
CSU = known concentration of surrogate added to extract 

Analyte concentration corrections (Ccorrected) for surrogate recovery are calculated using 

the following equation: 

100x 

SU

C C

Recovery

Corrected

5.0 QUALITY CONTROL (QC) 

Samples are analyzed in analytical batches consisting of 19 samples or fewer and QC 

samples. The QC samples are a method blank, laboratory duplicate, matrix spike, matrix 

spike duplicate, and standard reference material (SRM). A method blank is a reagent 

blank prepared in the laboratory. A duplicate is a sample for which a second aliquot is 

analyzed. Matrix spikes are samples that are spiked with known analyte concentrations. 

The SRM is a reference material with known analyte concentrations. All SRMs are 

certified and traceable to National Institute of Standards and Technology (NIST). The 

SRM used depends upon availability, matrix, and analytes. 

The validity of the data is monitored using defined QC criteria. The following QC 

criteria are used to evaluate analytical batches: 

1) Calibration 

• The calibration criteria (Section 3.2) must be met prior to data analyses. If the 

calibration criteria are not met, then the run is aborted and the instrument recalibrated before further sample analysis. 

2) Method Blank 

• No more than two target analytes may exceed 3 times the concentration of the 

MDL. 

• Exceptions are that if an analyte detected in the method blank exceeds 3 times the 

concentration of the MDL but is not present in the associated samples, or if a 

sample analyte concentration is greater than 10 times that analyte concentration in 

the method blank, the result is qualified and reported. 

• If a method blank exceeds these criteria then the source of contamination is 

determined and corrective action is taken before further sample analysis. 

3) Matrix Spikes 

• Analytes spiked into a matrix are considered valid only if they are spiked at 

concentrations equivalent to levels found in the sample. 

35
• The average recovery for all valid spiked analytes in a matrix spike is between 

60% and 120%. No more than two individual spiked analyte (valid) recoveries 

may exceed 40%-120%, with the exception of decalin and biphenyl. 

• If the QC criteria are not met then the matrix spike sample failing the criteria will 

be re-analyzed and if the re-analyzed spike meets the criteria then the data are 

reported. 

• If an analyte exceeds the criteria and is not present in the associated samples 

analyzed with the analytical batch, the result is qualified and reported. 

• If upon re-analysis, QC criteria are still not met, the entire batch of samples is 

reanalyzed. If sufficient sample is unavailable to re-extract the matrix-spike, 

another sample may be selected or a blank-spike may be substituted. 

• The average RPD for a valid matrix spike/matrix spike duplicate or blank 

spike/blank spike duplicate pair is 30%. No more than two individual analyte 

RPDs may exceed 35%. 

4) Duplicate 

• The average RPD between the duplicate and original sample, for analytes greater 

than 10 times the concentration of the MDL, is 30%. The RPD for no more than 

two individual analytes may exceed 35%. 

• If the QC criteria are not met then the sample pair failing the criteria will be reanalyzed and if the re-analyzed samples meet the criteria then the data are 

reported. 

• If an analyte exceeds the criteria and is not present in the associated samples 

analyzed with the analytical batch, the result is qualified and reported. 

• If upon re-analysis, QC criteria are still not met, the entire batch of samples is 

reanalyzed. If sufficient sample is unavailable to re-extract the duplicate pair, 

another sample may be selected. 

5) Standard Reference Material 

• The average recovery for target analytes in a SRM should not exceed 30% of the 

upper and lower bounds of the mean certified values. No more than two target 

analytes should deviate more than 35% from the upper or lower bounds of the 

mean certified values. 

• If the QC criteria are not met then the SRM failing the criteria will be re-analyzed 

and if the re-analyzed SRM meets the criteria then the data are reported. 

36
• If an analyte exceeds the criteria and is not present in the associated samples 

analyzed with the analytical batch, the result is qualified and reported. 

• If upon re-analysis, QC criteria are still not met, the entire batch of samples is 

reanalyzed. 

6) Surrogates 

• The average recovery of surrogate compounds is between 50% and 150%. 

• Exceptions are analytical interferences with the surrogates and diluted samples. 

• If the average recovery of surrogates exceeds the criteria, and calculation and 

analytical errors are eliminated, the sample is re-analyzed. If sufficient sample is 

unavailable for re-extraction, the data are qualified and reported. 

7) Method Detection Limit 

• The method detection limit (MDL) is determined following the procedures 

outlined in Federal Register (1984). 

6.0 REFERENCES 

Federal Registry (1984) Vol. 49, No. 209:198-199. 

37
CHAPTER 6. EXTRACTION OF SEDIMENTS FOR BUTYLTINS 

2000 - 2006 

Juan A. Ramirez, Donell S. Frank, Susanne J. McDonald, and James M. Brooks 

TDI-Brooks International/B&B Laboratories Inc. 

College Station, Texas 77845 

ABSTRACT 

Determining organic contaminant levels in sediments require extraction, isolation, and 

concentration of analytes from the matrix. Sediments are dried, pulverized, and 

homogenized prior to extraction. Sediments are extracted in a tropolone/hexane or 

tropolone/dichloromethane mixture using sonication. The extracts are reduced in volume 

and hexylmagnesium bromide (Grignard reagent) is added, followed by neutralization 

with hydrochloric acid. The organic fraction is reduced in volume and purified using 

silica gel/florisil chromatography columns. The volume of the resultant eluent is reduced 

and analyzed for butyltins by gas chromatography/flame photometric detection. 

1.0 INTRODUCTION 

Butyltins (BTs), including monobutyltin, dibutyltin, tributyltin, and tetrabutyltin are 

contaminants of concern in the estuarine environment. The procedure described is used 

to extract, isolate, purify, and concentrate BT contaminants from sediments. 

Contaminant concentrations in parts per billion can be resolved in sediments. Sediment 

samples are homogenized, dried, and ground using a mortar and pestle. Approximately 

10 g of dry sediment are extracted in 60 mL of 0.1% tropolone/hexane or 0.1% 

tropolone/dichloromethane using a sonic probe. The liquid is decanted and the extraction 

procedure is repeated twice more. The combined extracts are reduced in volume. The 

samples are treated with hexylmagnesium bromide (Grignard reagent) and then 

neutralized with hydrochloric acid (HCl). The organic layer is drawn off, reduced in 

volume, and then purified using silica gel/florisil columns. The eluent is reduced to 10 

mL from which 2 mL is prepared for the analysis of BTs by gas chromatography/flame 

photometric detection (GC/FPD). 

2.0 APPARATUS AND MATERIALS 

2.1 EQUIPMENT 

Solvent reduction apparatus, Zymark TurboVap LV concentration workstation 

Sonicator, Tekmar TMX500 sonicator 

Balance, top loading, tare capacity to 300 g, capable of weighing to 1 mg 

Microbalance, capable of weight to 1 μg 

Calibrated weights, certified 

Combustion furnace, electric capable of combusting glassware at 400 °C for at least 4 hr 

38
Oven capable of maintaining 40 °C temperature 

Conditioning oven, electric, gravity convection, capable of maintaining a stable 

temperature of up to 200 °C 

Concentration tubes, Zymark® 60 mL borosilicate glass 

Concentration tubes, 60 mL certified pre-cleaned with open screw caps and Teflon lined 

septa 

Micropipettors, calibrated, 1% accuracy, disposable tips 

Beakers, 150 mL 

Erlenmeyer flasks, 250 mL 

Amber extract vials, 2.0 mL, with teflon-lined screw caps 

Glass wool 

2.2 REAGENTS 

Silica gel (CAS 1343-98-2)/Florisil (CAS 1343-88-0) columns, Resteck, 16 g florisil and 

5 g silica gel 

Dichloromethane (CAS 75-09-2), pesticide grade or equivalent purity 

Hexane (CAS 110-54-3), pesticide grade or equivalent purity 

Hydrochloric acid (CAS 7647-01-0), Tracepur® Plus or equivalent purity 

Copper (CAS 7440-50-8), granular, 20-30 mesh, ACS reagent grade, purified 

Tropolone (CAS 533-75-5), 98% purity 

Grignard reagent, hexylmagnesium bromide (CAS 3761-92-0) 

Nitrogen (CAS 7727-37-9), 99.8% purity 

3.0 PROCEDURE 

Sediment samples are frozen upon receipt at –20 °C until extraction. Prior to extraction, 

sediment samples are thawed and homogenized using a stainless steel spatula. A 

subsample is removed for percent moisture determination (McDonald et al., 2006). At 

least 10 g of sample are dried in an oven at 40 °C and then ground and homogenized 

using a mortar and pestle. Approximately 10 g of the dried sediment are extracted. 

Dried samples are weighed into 150 mL beakers to which 60 mL of 0.1% tropolone in 

either hexane or dichloromethane is added. Appropriate surrogates and spikes are added 

39
to beakers prior to extraction. The sediment/tropolone mixture is sonicated at 50% 

power, 1 second on and 0.5 second off, for a total of 3 minutes. The extract is decanted 

into a 250 mL Erlenmeyer flask. The extraction is repeated two more times. Quality 

control (QC) samples (e.g., blanks, duplicates, matrix spikes, and certified reference 

materials) are prepared in the same manner as samples. 

The combined extracts are reduced to 10 mL using a Zymark TurboVap LV 

concentration station set at 40 °C and 15 psi. An aliquot of extract (between 20 and 45 

mL) is added to a 60 mL concentration tube and reduced in volume. Additional aliquots 

of extract are added to the tube as the volume is decreased until the sample extract has 

been reduced to approximately 10 mL. Purified copper granules are carefully mixed with 

concentrated hydrochloric acid, followed by thorough rinsing with HPCL grade water. 

Copper is added to the concentration tube to remove sulfur. Copper is added until it no 

longer turns black, indicating that all sulfur has been bound. Samples extracted in the 

tropolone/dichloromethane mixture are back-extracted into hexane. The samples are 

quantitatively transferred to a clean 60 mL concentration tube and reduced to 10 mL. 

The butyltin in the sample extracts are then hexylated by adding 1 mL of 

hexylmagnesium bromide (Grignard reagent). The sample headspace is purged using 

nitrogen, the tubes are capped and shaken for 1 hour on a shaker table. After shaking, the 

Grignard reagent is neutralized by adding 40 mL of 10% HCl to each tube in an ice bath. 

The tubes are shaken and the upper organic layer is transferred to a 50 mL Zymark 

concentration tube. The acid fraction is rinsed twice more using 10 mL of hexane, each 

time transferring the organic layer to the concentration tube. The extract is then reduced 

to 2 – 4 mL using a Zymark TurboVap LV set at 40 °C and 15 psi. 

The concentrated extract is purified using silica gel/florisil chromatography columns. 

The chromatography columns contain 16 g of florisil and 5 g silica gel and are 

conditioned by flushing with 30 mL of hexane. The sample and solvent rinses are added 

to the top of column and 125 mL of hexane is added and eluted until the column is dry. 

The eluent is collected in 250 mL Erlenmeyer flasks. The eluent is quantitatively 

transferred to a Zymark concentration tube and reduced to a final volume of 10 mL. 

Approximately 2 mL of each extract is transferred to a clean 2 mL amber vial and stored 

at –20 °C until analysis. Internal standard is added immediately prior to instrument 

analysis. Figure 9-1 shows a flow chart of the extraction and purification procedure. 

4.0 QUALITY CONTROL (QC) 

Solvents are verified to be contaminant-free by lot tests prior to use. All equipment and 

glassware used to extract samples are thoroughly cleaned by solvent rinsing or 

combustion at 400 °C. The calibration and accuracy of balances, weights, pipettors, and 

thermometers are checked daily using certified weights and thermometers with 

calibrations traced to the National Institute of Standards and Technology (NIST). The 

calibration and accuracy of balances, weight, pipettors, and thermometers are verified 

yearly by independent sources. A series of quality control samples are processed with 

each batch of 20 samples or less. The following quality controls are used to ensure the 

accuracy and precision of tissue data. 

40
• Surrogates. Solutions containing analytes that do not interfere with the analytes 

of interest are prepared at concentrations approximately 5 to 10 times the method 

detection limit (MDL). Specified surrogates are added to each sample extracted, 

including QC samples, at a specified volume (typically 100 μL), immediately 

prior to extraction. 

• Method Blank. Method blanks are extractions of all support material used for 

extraction of samples, with the exception of sediment. A method blank is 

analyzed with each extraction batch of 20 or fewer samples. The method blank is 

extracted and analyzed in a manner identical to samples. 

• Matrix Spike. Matrix spikes are extractions of sample matrix fortified with spikes 

of selected target analytes. Spikes are prepared at concentrations approximately 

10 times the MDL. A matrix spike and matrix spike duplicate are analyzed with 

each extraction batch of 20 or fewer samples. Matrix spikes are extracted and 

analyzed in a manner identical to samples. 

• Laboratory Duplicates. A sample is analyzed in duplicate with each extraction 

batch of 20 or fewer samples. 

• Certified Reference Material (CRM). A standard reference material (CRM 

PACS-2) is analyzed with each extraction batch of 20 or fewer samples. 

41
SAMPLE RECEIPT

AND STORAGE

SAMPLE 

PREPARATION

SAMPLE 

EXTRACTION

HEXYLATION

WITH GRIGNARD REAGENT

NEUTRALIZATION

WITH HCL 

SAMPLE CLEANUP

VIA FLORISIL/SILICA GEL

ANALYSIS BY

GC/FPD 

% MOISTURE

DETERMINATION

Figure 6-1. Methodology for extraction, isolation, and quantification of sediment 

samples for butyltins. 

42
CHAPTER 7. EXTRACTION OF BIOLOGICAL TISSUES FOR 

BUTYLTINS 2000 - 2006 

Juan A. Ramirez, Donell S Frank, Susanne J. McDonald, and James M. Brooks 

TDI-Brooks International/B&B Laboratories Inc. 

College Station, Texas 77845 

ABSTRACT 

Determining organic contaminant levels in tissues require extraction, isolation, and 

concentration of analytes from the matrix. Bivalves are shucked and homogenized. 

Aliquots of homogenized sample are chemically dried using Hydromatrix® and extracted 

in a tropolone/ hexane or tropolone/dichloromethane mixture using a tissuemizer. The 

extracts are reduced in volume and hexylmagnesium bromide (Grignard reagent) is 

added, followed by neutralization with hydrochloric acid. The organic fraction is reduced 

in volume and purified using silica gel/florisil chromatography columns. The volume of 

the resultant eluent is reduced and analyzed for butyltins by gas chromatography/flame 

photometric detection. 

1.0 INTRODUCTION 

Butyltins (BTs), including monobutyltin, dibutyltin, tributyltin, and tetrabutyltin are 

contaminants of concern in the estuarine environment. The determination of these 

compounds at low concentrations in tissue is necessary to accurately monitor spatial and 

temporal changes in U.S. coastal waters. The procedure described is used to extract, 

isolate, purify, and concentrate BT contaminants from tissues. Contaminant 

concentrations in parts-per billion can be resolved in lipid-rich tissues. Shell length and 

shell volume are determined for specimens collected at each location. Bivalves are then 

shucked and multiple organisms are processed as one sample to ensure the sample is 

representative of a population at a given site and to have sufficient sample to complete 

the analyses. Tissue samples are homogenized using a stainless steel blender outfitted 

with titanium blades. Aliquots of approximately 10 g of wet tissue are chemically dried 

with Hydromatrix® or sodium sulfate. The tissue/drying agent mixtures are extracted in 

60 mL of 0.1% tropolone in either hexane or dichloromethane using a Tekmar®

tissumizer. The liquid is decanted and the extraction procedure is repeated twice more. 

The combined extracts are reduced in volume. The samples are treated with hexyl 

magnesium bromide (Grignard reagent) and then neutralized with hydrochloric acid 

(HCl). The organic layer is drawn off, reduced in volume, and then purified using 

silica/florisil columns. The eluent is reduced to 10 mL, from which 2 mL is prepared for 

the analysis of BTs by gas chromatography/flame photometric detection (GC/FPD). 

2.0 APPARATUS AND MATERIALS 

2.1 EQUIPMENT 

Solvent reduction apparatus, Zymark TurboVap LV concentration workstation 

43
Tissumizer, Tekmar®, with stainless steel probes 

Balance, top loading, tare capacity to 300 g, capable of weighing to 1 mg 

Microbalance, capable of weight to 1 μg 

Calibrated weights, certified 

Combustion furnace, electric, capable of combusting glassware at 400 °C for at least 4 

hours 

Oven capable of 40 °C temperature maintenance 

Conditioning oven, electric, gravity convection, capable of maintaining a stable 

temperature of up to 200 °C 

Concentration tubes, Zymark® 60 mL borosilicate glass 

Concentration tubes, 60 mL certified pre-cleaned tubes with open screw caps and Teflon 

lined septa. 

Micropipettors, calibrated to 100 μL, 1% accuracy, disposable tips 

Centrifuge bottles, 200 mL 

Erlenmeyer flasks, 250 mL 

2 mL amber extract vials with teflon-lines screw caps 

Glass wool 

2.2 REAGENTS 

Silica gel (CAS 7343-88-0)/Florisil (CAS 1343-88-0) columns, Resteck, 16 g florisil and 

5 g silica gel 

Dichloromethane (CAS 75-09-2), pesticide grade or equivalent purity 

Hexane (CAS 110-54-3), pesticide grade or equivalent purity 

Hydrochloric acid (CAS 7647-01-0), Tracepur® Plus or equivalent purity 

Hydromatrix® (CAS 68855-54-9/14464-46-1), conditioned by combustion at 400 °C for 

at least 4 hours and stored at 120 °C 

Sodium sulfate (CAS 7757-82-6), anhydrous granular powder, ACS reagent grade, 

purified by combusting at 400°C for at least 4 hours and stored at 120 °C. 

44
Hydrochloric acid (CAS 7647-07-0) 

Topolone (CAS 5533-75-5), 98% purity 

Grignard reagent, hexylmagnesium bromide (CAS 3761-92-0) 

Nitrogen, (CAS 7727-37-9) 99.8% purity 

3.0 PROCEDURE 

Shell length and volume are determined for all bivalves collected at each sampling site. 

The bivalves are then shucked and the soft tissue is homogenized using a stainless steel 

Waring® blender with titanium blades. Homogenized tissue samples are frozen at –20 °C 

until extraction. Prior to extraction, tissue samples are thawed and re-homogenized using 

a stainless steel spatula. A subsample is removed for percent moisture determination 

(McDonald et al., 2006). 

Approximately 10 g wet tissue and 20 g of prepared Hydromatrix® or prepared sodium 

sulfate are weighed into a 200 mL centrifuge bottle. The tissues samples must be 

thoroughly dry to optimize the extraction efficiency. Hydromatrix® and sodium sulfate 

chemically dry samples by binding moisture. Appropriate surrogates and spikes are 

added to the centrifuge bottle prior to extraction. The samples are extracted in 60 mL of 

0.1% tropolone, in hexane or dichloromethane, using a Tekmar® tissumizer. The samples 

are macerated for 2 min. The liquid is decanted into a 250 mL Erlenmeyer flask. The 

extraction is repeated two more times. Quality control (QC) samples (e.g., blanks, 

duplicate, matrix spikes) are prepared in the same manner as samples. 

The combined extracts are reduced to 10 mL using a Zymark TurboVap LV 

concentration station set at 40 °C and 15 psi. An aliquot of extract (between 20 and 45 

mL) is added to a 60 mL concentration tube and reduced in volume. Additional aliquots 

of extract are added to the tube as the extract volume is decreased until the sample extract 

has been reduced to approximately 10 mL. Samples extracted in the 

tropolone/dichloromethane mixture are back extracted into hexane. Samples are 

quantitatively transferred to a clean 60 mL concentration tube and reduced to 10 mL. 

BTs in the sample extracts are then hexylated by adding 1 mL of hexylmagnesium 

bromide (Grignard reagent). The sample headspace is purged using nitrogen and then the 

tubes are capped and shaken for 1 hour on a shaker table. After shaking the Grignard 

reagent is neutralized by adding 40 mL of 10% HCl to each tube, in an ice bath. The 

tubes are shaken and the upper organic layer is transferred to a 50 mL Zymark 

concentration tube. The acid fraction is rinsed twice more using 10 mL of hexane, each 

time transferring the organic layer to the concentration tube. The extract is then reduced 

to 2 – 4 mL using a Zymark TurboVap LV set at 40 °C and 15 psi. 

The concentrated extract is purified using silica gel/florisil chromatography columns. 

The chromatography columns contain 16 g of florisil and 5 g silica gel and are 

conditioned by flushing with 30 mL of hexane. The sample and solvent rinses are added 

to the top of the column and 125 mL of hexane is added and eluted until the column is 

45
dry. The eluent is collected in 250 mL Erlenmeyer flasks. The eluent is quantitatively 

transferred to a Zymark concentration tube and reduced to a final volume of 10 mL. 

Approximately 2 mL of each extract is transferred to a clean 2 mL amber vial and stored 

at –20 °C until analysis. Internal standard is added immediately prior to instrument 

analysis. Figure 10-1 shows a flow chart of the extraction and purification procedure. 

46
SAMPLE RECEIPT

AND STORAGE

SAMPLE 

PREPARATION

SAMPLE 

EXTRACTION

HEXYLATION

WITH GRIGNARD REAGENT

NEUTRALIZATION

WITH HCL 

SAMPLE CLEANUP

VIA FLORISIL/SILICA GEL

ANALYSIS BY

GC/FPD 

% MOISTURE

DETERMINATION

% LIPID

DETERMINATION

Figure 7-1. Methodology for extraction, isolation, and quantification of tissue samples 

for butyltins. 

47
4.0 QUALITY CONTROL (QC) 

Solvents are verified to be contaminant-free by lot tests prior to use. All equipment and 

glassware used to extract samples are thoroughly cleaned by solvent rinsing or 

combustion at 400 °C. The calibration and accuracy of balances, weights, pipettors, and 

thermometers are checked daily using certified weights and thermometers with 

calibrations traced to the National Institute of Standards and Technology (NIST). The 

calibration and accuracy of balances, weight, pipettors, and thermometers are verified 

yearly by an independent source. All samples are shipped and received under chain-ofcustody. A series of quality control samples are processed with each batch of 20 samples 

or less. The following quality controls are used to ensure the accuracy and precision of 

tissue data. 

• Surrogates. Solutions containing analytes that do not interfere with the analytes 

of interest are prepared at concentrations approximately 5 to 10 times the method 

detection limit (MDL). Specified surrogates are added to each sample extracted, 

including QC samples, at a specified volume (typically 100 μL) immediately prior 

to extraction. 

• Method Blank. Method blanks are extractions of all support material used for 

extraction of samples, with the exception of tissue. A method blank is analyzed 

with each extraction batch of 20 or fewer samples. The method blank is extracted 

and analyzed in a manner identical to samples. 

• Matrix Spike. Matrix spikes are extractions of sample matrix fortified with spikes 

of selected target analytes. Spikes are prepared at concentrations approximately 

10 times the MDL. A matrix spike and matrix spike duplicate are analyzed with 

each extraction batch of 20 or fewer samples. Matrix spikes are extracted and 

analyzed in a manner identical to samples. 

• Laboratory Duplicates. A sample is analyzed in duplicate with each extraction 

batch of 20 or fewer samples. 

48
CHAPTER 8. QUANTITATIVE DETERMINATION OF BUTYLTINS 

USING GAS CHROMATOGRAPHY/FLAME PHOTOMETRIC 

DETECTION 1999 -2006 

Donell S. Frank, Susanne J. McDonald, Juan A. Ramirez and James M. Brooks 

TDI-Brooks International/B&B Laboratories Inc. 

College Station, Texas 77845 

ABSTRACT 

Selected butyltins (BTs) are detected using a gas chromatograph/flame photometric 

detector. This method is capable of detecting low concentrations of monobutyltin, 

dibutyltin, tributyltin, and tetrabutyltin in complex matrices such as tissues and 

sediments. 

1.0 INTRODUCTION 

A gas chromatograph/flame photometric detector (GC/FPD), coupled to a capillary 

column, is used to resolve and detect butyltins (BTs) in tissues and sediments. Samples 

are injected into a temperature-programmed GC/FPD. The capillary column is a DB-5 

(30 m x 0.25 mm ID and 0.25 μm film thickness). The data acquisition system 

continuously acquires and stores all data for quantitation. This method is capable of 

producing data at parts per-billion-concentrations. 

2.0 APPARATUS AND MATERIALS 

2.1 EQUIPMENT 

Gas chromatograph equipped with large volume injector, electronic pressure control, and 

a flame photometric detector with a tin selective 610 nm filter, Thermoquest 2000 Series 

Trace GC 

Data acquisition system, Thermoquest Chromquest software, capable of continuous 

acquisition and storage of all data during analysis 

Autosampler capable of making 1 to 250 μL injections 

Capillary column, J&W Scientific DB-5 (30 m x 0.25 mm ID and 0.25 μm film 

thickness) 

Desolvation column, Supelco fused silica intermediate polarity (15 m long by 0.53 mm 

ID) 

Micropipetters, calibrated, 1% accuracy, disposable tips 

49
2.2 REAGENTS 

Hexane (CAS 110-54-3), pesticide grade or equivalent purity 

Helium (CAS 7440-59-7), 99.8% purity 

Hydrogen (CAS1333-74-0), 99.8% purity 

Nitrogen (CAS 7727-37-9), 99.8% purity 

Air (CAS 132259-10-0), 99.8% purity 

2.3 STANDARDS 

2.3.1 SURROGATE SPIKING SOLUTION 

The surrogate spiking solution is prepared with aliquots of pure compounds (Restek) 

diluted with hexane to a final concentration of 646 ng Sn/mL. The surrogate spiking 

solution includes tripentyltin chloride. The surrogate solution (100 μL) is added to all 

samples and quality control (QC) samples prior to extraction. The surrogate is resolved 

from, but elutes in close proximity to, the analytes of interest. The recovery of the 

surrogate is used to correct analyte concentrations based on retention time. 

2.3.2 INTERNAL STANDARD SOLUTION 

The internal standard solution is made from an aliquot of pure compound (Restek) and 

diluted with hexane to a final concentration of 1,015 ng Sn/mL. The internal standard 

compound is tetra-n-propyltin. The internal standard compound is resolved from, but 

elutes in close proximity to, the analytes of interest. The internal standard solution (100 

μL) is added to all samples and QC samples just prior to analysis. The internal standard 

is used to calculate relative response factors and specific analyte concentrations based on 

retention time. 

2.3.3 MATRIX SPIKING SOLUTION 

A certified solution containing monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT) 

and tetrabutyltin (TeBT) is purchased from a commercial vendor (Restek) and diluted 

with hexane to prepare the matrix spiking solution (Table 8-1). The matrix spike solution 

is diluted to approximately 10 times the MDL and is added to all matrix spike samples. 

2.3.4 CALIBRATION SOLUTION 

Calibrations solutions are prepared at 5 concentrations ranging from approximately 0.5 to 

50 ng Sn/mL (Table 8-2) by diluting a commercially prepared solution (Restek) 

containing the analytes of interest. 

50
2.3.5 RETENTION INDEX SOLUTIONS 

The mid-level calibration standard is used as a retention index solution to determine the 

retention times of analytes of interest. 

Table 8-1. Butyltins Contained in the Matrix-Spiking Solution. 

Analyte CAS Spiking Solution Concentration 

(ng Sn/mL)

Tetrabutyltin 1461-25-2 683.9 

Tributyltin chloride 1461-22-9 729.4 

Dibutyltin dichloride 683-18-1 781.4 

Butyltin trichloride 1118-46-3 841.4 

Table 8-2. Butyltins Contained in Calibration Solutions and their Approximate 

Concentrations. 

Compounds 

Contained in 

Calibration Solutions 

CAS Level 1 

(ng Sn/mL) 

Level 2 

(ng Sn/mL) 

Level 3 

(ng Sn/mL)

Level 4 

(ng Sn/mL)

Level 5 

(ngSnmL) 

Internal Standard

Tetra-n-propyltin 2176-98-9 10.15 10.15 10.15 10.15 10.15 

Surrogates

Tripentyltin chloride 3342-67-4 0.5 2.5 5.0 25 50 

Tri-n-propyltin 995-25-5 0.5 2.5 5.0 25 50 

Analytes

Tetrabutyltin 1461-25-2 0.5 2.5 5.0 25 50 

Tributyltin chloride 1461-22-9 0.5 2.5 5.0 25 50 

Dibutyltin dichloride 683-18-1 0.5 2.5 5.0 25 50 

Butyltin trichloride 1118-46-3 0.5 2.5 5.0 25 50 

3.0 QUANTITATIVE DETERMINATION OF BUTYLTINS BY GC/FPD 

3.1 INITIAL CALIBRATION 

A 5-point relative response factor (RRF) calibration curve is established for analytes of 

interest prior to the analysis of samples and quality control (QC) samples. An RRF is 

determined for each analyte for each calibration level using the following equation: 

( )( )

( )( AIS )

ISA

CA

CA = RRF

51
Where: 

AA = the area of the analyte to be measured 

AIS = the area of the specific internal standard 

CA = the known concentration of the analyte in the calibration solution (ng Sn/mL) 

CIS = the known concentration of the internal standard in the calibration solution (ng 

Sn/mL) 

The response factors determined for each calibration level are averaged to produce a 

mean relative response factor (RRFi) for each analyte. The percent relative standard 

deviation (%RSD) for the 5 response factors must be less than or equal to 15% for each 

analyte. 

100

RRFs the of Average

Deviation Standard the of RRFs %RSD = ×

Where: 

( )

1) - (n

2 n

1i

x - x

 Deviation Standard

∑ i

= =

Where: 

xi = each RRF value used to calculate the mean RRF 

x = the mean of n values 

n = total number of values (5) 

3.2 CONTINUING CALIBRATION 

A mid-level calibration standard is analyzed at the beginning and end of each analytical 

set, or every 10 samples (whichever is more frequent). The daily relative response factor 

for each compound is compared to the mean relative response factor from the initial 

calibration curve and the average percent difference (RPD) of all analytes must be less 

than 15%. If the calibration check does not meet this criterion then the initial five-point 

calibration is repeated. 

100x 

RRF

RRF RRF RPD

i

c − i

=

Where: 

RRFi = mean relative response factor from the most recent initial calibration (meeting 

technical acceptance criteria) 

RRFc = relative response factor from the continuing calibration standard 

52
3.3 GC/FPD ANALYSIS 

The initial calibration of the GC/FPD must meet the previously described criteria 

prior to sample analysis. Samples are analyzed in analytical sets that consist of 

standards, samples, and QC samples. Quality control samples are method blanks, 

laboratory duplicates, blank spikes, matrix spikes, and standard reference materials. 

An autosampler is used to inject 50 μL of all samples, standards, and QC samples into 

the capillary column of the GC using the following instrument conditions. Slight 

modifications may be necessary depending upon the analysis. 

Inlet: Large volume with solvent vent

Carrier gas: Helium, 1.9 mL/min 

Detector gas: Hydrogen 90 mL/min:Air 105 mL/min 

Make up gas: Nitrogen 20 mL/min 

Temperatures: 

Base Temp: 250°C 

FPD: 160°C 

Oven program: 

Initial oven temp: 65°C 

Initial hold time: 0.18 minutes 

Ramp rate: 10°C/min 

Final oven temp: 240°C 

Final hold time: 4 minutes 

3.4 ANALYTE IDENTIFICATION 

The retention time of a sample analyte must fall within 15 seconds of the retention time 

for the analyte in a calibration standard or retention index solution. 

Data not meeting established criteria are appropriately qualified or are re-analyzed. 

4.0 QUANTITATION CALCULATIONS 

Sample analyte concentrations are calculated based on the concentration and response of 

the internal standard. The equations in section 3.1 are used to calculate the RRF of each 

analyte relative to the concentration and area of the internal standard in the initial 

calibration. 

The mass (MA) of each target analyte (ng), is calculated using the following equation: 

( )

( iIS )

ISA

A

RRFA

MA M =

53
Where: 

AA = the area of the analyte measured 

AIS = the area of the specific internal standard 

MIS = mass of internal standard added to the extract (ng) 

RRFi = the average relative response factor for the analyte from the current 

calibration 

The concentration (C) of each target analyte in a sample (in ng Sn/g) is calculated using 

the following equation: 

( )

( ) WV

DFVM C

inj

EA

=

Where: 

Ve = final volume of the extract (μL) 

Vinj = volume of the sample injected onto the column (μL) 

W = the weight of the sample (g) 

DF = the dilution factor applied to the extract 

μL)(dilution make tousedextract of Volume

L)(Extract of Volume DF μ =

Analyte concentrations are reported as corrected for surrogate recovery. Percent 

surrogate recovery (SURecovery) is calculated using the following: 

x100

C

C SU

SU

ESU

Recovery =

Where: 

CESU = calculated surrogate concentration in the extract (ng Sn/mL) 

CSU = known concentration of surrogate added to extract (ng Sn/mL) 

Analyte concentration corrections (Ccorrected) for surrogate recovery are calculated using 

the following equation: 

100x 

SU

C C

Recovery

Corrected

5.0 QUALITY CONTROL (QC) 

Samples are analyzed in analytical batches consisting of 19 samples or fewer and QC 

samples. The QC samples are a method blank, laboratory duplicate, matrix spike, matrix 

spike duplicate, and standard reference material (SRM), if available. A method blank is a 

reagent blank prepared in the laboratory. A duplicate is a sample for which a second 

aliquot is analyzed. Matrix spikes are samples that are spiked with known concentrations 

of known analytes. The SRM is a reference material with known analyte concentrations. 

All SRMs are certified and traceable to the Canadian Research Council (NRC). A SRM 

54
is not available for tissues. The validity of the data is monitored using defined QC 

criteria. The following QC criteria are used to evaluate analytical batches: 

1). Calibration 

• The calibration criteria (Section 3.2) must be met prior to data analyses. If the 

calibration criteria are not met, then the run is aborted and the instrument recalibrated before further sample analysis. 

2). Method Blank 

• No more than one target analyte may exceed 3 times the concentration of the 

MDL. 

• Exceptions are that if an analyte detected in the method blank exceeds 3 times the 

concentration of the MDL but is not present in the associated samples, or if a 

sample analyte concentration is greater than 10 times that analyte concentration in 

the method blank, the result is qualified and reported. 

• If a method blank exceeds these criteria then the source of contamination is 

determined and corrective action is taken before further sample analysis. 

3). Matrix Spikes 

• Analytes spiked into a matrix are considered valid only if they are spiked at 

concentrations equivalent to levels found in the sample. 

• The average recovery for all valid spiked analytes in a matrix spike is between 

60% and 120%. No more than one individual spiked analyte (valid) recoveries 

may exceed 40%-120%, with the exception of monobutyltin. 

• If the QC criteria are not met then the matrix spike sample failing the criteria will 

be re-analyzed and if the re-analyzed spike meets the criteria then the data are 

reported. 

• If an analyte exceeds the criteria and is not present in the associated samples 

analyzed with the analytical batch, the result is qualified and reported. 

• If upon re-analysis, QC criteria are still not met, the entire batch of samples is 

reanalyzed. If sufficient sample is unavailable to re-extract the matrix-spike, 

another sample may be selected or a blank-spike may be substituted. 

• The average RPD for a valid matrix spike/matrix spike duplicate or blank 

spike/blank spike duplicate pair is 30%. No more than two individual analyte 

RPDs may exceed 35%. 

4). Duplicate 

55
• The average RPD between the duplicate and original sample, for analytes greater 

than 10 times the concentration of the MDL, is 30%. The RPD for no more than 

two individual analytes may exceed 35%. 

• If the QC criteria are not met then the sample pair failing the criteria will be reanalyzed and if the re-analyzed samples meet the criteria then the data are 

reported. 

• If an analyte exceeds the criteria and is not present in the associated samples 

analyzed with the analytical batch, the result is qualified and reported. 

• If upon re-analysis, QC criteria are still not met, the entire batch of samples is 

reanalyzed. If sufficient sample is unavailable to re-extract the duplicate pair, 

another sample may be selected. 

5). Standard Reference Material 

• The average recovery for target analytes in a SRM should not exceed 30% of the 

upper and lower bounds of the mean certified values. No more than two target 

analytes should deviate more than 35% from the upper or lower bounds of the 

mean certified values. 

• If the QC criteria are not met then the SRM failing the criteria will be re-analyzed 

and if the re-analyzed SRM meets the criteria then the data are reported. 

• If an analyte exceeds the criteria and is not present in the associated samples 

analyzed with the analytical batch, the result is qualified and reported. 

• If upon re-analysis, QC criteria are still not met, the entire batch of samples is 

reanalyzed. 

6). Surrogates 

• The average recovery of surrogate compounds is between 50% and 150% with the 

exception of monobutyltim. 

• Exceptions are analytical interferences with the surrogates and diluted samples. 

• If the average recovery of surrogates exceeds the criteria, and calculation and 

analytical errors are eliminated, the sample is re-analyzed. If sufficient sample is 

unavailable for re-extraction, the data are qualified and reported. 

7). Method Detection Limit 

• The method detection limit (MDL) is determined following the procedures 

outlined in Federal Register (1984). 

56
6.0 REFERENCES 

Federal Registry (1984) Vol. 49, No. 209:198-199. 

57
APPENDIX A. METHOD DETECTION LIMITS 

Table A-1. Reporting units for organic contamiants measured by the Mussel Watch 

Project. 

Analysis Reporting Unit

BT Analysis by GC/FPD ng Sn/dry g 

PAH Analysis by GC/MS ng/dry g 

PCB and PEST Analysis by GC/ECD ng/dry g 

Table A-2. Mussel Watch Project tissue butyltin method limits of detection (ng Sn/g dry 

weight). 

 2000 2001 2002 2003 2004 

Sample size 1.15 g 1.15 g 1.15 g 1.15 g 1.15 g 

Monobutyltin 1.95 1.95 1.95 0.56 0.56 

Dibutyltin 3.6 3.6 3.6 4.88 4.88 

Tributyltin 4.83 4.83 4.83 6.82 6.82 

Tetrabutyltin 2.73 2.73 2.73 6.53 6.53 

Table A-3. Mussel Watch Project sediment butyltin method limits of detection (ng Sn/g 

dry weight). 

 2003 2004 

Sample size 1.15 g 15.0 g 

Monobutyltin 2.54 0.71 

Dibutyltin 0.40 0.41 

Tributyltin 0.27 0.56 

Tetrabutyltin 0.21 0.49 

 

58
Table A-4. Mussel Watch Project tissue polycyclic aromatic hydrocarbon method limits 

of detection (ng/g dry weight). 

 2000 2001 2002 2003 2004 

Sample size 3 g 2.5 g 2.5 g 2.1 g 2.1 g 

Decalin 5.9 5.9 

C1-Decalin 5.9 5.9 

C2-Decalin 5.9 5.9 

C3-Decalin 5.9 5.9 

C4-Decalin 5.9 5.9 

Naphthalene 2.2 2.3 2.3 9.03 9.03 

C1-Naphthalenes 1.6 4.6 4.6 9.03 9.03 

C2-Naphthalenes 1.4 4.6 4.6 9.03 9.03 

C3-Naphthalenes 1.4 4.6 4.6 9.03 9.03 

C4-Naphthalenes 1.4 4.6 4.6 9.03 9.03 

Benzothiophene 3.94 3.94 

C1-Benzothiophene 3.94 3.94 

C2-Benzothiophene 3.94 3.94 

C3-Benzothiophene 3.94 3.94 

Biphenyl 1.5 2 2 2.45 2.45 

Acenaphthylene 0.7 0.5 0.5 2.22 2.22 

Acenaphthene 0.6 0.4 0.4 2.12 2.12 

Dibenzofuran 2.22 2.22 

Fluorene 0.7 0.6 0.6 2.48 2.48 

C1-Fluorenes 1.5 1.1 1.1 2.48 2.48 

C2-Fluorenes 1.5 1.1 1.1 2.48 2.48 

C3-Fluorenes 1.5 1.1 1.1 2.48 2.48 

Anthracene 2 1.7 1.7 1.18 1.18 

Phenanthrene 2.2 1.5 1.5 3.63 3.63 

C1-Phenanthrenes/Anthracenes 4.4 2.9 2.9 3.63 3.63 

C2-Phenanthrenes/Anthracenes 4.4 2.9 2.9 3.63 3.63 

C3-Phenanthrenes/Anthracenes 4.4 2.9 2.9 3.63 3.63 

C4-Phenanthrenes/Anthracenes 4.4 2.9 2.9 3.63 3.63 

Dibenzothiophene 3.5 0.5 0.5 1.78 1.78 

C1-Dibenzothiophenes 7.1 1.1 1.1 1.78 1.78 

C2-Dibenzothiophenes 7.1 1.1 1.1 1.78 1.78 

C3-Dibenzothiophenes 7.1 1.1 1.1 1.78 1.78 

Fluoranthene 2.1 0.8 0.8 9.02 9.02 

Pyrene 1.9 1.4 1.4 5.71 5.71 

C1-Fluoranthenes/Pyrenes 4.1 1.6 1.6 9.02 9.02 

C2-Fluoranthenes/Pyrenes 9.02 9.02 

C3-Fluoranthenes/Pyrenes 9.02 9.02 

59
Table A-4 (cont’d). Mussel Watch Project tissue polycyclic aromatic hydrocarbon 

method limits of detection (ng/g dry weight). 

 2000 2001 2002 2003 2004 

Sample size 3 g 2.5 g 2.5 g 2.1 g 2.1 g 

Naphthobenzothiophene 2.94 2.94 

C1-Naphthobenzothiophene 2.94 2.94 5.9 

C2-Naphthobenzothiophene 2.94 2.94 5.9 

C3-Naphthobenzothiophene 2.94 2.94 5.9 

Benz[a]anthracene 3.3 1 1 3.16 3.16 

Chrysene 3.8 0.9 0.9 5.08 5.08 

C1-Chrysenes 6.6 1.8 1.8 5.08 5.08 

C2-Chrysenes 6.6 1.8 1.8 5.08 5.08 

C3-Chrysenes 6.6 1.8 1.8 5.08 5.08 

C4-Chrysenes 6.6 1.8 1.8 5.08 5.08 

Benzo[b]fluoranthene 1.6 0.8 0.8 3.83 3.83 

Benzo[k]fluoranthene 1.2 0.7 0.7 2.82 2.82 

Benzo[e]pyrene 1.6 1.9 1.9 2.68 2.68 

Benzo[a]pyrene 1.2 1.3 1.3 1.63 1.63 

Perylene 1.5 2 2 5.35 5.35 

Indeno[1,2,3-c,d]pyrene 1.2 1 1 3.34 3.34 

Dibenzo[a,h]anthracene 1 0.4 0.4 2.36 2.36 

C1-Dibenzo[a,h]anthracene 2.36 2.36 

C2-Dibenzo[a,h]anthracene 2.36 2.36 

C3-Dibenzo[a,h]anthracene 2.36 2.36 

Benzo[g,h,i]perylene 1.4 0.7 0.7 2.39 2.39 

2-Methylnaphthalene 1.1 0.6 0.6 

1-Methylnaphthalene 0.7 0.3 0.3 

2,6-Dimethylnaphthalene 0.7 0.5 0.5 

1,6,7-Trimethylnaphthalene 1.2 1 1 

1-Methylphenanthrene 1.2 1.3 1.3 

 

60
Table A-5. Mussel Watch Project sediment polycyclic aromatic hydrocarbon method 

limits of detection (ng/g dry weight). 

 2003 2004 

Compound 15.0 g 15.0 g 

Decalin 0.17 0.17 

C1-Decalin 0.35 0.35 

C2-Decalin 0.35 0.35 

C3-Decalin 0.35 0.35 

C4-Decalin 0.35 0.35 

Naphthalene 0.17 0.17 

C1-Naphthalenes 0.33 0.33 

C2-Naphthalenes 0.35 0.35 

C3-Naphthalenes 0.35 0.35 

C4-Naphthalenes 0.35 0.35 

Benzothiophene 0.17 0.17 

C1-Benzothiophene 0.35 0.35 

C2-Benzothiophene 0.35 0.35 

C3-Benzothiophene 0.35 0.35 

Biphenyl 0.14 0.14 

Acenaphthylene 0.19 0.19 

Acenaphthene 0.13 0.13 

Dibenzofuran 0.2 0.2 

Fluorene 0.19 0.19 

C1-Fluorenes 0.39 0.39 

C2-Fluorenes 0.39 0.39 

C3-Fluorenes 0.39 0.39 

Carbazole 0.33 0.33 

Anthracene 0.19 0.19 

Phenanthrene 0.14 0.14 

C1-Phenanthrenes/Anthracenes 0.29 0.29 

C2-Phenanthrenes/Anthracenes 0.29 0.29 

C3-Phenanthrenes/Anthracenes 0.29 0.29 

C4-Phenanthrenes/Anthracenes 0.29 0.29 

Dibenzothiophene 0.15 0.15 

C1-Dibenzothiophenes 0.31 0.31 

C2-Dibenzothiophenes 0.31 0.31 

C3-Dibenzothiophenes 0.31 0.31 

Fluoranthene 0.21 0.21 

Pyrene 0.19 0.19 

C1-Fluoranthenes/Pyrenes 0.39 0.39 

C2-Fluoranthenes/Pyrenes 0.39 0.39 

C3-Fluoranthenes/Pyrenes 0.39 0.39 

 2003 2004 

Compound 15.0 g 15.0 g 

Naphthobenzothiophene 0.2 0.2 

C1-Naphthobenzothiophene 0.41 0.41 

C2-Naphthobenzothiophene 0.41 0.41 

C3-Naphthobenzothiophene 0.41 0.41 

Benz[a]anthracene 0.13 0.13 

Chrysene 0.17 0.17 

C1-Chrysenes 0.35 0.35 

C2-Chrysenes 0.35 0.35 

C3-Chrysenes 0.35 0.35 

C4-Chrysenes 0.35 0.35 

Benzo(b)fluoranthene 0.29 0.29 

Benzo(k)fluoranthene 0.23 0.23 

Benzo(e)pyrene 0.31 0.31 

Benzo(a)pyrene 0.22 0.22 

Perylene 1.38 1.38 

Indeno[1,2,3-c,d]pyrene 0.28 0.28 

Dibenzo(a,h)anthracene 0.15 0.15 

C1-Dibenzo[a,h]anthracene 0.31 0.31 

C2-Dibenzo[a,h]anthracene 0.31 0.31 

C3-Dibenzo[a,h]anthracene 0.31 0.31 

Benzo[g,h,i]perylene 0.14 0.14 

2-Methylnaphthalene 

1-Methylnaphthalene 

2,6-Dimethylnaphthalene 

1,6,7-Trimethylnaphthalene 

1-Methylphenanthrene 

61
Table A-6. Mussel Watch Project pesticide tissue method limits of detection (ng/g dry 

weight).

 2000 2001 2002 2003 2004 

Sample size 3 g 2.5 g 2.5 g 2.1 g 2.1 g 

Aldrin 0.23 0.25 0.25 0.24 0.24 

Dieldrin 0.27 0.37 0.37 0.22 0.22 

Endrin 0.38 0.24 0.24 0.21 0.21 

Heptachlor 0.33 0.28 0.28 0.25 0.25 

Heptachlor-Epoxide 0.28 0.3 0.3 0.23 0.23 

Oxychlordane 0.52 0.27 0.27 0.28 0.28 

Alpha-Chlordane 0.59 0.29 0.29 0.23 0.23 

Gamma-Chlordane 0.71 0.29 0.29 0.27 0.27 

Trans-Nonachlor 0.44 0.22 0.22 0.22 0.22 

Cis-Nonachlor 0.4 0.26 0.26 0.24 0.24 

Alpha-HCH 0.43 0.25 0.25 0.23 0.23 

Beta-HCH 0.27 0.26 0.26 0.23 0.23 

Delta-HCH 0.43 0.23 0.23 0.23 0.23 

Gamma-HCH 0.37 0.25 0.25 0.22 0.22 

2,4'-DDD 0.34 0.29 0.29 0.22 0.22 

4,4'-DDD 0.57 0.34 0.34 0.2 0.2 

2,4'-DDE 0.28 0.25 0.25 0.21 0.21 

4,4'-DDE 0.51 0.24 0.24 0.22 0.22 

2,4'-DDT 0.3 0.25 0.25 0.25 0.25 

4,4'-DDT 0.47 0.25 0.25 0.21 0.21 

1,2,3,4-Tetrachlorobenzene 0.26 0.29 0.29 0.33 0.33 

1,2,4,5-Tetrachlorobenzene 0.38 0.27 0.27 0.3 0.3 

Hexachlorobenzene 0.51 0.24 0.24 0.25 0.25 

Pentachloroanisole 0.32 0.28 0.28 0.18 0.18 

Pentachlorobenzene 0.28 0.26 0.26 0.22 0.22 

Endosulfan II 0.72 0.26 0.26 0.25 0.25 

Endosulfan I 0.72 0.26 0.26 0.25 0.25 

Endosulfan Sulfate 0.72 0.29 0.29 0.27 0.27 

Mirex 0.42 0.28 0.28 0.23 0.23 

Chlorpyrifos 0.47 0.28 0.28 0.25 0.25 

 

62
Table A-7. Mussel Watch Project pesticide sediment method limits of detection (ng/g dry 

weight). 

 2003 2004 

Sample size 15.0 g 15.0 g 

Aldrin 0.1 0.1 

Dieldrin 0.06 0.06 

Endrin 0.12 0.12 

Heptachlor 0.09 0.09 

Heptachlor-Epoxide 0.18 0.18 

Oxychlordane 0.05 0.05 

Alpha-Chlordane 0.04 0.04 

Gamma-Chlordane 0.05 0.05 

Trans-Nonachlor 0.04 0.04 

Cis-Nonachlor 0.07 0.07 

Alpha-HCH 0.09 0.09 

Beta-HCH 0.07 0.07 

Delta-HCH 0.08 0.08 

Gamma-HCH 0.05 0.05 

2,4'-DDD 0.07 0.07 

4,4'-DDD 0.11 0.11 

2,4'-DDE 0.05 0.05 

4,4'-DDE 0.04 0.04 

2,4'-DDT 0.1 0.1 

4,4'-DDT 0.08 0.08 

1,2,3,4-Tetrachlorobenzene 0.12 0.12 

1,2,4,5-Tetrachlorobenzene 0.08 0.08 

Hexachlorobenzene 0.05 0.05 

Pentachloroanisole 0.06 0.06 

Pentachlorobenzene 0.06 0.06 

Endosulfan II 0.1 0.1 

Endosulfan I 0.1 0.1 

Endosulfan Sulfate 0.11 0.11 

Mirex 0.04 0.04 

Chlorpyrifos 0.1 0.1 

 

63
Table A-8. Mussel Watch Project polychlorinated biphenyl tissue method limits of 

detection (ng/g dry weight). 

 2000 2001 2002 2003 2004 

Sample size 3 g 2.5 g 2.5 g 2.1 g 2.1 g 

PCB8 0.77 0.39 0.39 0.36 0.36 

PCB18 0.8 0.52 0.52 0.44 0.44 

PCB28 0.54 0.45 0.45 0.22 0.22 

PCB44 0.63 0.35 0.35 0.4 0.4 

PCB52 0.26 0.48 0.48 0.24 0.24 

PCB66 0.67 0.43 0.43 0.34 0.34 

PCB101 0.43 0.66 0.66 0.32 0.32 

PCB105 0.85 0.41 0.41 0.33 0.33 

PCB118 0.35 0.67 0.67 0.25 0.25 

PCB128 0.83 0.43 0.43 0.54 0.54 

PCB138 0.58 0.87 0.87 0.43 0.43 

PCB153 0.97 0.67 0.67 0.49 0.49 

PCB170 0.51 0.81 0.81 0.32 0.32 

PCB180 0.84 0.58 0.58 0.24 0.24 

PCB187 0.45 0.62 0.62 0.31 0.31 

PCB195 0.34 0.33 0.33 0.27 0.27 

PCB206 0.33 0.31 0.31 0.29 0.29 

PCB209 0.32 0.36 0.36 0.25 0.25 

 

64
65

Table A-9. Mussel Watch Project polychlorinated biphenyl sediment method limits of 

detection (ng/g dry weight). 

 2003 2004 

Sample size 15.0 g 15.0 g 

PCB8 0.1 0.1 

PCB18 0.06 0.06 

PCB28 0.05 0.05 

PCB44 0.1 0.1 

PCB52 0.05 0.05 

PCB66 0.04 0.04 

PCB101 0.04 0.04 

PCB105 0.1 0.1 

PCB118 0.06 0.06 

PCB128 0.11 0.11 

PCB138 0.06 0.06 

PCB153 0.06 0.06 

PCB170 0.06 0.06 

PCB180 0.06 0.06 

PCB187 0.04 0.04 

PCB195 0.04 0.04 

PCB206 0.04 0.04 

PCB209 0.07 0.07 

 

United States Department of Commerce 

Carlos M. Gutierrez 

 Secretary 

National Oceanic and Atmospheric Administration 

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

Under Secretary of Commerce for Oceans and Atmospheres 

National Ocean Service 

John H. Dunnigan 

Assistant Administrator for Ocean Service and Coastal Zone Management