United States Offtce of EPA 560/ 5 86 036 Environmental Protection Toxic Substances December. 1986 Agency Washington DC 2046G Toxic Substances SEPA BROAD SCAN ANAbYStS OF THE FY82 NATIONAL HUMAN ADIPOSE TISSUE SURVE  SPECIMENS VOLUME II  VOLATILE ORGANIC C MPOUNI 0P? 7 2wz QO 66 002 3 REPROWCEOBY US. DEPARTMENTOFCOMMERCE HAfloNplLrrcHMcAL 000001 INFFORMATIONSEWNICE SPRINGFELD. VA22161 PREFACE This report is the second of a five volume series that details the broad scan chemical analysis of composite adipose tissue samples. These composite samples were prepared from individual specimens obtained from the Environmental Protection Agency's ( EPA) National Human Adipose Tissue Survey ( NHATS) fiscal year 1982 ( FY82) repository. . .... .

This volume summarizes data generated from the analysis of the composited samples for volatile organic compounds. Volume I, the executive summary presents a synopsis of a71 analysis efforts completed under the broad scan program. Volumes I11 through V deal specifically with the chemical analy sis of the NHATS composites for general semivolatile organic compounds, polychlorinated dibenzo e dioxins ( PCDD), and dibenzofurans ( PCDF), and trace elements The statistical analyses of the data reported in these volumes will be reported separately by the EPA's Office of Toxic Substances ( OTS) Design and Development Branch contractor, Battelle Columbus Laboratories.

The entire series of reports are referenced as follows: Stanley JS. 1986. Broad scan analysis of human adipose tissue: Volume I: Executive summary.  PA 560/ 5 86 035. Stanley JS. 1986. Broad scan analysis of human adipose tissue: Volume 11: Volatile organic compounds. EPA 560/ 5 86 036. Stanley JS. 1986. Broad scan analysis'of human adipose tissue: Volume 111: Semivolatile organic compounds. EPA 560/ 5 86 037. Stanley 35.. 1986. Broad scan analysis of human adipose tissue: Volume IV: Polychlorinated dibenzo p dioxins ( PCDD) and polychlorinated dibenzofurans ( PCDF).  PA 560/ 5 86 038.

 Stanley JS, Stockton RA. 1986. Broad scan analysis of human adipose tissue: Volume V: Trace elements. EPA 560/ 5 86 039.

These method development, sample analyses, and reporting activities were completed for the EPA/ OTS Field Studies Branch ( FSB) broad scan analysis of human adipose tissue program ( EPA Prime Contract Nos. 68 02 3938and 68 024252 Work Assignments 8 and 21, respectively, Ms. Janet Remmers, Work Assignment Manager, and Dr. Joseph Breen, Project Officer).

The samples were prepared with the assistance of Ms. Leslie Moody and Mr. Steven Turner. The HRGC/ MS methods development and sample analyses were conducted by Mr. Steven Turner, Ms. Margaret Wickham, and Mr. Gil Radolovich. The cornpositing scheme used to prepare the samples from the NHATS repository was provided by Dr. Gregory Mack, Battelle Columbus Laboratories, under contract to the EPA/ OTS Design and Development Branch ( Mr. Philip Robinson, Task Manager, and Ms. Cindy Stroup, Program Manager).

MIDWEST RESEARCH INSTITUTE . AhnE. Going Paul C. Constant Director Program Manager Chemical Sciences Department iii 088002 DISCLAIMER i This document has been reviewed and approved for publication by the Office of Toxic Substances, Office of Pesticides and Toxic Substances, U. S.  Environmental Protection Agency. The use of trade names or commercial products does not constitute. Agency endorsement or recommendation for use. i ...*

TABLE OF CONTENTS Pagc!

Executive Summary .......................... xi 1. I ntroducti. on ........................ 1 A . Broad Scan Analysis Strategy ........... 1 B. Work Assignment Objectives ............. 1 C. Background Information ............... 2 I1. Recommendations ....................... 3 111 Experimental........................ 3 A .  3 C .

0.

E.

F. G.

H . Collection and Storage of NHATS Specimens..... 4 Sample Compositing Activity............. 4 Sources and Preparation of Analytical Reagents . . 5 1. Internal Quantitation Standards ....... 5 2 . Reference Compounds ............. 6 3 . Spiking Solutions .............. 7 Apparatus .................... 8 1. Dynamic Headspace Purge and Trap System ... 8 2 . High Resolution Gas Chromatography/ Mass . Spectrometry ................ 8 Analysis Procedures................ 10 1. Spiking Procedure .............. 10 2. Method Blank Analysis ............ 10 3 . Calibration Curves for Reference Standards. . 10 4 . Composited Sample Analysis.......... 11 Quality Control Procedures ............ 11 Data Interpretation................ 11 1.. Qualitative Identification.......... 11 2. Quantitation................. 14 Method Val idation................. 14 IV . Resu1 ts .......................... 23 V . Quality Assurance/ Quality Control ............. 75 A . Instrument Performance Checks........... 75 1. Internal QC ................. 75 2. External QC ........... .... 75 8. Spiked Adipose Tissue Samples...... .... 76 C. Internal Standards .......... .... 76 VI . References................... ..... a5 Append x A . Analyticat Method for the Determination of Vo ati le Organic Compounds in Human Adipose Tissue. . ..... A 1 Amendix 8 . Volatile Organic Compound Data from the NHATS FY82 .. Composite Human Adipose Tissue Samples ........  3 3 Appendix C . Cornpositing Scheme for the NHATS FY82 Specimens..... c 1 V 00Q004 Preceding page blank LIST 06 FIGURES Figure Page 1 Schematic of apparatus for volatile organic analysis ... 9 .2 Analytical scheme for analysis of volatile organic compounds from human adipose tissue. ......... 12 .

3 Purging efficiency of three internal standards ( 1 pg each) from 20 g of human adipose tissue, over four sequential, 40 min heating and purging cycles. ..... 19 4 Purging efficiency of eight stable isotope labeled internal standards ( 1pg each) from 20 g of human adipose tissue over four sequential 40 min heating and purging cycles ................... 20  5 Purging efficiency of seven target analytes ( 0.20 pg each) spiked into 20 g of human adipose tissue, over four sequential 40 min heating and purging cycles. ... 21 Purging efficiency of six target analytes ( 0.20 pg each) spiked into 20 g. of human adipose tissue, over four sequential 40 min heating and purging cycles ...... 22 Comparison of purging efficiencies for target analyte/ deuterated analog pairs: ( a) benzene/ d, benzene and ( b) toluene/ d, toluene .................... 24 Comparison of purging efficiencies for target ana? yte/ deuterated analog pairs: ( a) chlorobenzene/ d5 chlorobenzene and ( b) ethylbenzene/ d,, ethylbenzene. .. 25 Comparison of purging efficiencies for target analyte/ deuterated analog pairs for ( a) 1,1,2,2 tetra chloroethane/ d2 l, l, 2, Z tetrachloroethane and ( b) 1,2 dich10robenzene/ d4 1,4 dich10robenzene ..... 26 10 Purging efficiency of seven volatile organic analytes . endogenous to human adipose tissue over four sequential 40 min heating and purging cycies ...... 27 11 HRGC/ MS chromatogram of purge tower standard analyzed daily to document instrument performance ........ 28 12 HRGC/ MS chromatogram for the volatile organic analysis of a bulk adipose ( tissue standard) spiked with 1.0 pg of each internal standard and 0.20 pg of each target analyte. ........................ 29 13 HRGC/ MS chromatogram of volatile compounds from the NHATS FY82 composite of the 0 14yr age group from the Middle Atlantic census division. ............ 30 vii Precedingpage blank OQIQOBS LIST OF FIGURES ( concluded)

Figure Page 14 Comparison of the HRGC/ MS chromatograms for the volatile organic analytes for three composite age groups from the Middle Atlantic census division. .......... 31 15 Comparison of the HRGC/ MS chromatograms for the volatile organic a. nalytes'for three composite age groups from the East North Central census division ........... 32 16 Comparison of the HRGC/ MS chromatograms of the volatile organic analysis of three composite specimens repre senting the 15 44 age group from three census divisions. 33 17 Comparison of the HRGC/ MS chromatograms of the vol atile organic analysis of three composite specimens repre senting the 0 14 age group from three census divisions . 34 18 Incidence of detection of volatile organic compounds in cornposited human adipose tissues from the Northeast census region. ..................... 36 19 Incidence of detection of volatile organic compounds in cornposited human adipose tissues from the South census region. ..................... 37 20 Incidence of detection of volatile organic compounds in cornposited human adipose tissues from the North Central census region. ..................... 38 21 Incidence of detection of volatile organic compounds in composited human adipose tissues from the West census region. ..................... 39 2.2 Observed HRGC/ MS responses for the internal standards d chloroform and d6 benzene, from method blanks, spiked tissue samples and the NHATS composite samples. . 81 23 Observed HRGC/ MS responses for the internal' standards d8 toluene and d, chlorobenzene, from method blanks, spiked tissue samples and the NHATS composite samples. .. 82 24 Observed HRGC/ MS responses for the internal standards dIo p xylene and d, o ethylbenzene, from method blanks, spiked tissue samples and the NHATS composite samples. . a3 25 Observed HRGC/ MS responses for the internal standards d2 1, l, 2,2 tetrachloroethane and d4 dichlorobenzene, from method blanks, spiked tissue samples and the NHATS composite samples. ................ 84 viii . LIST OF TABLES Table Page  1 Characteristic Ions, Internal Standards, and Relative Retention Times Used to Identify and Quantitate Target Analytes. . . . . . . . . . . . . . . . . . . . . 13 2 Summary of Spiked Recovery ( X) Experiments for Volatile Analytes from 20 g of Human Adipose Tissue . . . . . . . 16 3 Summary of Spiked Recovery (%) Experiments Based on a Single Internal Standard Bromoch? oropropaneversus Multiple Deuterated Standards ( Isotope Dilution) . . . 17 4 Data Report  Chloroform ( CAS No. 67 66 3)  FY82 Composite Adipose Tissue Samples. . . . . . . . . . 40 5 Data Report  l, l, l Trichloroethane ( CAS No. 67 66 3) FY82 Composite Adipose Tissue Samp les. . . . . . . . . . 42 6 Data Report  Bromodichloromethane ( CAS No. 75 27 4)  FY82 Composite Adipose Tissue Samples. . . . . . . . . 44 7 Data Report  Benzene ( CAS No. 71 43 2) FY82 Composite Adipose Tissue Samples. . . . . . . . . . 46 8 Data Report  Tetrachloroethene ( CAS No. 127 18 4)  FY82 Compbsite Adipose Tissue Samples. . . . . . . . . 48 3 Data Report  Dibromochloromethane  FY82 Composite Adipose Tissue Samples. . . . . . . . . . 50 10 Data Report  l, I, Z Trichloroethane ( CAS No. 79 00 5)  FY82 Composite Adipose Tissue Samples. . . . . . . . . . 52 11 Data Report  Toluene ( CAS No. 188 88 3). FY82 Composite Adipose Tissue Samples. . . . . . . . . 54 12 Data Report  Chlorobenzene ( CAS No. 108 90 7)  FY82 Composite Adipose Tissue Samples. . . . . . . . . 56 13 Data Report  Ethylbenzene ( CAS No. 100 41 4)  FY82 Composite Adipose Tissue Samples. . . . . . . . 58 14 Data Report  Bromoform ( CAS No. 75 25 2)  FY82 Composite Adipose Tissue Samples. . . . . , . . . . 60 15 Data Report  Styrene ( CAS No. 100 42 5)  FY82 Composite Adipose Tissue Samples. _.. . . . . . . 62 16 Data Report  1,1,2,2 Tetrachl. oroethane ( CAS No. 79 34 5)  FY82 Composite Adipose Tissue Samples. . . . . . .  64 ix LIST OF TABLES ( concluded)

Table Page  17 Data Report  1,2 Dichlorobenzene ( CAS No. 95 50 1)  FY82 Composite Adipose Tissue Samples. .......... 66 18 Data Report  1,4 Dichlorobenzene ( CAS No. 106 46 7)  FY82 Composite Adipose Tissue Samples. ......... 68 19 Data Report  Ethyl Phenol ( CAS No. 25429 37 2)  FY82 Composite Adipose Tissue, Samples. ......... 70 20 Data Report  Xylene ( CAS No. 1330 20 7)  FY82 Composite Adipose Tissue Samples. ......... 72 21 Incidence of Detection of Selected Volatile Organic Compounds in the NHATS FY82 Composite Samples. ..... 74 22 Summary of the Internal QC Instrument Performance Checks for Selected Volatile Organic Analytes ......... 77 23 Average Recovery for InternaJ QC Instrument Performance Checks ......................... 78 24 Summary of Results  QC Performance Audit Samples. .... 79 .25 Summary of Method Recovery (%) of Selected Volatile Organic Analytes Spiked into 20 9 Aliquots of Human Adipose Tissue  Internal QC ........... 80 X 000008 EXECUTIVE SUMMARY The U. S. Environmental Protection Agency's Office of Toxic Substances ( EPA/ OTS) maintains a unique program for monitoring human exposure to potentially toxic substances. The National Human Adipose Tissue Survey ( NHATS) is a statistically designed annual program to collect and analyze a nationwide sample of adipose tissue specimens for toxic compounds. The primary focus for NHATS has been to document trends in human exposure to environmentally persistent contaminants, specifically organochlorine pesticides and polychlorinated biphenyls ( PCBs).

EPA/ OTS has recognized the need to provide a more comprehensive assessment of the toxic substances that accumulate in adipose tissue. Thus, the NHATS specimens collected during fiscal year 1982 ( FY82) were designated for " broad scan analysis'' to determine volatile and semivolatile organic compounds and trace elements.

This volume of the final report deals specifically with the measurement of volatile organic chemicals in composited adipose tissue specimens from the FY82 NHATS repository. The objectives of this part of the study were ( 1) to develop an analytical method based on high resolution gas chromatography/ mass spectrometry ( HRGC/ MS) for determination of volatile organic chemicals in human adipose tissue and ( 2) to complete the analysis of the FY82 NHATS specimens as composited for volatile organic compounds.

The analytical method developed to sample volatile organic compounds from human adipose tissue is based on a heated dynamic headspace purge and trap technique. The volatile organic compounds were separated and detected using HRGC/ MS. HRGC was selected to achieve the best possible separation of volatile components, and MS was selected to provide the necessary specificity to identify positively the volatile compounds present in the tissue. Target analytes were quantitated based on a multiple internal standard technique. The method evaluation studies and daily quality control checks demonstrated ' that method accuracy was improved for analytes which had a corresponding deuterated analog as an internal quantitation standard.

Forty six composite samples were prepared from the FY82 NHATS repository according to a study design prepared by the EPA/ OTS Design and Development Branch contractor, Battelle Columbus Laboratories. The composite samples represent the nine U. S. cen~ usdivisions, stratified by three age groups ( 0 14,15 44, and 45 plus).

The HRGC/ MS analysis of the volatile compounds purged from the human adipose demonstrated a complex mixture of compounds consisting primarily of a? dehydes, ketones, hydrocarbons, and carboxylic acid esters. Additional corn pounds classified as aromatic, halogenated aliphatic, and halogenated aromatic . compounds were detected as minor constituents.

Quantitative efforts focused on target analytes classified as priority pollutants. Quantitative data are reported for 17 specific compounds. The predominant target analytes noted included chloroform, l, l, l trichloroethane benzene, tetrachloroethene, toluene, chlorobenzene, ethylbenzene,

xi 080089 styrene, 1,1,2,2! tetrachloroethane, 1,4 dichlorobenzene, 1,2 dichlorobenzene, xylenes, and ethyl phenol. In all composite samples several compounds were detected including styrene, the xylene isomers, 1,4 dichlorobenzene, and ethyl phenol. The frequencies of detection for each of the compounds by age groups and census divisions are detailed in the report. The volatile organic compounds were detected in the composites from all census divisions and age group.

The quantitative data for the 17 specific compounds have been submitted along with all supporting quality control data to the OTS design and development contractor, Battelle Columbus Laboratories, for statistical analysi s.

Characterization of additional chromatographic peaks in the HRGC/ MS data to identify other compounds of interest to the Agency has been initiated under a separate work assignment ( Contract No. 68 02 4252, Work Assignment No. 23).

xi i I. INTRODUCTION The National Human Adipose Tissue Survey ( NHATS) is the main operative program of the National Human Monitoring Program. The National Human Monitoring Program was first established by the U. S. Public Health Service in 1967 and was subsequently transferred to the U. S. Environmental Protection Agency ( EPA) in 1970. During 1979 the program was transferred within EPA to the Exposure Evaluation Division ( EED) of the Office of Toxic Substances ( OTS).

NHATS is an annual program to collect a nationwide sample of adipose tissue specimens and to chemically analyze them for the presence of toxic compounds. The objective of the NHATS program is to detect and quantify the prevalences of the compounds in the general population. The NHATS data are used to address part of OTS's mandate under the Toxic Substances Control Act ( TSCA) to assess chemical risk to the U. S. population. The specimens are collected from autopsied cadavers and surgical patients according to a statistical survey design ( Lucas, Pierson, Meyers, Handy 1981). The survey design ensures that specified geographical regions and demographic categories are appropriately represented to permit valid and precise estimates of baseline ' levels, time trends, and comparisons across subpopulations.

The data for the NHATS are generated by collecting and chemically anafyzing adipose tissue specimens for selected toxic substances. Historically organochlorines and polychlorinated biphenyls ( PCBs) have been selected for evaluation.

A. Broad Scan Analysis Strategy *

EPA/ OTS has recognized the need to provide a more comprehensive assessment of the toxic substances that accumulate in adipose tissue. An aggressive strategy to assess TSCA related substances that persist in the adipose tissue of the general U. S. population has been developed by  ED. The NHATS specimens col7ected during fiscal year 1982 ( FY82) were selected for a broad scan analysis of volatile and semivolatile organic TSCA related chemicals and trace ' elements ( Mack, Stanley 1984).

The initiative to achieve a more comprehensive assessment necessitated either the development of new methods or the modification of the existing analytical procedures, specifically high resolution gas chromatography/ mass spectrometry ( HRGUMS). Data on organochlorine pesticides and PCBs reported for the NHATS specimens. up to the FY82 collection are based on packed column gas chromatography/ electron capture detector ( PGC/ ECD) analysis.

8. Work Assignment Objectives The objectives of this phase of the work assignment were ( 1) to identify appropriate analytical methods for a broad scan analysis of volatile organic compounds in human adipose tissue based on HRGC/ MS detection; ( 2) to conduct preliminary evaluation of the analytical procedures, and ( 3) to complete the sample workup and HRGC/ MS analysis of 46 composite samples prepared from the NHATS specimens collected during FY82. The target detection range far analytes by the HRGC/ MS as specified in the current NHATS strategy ( Mack, Stanley 1984) was 0.05 to 0.10pg/ g C. Background Information The exposure of the general U. S. population to volatile organic compounds has not previously been addressed through a national sampling of b. iologica1 matrices ( breath, blood or tissue). Specific studies have been conducted, however, to determine the effects of exposure to chemical solvents, monomers such as vinyl chloride and styrene in the plastics industry, and anesthetics ( Wolff 1976; Wolff,. Loumer, Selikoff, Aubrey 1977; Engstrom, Riihimaki 1979; Engstrom 1984).

The fact that blood and breath levels of volatile organics can be detected at declining levels from several hours to several days after a specific exposure incident indicate tissue retention ( Whitcher, Cohen, Trudell 1971; Corbett 1973; Wolff 1976). Human adipose tissue has been evaluated as a depot for storage and release of volatiles in specific exposure studies of workers to styrene and ethylbenzene in the polymerization industry ( Wolff 1976; Wolff, Daum, loumer, Selikoff, Aubrey 1977; Engstrom 1984).

Several analytical procedures have been developed for the measurement of specifjc compounds from these exposure scenarios, Analytical techniques have required dissolution of milligram quantities of adipose into carbon disulfide, or other suitable solvent, followed by direct injection onto gas chromatographic ( GC)~ columnswith flame ionization/ electron capture, microcoul ometri c or mass spectrometer ( MS) detectors. Other analytical procedures combined with these detection systems have included: ( a) static head space analysis of heated tissue, ( b) stripping volatiles from heated tissue by flowing an inert gas directly through the rendered sample, ( c) vacuum distillation and cryogenic focusing of the analytes on a high resolution gas chromatography ( HRGC) column, and ( d) dynamic solution purge and trap and headspace analysis of heated tissue water mixtures combined with cryogenic focusing and HRGC analysis. Each of these techniques has certain advantages and disadvantages in terms of complexity of the actual sampling procedure and sensitivity and selectivity of the detector ( Novotny, McConnell, Lee, Farlow 1974; Politzer, Githens, Dowty, Laseter 1975; Luskus, Kilian, Lackey, Biggs 1977; Snyder, Erlichman, Goldstein, Laskin 1977; Karbowski, Braun 1978; Peoples, Pfaffenberger, Enos, Shafik 1978; Balkon, Leary 19a; Peoples, Pfaffenberger,  Shafik, Enos 1979; Pfaffenberger, Freal 1979; Michael, Erickson, Parks, Pellizzari 1980; Pantarotto, Fanelli, Belletti, Bidoli 1980; Reddrop, Riess, Slater 1980; Vogt, Liao, Sun 1980; Zuccato, Marcucci, Mussini 1980; Lin, Fu, Bruckner, Feldman 1982; Hiatt 1981; Reinert, Hunter, Sabatino 1983).

An analytical method for the measurement of volatile organic chemicals in human adipose tissue has been developed as a result of this work assignment. This analytical method is a modification of a dynamic headspace purge and trap procedure ( Michael, Erickson, Parks, Pellizzari 1980) combined with high resolution gas chromatography and mass spectrometry. This report describes the method and summarizes the volatile organic data for 46 composite specimens from the FY82 NHATS repository determined with the method.

Following this introductory section, Section I1 presents recommendations for further activities for volatile organics. Section III is the 2 QQ( BO3.2 experimental section and describes the coliection and storage of the NHaTS specimens, the adipose tissue cornpositing procedures, the development of the analytical method, and the routine analytical procedures. Section IV presents the results of the composite tissue analyses. Section V summarizes the quality control ( QC) procedures and results. Section VI contains references. Appendices A, E, and C provide, respectively, the volatile organic analysis method in detail, additional data tables on the results of the analyses, and the exact compositing scheme used for the FY82 NHATS specimens.

Ir  RECOMMENDATIONS Further analytical development should be pursued to improve the determination of volatile organic compounds in human adipose tissue samples. These improvements should specifically include smaller sample sizes ( 1.0 to 5.0g), more efficient transfer of volatile organics onto the HRGC* column, and further development of the isotope dilution quantitation technique. These modifications can possible be achieved by using widebore HRGC column's and/ or cryofocusing techniques.

The analytical method should be modified to provide quantitative information on compounds of greater volatijity than chloroform ( such as methylene chloride, vinyl chloride, etc.). This possibly could be accomplished by conducting two analyses on each tissue sample. The first analysis should be conducted for the more volatile compounds with the sample heated in the range of 50 8OoC and the headspace sampled for 15 min or less. The second sample analysis should be conducted with the procedures specified in this report to provide quantitative data for compounds ranging in volatility from  chloroform through the dichlorobenzene isomers.

Stability studies should be conducted to determine the effects of long term storage at subzero temperature, and repeated thawing and freezing on the integrity of the volatile organic content in the sample. The results of the sample analysis conducted for the FY82 composites indicate considerable differences in the absolute quantities of the major volatile constituents ( hydrocarbons, aldehydes, ketones, etc.) for samples analyzed within 6 mo of collection and the NHATS specimens that have been archived prior to analysis for up to 2 yr.

I1I. EXPERIMENTAL This section of the report describes:

A. collection and storage of NHATS specimens; 8. sample compositing activity; c. sources and preparation. of analytical standards;  D. apparatus; E. analysis procedures; . F. quality control procedures; G. data interpretation; and H. method validation.

3 .

A. Collection and Storage of NHATS Specimens The adipose specimens were . originally collected during FY82 ( October 1, 1981, through September 30, 1982) for determination of organic chlorine pesticide and PCB residues. The specimens were col? ected during surgical procedures or as part of postmortem examinations. The cooperating physicians and pathologists were requested to acquire at least 5 g of high lipid adipose ( subcutaneous, perirenal, or mesenteric), taking precautions to avoid Contamination that might result in direct contamination from chemicals such as solvents, paraffin, disinfectants, preservatives, or plastics. The cooperators were given no specific instructions to avoid potential contamination that might arise from background contribution of solvents or metals.

The adipose tissue specimens were sealed in glass jars and frozen '

( 20OC) following collection. The specimens were shipped in insulated coolers packed on dry ice. The FY82 specimens were originally received and stored at EPA's Toxicant Analysis Center ( TAC) at Bay St. louis, MS. The NHATS repository was transferred to Midwest Research Institute ( MRI) during September 1982. The specimens were shipped in insulated coolers and packed on dry ice. The specimens were inventoried at MRI upon receipt and were then stored in freezers ( 20OC). Precautions were taken to ensure that the specimens remained frozen during all inventory and sample handling procedures.

8. Sample Compositing Activity The NHATS FY82 adipose tissue specimens were subsampled and composited as specified by EPA's Design and Development Branch contractor, Battelle Columbus Laboratories ( BCL). Prior to preparation of the composites, all of the FY82 specimens were retrieved from the NHATS repository and the individual specimens bott? es were grouped according to the designated cornpositing scheme provided by BCL. Care was taken to ensure that the specimens were not allowed to reach room temperature. The specimen were stored on dry ice during this process and were returned to a freezer ( 20 ° ) once all individual specimen for a specific composite had been located.

All specimens for a specific composite were removed from the freezer at the same time for the cornpositing effort. The specimens were placed on dry ice so they would remain frozen during the cornpositing effort. Each specimen was handled separately and each was subsampled for the composites for both volatile and semivolatile organic anlaysis. This resulted in minimum handling of each specimen. Once the speciment had been subsampled for each composite it was placed on dry ice. After all specimen had been added to the composites, the batch was returned to the freezer.

All samples were handled in a positive pressure Plexiglas hood of approximately 94.5 L volume to prevent contamination from laboratory air. Compressed air was filtered through a charcoal trap to remove potential volatile contaminants from air supply before it entered the hood. The subsamples were manipulated with the rounded end of a lab spoon type stainless steel spatulas and placed in 40 mL vials with TFE septa caps. Each specimen was manipulated with a separate clean spatula. All weighings were performed to k 0.1 g on a Mettler open pan balance placed in the hood.

4 .~ . .. .. ' 3 3'.

The nominal mass of each individual specimen necessary to achieve a final composite mass of 20 g was determined before proceeding with the physical compositing. For example, if a composite consisted of 20 specimens, 1.0g of each specimen was necessary to achieve a final composite mass of 20 g Separate composite samples were prepared for both semivolatile ( Stanley 1986c) and volatile organic analysis. The individual specimens were added first to the composites for the semivolatile organic analysis. This resulted in the addition of a total available specimen, in some cases, to the semivolatile organic composite only. As a result, several of the volatile organic composites contain somewhat less than the target 20 g of tissue mass. The samples resulting from the 46 volatile organic composites ranged from 5.1 to 25.6 g total mass with an average mass of 19 g. Appendix C provides a summary of the exact compositing scheme for both the volatile and semivolatile organic analyses.

Prior to the compositing effort, the vials were washed in soap and water, rinsed thoroughly with tap water, deionized water, bulk acetone, Burdick and Jackson (%& J) acetone, and B& J hexane, and then placed in an oven at 200OC for 48 h before use. The spatulas were washed and rinsed as above and were dried for at least 5 min in the oven.

All composites were stored on dry ice until transfered to a freezer ( 20OC). Before being placed in the freezer in the 40 ml vials, the composited samples were grouped by census division and placed in 1.0 qt jars ( cleaned as above) containing a layer of activated charcoal and closed with a TFE lined cap. The samples were stored in the freezer until analysis.

As a QA/ QC check, six of the empty vials used for compositing were placed in the hood on the weighing balance for approximately 15 min ( approximating the time needed for compositing), and then were capped, sealed, and stored with the samples. These blank vials were included with the analysis of the composited specimen as method blanks.

The samples resulting for the 46 composites ranged from 5.1 to 25.6 g total mass with an average mass of 19 g. Appendix C provides a summary of the exact compositing scheme.

C. Sources and Preparation of Analytical Reagents The sources of the internal standards and reference compounds and the procedures for preparing spiking and calibration standards are described below.

1. Internal Quantitation Standards A three. component internal quantitation standard mixture was obtained from Supelco, lnc. ( Catalog No. 4 8823, Lot No. LA 11802). This mixture contained bromochloromethane, 1,4 dichlorobutane, and 1 chloro Z bromopropane at 20 mg/ mL each in 1.0mL of methanol. This mixture was used as received and was stored in the freezer in its original resealable ampule.

5 080015 A second stock solution of deuterated internal quantitation standards was prepared from the compounds listed below by aliquoting each with a 100 pL syringe into a IO mL volumetric flask and diluting to the mark with methanol ( B& J high purity, Lot No. 43029). The volume of each compound necessary to achieve the desired concentration was determined by the density of the specific compound. The final concentration was approximately 10 mg/ mL for each deuterated internal standard. Because the 1,4 d4 dich1orobenzene is a solid, a separate stock was prepared in methanol before addition to the solution containing the other deuterated analogs. The final mixture was sealed in two 5 mL vials and stored in the freezer until just prior to use.

Compound d6 benzene d chloroform l, l, Z, Z d, tetrachloroethane d, methylene chloride d5 chlorobenzene 1,4 d4 dichlorobenzene dIo ethylbenzene de to1uene d,, e xylene 2. Reference Compounds Suppl ier Aldrich Gold Label ( 99.5% 0) No. 15, 181 5 Aldrich Gold Label ( 99.8% D) No. 15, 182 3 M and D Isotopes No. MD 1416 M and D Isotopes No. MD 53 KOR Isotopes ( 99% DS) NO. 521510 KOR Isotopes ( 98% D4) No. 521530 KOR Isotopes ( 98% Dlo) No. 521443 KOR Isotopes ( 99.9% D8) No. 510041 KOR Isotopes ( 98% DIo) No. 521133 Several compounds characteristic of aromatic compounds, chlorinated and brominated a1 iphatic compounds, and chlorinated aromatic compounds were selected for preparation of analytical standards. These reference compounds were also selected as a preliminary list of target analytes as a result of their presence in tissue from the preliminary method development and their classification as priority pollutants.

The compounds listed below were aliquotted with a 100 pL syringe into a 10 mL volumetric flask that was partially filled ( approximately 5 mL) with methanol. The volume of each compound necessary to achieve the desired concentration was determined from the density of each specific compound. The solution was diluted to the mark and stored in the same manner as the internal quantitation standard solutions. The compounds were a1 iquotted to obtain a final stock concentration of approximately IO mg/ mL for each compound.

Compound bromoform dibromochloromethane to1uene 1,1,2 trichloroethane styrene tetrachloroethylene bromod i chl oromethane chlorobenzene 1,2 dichlorobenzene ethylbenzene Supplier Aldrich Gold Label ( 99%/ B EtOH) Columbia No. 01843 B& J H. P. No. A1 857 Aldrich No. JB 070177 ( 95%) Eastman No. 1465 Aldrich Gold Label No. 120457 Aldrich No. 7628AH ( 98%) Aldrich No. 120277 ( 99%) Aldrich No. 05 680 2 ( 98%) Aldrich No. E1 250 8 ( 99%)

6 I I _,. .. .. I.,,, i . c ...,.. ::,.. , , . i ,. . i.! . ,

Compound Supplier 1,1,2,2 tetrachloroethane   Baker No. 017386 l, l, l trichloroethane Fisher No. 775974 ch 1or0form B& J H. P. ( XEtOH preservative) No, AG594 benzene MCB Pest. Grade No. U2738 carbon disulfide Mal 1i nckrodt No. KMEE 3. Spiking Solutions The reference stock solution and the two internal quantitation standard stock solutions ( Supelco mixture and the deuterated analog mixture) were . used to make up the spiking solutions necessary, for HRGC/ MS analysis. The three separate stock solutPons allowed manipulation of the concentrations of the spiking levels to verify method calibration and accuracy of the analyses from spiked tissue and blank samples. ,

 Spiking solutions were prepared fresh weekly from the three stock solutions. The spiking solutions were prepared. in 1.0 mL microreaction vials ( Supelco No. 3 3292) with Mininert Valves ( Supelco No. 3 301). Tetraglyme was used as the solvent for the spiking solutions to enhance their stability. All spiking solutions were stored in the refrigerator and transported between the GC/ MS facility and the laboratory on ice in a cooler. Following sample spiking, the. solutions were immediately returned to the cooler.

The spiking solutions were prepared by aliquoting tetraglyme into the 1.0 mt reaction vials and then spiking the tetraglyme with the stock solutions For examp le, a 500 ng/ pL internal standard spiking solution was made /  by injecting 975 pL of tetraglyme with a 1.0 mL syringe ( availab ie from Supelco, Inc.) into the reaction vial and then injecting 25.0 pL of the three component ( 20 mg/ mL) Supelco internal quantitation standard stock solution into the tetraglyme, A 500 ng/ vL spiking solution of the deuterated internal  standards was similarly prepared by spiking 950 pL of tetraglyme with 50.0 pL of the 10 mg/ mL deuterated internal standard stock solution.

The target analyte spiking solution was prepared in the same manner but at 100 ng/ pL. This was accomplished by spiking 990 pL of tetraglyme with 10.0 pL of the target analyte stock solution.

When the actual sample analyses were initiated, only two spiking solutions were necessary. A 500 ng/ pL solution of the three internal standards from the Supelco mixture and the nine deuterated internal standards were combined. This mixed internal quantitation standard solution was prepared with 925 pt of tetraglyme, 25.0 pt of the three component Supelco standard, and 50.0 pL of the deuterated internal standard stock solution. The target analyte spiking solution was prepared at the 100 ng/ vL level as described above.

7 D. Apparatus . The design of the dynamic headspace purge. and trap system used to collect the volatile organics and the HRGC/ MS system used for separating and identifying these compounds are described below.

1. Dynamic Headspace Purge and Trap System Method development for volatile organic analytes in human adipose tiss ue led to the system shown in Figure 1. Several Wheaton purge and trap vessels ( No. 991765) were modified for the analyses. The thermometer and funnel arms were cut near the vessel and replaced by 1/ 4 in. Kovam to PX seal tubes. ( Ace Glass, Inc., No. PT. 976). This allowed the inlet and outlet lines to be connected with standard 1/ 4 in. Swagelock@ fittings and provided leak tight connections.

The Wheaton vessel was connected to a hot water circulating bath ( Haake, No. F4391) maintained at 95OC. Approximately 5 min was required for the solution within the vessel to reach the maximum purge temperature. The vessel was placed on a magnetic stirrer ( Ace Glass, Inc., No. 12064 08), and a 1. O in. TFE stirring bar agitated the solution. Helium was directed into the vessel to displace the headspace at 40 mt/ min. All metal gas carrier lines beyond the vessel outlet were wrapped with heat tape maintained at 15OOC to prevent condensation of the target analytes and internal standards. The effluent from the vessel line flowed into a column equipped with a stopcock and frit that contained 1.0 mL of volatile free water. This column was used as a condenser to remove excess moisture from the purge gas. The outlet line from this purge tower was attached to a six way Carle valve. A GC carrier gas line of helium was attached to the Carle valve. The Carle valve was attached to a glass lined U tube ( 1/ 8 i. d.) packed with a 1.0 in. plug of Tenax. Glass wool was used to maintain the position of the Tenax in the center of the U tube. The U tube was rapidly heated ( within 5 to 8 s) to 25OOC. A resistance circuit with a thermocouple was used to heat and regulate the Utube's temperature. In the purge mode the Carle valve directed the purge gas and analytes into the U tube, which was at ambient temperature. The analytes were trapped and the purge gas vented. The helium carrier gas was directed onto the HRGC column during the purge mode. After the purge time had elapsed, the Carle valve was switched to the desorb mode and the U tube was heated to flash volatilize the analytes. The helium carrier gas was then routed through the U tube in the opposite direction of the purge mode and directed onto the HRGC column.

2. High Resolution Gas Chromatography/ Mass Spectrometry The volatile organic compounds were analyzed using a finnigan 9610 GC and a finnigan 4000 quadrupole MS equipped with an INCOS data system. Separation of the volatile organic compounds was achieved with a Durabond DB 5 fused silica capillary column, 30 m x 0.25 mm, 0.25 pm film thickness ( Jaw Scientific, Rancho Cordova, CA). The capillary column was routed directly into the ion source. The helium carrier gas was adjusted at 12 psi head pressure The GC was equipped with a Grob type split/ splitless injector. The effluent from the adsorbent trap was adjusted to 5 to 10 mL/ min and directed 8 Purse Tower with Gioss Frit 1Water Ftow Cmtr3l fer for He .

H20 in Figure 1. Schematic of apparatus for volati? e. organic analysis.

9 000019 into the Grob injector with a syringe needle attached to the stainless steel tubing from the adsorbent trap. The injector was operated in the split mode with a 1O: l split ratio.

The GC was held isothermally at 3OoC for 5 min and then programmed at 6OC/ min up to 125OC where it was held for 10 min. Mass spectral data were acquired across the mass range of 35 275 amu for 20 min from initiation of the program. The HRGC column was programmed to 2OO0C between sample analyses as a precaution to ensure that all materials desorbed from the Tenax adsorbent had been eluted from the column prior to the next analysis.

E Analysis Procedures The procedures for spiking internal quantitation standards, the method blank analysis, generation of calibration curves, and composited sample analys s are described below.

1. Spiking Procedure Internal quantitation standards and target analytes were added to the purge tower, method blanks, spiked adipose tissue matrix and composite samples using the following procedure. A 35 mm length of TFE tubing was inserted over the needle port on the Leur lock assembly of a 10.0 mL syringe. The syringe was filled with 3.0 mL of volatile organic free water and the T, FE tubing removed. The water in the syringe was spiked with the working standards ( using a 5.0 pL syringe and needle) through the needle port, and the TFE tubing was replaced. An additional 2 mL of water and 1.0 mL of air was then drawn into the 10 mL syringe, and the syringe was inverted several times to ensure mixing of the tetraglyme and water solution. The contents of the syringe were quantitatively transferred to the sample vessel. The sample vessel was tightly capped and the sample was allowed to sit at room temperature for 30 min prior to initiating the analysis.

2. Method Blank Analysis The Wheaton vessel was filled with 80 mL of the volatile organic free water and spiked with 1.0 pg of each of the internal quantitation standards The vessel was quickly capped and the gas flow turned on. The purge tower valve was kept in the off position, and the vessel and lines were leak checked with vo? atile organic free water. Following the leak check, the purge tower valve was opened, the hot water bath valves opened, and the stirring bar turned on. The headspace above the stirred mixture was purged for 40 min and collected on the Tenax trap.

3. Calibration Curves for Reference Standards Calibration curves for the compounds selected as reference standards were developed using a spiked human adipose tissue matrix. The spiked adipose tissue matrix is referred to as the tissue standard. A separate 20 9 aliquot of a bulk adipose matrix was used to prepare each tissue standard. These tissue standards were prepared immediately prior to analysis. Preparation of the spiked tissue samples as homogenize4 matrices for repeated analysis was not evaluated in this study.

10 000020 . . , ,._ :. T '. . ..:,<.. 2 :, :.

The analysis of the tissue standard was performed in'the same way as the analysis of the blank but with 20 g of human adipose tissue added to the Wheaton vessel. A spatula was used to remove the frozen tissue from the sample vials. The tissue samples were spiked with 1.0 pg of each of the internaf standards and 0.2 to 1.4 pg of each of the target analytes. Purge time was 40 min.

4. Composi ted Sample Analysis The composited samples were analyzed in exact'ly the same manner as the tissue standard except that only the internal standard spiking solution wasxadded to the aqueous mixture.

F. Quality Control Procedures Figure 2 shows the general volatire organic analysis scheme and the related quality control ( QC) procedures. Daily QC procedures included the analysis of reference standards and internal standards spiked in the purge tower ( instrument performance check); a system blank with internal standards; a reference adipose tissue spiked with standards; and a QC sample. The QC sample consisted of either an additional instrument performance check to verify calibration or an adipose'tissue spiked at a known level by the MS analyst. These QC samples were included for several days at the initiation . of the analysis of the composites. Also, the MRI quality control coordinator ( QCC) periodically submitted blind spikes to the MS analyst to assess instrument performance. These blind spikes were added to the purge tower before proceeding with the anlaysis.

The results of these analyses are provided in Section V. Before proceeding with the FY82 compositesspecimens, the MS analyst was required to complete successfully the analyses of check samples submitted by the QCC.

G. Data Interpretation The HRGC/ MS data for each sample were interpreted with computer assisted quantitation routines. A mass spectral library and quantitation list of the target analytes, based on relative retention times and the primary characteristic ion, were used to search each data file.

1. Qualitative Identification The automated search quantitation routine identified positive re. sponses based on the primary characteristic ion for each of the target analytes Table l provides a list of the target analytes, primary characteristic quantitation ions, internal standards, and the relative retention times. In addition to the automated search, the MS analyst identified compounds by reviewing the total mass spectra at the specified HRGC/ MS scan number. This effort was required to avoid the inclusion of false positives in the sample set. The HRGC/ MS data were also reviewed at 2 30 s of the retention time of each. of the compounds to avoid reporting of false negatives.

13 f Purge Tower Standard 1 I * Purge Tower QC Internal OC Spike  Daity Blind OC Spike  Weekly Instrument Performance Eva I uation Analytical Method Calibration and Evaluation System Blank Spiked Adipose Tissue Standard ( 20 g) ( 0.2 pg  1.4 pg)

Spiked Adipose Tissue Sample ( 20 g) lnterna? QC Composlte FY82 Adiwse Tissue Samples ( 5  20 g),

11 2  5 SampieslDay I 1 i t Figure 2. Analytical scheme for analysis of volatile organic compounds from human adipose tissue.

12 000022 Table 1. Characteristic Ions, Internal Standards, and Relative Retention Times Used to Identify and Quantitate Target Analytes Primary characteristic quantitation Target analyte ion ( m/ z)

Bromochl oropropane Chloroform d Chlor0form l, l, l Trichloroehtane Bromodichloromethane Benzene d, Benzene fetrachloroethene Dibromochloromethane 1,1,2 Tri chl oroethane To 1uene d8 Toluene Chlorobenzene ds Chl orobenzene Ethylbenzene dlo fithylbenzene Bromoform 5 tyrene . 1,1,2,2 Tetrach? oroethane d, l, l, 2,2 Tetrachl oroethane 1,2 Dichlorobenzene d,  1 4 Dich1orobenzene 1,4 Dichlorobenzene Xylene dIo p Xylene Ethylpheno1 77 79 82 97 129 78 a4 166 129 97 91 /

100 112 117 106 116 173'

104 83 84 146 150 146 106 116 122 ~

Internal quakitation standardb RRT~

Bromochloropropane 1 00 d Chl oroform 0.20 0.43 0.20 0.43 Bromochl oropropane 0.35 0.58 Bromochloropropane 0.53 0.76 ds Benzene 0.39 0.62 0.39 0.62 B romoch1oropropane 1.16 1.39 Bromoch1 oropropane 1.05 1.2a Brornochloropropane 0.88 1.11 d8 Toluene 0.83 1.07 0.83 1.07 d, Chl orobenzene 1.48 1.72 1 48 1.72 d,, Ethylbenzene 1.62 1.85 1.62 1.85 Bromochloropropane 1.79 1.96 Bromochloropropane 1.85 2.08 d2 1,1,2,2 Tetrachloroethane 2.05 2.28 2.05 2. za d4 1,4 Dich1orobenzene 2.88 3.11 2.73 2.96 d4 1,4 Dichlorobenzene 2.73 2.96 d,, g Xylene 1.65 1.88 1.65 1 88 dIo Ethylbenzene 2.19 2.43 aRelative retention times calculated versus the internal want tat on standard, bbromochloropropane. Designation indicates the pairing of the target analytes with the respective internal standards.

13 000023 2. Quantitation Data were quantitated based on either the internal standard or the isotope dilution principle. The isotope dilution principle applies to the quantitation of target analytes for which a deuterated analog was available as an internal standard. Where possible, the deuterated analog of a target analyte was used for quantitation. Other compounds' concentrations were cal culated versus the internal standard, brornochloropropane ( 1 chloro 2 bromo propane).

The HRGC/ MS data were quantitated using the following equation:

A, x IS 3 Target analyte, 1.19= AIS x RRF where As = area of the characteristic quantitation ion of the target analyte,

= area of the characteristic quantitation ion of the internal standard,

IS = amount of the internal standard in micrograms ( pg),

RRF = relative response factor of the target analyte versus the respective internal standard The concentration ( vg/ g) of the target analyte in the original sample was calculated by dividing the total micrograms of target analyte detected by the composite weight. In this report concentration data are expressed in either micrograms per gram ( pg/ g) or nanograms per grain ( ng/ g).

The quantitative data were qualified based on the observed response for each target analyte and the corresponding internal standard. Compounds for which no positive response was observed are reported as not detected ( ND).

, An estimated limit of detection ( LOD) was calculated based on the observed instrumental response for the specific target analyte and corresponding internal standard. in each sample. The LOD was estimated as the amount of a specific analyte that must be present to give rise to a signal at least 2.5 times the background signal to noise.

Compounds detected for which the observed response of the characteristic quantitation ion was greater than 2.5 times but less than 10 times the background signal to noise are reported as trace ( tr) values. Target analytes detected at greater than 10 times the background signal to noise, limit of quantitation ( LDQ), are reported as positive quantifiable values.

All data were corrected for blank values observed for the system blank run daily with each set of samples.

H. Method Validation The analytical method was evaluated by analyzing adipose tissue specimens fortified with known levels of specific volatile organic compounds.

14 000024 I ..

These experiments were designed to evaluate the use of the internal standards recommended for  PA Method 624 ( USEPA 1984) for determination of volatile organics in water and wastewater ( bromochloromethane, bromochloropropane, and 1,4 dichlorobutane), as well as multiple deuterated analogs of specific target analytes ( isotope dilution). Preliminary studies demonstrated that the internal standards, bromochloromethane and 1,4 dichlorobutane, were not adequately recovered from the adipose tissue/ water mixtures. Bromochloropropane provided reasonable response from the spiked adipose matrix and was further evaluated as a quantitation standard.

The results of the analysis of adipose tissue spiked with nine volatile compounds at four concentration levels ranging from 5 to 75 ng/ g ( ppb) are presented in Table 2. In general, the average recovery of the analyses for these compounds was good, except for chloroform, which demonstrated an average recovery of 264%. Although the average recovery for the other compounds was quite good ( 62 to 116%), wide variability was noted for the individual analyses at each spike level.

The high recovery of chloroform from this study may be a result of a difference in the background chloroform concentration in the adipose tissue used for this spiking experiment as compared to the tissue used for calibration. An absolute value for the background contribution of each target analyte was not determined from unspiked tissued used in these experiments Another factor that may have resulted in the high recovery of chloroform is the possible conversion of trichloroacetic acid ( present as a metabolite of tetrachlroethylene, trichloroethane, etc.). to chloroform at the elevated temperatures required to complete the analyses ( Peoples, Pfaffenberger, Shafik, Enos 1979).

The impact of the use of multiple internal standards versus a single internal standard was evaluated as a separate experiment. Deuterated analogs of several target analytes were added to fortified tissue samples in addition to bromochloropropane, Table 3 presents the comparison of the single versus multiple internal standard quantitation techniques. As noted in Table 3, a deuterated analog was not available for tetrachloroethene. The data generated for the target analytes demonstrate average recoveries ranging from 82 to 136% for the multiple internal standard quantitation as compared to average recoveries of 74 to 143% for the same compounds quantitated versus bromochloropropane. The somewhat tighter range of recoveries or accuracies of quantitation with incorporation of the multiple internal standards results from the fact that the deuterated ana logs behave similarly to the target analytes.

The purging efficiencies of the target analytes and the internal standards were evaluated from replicate analyses of a single fortified adipose tiss6e. This experiment was necessary to ( 1) demonstrate. the difference in partitioning of the various target anafytes from the water/ tissue mixture to the headspace and ( 2) compare the partitioning characteristics of the available internal standards versus the target analytes. A 20 9 aliquot of tissue was spiked with 1.0 yg each of the internal standards and 0.20 pg of the target analytes. The sample was heated to approximately 8OoC, and the headspace above the stirred mixture was sampled for 40 min. The volatile compounds were desorbed from the Tenax adsorbent and analyzed by HRGWMS. The heating, purging sampling, and analysis procedures were repeated an additional three times.

15 a3 L N 0 Q cv m m m  4 0  t m In CD m 4 p\ I rl + I + I + I + I + I + I + I + I 0 00 0 Iv m l 0 a3 N tD m rn 0 QI d m +

h h M 0 m rlco d m m cv m In 3 0 0 0 0 0 0 0 0 m m Ln m m m m m 0 0 0 0 0 0 0 0 0 rl 0 co r l m I. m Lo ( v * m co d 0 m r( 0 0 0 0 0 0 0 co a W W co W W co 0 0 0 0 0  0 0 0 * IA W d * d m 0 0co d 0 r( ? I 0 l l + 0 0 0 0 0 0 0 0 d d r( l l I4 d 4 7 4 0 0 0 0 0 0 0 0 rl h 0 u) * 0 03 m n m m rr) ri h ( v N ri 4 0 0 l i 4 ri m In m Lo m m In rc, rl. ri rl r l ri r) I4 4  mo5 mN W  9 c 2caJ a, 3 0,  7 I I c 0) N  L0 0 aJ c c E L L N W aJL OQ, 0 C N a0  c  aJ aJ C% Q) cm 0) 2s 5 Q, 0 0 C VT C VaJ 0 PL a 03 aJ m. c L P  0 0 N Ea 3 L4J 0 h rc C OE c c, QI 7 c L1 0) m L I 0 I Q, JZ 4 . V V rn 0 w n 16 W L W in 0 rl cy ri 03 4 + I 1 + I + I + I N 0, cn Wa 0 m mrf rl W I 03 0 Wh Q1 m zf r(

f. t rl w f. m rl 0 m rl 4 4 2 I PI Ln ul ll 0 cvd rl rf rl a m zf w w b In F? + t + I + I + I + I d rf 0 cv m b W rl 0 d ll 4 rl 0 3 In W h QI ro h 0 m d m PI 4 4 4 U 1 d a l CI) fi u) W In u, ri 0 u) 0 4 rl FI ..

' 0 teL 0) OQ 1 0 u) r) m In  LO N 0 co 0 0 0 a 0 4 d l 4 l 4 E= EW 30 WE 0 a, W L c cW a 0, m . a 1= r S + J c, aJ Q, aJ a W N 1 0 0 c 0 c Q, L aJ n c  0 N 0 a I m 3 c c 00 P W t . t= L E S U  n U 0 a m 7 u 0  3 L A L c c1 . c c, 0 a, & W '? U I I I I w # 13 a 17 The absolute area counts for the characteristic quantitation ion for each com pound were recorded. The data for each compound monitored are provided in Figures 3 to 6 and are plotted as absolute area counts versus the sample run number.

Figure 3 is a plot of the three internal standards used in EPA Method 624 for volatiles in water and wastewater. Three distinctive plots for the three internal standards are presented. These data demonstrate that bromochloromethane is not an acceptable internal standard due to low response. The breakthrough volume for the volatile components is based on the total sample volume mass of adsorbent and sampling temperature. Based on sampling volumes extrapolated by Brown and Purnell ( 19771, it is expected that the optimum sampling volume for the more volatile components, such as bromochloromethane are approximately 9 to 10 L per gram of Tenax adsorbed. The total sample volume used in these experiments was 1.6 L ( 40 mL/ min for 40 min) and the total mass of Tenax was approximately 0.2 g. Hence, the low response observed for bromochloromethane is probably due to the low capacity of the Tenax adsortjent system for the more volatile compounds rather than to the purging efficiency from the matrix.

The plot of bromochloropropane indicates that this internal standard is effectively partitioned from the adipose/ water mixture to air for all four analyses. The plot for 1,4 dichlorobutane, on the other hand, indicates that this compound is fairly soluble in the adipose/ water matrix. The adipose/ air partitioning constant is obviously low. Hence, this compound was not considered as a quantitative internal standard for further sample analyses.

Figure 4 presents additional plots of the observed purging efficiencies for eight deuterated internal standards spiked initially at 1.0 pg in the 20 9 adipose tissue sample. Again it is noted that the deuterated analog of chloroform was detected only in the first sample analysis. This observation is possibly due to the fact that chloroform has the lowest boiling point ( b. p. 6O. 5 61, S0C), hence greatest volatility, of the compounds analyzed. Deuterated methylene chloride ( d, CH2C12) was also added to these samples but was not detected even in the first analyses. The plots of the other internal standards appear to reflect the effect of boiling point on purging efficiency from the heated mixture. The d6 benzene and d, toluene demonstrate the most significant differences in absolute response for the sequential analysis of the spiked adipose sample. The boiling points for these compounds range from approximately 80 to lll ° C as compared to 135 to 173OC for d,, R xylene and d4 l, 4 dichlorobenzene.

The absolute responses of several target analytes spiked ( 0.20 pg each) into the same 20 9 aliquot of human adipose are plotted for the four sequential analyses ( Figures 5 and 6). The plots for these target analytes also appear to correspond with the boiling points of the specific compound. The more volatile cempounds such as chloroform ( b. p. 60.5 61. S" C), l, I, l trichloroethane ( b. p. 74 76OC), and dibrornochloromethane ( b. p. 119 12OoC) were detected in only the first and second analyses, indicating better purging efficiency a1though less retention on the Tenax adsorbent, than demonstrated for the other compounds. The response profiles noted for compounds such as benzene, toluene, chlorobenzene, tetrachloroethene, ethylbenzene, and dichlorobenzene are very similar to the plots of the deuterated analogs presented in Figure 4.

18 _... ..

Q) C 0 a a, OSwv h EU 0 Lrn +. I= .

1P 0  w m ~

c, L Y 13 i 00 19 c m E 3 c uwE5 3 20 st. a 0 0 0 * 0 0A 000031 0' v 22 P . C c * L Q7 ma 040 nn urn f C 3 er:

XL. 3  0 4le OL 22 000032 I , I The similarity in the response profiles for several target analytes and the corresponding deuterated analogs are presented in Figures 7 to 9. These plots reflect that the purging efficiencies of each pair of compounds are equivalent from the spiked adipose tissue. These plots demonstrate the advantages of the isotope dilution ( target analyte/ deuterated internal standard pair) technique versus a single internal standard for quantitation of volatile organics in human adipose tissue. With the exception of toluene, the differences of responses noted for these pairs are roughly a factor of 5 for each analysis,. which corresponds to the spike ratio of 0.20 pg of target analyte versus 1.0 pg for the deuterated internal standard. The difference noted for the toluene/ d8 toluene plot is the result of the endogenous toluene concentration in the tissue sample used for these analyses.

Figure 10 provides a plot of the obse. rved responses for several com pounds not spiked into the tissue sample. These compounds include 1,4 dichlo robenzene, two xylene isomers, and three C3 alkyl benzenes ( trimethyl, methyl ethyl, or propyl isomers). These results indicate that the. method might be capable of detecting and quantitating compounds with boiling points ranging up to approximately 175OC.

IV. RESULTS The analyses for the NHATS FY82 composite samples were completed following the method evaluation studies. Figures 11 to 17 provide examples of the HRGC/ MS chromatograms for the analysis of instrument performance checks, spiked tissue samples, and actual composite samples.

Figures 12 and 13 compare the HRGC/ MS chromatogram of the bulk adipose tissue used to establish method performance and one of the composite samples. The HRGC/ MS chromatogram of the composite sample appears to have considerably more volatile material than the bulk adipose used to prepare the tissue standard. Tentative identifications for some of the major peaks in Figure 13 were assigned based on the comparison of the mass spectra to library spectra.

figures 14 and 15 provide comparisons of the composite specimens for three age groups within the same census division. All of the samples from each census division were analyzed on the same day. The results demonstrate that the elution profiles of all samples are comparable although the absolute responses of the peaks vary.

Figures 16 and 17 provide comparisons of three composite samples representing the 15 44and 0 14age groups from three census divisions. Again the general HRGC/ MS profiles appear to be consistent although the absolute response of each peak varies from sample to sample.

23 cp 0 . a C m  5 aJ CJ m L ii m 80 000034 D"

,

.. v) L S8 l 80 I 0 r4 o

It 0 ao B s z C PL N v

000035 m . a . I.

/

26 * W ' C 0, N c a2 5 I hc U a E 3 r 0 4. cmou40.

0 I 7 f c v) 3 Yoc OL U 3 A0 .

ILL 27 000037 .

1. Benzene/ dg benzene 2. 3romodichloromethane~ 3. Toluene/ d8 toluene 4. Bromochloropropane/ 1, l , Z trichJoroethane 5. DibromochI orometha ne 6. Tetrachloroethene 7. Chlorobenzene/ dg chlorobenzene 8. d10 ethy1benzene 9. Ethylbentene 10. dlo p xylene 11, 8romoform 12. Styrene 13. Dichlorobutone 14. 1,1,2,2 tetrachloroethane 15. d4 1,4 dichIorobenzene 16. 1,2 dichlorobenzene Purge Tower 5tandard 14 r 16 I i * lor80 Fiqure 11. HRGC/ MS chromatoqram of purge tower standard analyzed daily to document instrument performance.

28 1 . Chioroform/ d chloroform 2, Bromochtororqethane 3. 1, I, I trichloroethane 4. BenzeneJdg benzene 5. Bromodichloromethane 6. Toluene/ dg to 1uene 7. 1, ? ,2 trichloroethane 8. Di bromoch1 oromethane 9. Tetroch Ioroethene . 10. Chlorobenzene/ . dg ch 1 orobenzene 11. dl0 ethylbentene 12, Ethylbenrene 13. dl~ p xyJene 14. Bromoform 15. Styrene 16. 1,1,2,2 tetrachloroethane/ d2 lf1,2,2 tetrachloroethane 17. d4? ,4 dic h lorobentene 18. 1,2 dichlorobenzene Tissue Standard lee.

BfE 3I2e . & e . .

Figure 12. HRGC/ MS chromatogram for the volatile organic analysis of a bulk adipose ( tissue standard) spiked with 1.0 pg of each internal standard and 0.20 pg of each target analyte.

29 '

000039 Tentative Identification of Major Peaks 1. Acetic acid ethyl ester/ Propanoic acid propyl ester 2. Heptanal 3. Decane 4. Dimethyfoctane 5. Trimethylcycfohexane 6. Nonanal 7. Undecene 8. Ethyl ester, carboxylic acid 9. Nonadienul Middie Atlantic, 0 14 years 5 7 Figure 13. HRGC/ MS chromatogram of volatile compounds from the NHATS FY82 composite of the 0 14 year age group from . the Middle Atlantic census division.

30 080040 Middle Atlantic 45 plus years Middle Atlantic 15 44 years Middle Atlantic 0 14 years Figure 14. Comparison of the HRGC/ MS chromatograms'for the volatile organic analytes for three composite age groups from the Middle Atlantic census division. 31 000041 East North Central 45 plus years East North Central 15 44 years .

East North Central 0 14 years , 1llY Figure 15. Comparison of the HRGC/ MS chromatograms for the volatile organic analytes for three composite age groups from the East North Central census division. 32 000042 Middle Atlantic 15 44 years East North Central 15 44 years West North Centrai 15 44 years . Figure 16. Comparison of the HRGC/ MS chromatograms of the volatile organic analysis of three composite specimens representing the 15 44 age group from three census divisions. 33 000043 Middle Atlantic 0 14 years LJ rt 7 East North Central 0 14 years West North Centra1 0 14 years . Figure 17. Comparjson of the HRGC/ MS chromatograms of the volatile organic analysis of three composite specimens representing the 0 14 age group from three census divisions. 34 000044 Seventeen target analytes were determined using the automated HRGC/ MS search routines and the mass spectral library established in the method evaluation studies. Figures 18 to 21 summarize the incidence of detection of each of these analytes by age and by census division within the four regions. A plus indicates that the compound was detected in the analysis of a particular composite. A minus indicates the compound was not detected above the estimated limit of detection. The number of symbols shown under each age category for a specific compound indicates the total number of composi tes analyzed. . The quantitative data for the 17 target analytes are shown in Tables 4 through 20 b) census division and age group. The tables provide the total wet tissue weight composited, the total mass of the specific analyte detected, the concentration based on the original wet tissue weight, and the analysis date. The tables provide estimated limits of detection for composites for which a compound was not detected ( ND) and for composites for which the compound is reported as a trace ( tr) value. Trace values are reported for. responses observed at greater than 2.5 times the signal to noise but less than 10 times signal to noise. Data reported with no qualifier indicate a positive response detected above the limit of quantitation ( LOQ) or greater than 10 times the background signal to noise.

Upon reviewing the data in Tables 4 through 20 reported as not detected ( ND), it is noted that the level of detection ( total micrograms) varies from one sample to another. This results from a combination of the observed background in a sample at the characteristic ion for a specific analyte and the intensity of the response of the characteristic ion for the corresponding internal standards. Hence, the calculated limits of detection were higher in some instances due to high background at the charasteric ions for the target analytes and/ or low recovery ( observed as intensity of repsonse) of the associated internal standards.

4. Table 21 presents the frequency of observation of the target analytes from the 46 composite samples. Styrene, 1,4 dichlorobenzene, xylene isomers, and ethylphenol were detected in all 46 composites. Two responses were observed that correspondedLto xylene isomers. However, the isomers are not specified since the order of elution of the three possible compounds was not determined. Benzene, chlorobenzene, ethylbenzene, and toluene were detected in nearly all composites. Tetrachloroethane, 1,2 dichlorobenzene, and l, l, l trichloroethane were detected in approximately 50 to 60% of all composite specimens. 1,1,2,2 Tetrachloroethane was detected in less than 10% of all composite samples ( 4 of the 46). The three brominated target analytes and L, l, 2 trichloroethane were not detected in any of the composite specimens.

The quantitative data for all compounds detected in a specific com posite are reported in Tables B l to B 18 in Appendix B.

35

   Census Region Census division: Age group:

Compound Northeast New England Middle Atlantic 0 14 15 44 45+ 0 14 15 44 45+

Chloroform + + + +++ ++

1, l, l Trichloroethane  + ++

Bromodi chloromethane Benzene ++ ++

Tetrachloroethene ++ ++ ++

 Dibromochloromethane l, l, 2 Trichloroethane ,

Toluene + + + ++ ++ ++

Chlorobenzene' . ' + + + ++ ++ ++

Ethylbenzene + + + ++ ++ ++

 Bromoform e + + + ++

+ + ++ ++ ++.

++ ++ Styrene 1,1,2,2 Tetfachloroethane 1,2 Dichlo. robenzene . 1,4 Dichlorobenzene + + + ++ ++ ++

X~ I enea + + + ++ ++ . ++

Ethylphenol + + + ++ ++ ++

afhe exact i somers were not determined.

Figure 18. Incidence of detection of volatile organic compounds in composited human adipose tissues from the Northeast census region. The total number of + and  symbols for a specific compound indicates the number of composites analyzed for each age group.

36

 Census Region Census division: Age group:

ComQound Chloroform 1.1 , l Trichloroethane.

Bromodichloromethane Benzene Tetrachl oroethene Dibromochloromethane l, l, Z Trichloroethane To1uene Chlorobenzene Ethylbenzene Bromoform Styrene l, l, Z, Z Tetrachloroethane 1,2 Dichlorobenzene 1,4 Dithlorobenzene X~ I mea Ethyl phenol a  The exact isomers were not determined. South Atlantic 0 14 15 44 45+

++ +++

+++

++++

++++

..

+++++

++ *+++

++ ++++

++ ++++

..

+

++++

++ ++++

++ ++++ South  East South Central West South Cent 0 14 15 44 45+ 0 14 15 44 + ++

+ +

++ ++

++ +

.. c ++ +

++ ++

++ ++ ..

++ ++ .

+ +

+

++

++ ++.

++ ++

Figure 19. Incidence of detection of volatile organic compounds in composited human adipose tissues from the South census region. The total number of + and  symbols for a specific compound indicates the number of composites analyzed for each age group.

37 000047

 *. .

~

Census Region Census division: Age group:

Compound Chloroform l, l, l Trich? oroethane Bromodichloromethane Benzene Tetrachloroethene Dibromochloromethane 1,1,2 Trichloroethane To7uene Chlorobenzene Ethyl benzene i3romoform Styrene Northcentral East North Central West North Central 0 14 15 44 45+ 0 14 15 44 45+

++ +++ +++ +

++ +

++ +++ ++++

+++ ++ +*

++ +++ +++ ++

++ +++ +++ ++

++ +++ +++ ++

++ +++ +++ ++

l, l, Z, Z Tetrachloroethane +

1,2 Dichlorobenzene . ++ ++++ +

1,4 Dichlorobenzene ++ ++ f +++ ++

xyl enea ++ ++ f +++ ++

Ethy1pheno1 ++ +++ +++ ++

~~ a The exact isomers were not determined.

Figure 20. Incidence of detection of volatile organic compounds in composited human adipose tissues from the North Central census region. The total number of + and  symbols for a specific compound indicates the number of composites analyzed for each age group.

38 Census Region  Census division: Mountain Pacific Age group: 0 14 15 44 45+ 0 14 15 44 45+ Compound Chloroform + + + + + + West 1,1, l Tri chloroethane + + + +

Benzene 4. + .+ + +. +  Tetrach1 oroethene + 4. +  Dibromochl oromethane   l, l, Z Trichloroethane ' *

To 1 uene + + + +. + +

Chlorobenzene +  5+ + + +

Ethylbenzene + + + + + +  5romoform Styrene + i +  i + +'  l, l, Z, Z Tetrachloroethane ,

1,2 Dich1 orobenzene * + + + + +

1,4 Dichlorobenzene + + i + + i eneaxy~ + + + + + +

Ethyl pheno1 + + + + + +

, I aThe exact isomers were not determined.

Figure 21 Incidence of detection of volatile organic compounds in composited human adipose tissues from the West census region. The total number of + and  symbols for a specific compound indicates the number of composites analyzed for each age group,

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73 000083 Table 21. Incidence of Detection of Selected Volatile Organic Compounds in the NHATS FY82 Composite Samples Compound Chloroform l, l, l Trichloroethane Bromodichloromethane Benzene Tetrachloroethene Dibromochloromethane 1,1, P Trichloroethane To1uene Ch7orobeniene Ethy1 benzene Bromoform Styrene 1,1,2,2 Tetrachloroethane 1,2 Dichlorobenzene 1,4 Dichlorobenzene Xylene Ethylphenol Frequency of observation (%)

76 48 0 96 61 0 0 91 96 96 0 100 9 63 100 100 100 74 000084 V. QUALITY ASSURANCE/ QUALITY CONTROL As mentioned in Section IV, several procedures were included with the analysis of the composite specimens to document the quality of the data reported. These procedures included instrument performance checks using re agent spikes at the purge tower. These were performed daily following initial instrument cal ibration by the HRGC/ MS analyst. The quality control procedures also required that the analyst demonstrate background contribution of the headspace analysis system through the analysis of system blanks. Adipose tis sue samples were spiked with known amounts of the target analytes and internal standards to verify consistency of response factors before proceeding with the sample analyses. When necessary, the analyst completed the analysis of a three point calibration curve before proceeding with sample analysis. Typical . spike levels of the target analyte ranged from 0.2 to 1.4 1.19per 20 g aliquot of tissue. These spike. levels were equivalent to concentrations from 0.010 to 0.070 pg/ g.

A. Instrument Performance Checks Instrument performance checks were completed using quality control samples prepared by the MS analyst ( internal QC) or by the project quality control coordinator ( external QC). These instrument performance checks were completed to demonstrate that the HRGC/ MS system was properly calibrated and to document method performance ( precision) over time. The instrument performance checks were achieved by spiking a solution of the reference compounds into the purge tower and then proceeding with the sampling and analyses events as described earlier in the experimental section.

1. Internal QC Table 22 presents a summary of the internal QC instrumental performance check completed for 10 different analysis days. Spike levels for these instrument performance checks were from 0.20 to 0.40 pg for each of the specified target analytes. Table 23 provides the average recovery and range of measured recoveries for each compound. As noted, the method recoveries for   all compounds were typically within the range of 80 to 120% with the exception of bromochloromethane. The method precision for the internal QC measurements was generally within f 15%. These accuracy and precision estimates reflect day to day performance of the analytical method.

2. External QC A mixed volatile stock standard was prepared containing chloroform, benzene, bromodichloromethane, toluene, chlorobenzene, ethylbenzene, 1, Z dichlorobenzene 1,1,2,2 tetrachloroethane, and tetrachloroethene at a concentration of 10 mg/ mL for each compound. One portion of the stock solution was diluted and used for internal QC, and another was diluted and used for performance audit samples.

Five dilutions of the stock solution were prepared in tetraglyme, sealed in microreaction vials sealed with Mininert valves, and stored in a freezer until needed. The concentration of each compound per dilution was:

75 Audit sample no. VOA 1 VOA 2 VOA 3 VOA 4 VOA 5 Concintration 0.41 pg/ pL 0.80 pg/ pL 0.80 pg/ pL 0.68 crn/ uL 0.81 & bL The results of the QC performance audit samples are founG in Table 24.

B. Spiked Adipose Tissue Samples An additional QC control check included the analysSs of spiked adipose tissue samples. Table 25 summarizes the results for five spiked adipose tissue specimens analyzed with the first sample batches. The mean accuracy of these measurements ranged from 94% for l, l, Z trichloroethane to 14% for 1,1,2,2 tetrachloroethane. The range of recoveries for each compound is somewhat broader than noted for the instrument performance checks due to the influence of the matrix on recovery efficiency and background contribution.

C. Internal Standards The absolute responses of the internal standards were noted for each QC check sample and composite adipose tissue sample to document instrument operating parameters. The absolute responses of the internal standards in the first composite sample analyzed were observed to decrease markedly in comparison to the responses observed for the instrument performance check and system blank. A reduction in the response can be attributed to the adipose Obviously, the presence of oily lipid materials affects the tissue matrix. aqueous to air partitioning of the volatile internal standards.

Differences in the recovery of the internal standard from the FY82 composite specimens and the bulk adipose tissues used for method calibration were also observed. The absolute responses of the internal standards from the bulk adipdse tissue samples were nearly always greater than the responses for the NHATS composites. This indicates that the recoveries or observed partitioning of the . internal standards from the spiked samples to the headspace was greater for the QC samples than the FY82 composites. This observed recovery might indicate some difference in the two sources of adipose tissues. This bulk adipose tissue had been collected within 6 mo of the volatile organic analysis as compared with the FY82 specimens, which had been stored for up to 2 yr.

After observing the reduction in response with the first composite sample, the instrument performance was verified through reanalysis of a vessel blank ( water plus internal standards). This analysis demonstrated that the instrument was properly calibrated and that the matrix was responsible for the reduction in response of the internal standards.

Figures 22 to 25 are plots of several of the internal standard responses from the system blanks, spiked reference tissue samples, and NHATS composite samples. These plots demonstrate that the differences in the observed internal standards responses for. the method blanks, spiked reference tissues, and the NHATS composites were noted for each day that the composites were analyzed.

76 000086 w * Q~ uOOmONOOdd n mmor? wmmoo~ ww m* owdwuowwdoo ln CvmNN d4NNd440 d4NN~" N" NNN . c 000000000000= 000000000000d .& c, lnL XO I m omm* ooooom~ ddN"~ NNtwN" HN 0000000000000 NdWWNNWNNNNNN 00000000000d0 77 000087 fable 23. Average Recovery for Intsrnal QC Instrument Performance Checks Compound Average recovery (%) Range (%)

l, l, l Trichloroethane Bromochloromethane 105 2 11.8 121 k 71 90 125 50 280 Benzene 98.3 It 4.9 90 105 Tetrachloroethene 99.3 k 8.9 88 115 Dibromochloromethane 98.8 f 6.1 90 110 1,1,2 Trichloroethane . To1uene 101 f 5: 7 103 2 12.1 90 110 88 120 Chlorobenzene 103 k 6.7 95 120 Ethylbenzene Bromoform 100 +, 5.3 99.5 k 12.3 90 110 80 115 Styrene l, I, Z, Z Tetrachloroethane 1,2 Dichlorobenzene 103 rt 14.6 101 k 9.4 102 k 7.9 90 140 80 110 90 115 . aThese data were summarized to include accuracy for isotope dilution measurements where possible.

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Method Blonk 0 SpILed Refettnce firrue 0 Spiked Reference Tissue  Internet OC . A NHATS FY 82 Composite Spcimns d chlaoform I 1 I. I t I I I 3I 9I io 11 izI 13 16 17 ia J 0 6/ 19 21 zz 26 27 28 29 7/ 2 I I I I I I Analysis Dote ( 1984)

Atb2 a 0 r 0 A i J000091 0 : * r de toluene A A A b A 8 A A A a A b A b A A 1 I 1 1 I I I . I I I 6/ 19 21 22 2s ,27 29 29 712 3 9 Amlyrir Dole ( 1981)

b A L A H 3 Ie 3

3 Method Blank 0 Spiked Refereme Tissue 0 Spiked Reference Tiwue  Internol QC A NHATS FY 82 Campolite Specimens A A A AA A A A8A A A A A 8 A A A A I I i I t I I J IO II 12 13 16 17 ie kth+ Blank a Splked Reference Tirrue 0 Splked Rclercncs litru.  Interno1 QC A NHATS FY 82 Comporlte Specimens Figure 23. Observed HRGC/ MS responses for the internal standards dg toluene and dg chlorobenzene, from method blanks, spiked tissue samples and the NHATS composite samples.

82 000092 20 kthod 8lonL 0 Spiked Reference Tissue 0 Spiked Reference Tirwe  lntcrnol QC A NHATS FY 82 Compoiite Spcc; meny 4r 01 1 I I 1 I I I I I I 1 I I I 1 1 f J 6/ 19 21 22 26 27 28 29 7/ 2 3 9 10 II 12 13 16 17 18 20 Anolysii Dote ( 1984)

Method BIonk 0 SpIked Reference Tirsue 0 Spiked Reference Tiiswe  Internal QC A NHATS FY 82 Compoiite Specimens d 10 ethyibenzene A A 01 I f I I I I I I I I I I I I I I I I 6/ 19 21 22 26 27 28 29 7/ 2 3 9 IO I1 12 13 16 17 IS 20 Amlyrii Dote ( 1984)

Figure 24. Observed HRGC/ MS responses for the internal standards dig p xylene and dipethylbenzene, from method blanks, spiked tissue samples and the , NHATS composite samples.

83 000093 20 A b I 1 I 1 I I I I I I I t I 1 I I 1  I IF 01 6/ 19 21 22 26 27 28 29 7/ 2 3 9 10 11 I2 13 16 17 18 J Analysis Doh ( 1964)

Mekhod Blonk 0 Spiked Reference Tittw 0 Spiked Reference Tioue  lntcrnol QC a " ATS FY E2 Conlpogiie Specinlcns dq dichlorobenzene *.* .*** */*\. I C..** .

m 2 0 I I I 1 I I, I I I I 1 t I I I I lj 6/' l9 21 22 26 27 28 29 7/ 2 3 9 IO It 12 13 I6 li 18 10 Anolysis Date ( 1964)

Figure 25. Observed HRGC/ MS responses for the internal standards d2 l , l, Z, Z tetrachloroethane and dq dichl orobenzene, from method blanks, spiked tissue samples and the NHATS composite samples. 84 880094 VI. REFERENCES Balkon 3, Leary JA. 1979. An initial report on a comprehensive, quantitative screening procedure for volatile compounds of forensic and environmental interest in human biofluids by GC/ MS. J Anal Toxicol 3: 213 215.

Brown RH, Purnell CJ, 1979. Collection and Analysis of Trace Organic Vapor Pollutants in Ambient Atmospheres. The Performance of a Tenax Adsorbent Tube. 178: 79 90.

Corbett TH. 1973. Retention of anesthetic agents following occupational exposure Anesth Anal 52: 614.

Engstrom K. 1984. Styrene. Chapter 10. In: Biological monitoring and surveillance of workers exposed, to chemicals. Antero A, Riihimgki V, Vainio H, eds. Hemisphere Pub1ishing Corp, pp. 99 110.

Engstrom J, Riihim'dki V. 1979. Distribution of m xylene to subcutaneous adipose tissue in short term experimental human exposure. Scand 3 Work Envi ran Health 5: 126 134.

Hiatt MH. 1983. Determination of volatile organic compounds in fish samples by vacuum distillation and fused silica capillary gas chromatography/ mass spectrometry. Anal Chem 55: 506 516.

Karbowski RJ, Braun WH. 1978. Quantitative determination of styrene in biological samples and expired air by gas chromatography/ mass spectrometry ( selected ion monitoring). J Chromatog 160: 141 145.

Lin S N, Fu FW Y, Bruckner JV, Feldrnan S. 1982. Quantitation of 1, l and 1,2 dichloroethylene in body tissues by purge and trap gas chromatography. J Chromatog 244: 311 320.

Lucas RM, Pierson SA, Myers DL, Handy RW. 1981. National Human Adipose Tissue Survey Quality Assurance Program Plan, Preliminary Draft, RT1/ 1864/ 21 11.

Luskus LJ, Kilian HJ, Lackey WW, Biggs JD. 1977: Gases released from tissue '

and analyzed by infrared and gas chromatography/ mass spectroscopy techniques. 3 Forsenic Sci 22( 3): 500.

Mack GA, Stanley J. 1984. Preliminary strategy on the National Human Adipose Tissue Survey. Washington, DC: Office of Toxic Substances, Contracts 68 016721 ( Task 21) and 68 02 3938( Work Assignment 8).

Michael LC, Erickson MD, Parks SP, Pellitzari ED. 1980. Volatile environmenta pollutants in biological matrices with a headspace purge technique. Anal Chem 52: 1836 1841.

Novotny My McConnell ML, Lee Mt, Farlow R. 1974. High resolution gas chromatographic analysis of the volatile constituents of body fluids, with use of glass capillary columns. Clin Chem 20( 9): 1105 1110. . Pantarotto Cy Fanelli R, Belletti I, Bidoli F. 1980. Determination of styrene in biological specimens by gas chromatography selected ion monitoring: distribution in mice. Anal Biochem 105: 340 347.

Peoples AJ, Pfaffenberger CD, Enos HF, Shafik TM. 1978. An LSC/ GLC/ ECD method for quantifying human serum and adipose tissue levels of volatile purgeable halogenated hydrocarbons. Am Chem SOC Div Environ Chem 18( 2): 485 486.

Peoples AJ, Pfaffenberger CD, Shafik TM, Enos HF. 1979. Determination of volatile purgeable halogenated hydrocarbons in human adipose tissue and blood serum. Bull Environm Contam Toxicol 23: 244 249.

Pfaffenberger CD, Freal 53, 111. 1979. A convenient purging device for analyzing adipose tissue for volatile components. J HRC & CC 10114.

Politzer IR, Githens S, Dowty BJ, Laseter JL. ' 1975. Gas chromatographic evaluation of the volatile constituents of lung, brain and 1i" ver tissues. J Chromatog Sci 13: 378 379.

Reddrop CJ, Riess W, Slater TF. 1980. Two rapid methods for the simultaneous gas liquid chromatographic determination of carbon tetrachloride and chqaroform in biological material and expired air. J Chromatog 193: 71 82.

Reinert KH, Hunter JV, Sabatino T. 1983. Dynamic heated headspace analyses of volatile organic compounds present in fish tissue samples. J Agric Food Chem 31: 10' 57 1060.

Stanley JS. 1986a. Broad scan analysis of human adipose tissue: Volume I; Executive Summary. EPA 560/ 5 86 035.

Stanley JS. 1986b. Broad scan analysis of human adipose t ssue: Volume 11: Volatile organic compounds. EPA 560/ 5 86 036.

Stanley 35. ' 1986~. Broad scan analysis of human adipose t ssue: Volume 111: Semivolatile organic compounds. EPA 560/ 5 86 037.

Stanley JS. 1986d. Broad scan analysis of human adipose t ssue: Volume N: Polychlorinated dibenzo pdioxins ( PCDDs) and polychlorinated dibenrofurans ( PCDFs). EPA 560/ 5 86 038.

Snyder CAY Erlichman MN, Goldstein BD, Laskin S. 1977. An extraction method for, determinationof benzene in tissue by gas chromatography. Am Ind Hyg ASSOC J 38: 272 276.

USEPA. 1984. U. S. Environmental Protection Agency. Method 624 purgeables. ( 49 FR 141 152).

Vogt CR, Liao JC, Sun AY. 1980. Extraction and determination of chloroform in rat blood and tissues by gas chromatography electron capture detection:, distribution of chloroform in the animal body. Clfn Chem 26( 1): 66 68.

86'

000096 Whitcher CE, Cohen EN, Trudell JR. 1971. Chronic exposure to anesthetic gases in the operating room. Anesth 35: 349.

Wolff MS. 1976. Evidence of existence in human tissues of monomers for plastic and rubber manufacture. Environ Health Perspect 17: 183 187.

Wolff MS, Daum SM, Loumer WV, Selikoff 13, Aubrey BB. 1977. Styrene and related hydrocarbons in subcutaneous fat from polymerization workers. Toxicol Environ Health 2: 997 1005.

Zuccato E, Marcucci F, Mussini E. 1980. GLC determination of ethylene dichloride ( EDC) in biological samples. Anal Ltr 13( B5): 363 370.

87 00009' 7 APPENDIX A ANALYTICAL METHOD FOR THE DETERMINATION OF VOLATILE ORGANIC COMPOUNDS IN HUMAN ADIPOSE TISSUE A I ANALYTICAL METHOD FOR THE DETERMINATION OF VOLATILE ORGANIC COMPOUNDS IN HUMAN ADIPOSE TISSUE 1.0 SCOPE AND APPLICATION 1.1 This method covers the determination of volatile organic compounds in human adipose tissue: The following compounds have been evaluated and determined by this method.

Compound Chloroform l, l, l Trichloroethane Bromodichloromethane Benzene Tetrachloroethene Dibromochloromethane l, l, Z Trichloroethane To 1 uene Chlorobenzene Ethylbenzene Bromoform Styrene 1,1,2,2 Tetrachloroethane 1,2 Dichlorobenzene 1,4 Dichlorobentene Ethyl phenol Xylene CAS no.

67 66 3 71 55 6 75 27 4 71 43 2 127 18 4 124 48 1 79 00 5 108 88 3 108 90 7 100 41 4 75 25 2 100 42 5 79 34 5 95 50 1 106 46 7 25429 37 2 1330 20 7 ~

1.2 This is a dynamic headspace high resolution gas chromatography/ . mass spectrometry ( HRGC/ MS) method applicable to the determination of volatile organic compounds in human adipose tissue.'

1.3 The method detection limit ( MDL) for each parameter is estimated to range from 0.001 0.10pg/ g for a 20 9 sample. The MDL for a specific sample of human adipose tissue may differ depending upon the nature of interferences in the sample matrix and the specific analyte determined.

2.0 METHOD SUMMARY Helium gas is swept through the head space of a special. 1ydesigned purging chamber containing water and the adipose tissue sample. The water and sample are heated and stirred to aid in the efficient transfer of the analytes from the adipose tissue and water mixture to the gaseous phase. The vapor is swept through a cooling tower, atambient temperature to remove excess water vapor and onto a Tenax sorbent trap where the volatile analytes are captured. After the sampling is complete the sorbent trap is heated and backflushed with helium to desorb A 2 000099 the analytes onto a HRGC column. The gas chromatograph is temperature programmed to separate the compounds which are then detected by the mass spectrometer. Figure A 1 provides a schematic of the dynamic head space purge and trap HRGC/ MS analysis system.

3.0 CONTAMINATION AND INTERFERENCES 3.1 ' Impurities in the purge gas, organic compounds out gassing from the plumbing upstream of the trap, and solvent vapors in the laboratory account for the majority of contamination problems. The analytical system is demonstrated to be free from interferences under conditions of the analysis by analyzing blanks initially and with each sample set analyzed daily.

3.2 Samples can be contaminated by diffusion of volatile organic compounds through the bottle seal during preparation, handling, or storage. A sample container blank carried through the sampling and handling protocol serves as a check on such contamination.

3.3 Contamination by carry over can occur when high level and low level samples are analyzed sequentially. To reduce carry over, a clean headspace chamber is used for each sample analysis and sample syringes are rinsed between samples with reagent water. Instrument performance checks and QC samples are followed by analysis of a reagent water blank to check for carry over'. The headspace device is washed with soap solution,. rinsed with tap and distilled water, and dried in an oven at 10O 12S0C overnight. The Tenax adsorbent trap and other parts of the system are also subject to contamination; therefore, frequent bakeout and purging of the entire system may be required.

3.4 Interferences resulting from samples may vary considerably, depending on the length of sample storage.

4.0 SAFETY 4.1 The toxicity or carcinogenicity of each compound used in this method has not been precisely def ined; however , each compound should be treated as a potential health hazard. Exposure to these chemicals should be maintained at the lowest possible level.

4.2 The glass purging apparatus used in this method is of fairly recent design and has apparent limitations which must be observed. The pressure in the system should not exceed 10 psi. Excessive pressure above this limit may result in the possible explosion of the purging vessel or purge tower. Monitoring the gas pressure is easily accomplished by placing a pressure gauge between the helium source and the gas inlet at the vessel. At no time should any glassware be pressurized without the analyst wearing approved safety goggles.

A 3 A 4 5.0 APPARATUS AND MATERIALS 5.1 Gas pressure gauge  Supelco No. 2 0392 or equivalent.

5.2 Spatulas, stainless steel  Fisher No. 14 375 10.

5.3 Volumetric flask 5.4 Syringes,

5.4.1 Syringe 5.4.2 Syringe 5.4.3 Syringe 5.4.4 Syringe 5.4.5 Syringe 5.5 Vials  2 x 5.0 mt 10.0 mL  Ace No. 7100 02.

10.0 mL  Hamilton No. 1010.

1.0 mL  Hamilton No. 1001.

100.0 pL  Hamilton No. 710N.

25.0 yL  Hamilton No. 702N.

2 x 5.0 pL  Hamilton No. 75N.

microreaction vessels  Supelco No. 3 3298.

5.6 Vials  2 x 1.0 mL microreaction vessels  Supelco No. 3 3292.

5.7 Mininert valves ( x2)  Supelco No. 3 3301.

5.8 leflon@ tubing ( 1/ 8 in. ID)  Ace No. 12687 08.

5.9 Funnel ( 1.0 L)  Ace No. .7249 40.

5.10 Three way stopcock  Ace No. 8143 05.

5.11 Vinyl tubing  Ace No. 12679 24.

5.12 Hot water bath/ circulator  HAAKE No. F 4391.

5.13 Teflon@ stir bars  Ace No. 13654 10.

5.14 Stirrer  Ace No. 13635.

5.15 Heat tape  Ace No. 12064 08.

5.16 Powerstat  Ace No. 12077.

5.17 Headspace vessel  Wheaton No. 991765.

5.18 Kovar to PX seal tubes  Ace No. Pt. 976.

5.19 Stainless steel tubing  Supelco No. 2 0526. .

5.20 . Six way. Carle valve.

A 5 000102 5.21 Stainless steel glass lined U tube ( 1/ 8 in. ID).

5.22 Chromatography gravity flow column  Howe Scient. Inc., No. 3060 13 " purge tower".

5.23 Heat controller and thermocouple. I 5.24 Glass wool " silanized".

5.25 HRGC column ( 30 m x 0.25 mm ID) Durabond DB 5, 0.25 pm film thickness ( J& W Scientific, Rancho Cordova, CA) or equivalent.

5.26 Gas chromatograph  Finnigan 9610 or equivalent capable of maintaining 3OoC isothermal and programmable to 200OC. The gas chromatograph must be equipped with a Grob split/ splitless type injector.

5.27 Mass spectrometer  Finnigan 4000 quadrupole or equivalent, 70 eV electron impact ionization; capable of repetitive scan from 35 275 amu every 2 3 s, and produce a unit resolution ( valleys between m/ z 174 176 less than 10 percent of the height of the m/ z 175 peak), background corrected mass spectrum from 50 ng 4 bromo fluorobenzene ( BFB) injected into the GC. The BFB spectrum must meet the mass intensity criteria in Table A 1. All portions of the HRGC column, transfer lines, and the HRGC column routed directly to. the ion source shall remain at or above  the column temperature during analysis to preclude condensation of less volatile compounds.

Table A 1. BFB Mass Intensity Specifications Mass 50 75 95 96 173 174 176 177 __

Intensity required 15 to 40 percent of mass 95. 30 to 60 percent of mass 95. base peak, 100 percent. 5 to 9 percent of mass 95. < 2 percent of mass 174. > 50 percent of mass 95. 95 to 100 percent of mass 174. 5 to 9 percent of mass 176.

5,28 Data system  Capable of collecting and recording MS data, storing mass intensity data in spectral libraries, processing HRGC/ MS data and generating quantitation reports, and calculating and recording response factors.

\

A 6 000103 4 ' i I ..: .. ,. . _.. . , I' . ,.~ . ,.

5.28.1 Data acquisition  Mass spectra shall be collected continuously throughout the analysis and stored on a mass storage device.

5.28.2 Mass spectral ' libraries  User created libraries containing mass spectra obtained from analysis of authentic standards must be used to reverse search GUMS runs for the compounds of interest.

5.28.3 Data processing  The data system shall be used to search, locate, identify, and quantitate the compounds of interest in each HRGC/ MS analysis. Software routines shall be employed to compute retention times and extracted ion current plot ( EICP) areas. Displays of spectra, mass chromatograms, and library comparisons are required to. verify results.

5.28.4 . Response factors and multi point calibrations  The data system shall be used to record and. maintain lists of response factors ( response ratios for isotope dilution) and generate multi point calibration curves. Computations of relative standard deviation ( coefficient of variation) are useful for testing calibration linearity.

5.29 Balance  Ana'lytical, capable of weighing 0.1mg.

6.0 REAGENTS 6.1 Volatile organic free water  prepare as described in Section 8.1,

6.2 Prepurified nitrogen.

6.3 Prepurified he1i urn.

6.4 Tenax absorbent.

6.5 Internal standard compounds 6.5.1 Bromochloromethane, 1,4 dichlorobutane, and I chloro 2bromopropane ( a ZO mg/ mL mixture, Supelco, Inc., No. 4 0823).

6.5.2 . d6 Benzene  Aldrich gold label ( 99.5% 0) No. 15, 181 5.

6.5.3 d Chloroform  Aldrich gold label ( 99.8% 0) No. 15, 182 3.

6.5.4 d2 1,1,2,2 Tetrachloroethane  M and D Isotopes No. MD 1416.

6.5.5 d, Methylene chloride  M and D Isotopes No. MD 53.

A 7 000104 6.5.6 d, Chlorobenzene  KOR Isotopes ( 99% D,) No. 521510.

6.5.7 1,4 d4 Dichlorobenzene KOR Isotopes ( 98% 0,) No. 521530.

6.5.8 dlo Ethylbenzene KOR Isotopes ( 98% Dlo) No. 521443.

6.5.9 d8 Toluene  KOR Isotopes ( 99.9% D,) No. 510041.

6.5.10 dlo p Xylene KOR Isotopes ( 98% Dlo) No. 521133.

6.6 Target analytes 6.6.1 Bromoform  Aldrich gold label ( 99%/ l% EtOH) ( d 2.894 g/ mL).

6.6.2 Dibromochloromethane  Columbia No. D1843 ( d 2.45 g/ mL).

6.6.3 Toluene  Burdick and Jackson H. P. No. A1 857 ( d 0.867 g/ mL).

6.6.4 1,1,2 Trichloroethane Aldrich No. J6 070177 ( 95%) ( d 1.435 g/ mL).

6.6.5 Styrene  Eastman No. 1465 ( d 1.34g/ mL). I 6.6.6 Tetrachloroethene  Aldrich gold label No. 120457 ( d 1.54 g/ mL).

6.6.7 Bromodichloromethane  Aldrich No. 7628AH ( 98%) ( d 1.49 g/ rnL 1.

6.6.8 Chlorobenzene  Aldrich No. 120277 ( 99%) ( d 1.107 g/ mL).

6.6.9 1,2 Dichlorobenzene Aldrich No. D5 680 2( 98%) ( d 1.306 g/ mL).

6.6.10 Ethylbenzene  Aldrich No. E1 250 8( 99%) ( d 0.867g/ mL).

6.6.11 1,1,2,2 TetraChloroethane  Baker No. 017386 ( d 1.586 g/ mL).

6.6.12 l, l, l Trichloroethane Fisher No. 775974 ( d 1.338g/ mL).

6.6.13 Chloroform  Burdick and Jackson H. P. No. AG594 ( d 1.492 g/ mL).

6.6.14 Benzene  MCB pest. grade No. U2738 ( d 0.874g/ mL).

6.7 Methanol  Burdick and Jackson, high purity distilled in glass.

6.8 Tetraglyme  Aldrich 17 240 5.

6.9 Acetone  Burdick and Jackson, high purity distilled in glass.

6.10. Hexane  Burdick and Jackson, high purity distilled in glass.

A a 000105 I_ 7.0 8.0 HEADSPACE APPARATUS PREPARATION 7.1 . Glassware preparation  All glassware is washed with a laboratory grade soap ( i. e., Sparkleen or equivalent) and rinsed with deionized water, bulk grade acetone, B& J. acetone, and finally B& J hexane. The glassware is air dried to remove traces of hexane and then dried in an oven at 2OO0C for 48 h before use.

7.2 Wheaton purge vessel  The Wheaton purge vessel thermometer and funnel arms were modified as follows. The threaded arms were cut near the vessel and replaced by 1/ 4 in. Kovar@ to PX seal tubes ( Ace Glass, Inc., No. PT. 976). The KovarB length was 1.0 in. The arms were made as short as possible to minimize dead volume. The vessel is integrated in the system as in Figure A 1.

7.3 Chromatographic gravity flow column ( purge tower)  The column is connected to the system as displayed in Figure A 1. Two milliliters of volatile organic free water is placed in the tower to trap excess water vapor.

7.4 Glass lined U tube sorbent trap  A 1.0 in. plug of Tenax is placed in the tube, and a plug of silanized glass wool placed at both ends. Do not overpack the Tenax. Pack only tight enough to avoid any dead volume in the Tenax. Overpacking will make it difficult to maintain a 40.0 mL/ min helium flow under the 10.0 psi safety pressure.

Refer to'Figure A 1 for a detailed orientation of all system components

REAGENT PREPARATION 8.1 Volatile free water  Volatile organic compounds are purged from water, taken from the Millipore system; by bubbling the prepurified nitrogen through the water for 24 h prior to use.

8.2 Internal standard preparation .

8.2.1 Internal standard stock solution  The internal standard stock solution is prepared from compounds 6.5.2 through 6.5.10 by a1iquotting each compound with a 100.0 pL syringe into a 10.0 mL volumetric containing 5.0 mL of methanol. The exact volume of each target analyte necessary to achieve a final concentration of 10 mg/ mL for each compound can be determined from the density of the compound. After aliquotting all compounds, dilute to the mark with the methanol. Transfer the 10.0 mt solution to two 5.0 mL vials and seal tightly with the TFE lined caps. Store in the freezer until needed.

A 9 8.2.2 Internal standard spi king solution  A 500 ng/ yL internal standard spiking solution is prepared in tetraglyme by injecting 925.0 pL of tetraglyme into a 1.0 mL reaction vial with mininert cap. This is then spiked with 25.0 pL of the 3 component ( 20 mg/ mL) Supelco standard and 50.0 pL of the lO. O mg/ mL internal standard stock of 8.2.1. Store in the freezer until needed.

8.3 Target analyte preparation 8.3.1 Target analyte stock solution  A lO. O mg/ mL each solution of the analytes from 6.6.1 through 6.6.14 is prepared as described for the internal standard stock solution in 8.2.1. Store in the freezer.

8.3.2 Target analyte spiking solution  A 100.0 ng/ pL solution of each of the target analytes is prepared by injecting 990.0 VL of tetraglyme into a 1. O mL reaction vial with mininert cap and spiking the tetraglyme with 10.0 pL of the stock solution of 8.3.1. Store in the refrigerator.

ANALYSIS ROUTINE This routine must be followed after any system shutdown or at least once every 8 h prior to any sample analysis.

9.1 Perform an instrument mass calibration using bromofluorobenzene ( BFB) to achieve'the mass intensity specifications in Table A 1.

9.2 Conduct a purge tower instrument evaluation with the system set up as in Figure A 1.

9.2.1 Conduct a leak test by squirting VOA free water around , all connections, vessel cap, etc. Correct as needed. Check the bubble meter for the correct 40.0 rnL/ min flow. The Carle valve must be in the purge position.

9.2.2 Turn off the purge tower stopcock and immediately turn the helium supply off. Check the pressure gauge for a slow pressure falloff. If the gauge reading drops, check again for leaks.

9.2.3 Remove the purge tower cap.

9.2.4 Connect a 30 cm length of Teflon@ tubing to the 10.0 mL syringe. Draw 3.0 mL of VOA free water into the syringe. Remove the Teflon@ tubing and draw 1.0 mL of air into the syringe. Using two separate 5.0 pL syringes, withdraw 2.0 pL of each working solution of 8.2.2 and 8.3.2. Spike the 10.0 mL syringe with each of the standard spiking A 10 . 000107 9.0 solutions. Be sure to inject directly into the water. Reseal the mininert valves and return the standards to a refrigerator. Replace the Teflon@ tubing on the 10.0 mL syringe. Invert the syringe several times to ensure adequate mixing of the tetraglyme and water.

9.2.5 Inject the contents of the 10.0 mL syringe directly into the water at the bottom of the purge tower. The air in the syringe should push the water solution completely out, but should not be allowed to bubble out the end of the Teflon@ tube. Immediately withdraw the Teflon@ tube and recap the purge tower.

9.2.6, With the Carle valve in the purge mode, the Tenax trap at ambient temperature, and all heat' tape lines at 15OoC, turn on the helium supply valve and then the stopcock at the purge tower.

NOTE: If the stopcock on the purge tower is left closed, the helium gas . may rupture the purge vessel.

Check the system for leaks. Ifa leak is detected, the system must be shut down and the purge tower cleaned as out1ined below.

9.2.7 Purge the purge tower, for 12 min at 40 mL/ min.

9.2.8 After the purge is complete, turn the Carle valve to. the desorb mode, heat the Tenax trap to approximately 25OoC and initiate the HRGC/ MS data acquisition. These three procedures are done as quickly as possible to maintain the absolute retention times between analysis. The gas ~

chromatograph should be held isothevmal at 3OoC for 5 min then programmed to 125OC at 6OC/ min. Hold at 125 ° C for 10 min and then program to 2OO0C before returning to 3OOC.

4 9.2.9 During the HRGC/ MS data acquisition, remove the purge tower cap and rinse the tower. Using the 10.0 mL syringe and Teflon@ tube, alternately add and withdraw 10, O mL portions of VOA free water. Repeat this procedure a minimum of six times and discard each rinse. Reassemble the tower with 2.0 mL of VOA free water.

9.2.10 After data acquisition is complete, check the HRGC/ MS response to the internal standards. Note that often the initial analysis of the system following instrument shutdown will give variable responses. If this is observed and the responses are not equivalent to that previously regarded as optimum performance, the purge tower instrument evaluation must be repeated. Refer to Calculations ( Section 11.0) for response factor calculations. Figure A 2 is an example of an HRGC/ MS chromatogram obtained for an instrument performance check.

A 11 0003.08 1. Benzene/ dg benrene 2. Bromodichforomethane 8. 9. d 10 e thy1benzene Ethylbentene 3. To Iuene/ d8 to Ive ne 10. dio p xyle ne 4. Bromochloropropane/ 11. Brornoform 1, i, 2 trichloroethane 12. Styrene 5. Dibrornochloromethane 13. Dic hlorobutane 6. Tetrachloroethe ne 7. Ch lorobentene/ 14. 15. 1,1,2,2 tetrochloroethane d4 1,4 dichlorobenzene dg chlorobenzene 16. 1,2 dichlorobenzene Purge Tower. 5 tandurd 1W. l 1 1 .' ,

4 4 f 16 12 1 6 Figure A 2. HRGC/ MS chroma togram of purge tower standard analyzed daily to document instruhent performance.

A 12 000109 9.3 External quality control check  External quality control checks ( blind samples) should be conducted by the project Quality Control Coordinator ( QCC) to demonstrate proper instrument calibration. These QC checks are performed periodically ( typically on the first analysis day of each week).

9.3.1 Allow the Tenax trap to return to room temperature.

9.3.2 The Q'C check is conducted in the same manner as the purge tower instrument check, but the target analyte spiking solution is provided by the project Quality Control Coordi nator ( QCC 1.

9.3.3 The recoveries of the target ana? ytes are calculated and provided to the QCC for evaluation. If the evaluation is adequate, the analyst proceeds with the system blank after completing the system rinse as in 9.2.9. If the QC check does not demonstrate acceptable performance, the analyst must recalibrate the system and repeat the QC check.

9.4 System blank 9.4.1 With the system set up as in Figure A 1, with the Carle valve in purge mode, add 80.0 mL of VOA free water in the Wheaton vessel and check for leaks.

9.4.2 Turn the purge tower valve off and helium supply off.

9.4.3 Remove the Wheaton vessel cap and inject 2.0 pL of the internal standard solution. Follow 9.2.4, but substitute the vessel for the purge tower and omit the analyte solution

9.4.4 Reseal the vessel, turn on the helium at the helium supply valve. When a pressure reading is observed in the system, open the stopcock at the purge tower; this will prevent water from seeping into the lines at the bottom of the purge tower. Check for leaks.

9.4.5 Turn on the stirrer and the valve at the three way hot water junction, so that the heated ( 95OC) water will flow through the vessel jacket. Purge the system for 40.0 min.

9.4.6 After the 40.0 min purge, desorb as in 9.2.8.

9.4.7 Remove the modified Wheaton vessel and replace with a clean headspace apparatus containing 80.0 mL VOA free water and clean stirring bar.

A 13 9.4.8 Determine the level of contaminants and record. If excessive background is observed, clean the purge tower, the transfer lines and repeat the system blank using a clean headspace apparatus.

9.5 Tissue standard  A tissue sample ( 20 g) spiked with known levels of the series of target analytes and internal standards should be analyzed daily to document relative response factors from the tissue matrix. Prior to initiating actual sample analysis, the analyst should analyze a series of adipose tissue samples spiked with 0.20 to 1.4 pg of each of the target analytes and 1.0 pg of each internal standard. The analyst should require a relative response factor consistency of It 40% variability for the Calibration curve and for day to day verification of the calibration. If the measured response factors are determined to vary by more than 40% of the average, the calibration curve must be re established The analyst must be aware that the background contribution of the tissue selected for spiked samples will effect the measured response factors. If the background contribution is significant, the analyst must analyze an unspiked sample to determine the level of background. Figure A 3 is an example of a spiked tissue standard.

9.5.1 With the system as in Figure A 1, turn off the purge tower stopcock and helium supply valve. Remove the Wheaton vessel cap and place 20.0 g of frozen adipose tissue in the 80.0 mL of water. Use the Fisher spatula to manipulate the frozen tissue. The adipose tissue used for the tissue standard analysis should be from a bulk supply so that each tissue standard will be subjected to the same matrix effects. Recap the vessel.

9.5.2 Spike this solution with 2.0 pL of each spike solution as in 9.2.4. Quickly recap the vessel.

9.5.3 Turn on the helium supply valve and open the purge tower stopcock. Be sure the Carle valve is in the purge position and the Tenax trap is at ambient temperature. Check for leaks.

9.5.4 Purge the vessel for 40 min. After the 40 min, desorb as in 9.2.8.

9.5.5 During data acquisition, clean and replace the Wheaton vessel.

9.5.6 Check the instrument ' response to the internal standard compounds. Calculate the response factor for each target analyte versus the respective internal standard and record.

A 14 . . .. . '. :..' , ,.. . ..... , .. .', ;. , . ...

1 . Ch1oroform/ d c hI oroform 2. Bromachloromethans 3. 1,1, I trichloroethane 4. Benzene/ dg benzene 5. Bromodi c hIorom& hane 6. ToI ueneidg to I we ne 7. 1,1,2 trichloroethane 8. DibromochIoromethane 9, Tetrachloroethene 10. Chlorobenzene/ dg ch lorobenzene 11. dlpethylbenzene 12. Ethylbenzene 13. dlpp xylene 14. Bromoform 15. Styrene 16. 1,1,2,2 tetrachioroethane/ d2 l11,2,2 tetrachloroethane 17. d4 1 , I dichlorobenzene 18. I ,2 dichlorobenzene Tissue Stondard 9 BIC.

P 8 C F i 19lee Figure A 3. HRGC/ MS chromatogram for the volatile organic analysis of a bulk ' adipose tissue spiked ' with 1 . O UCJ of each internal standard and 0.20 uq of each target analyte.

A 15 080112 . 9.6 Sample analysis 9.6.1 Sample analysis is achieved exactly the same manner as the tissue standard with the exception that the target analyte spiking solution is omitted. Figure A 4 is an example of the volatile organic analysis of a composite adipose tissue sample analyzed with the National Human Adipose Tissue Survey Fiscal Year 1982 samples.

9.6.2 Checking the instrument response after each acquisition and before proceeding to the next analysis will help prevent the loss of valuable samples.

10.0 QUALITATIVE IDENTIFICATION Obtain EICPs for the primary quantitation ion and at least one secondary masses for each parameter of interest. The following criteria must be met to make a qualitative identification. Table A 2 provides a summary of the primary quantitation ions and the HRGC characteristics for each of the target analytes and internal standards.

10.1 The characteristic masses of each parameter of interest must maximize in, the same or within one scan of each other.

10.2 The retention time must fa. 11 within ? I 30 s of the retention time of the authentic compound.

10.3 The relative peak heights of the characteristic masses in the EICPs must fall within & 20% of the relative intensities of these masses in a reference mass spectrum. The reference mass spectrum can be obtained from a standard analyzed in the HRGC/ MS system or from a reference library.

11.0 CALCULATION 11.1 Relative response factors  Analyze each instrument performance check standard and spiked adipose tissue sample. Tabulate the area response of the characteristic quantitation ion against the concentration for each target analyte and its corresponding internal standard ( Table A 2) and calculate relative response factors ( RRF) using Equation 1: I A I6 000113 Tentative Identification of Major Peaks 1. Acetic ucid ethyl ester/ Propanoic acid propyl ester 2. Heptanal 3. Decane 4. Dimethyfoctane 5. Tr imethylcyc lohexane 6. Nonanal 7. Undecene 8. Ethyl ester, _ carboxylic acid 9. Nonadienal Middle Atlantic, 0 14 years 4 3 ll 7 3: 2Y Figure A 4. HRGC/ MS chromatogram of volatile compounds from the NHATS FY82 composite of the 0 14 yr age grou$ from the illddle Atlantic Census Division.

A 1 7 000114 Table A 2. Characteristic Ions, Relative Retention Times ( RRT), and Internal Standards Used to Quantitate Target Volatile Organic Analytes Characteristic quantitation Compound ion ( m/ z) RRT~

Bromochloropropane Chloroform d Ch1orof orm l, l, l Trichloroehtane Bromodichloromethane Benzene d6 Benzene Tetrachloroethene Dibromochloromethane l, l, 2 Trichloroethane To1 uene dg Toluene Chlorobenzene d5 Chlorobenzene Ethy1benzene dlo Ethylbenzene Bromoform Styrene 1,1,2,2 Tetrachloroethane d2 1,1,2,2 Tetrachloro ethane 1,2 Dichlorobenzene de 1,4 Dichlorobenzene 1,4 Dichlorobenzene Xylene dlo e Xylene Ethylphenol 77 . 1.00 79 0.20 0.43 82 0.20 0.43 97 0.35 0.58 129 0.53 0.76 78 0.39 0.62 84 0.39 0.62 166 1.16 3.39 129 1.05 1.28 97 0.88 1.11 91 0.83 1.07 100 0.83 1.07 112 . 1.48 1.72 117 1.48 1.72 106 1.62 1.85 116 1.62 1.85 173 1.79 1.96 104 1.85 2.08 83 2.05 2.28 84 2.05 2.28 146 2.88 3.11 150 2.73 2.96 146 2.73 2.96 106 1.65 1.88 116 1.65 1.88 122 2.19 2.43 Internal standard Bromochloropropane d Chloroform  Bromochlororopane Bromochloropropane de Benzene .

Bromochloropropane Bromochloropropane Bromochloropropane da Tol uene *

d5 Chlorobenzene  d 0  Ethy1benzene  Bromochloropropane Bromochloropropane d2 1,1,2,2 Tetrachloroethane

d4 1,4 Dichlorobenzene  d4 1,4 Dichlorobenzene d 0 E Xy 1ene dlo thylbenzene dlo FXylene aRelative retention times calculated versus the internal standard, brornochloropropane A 18 000115 Equation 1.

= area of the characteristic quantitation ion for where: As the target analyte.

Ais = area of the characteristic quantitation ion for the internal standard.

Cis = Concentration of the internal standard ( pg).

Cs = Concentration of the compound to be measured.

If the RRF value over the working range is a constant (< 40% RSD), the RRF can be assumed to be invariant and the average RRF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, AS/ AiS vs. RRF.

11.. 2 When a target analyte has been identified, the quantitation of that compound should be based on the integrated abundance from the EICP of the primary quantitation ion given in Table A 2.

Calculate the concentration in the sample using the relative response factor ( RRF) determined in Section 11.1 and Equation 2.

Equation 2.

Concentration ( pg/ g) =

where: AS = area of the character stic quantitation ion for the target analyte.

Ais = area of the characteristic quantitation ion for the internal standard.

Cis = concentration of the corresponding internal standard ( w>.

Wt = wet tissue weight ( 9).

12.0 REPORTING 12.1 Report target analyte results are demonstrated in Figures A 5 and A 6.

12.2 Quantitative data must be qualified to provide an indication of the intensity of response. Quantitative data for target analytes A 19 m f m .. U f 3 0 ci E 0 P u . E. m X w W L 3 0 A 20,

000117 Report Date: Prepared By:

Reviewed By:

HUMAN ADIPOSE TISSUE DATA REPORTING FORM  VOLATILE ORGANICS Census Division Composite No. Age Group Sample Weight Compound Chloroform l, l, l Trichloroethane Bromodichloromethane Benzene Tetrachloroethene Dibromochl oromethane ' 1,1,2 Trichloroethane To1 uene Chlorobenzene Ethyl benzene Bromoform Styrene 1,1,2,2 Tetrachloroethane 1, Z Di chlorobenzene 1,4 Dichlorobenzene Xy 1ene Ethy1pheno1 Total pg/ Cornposite Specimen ES  East South Central ( 1) ( 1) ( 2) ( 1) ( 2) 0 14 15 44 15 44 45+ 45+ 25.6 g 19.0 g 24.3 9 20.6 g 19.3 g Figure A 6. Example data report format, for vofat. iIe organic analytes '

detected in composite samples from a specific census division.

A 21 000118 13.0 responses of less than 2.5 times background signal to noise ( limit of detection, LOD) are labeled as not detected ( ND). Target analyte responses ranging from 2.5 to 10 times the background signalto noise are reported as trace ( tr) values. Target analytes with response greater than 10 times the background signal to noise ( limit of quantitation, LOQ) are considered positive quantifiabie ( PO) values.

METHOD PERFORMANCE The method performance as determined by a single laboratory for instrument performance checks and spiked adipose tissue specimens are presented in Tables A 3 and A 4. These data were generated using a Finnigan 4000 quadrupole mass spectrometer for the analysis of 46 composite specimens from the National Human Adipose Tissue Survey ( NHATS) Fiscal Year 1982 collection. The method detection limits are estimated to range from 0.001 to 0.10 pg/ g from 20 9 tissue samples depending on the specific analyte and the observed interferences.

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