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Polychlorinated Biphenyls in Human Adipose Tissue by Francis J. Biros, Annita C. Walker and Angela Medbery Perrinc Primate Research Branch Division of Pesticides, Food and Drug Administration Department of Health, Education, and Welfare Perrine, Florida Recent reports have demonstrated that chlorinated hydrocarbons are among the most abundant synthetic pollutants in the environment (1). Concern over the pollution hazard presented by this class of organic compounds has been deepened by the detection of polychlor inated biphenyls (FCB) in animal and environmental media. Since the first reported presence of FCB residues in wildlife tissues by Jen sen (2), attention has been focused on elucidating the extent of dis tribution of these materials in wildlife species, especially in fish and birds (3). The occurrence of PCB in any sample, in addition to being toxicologically significant in living systems (4), presents a difficulty in the analysis of Certain chlorinated pesticidal chemi cals (5). Adequate discussions of the properties and use of PCB compounds, their distribution in the environment, and their inter ference with pesticide residue analysis have been presented previously (1-6). The analytical evidence for the presence of PCB in environ mental samples has been based on mass spectrometric analysis (7), re tention times in gas chromatographic analysis (1, 3, 6, 8), chlorine content by mlcrocoulometric analysis (6), relative inertness to ni tration and dehydrochlorination procedures which are used to confirm common chlorinated pesticides (9), and indirect evidence from the re lative distribution of these compounds in the environment (1). The most conclusive spectroscopic data have been the mass spectra of these compounds obtained with a combined gas chromatograph-mass spec trometer from extracts of fish, seabirds, conifer needles, and human depot fat (7). The present work was initiated because of the lack of detailed information about the mass spectral properties of PCB. The relative chemical and thermal inertness of these materials renders them par ticularly suitable for analysis by gas chromatography-mass spectro metry. Furthermore, recent success utilizing combined gas chromato graphy-mass spectrometry for pesticide residue analysis (10) and the availability of human tissue suspected to contain PCB prompted us to examine the behavior of PCB during combined gas chromatography-mass spectrometry and, if possible, to a prelimiry TKfgcrtptlon",Ol~t.iie gas cHTWnatographic-mass spectrometric be havior of PCB and of the analytical methodology applicable to the analysis of these compounds in human tissue by gas chromatographymass spectrometry. . Experimental The combined gas chromatograph-mass spectrometer system used in this study has been described previously (10). The gas chromatographic 317 Bulletin ol Environmental Contamination A Toxicology, Vol. S, No. 4,1970, pubiiahed by Springer-Verlag New York, Inc. . ' - STLCOPCB4090754 column was a stainless steel capillary, 100 feet x 0.020 inches i.d. and coated with 0V-1 silicone oil, obtained from the Perkin-Elmer Corp., Norwalk, Conn. Programmed temperature analyses were made both for the six Aroclor 1200 series standard materials^ and the tissue extracts. Figures 1 and 2 depict chromatograms obtained for Aroclors 1254 and 1260 of these series and detail the temperature programming conditions used. The molecular separator and gas inlet temperatures were maintained at 210C and 215C, respectively. All mass spectra were recorded at 80 ev electron energy with 2300 v ac celerating voltage; the filament emission current was 100^. Chro matograms were recorded from the total ion current monitor. Helium carrier gas flow rate was 4 ml/min. The injector temperature was 175 C. Mass spectra were scanned magnetically over the range m/e 5 to m/ 500 in 6 seconds. Two samples of human adipose tissue were examined by gas chro matography-mass spectrometry. Previous analysis by microcouloraetric and electron-capture gas chromatography had indicated the presence of PCB residues. The final analytical scheme used for preparation Figure 1. Total ion current monitor chromatogram of standard Aro clor 1254 mixture of polychlorinated biphenyls. Programmed temper ature analysis: 2 min at 185*C, to 210C at 5aC/min, isothermal at 210C. (See text for remaining instrumental parameters and partial peak identification.) The Aroclor 1200 series PCB standard materials were provided by the Monsanto Chemical Co., St. Louis, Mo. as a gift to the Pesti cides Repository of this laboratory. 318 STLCOPCB4090755 Figure 2. Total ion current monitor chromatogram of standard Aroclor 1260 mixture of polychlorinated biphenyls. Programed temper ature analysis: 2 min at 200"C, to 230"C at 5*C/min, isothermal at 230*C. (See text for remaining instrumental parameters and partial peak identification.) Approximately 2 g portions of ether, partitioned with ace tonitrile, and subjected to fractionation and cleanup by Florisil column chromatography. All of the FOB compounds were eluted in the first fraction (6% ethyl ether in petroleum ether) and this volume of eluate (200 ml) was carefully concentrated to a final volume of 10 pi. A 2 pi aliquot was then analyzed by gas chromatography-mass spectrometry. The results of the analyses for samples A and B are illustrated in Figures 3 and 4, respectively. Figure 3. Total ion current monitor chromatogram of human adipose tissue extract A. Programed temperature analysis: 5 min at 180*C, to 210'C at 5*C/min, isothermal at 210*C. 319 0S\N &52339 STLCOPCB4090756 Figure 4. Total ion current monitor chromatogram of human adipose tissue extract B. Programmed temperature analysis: 2 min at 190C, to 230C at 58C/min, isothermal at 230C. Results and Discussion Separation efficiency for the individual PCB compounds was in creased when the ArocLor standards were analyzed by capillary column gas chromatography-mass spectrometry. Resolution was better than that reported in the literature for standard analytical packed columns. No evidence of thermal degradation of any of the FCB compounds was found. It is apparent from the non-symmetrical nature of several of the peaks in both standard Aroclor chromatograms that all PCB isomeis and/or chlorine homologs were not completely separated and resolved. How ever, satisfactory mass spectra were obtained for all of the major numbered peaks in Figures 1 and 2. All seriously overlapping peaks appeared to be isomers of the same PCB. No attempt was made to elu cidate the structure of individual PCB components other than to es tablish the molecular weight and number of chlorine atoms. All com ponents of the Aroclors gave molecular ion groups of high intensity, as would be expected from highly chlorinated biphenyl structures. In addition, the characteristic isotopic distribution pattern (12) corresponding to the number of chlorine atoms in the parent ion and chlorine-containing fragment ions, was observed. Two noteworthy features of the spectra were the relatively intense fragment ions produced by consecutive loss of chlorine atoms from the parent ion and the presence of intense doubly charged fragments within the mass spectra of most of the PCB compounds. Figures 5 and 6 illustrate mass spectra obtained for a trichlorobiphenyl isomer in Aroclor 1232 and a heptachlorobiphenyl isomer in Aroclor 1260, respectively. Thus, peaks 1 and 2 in the chromatogram of Aroclor 1254 were shown to be tetrachlorobiphenyls; peaks 3 through 6, pentachlorobiphenyls; and peaks 7 through 11, hexachlorobiphenyls. Similarly, peaks 1 and 2 in Aroclor 1260 were identified as pentachlorobiphenyls, peaks 3 through 6 as hexachlorobiphenyls, peaks 7 and 9 through 12 as heptachlorobiphenyls, peaks 13 and 14 as octachlorobiphenyls, peaks 15 and 16 as nonachlorobiphenyls, and peak 17 as decachlorobiphenyl. 320 DSW 552340 STLCOPCB4090757 1 (-,! uimm uum i Figure 5. Hass spectrum of a trichlorobiphenyl contained in standard Aroclor 1232. Figure 6. Mass spectrum of a heptachlorobiphenyl found in standard Aroclor 1260. Examination of the mass spectra of the components of adipose tis sue sample extract A (Fig. 3) revealed that peaks a, b, c, and d were polychlorinated biphenyls whose mass spectra were indistinguishable from those obtained respectively for peaks 6, 7, 9, and 10 of Aroclor 1254 (Fig, 1). The mass spectrum of peak d (an isomer of hexachlorobiphenyl, mol. wt. 358) is reproduced in Figure 7. 2,2-Bis(p-chlorophenyl)-l,1-dichloroethylene (p,p'-DDE) was also confirmed in this adipose tissue sample. 1,l-Bis(p-chlorophenyl)-2 ,2 ,2-trichloroethane (p,p'-DDT), a chlorinated pesticide frequently found in human adipose tissue, was not detected. Under the conditions of the anal ysis, however, p,p'-DDT overlaps considerably with PCB peak d. Ad ditionally, p,p'-DDT is easily dechlorinated thermally to 2,2-bis (p-chlorophenyl)-1,1-dichloroethane (p,p'-DDD) on stainless steel gas chromatographic columns, particularly at the low concentration levels found in this sample. The remaining peaks observed in the total ion current monitor trace of adipose tissue extract A were non-chlorinated materials of lipid composition assumed to be natural constituents of adipose tissue carried through the analytical scheme. 321 DSW 552341 STLCOPCB4090758 U m iM la lla tilf 4 300 Figure 7. Mass spectrum of a hexachlorobiphenyl contained in human adipose tissue sample A (Peak d, Fig. 3). Injection of adipose tissue extract B into the gas chromat ograph-mass spectrometer resulted in the total ion current monitor trace illustrated in Fig. 4. Examination of the mass spectra obtained for peaks a through j showed that they were identical , to those obtained for peaks 7 and 9 through 17 in the Aroclor 1260 standard materials (Fig. 2). In addition, p,p'-DDE and p,p'-DDT, which were present at higher levels than those encountered in sam ple A, were also confirmed in this adipose tissue sample. The results detailed here demonstrate the ease with which FCB compounds can be identified in human adipose tissue by combined gas chromatography-mass spectrometry using existing analytical meth odology (12). Highly diagnostic mass spectra were obtained for each PCB component of Aroclor 1254 and 1260 standard materials and FCB isolated from two human adipose tissue samples. The presence of. p.p'-DDE and p,p'-DDT residues within the same samples were also . confirmed by mass spectrometry. High resolution capillary gas chro matographic columns may be used to separate pesticide residues such as p.p'-DDE and p.p'-DDT from PCB compounds. However, in those in stances where insufficient separation of residue components occurs, mass spectrometrie techniques are available for the identification of unresolved components in gas chromatographic effluents (13). Summary Among the chlorinated hydrocarbon synthetic pollutants in the environment, polychlorinated biphenyls represent a class of com pounds possessing mammalian toxicity and recently found to be pres ent as residues in the tissues of various wildlife species, although they are not in use as biocidal compounds. Two human adipose tissue samples examined .by combined gas chromatography-mass spectrometry were shown to contain substantial quantities of polychlorinated bi phenyls ranging from pentachlorobiphenyl to decachlorobiphenyl and including at least fourteen isomers and chlorine homologs. Although the origin of the PCB compounds found in the adipose tissue is un unknown, the data are presented as a description of the gas chromato graphic-mass spectrophotometric behavior of PCB and of the analytical methodology applicable to the analysis of these compounds in human tissues by gas chromatography-mass spectrometry. 322 OSVJ 552342 STLCOPCB4090759 Acknowledgments We thank Dr. Anne R. Yoba of the Division of Community Studies, Pesticides Program, Food and Drug Administration, Atlanta, Ga., and Dr. Harold Price of the Michigan State Department of Health for pro viding the human adipose tissue samples described in this study. References - 1. RISEBROUGH, R.W., RIECHE, P., HERMAN, S.G., PEAKALL, D.B. , and KIRVIN, M.N. Nature 220, 1098 (1968). 2. JENSEN, S. New Scientist 32, 612 (1966). 3. HOLMES, D.C. , SIMMONS, J.H. , and TATTON, J.O'G. Nature 216, 227 (1967). 4. LICHTENSTEIN, E.P., SCHULZ, K.R., FUHREMANN, T.W., and LIANG, T.T. J. Econ. Entomol. 62, 761 (1969). 5. REYNOLDS, L.M. Bull. Environ. Contain. Toxicol. 4, 128 (1969). 6. RISEBROUGH, R.W., RIECHE, P., and OLCOTT, H.S. Bull. Environ. Contam. Toxicol. 4, 192 (1969). " 7. WIDMARK, G. J. Assoc. Offic. Anal. Chemists 50, 1069 (1967). 8. HOLDEN, A.V. and MARSDEN, K. Nature 216, 1274 (1967). 9. ERRO, F., BEVENUE, A., and BECKMAN, H. Bull. Environ. Contam. Toxicol. 2, 372 (1967). 10. BIROS, F.J. and WALKER, A.C. In preparation. 11. MILLS, P. J. Assoc. Offic. Anal. Chemists 42, 734 (1959); Pes ticide Analytical Manual, Vol. I, Sec. 211, Food and Drug Admin istration, U.S. Department of Health, Education, and Welfare, Washington, D.C., 1969. 12. BEYNON, J.H. Mass Spectrometry and Its Applications to Organic Chemistry, p. 298 (1960), Elsevier Publishing Co., New York. 13. SWEELEY, C.C., ELLIOTT, W.H.` FRIES, I., and RYHAGE, R. Anal. Chem. 38, 1549 (1966). 323 DSW 552343 STLCOPCB4090760