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ig excreted in Heats that rat. Kb poIhaaa workers scraaaed slowly tels three /ldiphenol lydrolyted extract Is ilud, washed, 1 cleanup mol standard tool with hysn be oxidised rapare a pure ioa maintained r the 4,4'-sultheae reagents or this reason, ts are evapor* d and cleanup sen modified a method insilylation, detector Aie deterainand 4,4'-sulfortifled hu2 pp*. , LAOOMEA, (1968). 52 (1966). 1969). 68), d Chem. 15, 6, <408 (1968). BWi, C.R., Arch. Polychlorinated Biphenyls in Human Adipose Tissue by Francis J. Biros, Annita C. Walker and Ancela Medbexy . Perrine Primalr Rr/tcurrh Branch Division of Pesticides, Food and Drag Administration Department of Health, Education, and Welfare Perrine, Florida Recent reports have desunstrated that chlorinated hydrocarbons are among the most abundant synthetic pollutants In the environment (1). Concern over the pollution hasard 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 FCB in any sampla, in addition to being toxicologically significant in living systems (4) , presents a difficulty in the analysis of certain chlorinated pesticldal chemi cals (5). Adequate discussions of the properties and use of FCB compounds, their distribution in the environment, and their inter ference with pesticide residue analysis have been presented previously (1-6). The analytical evldanca for the presence of FCB in environ mental samples has been based on mass spectromstrlc analysis (7), re tention times in gas chromatographic analysis (1, 5, 6, 8), chlorine content by microcoulometric analysis (6), relative inertness to ni tration and dehydrochlorlnation 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 theso compounds obtained with a combined gas chroswtograph-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 FCB, The relative chemical and thermal inertness of theaa materials renders them par ticularly suitable for analysis by gas chromatography-mass spectro metry. Furthermore, recent success utilising combined gas chromato graphy-mass spectrometry for pesticide residue analysis (10) and the availability of human tissue suspected to contain FCB prompted us to examine the behavior of FCB during combined gas chromatography-mass spectrometry and, if posslMe, to ate presented as a prellmkry YWtiiptlon 01 the gas UlTUbatographic-mass spectromstrlc be havior of FCB 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 usad in this study has been described previously (10). The gas chromatographic *17 Bellctia of EavtrafunenUl Contamination A TixicaWsr, Val. 5, No. 4,1970, publubrd by Spriagar-Vei Uf New Yeffc, he. DSW 332375 STLCOPCB4078951 column was a stainless steel capillary, 100 feet x 0.020 lnchas l.d. and coated with 0V-1 cillcone oil, obtained from the Perkln-Elmer Corp., Norwalk, Conn. Programmed temperature analyaea were made both for the alx Aroclor 1200 aerlea atandard materials* and the tlaaue extracts. Flgurea 1 and 2 depict chromatograms obtained for Aroclora 1254 and 1260 of three series and detail the temperature programing conditions used. The molecular separator and gas Inlet temperatures were maintained at 210*C and 215*C, respectively. All mss spectra were recorded at 80 av 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%. Mass spectra were scanned magnetically over the range m/ 5 to m/e 500 in 6 seconds. Two samples of human adipose tissue were examined by gas chro matography-mass spectrometry. Previous analysis by microcoulomatrlc and electron-capture gas chromatography had indicated the presence of FCB residues. The final analytical scheme used for preparation until in Figure 2. clor 1260 ature ana 230* C. (: peak iden electron-! tissue we; tonltrlle column ch< first frai of eluate 10 pi. A spectrowe lllustraci Figure 1. Total Ion current monitor chromatogram of standard Aroclor 1254 mixture of polychlorinated biphenyls. Programmed temper ature analysis: 2 min at 185*C, to 210*C at 5*C/min, Isothermal at 210*C. (See text for remaining Instrumental parameters and partial peak identification.) *Tha Aroclor 1200 series PCB standard materials were provided by the Monsanto Chemical Co., St. Louis, Mo. as a gift to the Peat! cldea Repository of thlu laboratory. US ' u i mi ipiwwpir*npRpji Figure 3. tlaaue exi to 210* C s STLCOPCB4078952 laches i.d. in-Elmar re made end the telned (or pore tun gee Inlet lvely. All 2300 v oc tet. Chror. Helium cure woo ange m/e, .5 goo chrocoulometrlc e presence eparetloa e samples, d to con tained by t i T dard Aroed temperthermal at nd partial provided the Pestl- Figure 2. Total Ion current monitor chromatogram of standard Aroclor 1260 mixture of polychlorinated biphenyls. Programmed temper ature analysis: 2 min at 200*C, to 230*C at 5*C/min, isothermal at 230*C. (See text for reswining instrumental parameters and partial peak identification.) ___ Approximately 2 g portions of tissue were extrAdted with petroleum ether, partitioned with ace tonitrile, and subjected to fractionation and cleanup by Florisil column chroma'tografftiy. All of the PCB 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. i 2 |il aliquot was then analysed by gas chroma tography-msss spectrometry. The results of the analyses for samples A end B are illustrated in Figures 3 and 4, respectively. Figure 3. Total ion current monitor chromatogram of human adipose tisiue extract A. Programmed temperature analysis: 5 min at 180*C, to 210*C at 5`C/min, isothermal at 210*C. >19 wnw mm VIM "" qSV\! 3323?7 STLCOPCB4078953 I ci I I I s 44 Im Vi I M li Figure A. Total Ion current monitor chromatogram of human adipose tissue extract B. Programmed temperature analysis: 2 min at 190*C, to 230*C at 5*C/min, Isothermal at 230*C. 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 fra the non-symnetrical nature of several of the peaks In both standard Aroclor chromatograms that all PCB isomers and/or chlorine homologa were not completely separated and reaolved. How ever, satisfactory mats spectra were obtained for all of the major numbered peaks in Figures 1 and 2. All seriously overlapping paaks appeared to be isomers of the same PCB. No attempt was made to elu cidate the structure of individual PCB components other than to astabllsh 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 Iona 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 heptachloroblpheny1 isomer in Aroclor 1260, respectively. Thus, peaks 1 and 2 in the chromatogram of Aroclor 1234 were shown to be tetrachloroblphenyla; peaks 3 through 6, pentachloroblphenyls; and paaks 7 through 11, hexachlorobiphenyls. Similarly, peaks 1 and 2 in Aroclor 1260 were identified as pentachloroblphenyls, peaks 3 through 6 as haxachlorobiphenyls, peaks 7 and 9 through 12 as heptachlorobiphenyls, peaks 13 and 14 as octachlorobiphenyls, paaks 13 and 16 as nonachlorobipbanyls, and peaV^- 17 as decachlorobiphenyl. . no 1 '! ; ; I ; j Figura 5.. H_ Aroclor' 1232. Figure 6. I Aroclor 1260 Examine sue 8ample i polychlorin; from those i 1254 (Fig. 1 biphenyl, mi phenyl)--1,1adipoae ti_s; ethane (p,p adipose tis. ysis, howev. ditionally, (p-chlorophi. chromatograi found in thi current mom materials ot adipose tiss wain w m 332378 qSNN ' STLCOPCB4078954 tat ) . Adipose . t 190*C, Figure 5. Mass spectrum of a trichlorobiphenyl contained in standard Aroclor 1232. Is was inary column er than that columns. No Is was found. of the peaks s and/or *ed. How* _he major >ing peaks ad* tp eluin to es. Ml comintensity, iffcures. tern (12) it ion and iworthy ant Ions arant ion in: the mass lustra te roclor 1232 ively. thus, nown to be lyls; and s 1 and 2 peaks 3 ?as.heptapeaks 1$ and nyl. i I r ' ; ' 1 _ 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-chlorophenyD-l ,1-dlchloroethylene (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)-l,1-dichloroethane (p,p'-DDD) on stainless steel gas chromatographic columns, particularly at the low concentration levels fpund in this sample. The remaining peaks observed in the total ion current monitor trace of adipose tissue extract A were non-chlorineted materials of lipid composition assumed to be natural constituents of adipose tissue carried through the analytical scheme. 321 ................ mm ii win. j imp DSW 332379 STLCOPCB4078955 tllW Iia i lililtH figure 7. Mate pectrim of a hexachlorobiphenyl contained In human adipoae tissue sample A (Peak d,Fig. 3). Injection of adipose tissue extract B into the gas chromatograph-masa 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'-DDEand 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 FCB 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 eo separate pesticide residues such as p,p'-0DE and p,p'-DDT from FCB compounds. However^ in those in stances where insufficient separation of residue components occurs, mass spcctrometric 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 decachloroblphenyl and including at least fourteen isomers and chlorine homologs. Although the origin of the FCB compounds fouod in the adipose tissue is un unknown, the data are presented as a description of the gas chromato graphic-mass spectrophotometrie behavior of FCB and of the analytical methodology applicable to the analysis of these compounds in human tissues by gas chromatography-mass spectrometry. m ipfHUIll [ WJHIII) IJJJMPPI We tharu. Pesticides Pr Dr. Harold Pr viding the hu 1. RISEBR kirvi; 2. JENSEI 3. HOLME5 227 (J 4. LICHU T.T. 5. REYN01 6. RISE31 Codtas 7. WIDMAf 8. H0LDE1 9. ERRO, Toxict 10. BIROS, 11. MILLS, ticidc istra Washir i 12. BEYNOI Chemi: 13. SWEELi Chem. DSW 332380 STLCOPCB4078956 I_ ; Acknowledgments I ; We thank Dr. Anne R. Yobs of the Division of Comunlty Studies, j 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. in oraataonltor ;:ra cal or 1260 .'-DDT, in sam- ilch PCB >lned .cal meth1 for .els and usance |re also ` gas chrojiuas such chose in 's occurs, <cstion ;i3>. i) in the Y cost- l ibe presL although uliae tissue '.-ometry \ated bi;eyl and Although . is un . chromato. analytical \n human References RISEBROUGH, R.W., R1ECHE, P. , HERMAN, S.G., PEAKALL, D.B., and KIRVIN, M.N. Nature 220, 1098 (1968). 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. Contain. Toxicol. 4, 192 (1969). WIDMARK, G. J. Assoc. Offic. Anal. Chemists 50, 1069 (1967). 8. HOLDEN, A.V. and M^IS, K. Nature 216, 1274 (1967). 9. ERRO, F. , BEVENUE, IlHd BECKMAN, H. Bull. Environ. Contam. Toxicol. 2, 372 (196A|V* 10. BIROS, and WALKER, A.C. In preparation. .11 MILLS, P-AUP^oc Offic. Anal. Chemists 42, 734 (1959); Pes ticide AnmljF9cal Manual, Vo). 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. 36, 1549 (1966). 323 ,im pu ifr-pi^ ............................................. .. "r' DSW 332381 STLCOPCB4078957