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Anal. Chem. 1892, 64, 1176-1183
Determination of Methyl Sulfone-Substituted Polychlorobiphenyls by Mass Spectrometric Techniques with Application to Environmental Samples Hans-Rudolf Buser* Swiss Federal Research Station, CH-8820 Wadenswil, Switzerland Douglas R. Zook and Christoffer Rappe Institute of Environmental Chemistry, University of Umeci, S-90187 Umeci, Sweden A novel mlcroeyntherls method of methyl sulfonesubaltuted potychkroblphenyta (MeSO,-PCBs) and thelr analyrls by gas chromatography/mass spectrometry (oC/MS) udng both electron lonlzatlon (E1) and electron-capture negative-Ion lonlzatlon (ECNI) are dwcrlbed. The MeSO,-PCBs are syntheolzed vla methylthkPCBs (MeS-PCBs) prepared by 7 Irradlatbn of PCBs In dlmethyl dlwlflde. I n thk reactlon, a CI substituent In a PCB Is dlsplaced by a methylthlo (CH,S-) group wlth diaplacmmt preferentlal to chbrlnm the hlgher chiorhated phenyl rlng. Complete oxldatlon to the methyl sutfone b then easHy effected In a subsequent step. Thk fast and dmpk synthetic pathway results In small but, for GWMS analyds, sufflclent quantltles of numerous Identfflable PCB metabolltes. Chromatographic characterlstlcs of these MeS0,-PCBs are dercrlbed, and their mass spectrometric properHe8 are sunmarlzed. Marked dMerences are observed In both the E1 and ECNI ma88 spectra among various MeS02-PCB congeners which clearly allow dialnctlon of oMeSOJd-CI wb8tltut.d Isomers from others. Informatlon obtalned from thls assortment of synthetlc MeS0,-PCBs Is applled toward the analyrls of grey seal samples from the Batik Sea. ApproXLnatety 20 tetra- to heptachloro are detected In adipose and llver tissue, roughly one-thlrd the number of PCB congeners present, and the levels of MeSO,-PCBs In adipose are approrknetety onatenth those of the PCBs. I n comparkon to the hlgh numbers of MeS0,PCBs generated from the technkal formulatbns (A") by our synthedr method, thls lndlcates a hlgh speclflclty of the metabolism of PCBs by seal. Interestlngly, none of the MeS0,-PCB metabolltes detected In grey seal appear to be o-CH,SOJo'-CI substltuted.
oonoeners
INTRODUCTION Polychlorobiphenyls(PCBs) are among the most prevalent and important of toxic environmental contaminants. This group of chlorinated aromatic compounds has been used industrially for several decades as dielectric fluids, plasticizers, and nonflammable oils. Their presence in wildlife was first reported in the 1960s by Swedish Much attention has been directed toward the analysis of this compound class, and the global occurrence of PCBs has since been f m l y establishedH Nowadays PCBs are readily found at all trophic levels and are even present in human tissue and milk. The wide environmental distribution of PCBs has recently resulted in restrictions on their production, use, and industrial release. Due to their persistance in the environment there is now considerable interest in the environmental fate and the metabolism of these compounds. In particular, methyl sulfone-substituted PCBs (MeS02-PCBs)have received attention since their initial detection in seal by Jensen and ~o-workers.~
These sulfur-containing PCB metabolites have since been reported to be present in several aquatic and mammalian species (fish, whale, otter, clams, as well as a number of laboratory animals) and in man.&' Of medical interest waa their detection in the lung tissue of Yusho patienta.68 MeS02-PCBs are presumably formed in biological systems via glutathion and mercapturic acid conjugate^.^ Methyl sulfone metabolites of other chloro aromatic compounds, e.g. polychlorobenzenes (PCBzs), 1,l-bis(4-chlorophenyl)-2,2-dichlorothene(DDE), and polychloroterphenyls (PCTs), have also been ~bserved!J In addition dichloromethyl sulfone formed from industrial pulp bleaching is known to accumulate in aquatic organisms.1° The present interest in MeS02-PCBs has been bolstered by biochemical studies, including association to inhibition of aryl hydrocarbon hydroxylase (AHH) activity induced by 2,3,7,8-tetrachlorodibenzodioxin in human lymphoblastoid cells'l and in connection to a disease of the adrenal cortex called hyperadrenocorticism, or necrosis of the adrenocorticoid, studied in mice.12 Environmental analyses require reference and standard compounds, and data on their chromatographic and mass spedrometric properties. Synthesis methods for MeS02-PCBs are l ~ n o w n ~ J and ~ - ' ~can be used for the preparation of a number of these compounds; so far those for more than 115 individual MeS02-PCBs have appeared in the literature. These methods, however, are usually tedious and involve extensive isolation and purification. They are used for the preparation of milligram or gram quantities and generally lead to single compounds in high purity. Previously, we described a novel microsynthetic method for the preparation of small quantities of methylthio-substituted PCBs (MeS-PCBs) by y irradiation of PCBs in dimethyl disulfide (DMDS).l6 A general reaction scheme was deduced which allowed the assignment of the various isomers formed. In this report we describe the preparation of a number of MeS02-PCBsfrom the oxidation of these MeS-PCBs, as well as the chromatographic and mass spectral properties of these compounds. The analytical methods were then applied toward the analysis of environmental biological samples. Here we report resulta from Baltic grey seal.
EXPERIMENTAL SECTION Synthesis of Reference Compounds. A series of MeS-PCBs as single isomers or as mixtures of a few isomers was previously prepared by y irradiation of individual PCB isomers in DMDS.16 In addition complex mixtures with dozens of MeS-PCBs were likewise prepared from technical PCB formulations (Aroclors 1254 and 1260). These compounds were subsequently oxidized to MeS0,-PCBs with n-chloroperbenzoic acid (m-CPB; Fluka, Buchs, Switzerland). Oxidation is carried out with low-microgram quantities of MeS-PCBs dissolved in 200 p L of dichloromethane (DCM). To this solution, 200 p L of m-CPB solution (2% in DCM) is added. After 20 min at room temperature oxidation is complete and the
0003-2700/92/0384-1176$03.00/00 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL.
solution is then passed through a small silica column (0.5 g of silica gel 60,Merck, Darmstadt, Germany, packed in disposable Pasteur pipet). The column is washed with a total of 5 mL of DCM, and this eluate paased through a second silica column to further remove m-CPB. In order to remove unreacted PCBs from the reaction mixtures, the second column may be first washed with 5 mL of n-hexane; MeS02-PCBscan then be eluted with 5 mL of DCM as described above. After concentration, the samples are ready for GC/MS analysis,without actual isolation of the compounds. Oxidation was also carried out with independently synthesized 4-(methylthio)-2,2’,5,5’-tetrachlorobiphenyl (4-MeS-2,2’,5,5’-tetra-CB, courtesy of T. Mizutani, Kyoto Prefectural University, Kyoto, Japan). Preparation of Biological Samples (Grey Seal). Composite (pooled) tissues of adult grey seal (Halichoerwgrypw) collected from the Baltic Sea along the Swedish southeastern coast line were examined. These samples were extracted and prepared as follows: 30 g of tissue (liver or adipose) is mixed with 500 g of anhydrous sodium sulfate and homogenized in a high-speed blender. The sodium sulfate/tissue homogenate is then extracted with 500 mL of DCM/cyclohexane (1:l) to obtain a lipid extract. In this way, the percentage of lipids was determined as 4.5% for liver and 90% for adipose. Both polar and nonpolar organochlorine compounds are separated from the coextracted lipid material by means of polyethylene film/cyclopentane solvent dialysis. This dialysis technique is reported to allow for the efficient extraction of nonpolar organic contaminants with molecular weighta (MW) < 600 from the bulk lipid material.17*18 Approximately 0.2 g of lipid residue was recovered in the dialysate which was then further purified by gel permeation chromatography (GPC; ABC Laboratories, Columbia, MO) with SX3 Biobeads and elution with DCM/cyclohexane (1:l).The GPC eluate contained essentially no measurable lipids and was then further fractionated on a PX-21 carbon column to remove planar aromatic compounds such as the polychlorodibenzodioxins(PCDDs) and the polychlorodibenzofurans (PCDFs) for separate analysis. The eluate composed of the nonretained components from the carbon column contains nonplanar PCBs, MeS02-PCBs, and a wide array of persistent organochlorine compounds, both polar and nonpolar.18 The cleanup of this fraction may be concluded by chromatography using 8 g of Florisil (activated and then deactivated with 1.2% water), eluting with 38 mL of n-hexane, 30 mL of 15% and 30 mL of 50% DCM/n-hexane, and 25 mL of DCM, finally recovering MeS02-PCBswith 50 mL of methanol. All MeSOzPCB (0-, m-,and p-MeSO2-substituted)congeners and methyl sulfones of other chloro aromatic compounds (PCBzs, PCTs, DDE), if present in seal, are expected to be recovered in this fraction with similar recovery. It is noted that the above procedure wing polyethylene film dialysis and GPC was verified to extract MeS02-PCBs from lipids with uniform efficiency as conventional methods routinely employed by Haraguchi and co-w~rkers.’~ Aliquots of 2-pL corresponding to approximately 100 mg of seal tissue were used for analysis. Gas Chromatography/Mass Spectrometry (GC/MS) Analysis. A VG Tribrid double-focusingmagnetic sector hybrid mass spectrometer (EBEEqQ configuration, E = electric, B = magnetic, q,Q = quadrupole) (VG Analytical Ltd., Manchester, England) was used for analyte detection. The ion source was operated in either the electron ionization (EI, 70 eV, 180 “C) or electron-capture, negative-ionization mode (ECNI, argon/lO% mbar, 140 “C, 35 eV). The selection of these methane, 1 X ECNI conditions is briefly explained later under the Results and Discussion. E1 and ECNI mass spectra (m/z 35-535) were recorded (1.16 s/scan) at a resolution m/A m of 500. A Carlo Erba Model 5360 gas chromatographwith a 25-m SE54 high-resolution (HRGC) fused-silica (0.32-mm-i.d.) column was used and programmed as follows: 80 “C, 2 min isothermal, 20 “C/min to 200 OC, then at 5 “C/min to 280 “C, followed by an isothermal hold at thistemperature. All samples (2 pL in toluene) were on-column injected at 80 “C. Data acquisition and retention time measurements were started at 200 “C. Concentrations of MeS02-PCBs were estimated from E1 mass chromatograms in comparison to those of reference PCB compounds (e.g. 2,2’,4,4’,5,5’-hexa-CB) assuming equal responses for the molecular ions (M+) of corresponding compounds. The data thus generated are semiquantitative.
64, NO. 10, MAY 15, 1992 1177
Table I. Number of PCB and MeS02-PCBIsomers c1 substitution PCBs
MeS02-PCBs total no. of ortho” meta para MeS02-PCBs
monoditritetrapentahexahepta-
7 (1) 24 (8) 52 (27) 76 (51)
3
12
24 42 46
42 24 12
76 (60) 52 (46) 24 (23) 7 (7)
7 24 52 76 76 52 24
5 16 32 46 46 32 16 5
19 64 136 198 198 136 64 19 3
7 3 1(1) 1 1 nonadeca1 grand total 209 319 (224) 319 199 837 ” Number of o-MeSO,/o’-Cl substituted PCBs in Darentheses. octa-
Figure 1. Synthesis of three isomeric MeS02-pentaCBs by (1) y irradiatlon of 2,2‘,4,4‘,5,5‘-hexaCBin DMDS and (2)subsequent ox-
Mation to the methyl sulfones: (a) 2-MeS02-2‘,4,4‘,5,5‘-penta-CB (ortho-substituted,first eluting isomer);(b) 5-MeS0~-2,2’,4,4’,5’-penta-CB (meta-substituted); (c) 4-MeS02-2,2’,4‘,5,5-pentaCB (parasubstituted, last eluting isomer).
RESULTS AND DISCUSSION Synthesis of MeS02-PCBs. The MeS02-PCBsare composed of a series of mono- to nonachloro congeners with molecular ions mlz 266,300,334,368,402,436,470,504, and 538, respectively. The MeSOz group can be positioned at the ortho (2), meta (3))or para (4) position relative to the inter-ring bond of the biphenyl structure. In all there are 837 congeners with 319 0 - , 319 m-,and 199 p-MeSOz-PCBs (see Table I). The number of isomers in each congener group range from 3 (nonachloro compounds) up to 198 (tetra- and pentachloro compounds). The same numbers of isomers exist for methylthio- (MeS-) and methylsulfinyl- (MeSO-) PCBs. The MeS02-PCBreference compounds were prepared via oxidation of the MeS-PCBs previously synthesized in yields of 1-10% by y irradiation of PCBs in DMDS.16 This synthesis results in the displacement of a single C1 substituent by a methylthio (CH3S-) group. This is demonstrated from the reactions of symmetrically (3:3) substituted hexa-CBs for which the expected number of methylthio derivatives was observed. These numbers are lower in the cases of the unsymmetrically substituted penta- and hepta-CBs (3:2 and 5 2 C1 substitution, respectively) but still consistent with the numbers expected from a preferred displacement of C1 substituents in the higher chlorinated phenyl ring. Oxidation of these MeS-PCBs with m-CPB yields the same number of methyl sulfones. The oxidation is virtually complete within 20 min at ambient temperature with no MeS-PCBs left in the reaction mixtures. It appears that oxidation proceeds via the sulfoxides, as indicated by the analysis of an incompletely oxidized reaction mixture in which some MeSO-PCBs were observed. As shown in Figure 1,the reaction of 2,2’,4,4’,5,5’-hexa-CB leads to three possible MeSOpPCBs: (a) 2-MeS02-2’,4,4’,5,5’(o-MeS02-substituted), (b) 5-MeS02-2,2’,4,4’,5’- (mMeSOz-substituted), and (c) 4-MeS02-2,2’,4’,5,5’-penta-CB @-MeSOz-substituted). Assignment of these structures to the
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
Flgure 2. E1 mass chromatograms of Aroclor 1260 random methyl suifonatbn product showlng &tion of several dozens of MeSOrPCBs (25-m SE54 HRGC column; for exact condltlons, see text): (a, c, e, g) mass chromatograms m / r 370, 404. 438, and 472 for predominantly type I isomers of MeSOrtetra-, gente-,-hem-,and W-CBs, respecthrely; (b, d, f, h) mass chrometogams m/z 335,369,403 and 437 for predominantly type II isomers of MeS02-tetra-, -penta-, -hem-, and heptaCBs, respecthrely.
chromatographic peaks of a given reaction mixture are based on differences in the E1 and ECNI mass spectra of 0- and m/p-MeS02-PCBs(see below), on chromatographic elution patterns of PCBs and MeS02-PCBs reported earlier,’ and on the relative amounts of MeS-PCBs formed in the first synthetic step.l6 This set of information was self-consistent and allowed for isomer assignments with a high degree of confidence. In this way a total of 20 MeS02-PCBs have been obtained from the reaction of 6 individual PCB isomers, as shown in Table 11. The reaction sequence involving technical PCB formulations leads to complex mixtures of MeS02-PCBs. For example the reaction of Aroclor 1254 (a commercial PCB containing predominantly tri-,tetra-, penta-, and hexa-CBs) yields numerous di- to pentachlorinated MeS02-PCBs. Likewise from Aroclor 1260 (a commercial PCB with predominantly penta-, hexa-, hepta-, and octa-CBs) numerous tetra- to heptachlorinated MeS02-PCBs were obtained, comprised by many 0-,m-, and/or p-MeSOpwbstituted PCBs. The complexity of one of these mixtures is illustrated in Figure 2. Ae expected, the isomer profiles of the MeS02-derivatives thus obtained are more complex than those of the parent PCBs, because of the increased number of possible isomers (837 vs 209). As expected, several of the MeSOrPCBs derived from the Aroclors were also formed from the individual PCB isomers listed in Table 11, since some of these (e.g. 2,2‘,3,4,5‘- and 2,2’,4,4’,5penta-CB and 2,2‘,4,4‘,5,5’- and 2,2’,3,4,4’,5’-hexa-CB) are major components of Aroclor 1254 and 1260. In this way complex mixtures with numerous MeS02-PCBs can easily be
prepared from technical PCB formulations and used to eatablish chromatographic elution windows. EX Mass Spectraof MeS02-PCBs. E1 mass spectral data of a number of MeS02-PCBshave been published, and the new data’ presented in Table I1 are largely consistent with those reported.sJs16 The results discussed here include data from 11 previously unreported isomers. ECNI mass spectra described in the following section have however not been previously reported for any MeS02-PCBsand are provided in Table I1 as well. Due to the potential for increased sensitivity and selectivity to organochlorine compounds, and transparency to many otherwise interfering compounds, ECNI is an important tool for screening of environmental contaminants and their metabolites. On the basis of the E1 mass spectra the methyl sulfones can be placed into two groups. The first group, here referred to as type I isomers and later shown to be m- and p-MeS02PCBs, show strong, usually base peak molecular (M+) ions with the expected isotopic patterns due to the presence of chlorine and sulfur. Fragment ions for the type I isomers are typically observed at M+ - CH3 (M+- 15, minor), M+ - C H 8 0 (M+ - 63, medium, in one case the base peak), M+ - CH3S02 (M+ - 79, minor/medium), M+ - CH3S0 - CO (M+ - 91, medium), M+ - CH3S02- C1 (M+ - 114, major, particularily large for 3- and 4-MeS02-PCBs, perhaps attributable to a stable biphenylene structure), M+ - CH3SO2 - C12 (M+ - 149, minor), M+ - CH3S0 - CO - C12(M+ - 161, minor), and M+ - CH3S02- C13 (M+- 184, medium; not previously reported); doubly charged ions are also apparently present corresponding
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
Ei
8
1179
m
(D
I
El
111
LOO
2
a
b 70
8w
290
70
'T'
f(
60 50
40
30 20 10
Flgurr 9. E1 mass spectra of the three Isomeric MeS0,-penta-CBs (M+ = m / z 402) synthesized from the methyl sulfonatlon of 2,2',4,4',5,5'hexaCB: (a) 2-MeSO2-2',4,4',5,5'-pentaCB; (b) 5MeSO2-2,2',4,4',5'pentaCB; (c) eMeSO,-2,2',4',5,5'-penteCB.
to (M - 114)2+.The formation of some of these fragment ions requires rearrangement reactions, such as for M+ - 63 which is formed via a methyl sulfinate ester. The M+ - 63 ion appears to be particularily strong in the 4-MeS02-substituted isomers, possibly due to quinoid inter-ring stabilization. A minor ion at m/z 79 (CH3S02)was also observed for some isomers. Parts b and c of Figure 3 show E1 mass spectra of a m- and a p-MeS02-PCB, 5-MeS02-2,2/,4,4',5'- and 4MeS022,2/,4/,5,5/-penta-CB,respectively, typical for this group of compounds. The losses of C12and C1 from M+, which are predominant E1 fragmentations of PCBs, were not significant for these methyl sulfones. Furthermore, none of the type I methyl sulfones showed an intense M+ - CH3ion. These M+ - CH3cations are not as stable as the corresponding M- - CH3 anions observed with ECNI (see below). A single isomer (compound 7) had a small M+ - 62 ion (migration of H from the methyl group to the phenyl ring), previously reported for 3-MeS02-substitutedisomers without 0-C1 substituents.16 All type I isomers show ion intensity ratios M+/(M+- C1) >>l. A second group of MeS02-PCB isomers, referred to here as type II isomers and later shown to be o-MeS02/o'-C1PCBs, yield weak M+ and essentially no M+ - CH3 ions. The base peak from these isomers is usudly at M+ - C1 (M+- 35). This ion very likely has a cyclic sulfonium structure. A similar E1 fragmentation behavior was also observed for o-MeS/o'-Clsubstituted PCBe.16 Additional fragment ions occur at M+ - CH3S0 - C1 (M+- 98),ions with the exact mass and likely structure of polychlorodibenzofuran (PCDF) cations. As for the type I isomers other fragment ions were also observed at M+ - 114, M+ - 149, and M+ 184. In Figure 3a the E1 mass spectrum of 2-MeS02-2',4,4',5,5'-penta-CB is shown, typical for the type I1 isomers. This distinction of o-MeS02/o'-C1 PCBs from the other MeS02-PCBs was previously nokd.13 A small M+ - 78 ion is generated from some of these derivatives. All type 11isomers show ion intensity ratios M+/(M+ - C1)
