Environ. Sci. Technol. 1994, 28, 119-128
Isomer- and Enantiomer-Selective Analyses of Toxaphene Components Uslng Chiral High-Resolution Gas Chromatography and Detection by Mass Spectrometry/Mass Spectrometry Hans-Rudolf Buser' and Markus D. Muller
Swiss Federal Research Station, CH-8820 Wadenswil, Switzerland Achiral and chiral high-resolution gas chromatography (HRGC) in combination with electron ionization (EI)mass spectrometry/mass spectrometry (MS/MS) was used for the analysis of toxaphene components in a technical mixture and in tissue extracts of several aquatic vertebrate species collected from the Baltic Sea (herring, salmon, seal), the Arctic (seal), and the Antarctic (penguin). Enhanced isomer selectivity of some toxaphene components was obtained by selected-reaction monitoring (SRM) using ion transitions commonly not observed with other halogenated contaminants. All species showed extensive alteration of the original toxaphene mixture with only a few, but largely the same, polychlorobornanes present. Using chiral HRGC, the enantiomer resolution of several major toxaphene components was achieved. Analysis of the technical mixture revealed racemic or nearly racemic mixtures of these compounds, and analysis of the aquatic species showed some changes in enantiomeric composition for some components. However, the enantiomeric composition of the most accumulating nonachlorobornane, TOX9, was not much different in all the species, indicating little if any biological degradation of this component.
Introduction Toxaphene (polychlorinated camphene; camphechlor in the United Kingdom) was a widely used organochlorine insecticide, formerly manufactured in the United States and other countries (1). Although its use has been drastically limited in many countries, inputs into the environment likely continue at significant levels (2). Not surprisingly, toxaphene is now a major contaminant in environmental biological samples, particularily in aquatic samples, and can be found in specimens at all trophic levels from such remote areas as the Arctic and the Antarctic (3-5). Toxaphene is produced by the chlorination of camphene and consists of a complexmixture of chlorinated camphene derivatives, mostly polychlorobornanes (1, 6). Analyses readily reveal a complex pattern of compounds in technical toxaphene, but only a relatively small number of components are detected in biota, indicating extensive alteration of the original mixture (3-5,7,8). It is still largely unclear whether this is caused by chemical changes in the environment or by selective bioaccumulation or metabolism of some toxaphene components. Most of the polychlorobornanes are chiral and exist in two enantiomeric forms. As previously pointed out for other chiral marine pollutants (9, 10) biotic processes (uptake, metabolism, excretion) may be different for enantiomers whereas abiotic processes (chemical, photochemical, distribution, transport) will be the same. The drastic changes in congener and isomer composition of toxaphene in biota may be due to biotic and abiotic 0013-936X/94/0928-0119$04.50/0
0 1993 American Chemical Society
processes; however, possible changes in enantiomeric composition of chiral compounds would only be due to enantioselective biotic processes. In most previous studies on toxaphene, gas chromatography (GC) with electron-capture detection (ECD) or electron-capture, negative ionization mass spectrometry (ECNI MS) was used (11-16). ECNI MS allows a distinction among homologue groups in a toxaphene mixture, but no further differentiation among isomers is possible. Electron ionization (EI) MS yields extensive fragmentation and shows differences among isomers, but it was reported to provide insufficient sensitivity for the trace analysis of toxaphene (12). In this study we successfully applied E1 MS to the analysis of technical toxaphene and to tissue extracts of aquatic vertebrate species from the Baltic, the Arctic, and the Antarctic. Using MS/MS for detection, and achiral and chiral HRGC, various toxaphene components were first characterized in a technical mixture and then determined in the biological samples. The monitoring of specific ion transitions allowed isomer-specific, and in combination with chiral high-resolution (HR) GC, enantioselective analyses. The results document the capabilities of these techniques toward isomer and enantioselective analyses of complex mixtures, such as technical toxaphene and environmental samples.
Experimental Section Reference Compounds. Technical toxaphene (Ehrenstorfer GmbH, Augsburg, FRG) and the toxaphene components TOX8 and TOX9, previously isolated from harbor seal (Phoca uitulina; see ref 171, were available as reference compounds. Solutions in toluene were prepared at concentrations of 100 and 1000 ng/pL for technical toxaphene and at =l ng/pL for TOX8 and TOX9. Biological Samples. The aquatic samples investigated were the same as in previous studies and included salmon (Salmo salar) and herring (Clupea harengus) from the Baltic, harp seal (Pagophilus groenlandicus) from Greenland, and penguin (Pygoscelis adelis)from Antarctica (IO, 18,19). These samples were prepared at the Institute of Environmental Chemistry, University of Umeh, Sweden, or at the Norwegian Institute for Air Research, Lillestram, Norway. Aliquots of 1-2 pL corresponding to 200-400 mg of tissue were analyzed. HRGC-MSAnalysis. A VG Tribrid double-focusing magnetic sector hybrid mass spectrometer (VG Analytical Ltd., Manchester, England) was used in the E1 (70 eV, 180 "C) or in the ECNI (argon, 50 eV, 140 "C) mode under conditions as previously described (18,19). Full-scan E1 and ECNImass spectra ( m l z 35-535,1.16 s/scan, resolution MI AM = 500) were recorded for analyte identification. MS/MS experiments were carried out with the quadrupole analyzer set at nominal resolution (see also ref 20). The formation of specific daughter ions generated through lowEnvlron. Scl. Technol., Vol. 28, No. 1, 1894 119
Table 1. Compounds Investigated in Technical Toxaphene and in Environmental Biological Samples abbreviationa
compd, structure
TOX8 TOX9 TC1 TC2 TC3 TC4 TC5 TC6 TC7 TC8 TC9 TClO
octachlorobornane, Chart 1 nonachlorobornane, Chart 1 heptachlorobornane heptachlorobornane heptachlorobornene heptachloroisocamphane octachlorobornane octachlorobornane octachlorobornane octachlorobornane (toxicant A), Chart 1 octachlorobornene octachloroisocamphane
abbreviation TOX8 TOX9 TC 1 TC2 TC3 TC4 TC5 TC6 TC7 TC8 TC9 TClO
elut temp ('Qb 213.4 229.5 208.8 213.3 212.5 218.1 215.1 224.0
1.3 0.6 4.3 NA
225.8
0.0
224.7h 217.3 231.7
NA 4.2 NA
detectione SRM transition
MW
ECNIf
374+ 408+ 340+ 340t 374+ NA 374+ 374+ 374+ (374+ 408+ NA
410 444 376 376 374 376 410 410 410 410 408 410
375, Cl7 409, Cls 341, Cle 341, Cls 339, Ck 341, C& 375, c17 375, C17 375, Cl7 375, c17 373, Cl7 375, c17
------
278+ 312+ 244+ 210+ 244+
enant resol (R)obsc
occurrenced fish seal/penguin
techn
++ +++ ++ ++++ ++ ++ +++ +++ +++ +++ ++ +++
0.0 (0.7) 1.0 (1.1)
0.0
1.2
+++ ++++ + +++ + (+) ++++ +++ +++ + + (+)
++++ ++++ ++++ +++
ND ND
++++ ++++ +++
ND ND ND
MS data E18
375,374,339,327,303,291,278,267,255,329 408,373,361, (359), 337,327,325,312, 301,289,83 341,340,327,305,291,269,244,246 305,269,257,243,242,233,231,161 374,339,291,255,244,243,293 341,340,305,291,269,255,100 278+ 374,339,327,325,303,291,278,267,255,293 278+ 375, 374, (361), 339, 325,303,291, 289,278,280 312+ 375,339,327,312,303,291,279,195 278+)i 375, (361),339,327,303,291,278,277,267,161 278+ 408, (373), 325,305,289,277,267,195 375,361,339,325,303,291,289,377 a Refer to text and figures. Elution temperatures on SE54; for conditions, see text. Enantiomer resolution (R values) observed on chiral 20-m OV1701-BSCD column (values in parentheses from OV1701-BSCD/PECD; NA = not analyzed by chiral HRGC. d Occurrence (relative quantities) semiquantitatively indicated by the number of plus marks; ND = not detectable; (+) presence questionable; ECNI data us8d.e NA = not analyzed by SRM. f Most prominent monoisotopic ( W l ) ion, (M - C1)-; number of Cl's indicated. 8 Characteristic (monoisotopic W1) ions in higher mass range, weak ions in parentheses; base peak italicized. h Coelution with p,p'-DDT. i Nonoptimal detection.
*
energy (18 eV) collisions from selected parent ions was monitored (selected-reaction monitoring; SRM). Eight parent/daughter ion transitions (SRM chromatograms) were recorded simultaneously (1scads); the actual SRM chromatograms registered are listed in Table 1. Some analyses were also carried out by selected-ion monitoring (SIM). All samples were analyzed using achiral and chiral HRGC. An achiral25-m SE54 fused silica (0.32 mm i.d.) column was used. The chiral column was a 20-m OV1701 fused silica (0.25 mm i.d.) column with 30% tert-butyldimethylsilyl-6-cyclodextrin(BSCD; amount relative to OV1701) added as the chiral selector. Initially, chiral columns from other studies (18,19)were also investigated (PSO86-BSCD,PS086-PMCD, and OV1701-BSCD/PECD whereby PMCD = permethylated p-CD and PECD = perethylated a-CD), The columns were temperature programmed as follows: 100 OC, 2-min isothermal, 20 "C/ min to 140 "C, then at 5 OC/min to 280 "C (SE54) or at 3 OC/min to 250 OC (OV1701-BSCD), followed by isothermal holds at these temperatures. Enantiomers were denoted with annex-1 and annex-2 for earlier and later eluting enantiomers, respectively. Enantiomeric ratios (ER) and enantiomer resolution (R) were as defined in refs 18 and 19. Results and Discussion Achiral and Chiral Components in Technical Toxaphene. Technical toxaphene consists of several hundred compounds with an average elemental composition, CloHloCle, produced by the chlorination of camphene (I, 6). Polychlorobornanes (C~.oHl&l,; n = 5-12) are produced as the main components in a Wagner-Meerwein-type 120
Envlron. Sci. Technol., Vol. 28, No. 1, 1994
pclychiorobornanes
polychloro.2-bornenes
polychioroisocamphanes
Ri, RP, R p CH3, CHzCI, CHClz
4UPAC numbering of carbon skeleton used for bornane system. Hydrogens on ring carbons not indicated.
rearrangement reaction. Smaller amounts of other compounds, presumably polychlorobornenes (CloHls.,Cl,), polychlorobornadienes (CIOH1&ln) and polychloroisocamphanes (CloHle.,C1,), are also produced (see Scheme 1 for structures). In this study, hepta-, octa-, and nonachloro compounds were considered. Camphene is technically produced from a-pinene, a main constituent of terpene oil (I). Both compounds are chiral. In terpene oil from different provenances, a-pinene may occur in predominantly one or the other enantiomericform, in terpine oils from North America also as a more or less racemic mixture (21). Both (+)- and (-)-camphene are commercially available, but we do not know what actual
Chart 1. Structures of Some Chiral Polychlorobornanes Discussed in Study.
CI
I toxlcanl B (chiral)
toxicant A, (chlral)
n
TOX8
(chiral)
TOX9-1 (chlral)
I
TOX9-2 (chiral)
OThe structures of the two enantiomers of TOX9 are shown (arbitrarilyaeeignedasTOX9-1andTOX9-2,notnecessarilyidentical with elution order;mirror plane perpendicular through C-1, C-4, and C-7; H substituentson thealkyl substituentsnot indicated). Toxicant A reported as a mixture of the C-7 epimers (toxicantsA1 and A d and a nonachloro component, toxicant Ac.
forms of camphene were used in the technical synthesis of toxaphene. A synthesis from enantiomerically enriched camphene could still lead to racemic mixtures if symmetrical intermediates are involved. I t is expected that most polychlorobornanes contain at least one C1 each at C-2 and (2-10 and that C-4 is generally unchlorinated (6). Mainly CHB,CH2C1, and CHClz groups are present in the compounds of the technical mixture. Bornane (R1, R2, Rs = CH3, see Scheme 1)itself is achiral due to a symmetry plane through C-1, C-4, and C-7, but an initial chlorination product, 2-exo,l0-dichlorobornane (6),and most, but not all, of the polychlorobornanes are chiral. In case of the polychlorobornenes the C=C double bond can be assigned to C-2/C-3 (2-bornenes; double bond impossible at bridgeheads), and all these compounds are chiral. Similarily, all polychloroisocamphanes are chiral. A relatively small number of components have actually been isolated and identified from technical toxaphene. The most well known components are probably toxicant A and toxicant B (structures shown in Chart 1) (22-24) whereby toxicant A was later shown to be a mixture of three components (the two C-7 epimeric octachlorobornanes, toxicants A1 and A2, unresolved by capillary GC, and toxicant Ac, a nonachlorobornane) (23,25). Only very recently, two abundant congeners were isolated from biota (7, 8 ) and identified by lH nuclear magnetic resonance (NMR) spectroscopy as 2-exo,3-endo,5-exo,6-endo,9,9,10,lO-octachlorobornane (8) (TOX8 in this study, T2 in ref 8) and 2-exo,3-endo,5-exo,6-endo,8,9,9,10,10-nonachlorobornane (TOX9 in this study, T12 in ref 8) (for structures see also Chart 1; IUPAC numbering used; note that in some references a different numbering scheme with C-8 and C-9 reversed was used). Both compounds are chiral, and the structures of the two enantiomers of TOX9 are shown in Chart 1. Characterization of Toxaphene Components Investigated. In Table 1,we listed chromatographic and mass spectrometric data of the toxaphene compounds investigated. With the exception of TOX8, TOX9, and presumably TC8 (toxicant A, see below), the structures of these components are unknown. The 12 compounds selected are major components in the aquatic species and/ or in technical toxaphene, and they are briefly described below.
TOX9 is the main nonachlorobornane in all the aquatic species studied, and it is one of the components isolated from harbor seal and beluga whale (7,8). From retention time considerations and/or E1 MS data published, TOX9 is identical to the major component reported in fish, bird, and seal from Swedish waters and in human milk (peak 17 in refs 14 and 26), in fish from the North Sea and the Baltic Sea (peak 14 in ref 16),and in various environmental samples from the Canadian Arctic (peak D in ref 11).It corresponds likely to toxicant Ac, apersistent contaminant of toxicant A, for which a C-7 epimeric structure was probably erroneously assigned (25). TOX8 is the second component isolated from harbor seal and beluga whale (7, 8 ) . It is a major component in all the aquatic samples, but only a minor component of the technical product (see below). Components TC5, TC6, TC7, and TC8 are major octachlorobornanes in technical toxaphene and, except for TC8, in the aquatic samples. TC8 is tentatively identified as toxicant A by comparing its E1mass spectrum with those listed in refs 27 and 28, and its reported coelution with p,p'-DDT (28) (note that there is a conflicting mass spectrum reported in ref 29). TC8 eluted as a single peak on all our columns; it is not known whether it consists of two unresolved, epimeric components as reported for toxicant A (23). TC1 and TC2 are heptachlorobornanes and are present as major components in the technical material and in most aquatic species (see below). TC3 and TC9 are likely hepta- and octachlorobornenes, respectively, present in the technical material and at trace levels in fish. Finally, components TC4 and TClO are likely hepta- and octachloroisocamphanes, respectively, present in technical toxaphene but not in the aquatic samples.
So far, we were unable to locate toxicant B in our chromatograms, although it is listed as a major heptachlorobornane in technical toxaphene. The E1 mass spectra reported are conflicting. For instance, mass spectra in refs 12 and 27 show an ion cluster at rnlz 244-249 (mlz244 as an even-mass ion) whereas those in refs 28 and 29 show such a cluster at rnlz 278.
E1 Mass Spectra of Toxaphene Components. In Figure 1, panels a-d, we show E1 mass spectra of four major toxaphene components in penguin tissue [a hepta(TCl), two octa- (TOX8 and TC6), and the major nonachlorobornane (TOX9)I elutingfrom the achiral SE54 column. The E1 mass spectrum of a further octachlorobornane (TC8) in technical toxaphene is shown in Figure 2a. The mass spectra indicate extensive fragmentation which leads to a reduced sensitivity under E1 conditions. Generally, no molecular (M+) ions are observed, and the highest mass ions correspond to (M - C1)+ and/or (M HCl)*+. As shown by the E1 mass spectra of the three octachlorobornanes in Figures lb, Id, and 2a, there are significant differencesamong isomers. For instance, TOX8 (Figure lb) and TC8 (Figure 2a) show signals at rnlz 327 (M+ - 831, indicating the presence of CHC12 groups, whereas component TC6 (Figure Id) shows a signal at rnlz 325 (M+ - 85) from the loss of 'CH2C1 and HC1. The mass spectra of TOX8 and TOX9 from the penguin extract (Figures 1,panels band c) are similar to those of the isolates from harbor seal (17, 20). Environ. Sci. Technol., Vol.
28. No. 1, 1994
121
10
tx4*00t loo%, L
j
1 9
C
I 5 2
70
1
5
d 327
109
I li 0
I
~
4 0
M/Z I
Flgure 1. E1 mass spectra of toxaphene components Identified in Antarctic penguin: (a) component TC1 (hgptachlorobornane,M'+ = 376),(b) TOX8 (octachlorobornane,M'+ = 410; partially coeluting with component TC2), (c) TOX9 (nonachlorobornane,M'+ = 444), and (d) component TC6 (octachiorobornane,M + = 410). Even-mass RDA fragment ions indicated by arrows (see text). Note the vertical expansion as indicated.
Scheme 2. Fragmentation of Six-Membered Carbon Ring by RDA Mechanisms Leading Diagnostically Important Even-Mass Fragment Ionsa H
miz 408
'qCHck hc; m/Z 312
nonachlorobornane
H
>H%
CI
Cl's between the six-membered carbon ring, the bridge, and the bridgehead in the parent bornane structure (20). The components tentatively identified as polychlorobornenes (MW 2 amu lower than polychlorobornanes) yield weak M + ions, formally identical to (M- HC1)-+ions of polychlorobornanes. E1 fragmentation of the two series of compounds is similar, and the compounds are thus probably difficult to distinguish by EI. As an example, we show in Figure 2b the E1 mass spectrum of the octachloro compound TC9 from technical toxaphene. This component and the heptachlor0 compound TC3 yielded RDA fragment ions at mlz 244 and 278 via loss of C2HC13 (130 amu) from M + (see Scheme 2, center), suggesting three Cl's at carbon atoms C-5/C-6in the proposed bornene structure of these compounds. The compounds TC4, TC10, and others yielding ions at m / z 100and 134 (CsH&l'+ and CsH&12'+) were tentatively identified as polychloroisocamphanes (same MW as polychlorobornanes); one such a compound has actually been isolated from technical toxaphene (30). The formation of these ions can be rationalized by an RDA fragmentation pathwaystartingfrom (M-HCl)*+(see Scheme 2, bottom). As an example, the E1 mass spectrum of the octachloro compound TClO is shown in Figure 2c. ECNI Mass Spectraof Toxaphene Components. In ECNI, polychlorobornanes and polychlorobornenes yield intense, stable (M - C1)- anions from which the molecular composition is deducable, and the two series of compounds are distinguishable. ECNI can be used for congener group analysis. This is illustrated in Figure 3, panels a-f, where we show ECNI mass chromatograms (mlz 343,379, and 413) of hepta-, octa-,and nonachlorobornanes,respectively, in technical toxaphene and in the penguin extract analyzed
WCk 1
,+I
~
u13
CI
i
mizloa
H
CI CI
li'
mi2370 EHCb heptachlorolsocamphane
H mi2 340
mi2 100 (CSHSCI+.)
aIon transitions (SRM) correspondingto these fragmentations were used to monitor polychlorobornanes and polychlorobornenes. (top) Polychlorobornanes, (center) polychlorobornenes, and (bottom) polychloroisocamphanes.
Several of the compounds show the presence of evenmass radical ions (e.g., rnlz 244,278,312, and 278 in Figure 1, panels a-d, respectively). In particular, TOX8 and TOX9 show ion clusters with Cls- and C16-patterns at mlz 278 and 312, respectively, assigned to substituted cyclopentadiene ions and formed via a retro-Diels-Alder (RDA) fragmentation pathway of the six-membered carbon ring (see Scheme 2, top) (17,20). For TOX8 and TOX9, these data and data from additional, pseudo-RDA fragmentations allowed the determination of the distribution of the 122 Envlron. Sci. Technol.,Vol. 28, No. 1, 1994
"1 a 1 'is 90
175
70
269
I
305
of 49 and 83 amu, respectively) likely are characteristic to toxaphenes since these groups are not commonly present in other halogenated environmental contaminants. In the present study, eliminations of C2H4.,ClX ( x = 1-3) from (M- C1)+or (M- HCl).+were monitored which werejudged to be more selective toward individual congeners. Using the transitions listed in Table 1, SRM chromatograms showed single major peaks for TC1, TC2, TC3, TC7, TC9, and TOX9 in technical toxaphene. Components TC5, TC6, TC8, and TOXS were detected in one and the same SRM chromatogram (376+ 280+) but could be distinguished otherwise. In a previous MS/MS study, transitions from monoisotopic (3Wl and 12C)ions were preferred for diagnostic purposes (20). The isotopic transitions used here provide more signal abundance for polychloro compounds. Further isotopic transitions were sometimes monitored to confirm the presence of particular compounds. Enantiomer Separation of Toxaphene Components. The enantiomer resolution of TOX8 and TOX9 was studied on chiral columns from earlier studies (IO,18,191. A PS086/PMCD column showed no enantiomer resolution for TOX9, although this chiral column resolved several chlordane components enantiomerically (IO). Chiral columns with BSCD as the chiral selector, however, provided enantiomer resolution of TOX9 and some other components, and a OV1701-BSCD column was finally selected for most of the work reported. In particular, this column resolved components TC1, TC3, TC6, TC9, and TOX9 enantiomerically and partially TC2 ( R values in Table l), but it did not enantiomerically resolve TC5, TC7, and TOX8, so the chirality of TC5 and TC7 is actually unknown. The column showed coelution of some components (TC6, TC7, and TOX9), but this caused no problems because these components were detected selectively by monitoring different ion transitions (see Table 1). Partial enantiomer resolution of TOX8 was achieved on an OV1701-BSCD/PECD column. However, this column was less suitable for the analysis of environmental samples because of coelution of TOX8 and other components. In Figure 4, panels a-d, we show SRM chromatograms 374+ 278+ and 408+ 312+ for the TOX8 and TOX9 seal isolates. Whereas both components elute as single peaks from the achiral SE54 column (Figure 4, panels a and c), they are separated into pairs of enantiomers using the chiral OV1701-BSCD/PECD column (Figure 4, panels b and d). The enantiomeric ratios were determined as 1.12 f 0.04 and 1.08 f 0.04 for TOX8 and TOX9, respectively. Toxaphene Components in Environmental Samples. All aquatic species showed the presence of the heptachlorobornanes TC1 and TC2; the octachlorobornanes TC5, TC6, TC7, and TOX8; and TOX9 (see Figure 3, panels b, d, and f for data on penguin) at combined levels estimated to be in the hundreds of parts per billion (relative proportions indicated in Table I). Component TC8 is still present in fish to some degree but it is absent in the warm-blooded species such as seal and penguin (see Figure 3d). If identification of this component as toxicant A is correct, it suggests that compounds with geminaldichloro substitution and an unchlorinated carbon on the six-membered carbon ring are more rapidly metabolized. Other components of the technical mixture such as the tentatively identified polychlorobornenes (TC3 and TC9) and polychloroisocamphanes (TC4 and TClO), and several
-
n--x5.00
321
I
lcx5.00
~
Figure 2. E1 mass spectra of octachloro compounds identifled in technical toxaphene: (a) componentTC8 (presumably toxicant A an octachlorobornane, M'+ = 410), (b) component TC9 (an octachlorobornene, M'+ = 408), and ( 6 ) component TClO (an octachloroisocamphane, M + = 4101. Note the vertlcal expansion as indicated.
on the SE54 HRGC column. The technical mixture (Figure 3, panels a, c, and e) reveals complex patterns, but only a small number of congeners (three major hepta-, four major octa-, and the major nonachlorobornane, TOX9) are detected in the penguin extract (Figure 3, panels b, d, and f). The chromatograms of the other aquatic species were similar to those of the penguin. SRM versus SIM Detection of Toxaphene Components. In E1 and ECNI SIM analyses of polychlorobornanes, other components may interfere. For instance in ECNI, octa- and nonachlorobornanes are interfered with by octa- and nonachlordanes if ions at mlz 375 and 409 (M - C1 + 4)- are used. Interference is reduced by monitoring isotopic signals at mlz 379 and 413, respectively, as used in Figure 3. SRM can be expected to be more selective than SIM toward the detection of individual isomers since not only particular ions must be present but also a definite parent/ daughter ion relationship must exist. In general, SRM yielded much simpler chromatograms documenting an increased isomer selectivity in this detection mode. Fragmentations via elimination of CH2C1and CHC12 (loss
-
-
Environ. Sci. Technoi., Vol. 28, No. 1, 1994 123
to
Flgure 3. ECNI mass chromatograms showing elution of (a and b) hepta- (m/z 343), (c and d) octa- (m/z 379), and (e and f) nonachlorobornanes (mIz413) In technical toxaphene (left-side panels)and Antarctic penguin (right-side panels), using the achiral SE54 HRGC column. Note the reduced number of toxaphene components in the aquatic sample and, among others, the absence of component TC8 (presumably toxicant A) and the polychlorolsocamphanes TClO and TC4. Note the presence of late eluting peaks from hexa- and heptachloroblphenyls [M 191- In panels b and d, respectively. Retention tlme Is in minutes. Abbreviations are as shown In the text.
-
Flgure 4. E1 SRM chromatograms of mixed injections of Isolated TOX8 and TOXQ on the achlral SE54 (left-slde panels) and the chlral 10-m OV1701-BSCD/PECD HRGC column (right-side panels). (a and b) SRM chromatograms 376+ 280' showing elution of TOX8 (note the presence of signals for TOXQ)and (c and d) SRM chromatograms 410+ 314+ showing elution of TOXQ.Note the separation of the two Components Into enantlomers on the chlral column.
-
hepta-, octa-, and nonachlorobornanes are significantly reduced in the aquatic species analyzed. 124 Envlron. Scl. Technol.. Vol. 28, No. 1, 1994
-+
In Figure 5 we show SRM chromatograms (410+- 3149 of technical toxaphene, Baltic herring, Arctic seal, and
~
~~~~
-
~~~
~
Flgure 5. SRM chromatograms410+ 314+ of nonachlorobornanes in technical toxaphene and In the aquatic samples, using the achiral SE54 (left-side panels) and the chlral OV1701-BSCD HRGC column (right-side panels). (a and b) Technical toxaphene, (c and d) Baltic herring, (e and f) Arctic seal, and (g and h) Antarctic penguin. Note the separation of TOX9 into enantiomers on the chlral column. Earlier elutlng peaks are due to octachlorobornenes(technical toxaphene) and other, not necessarliy toxaphene, components (aquatic samples).
Antarctic penguin analyzed on the achiral SE54 and the chiral OV1701-BSCD column. Whereas the ECNI chromatogram of the technical mixture in Figure 3e shows at least five major peaks in the elution range of the nonachlorobornanes (17-21 min), the SRM chromatogram in Figure 5a now shows a major peak for TOX9. The chromatograms from the achiral column (Figure 5, panels c, e, and g) further show that TOX9 is a major component in all environmental samples. The chromatograms from the chiral column (Figure 5, panels b, d, f, and h) show enantiomer resolution of TOX9 with both enantiomers present in all the samples. The enantiomeric ratio determined for the technical product (1.13 f 0.02) is slightly different from the 1:l ratio for a racemic mixture. The enantiomeric ratios determined in the biological samples were 0.98 f 0.08 (Baltic herring), 1.19 f 0.03 (Arctic seal), and 1.34 f 0.02 (Antarctic penguin), indicating some small preference for the earlier eluting enantiomer (TOX9-1) in the warm-blooded species and a similar enantiomer composition of TOX9 in the Arctic seal as in the isolate of harbor seal (see above). For the same samples, we show in Figure 6 SRM 280+) selective for some occhromatograms (376+ tachlorobornanes. In panels a, c, e, and g of Figure 6 the achiral SE54 column is used. All aquatic samples showed the presence of components TC5, TC6, and TOX8. TOX8 is abundant in all aquatic species with the highest level observed in the Arctic seal (see Figure 6e). TOX8 is only a minor component in technical toxaphene (see Figure 6a). In panels b, d, f, and h of Figure 6, we show SRM chromatograms of the same samples, analyzed on the chiral
-
OV1701-BSCDHRGC column. The chromatograms show enantiomer resolution of component TC6, whereas TOX8 and TC5 are not enantiomerically resolved and partially coelute. The enantiomeric ratio of TC6 in the technical product (1.04 f0.03) suggests the presence of a racemate. In fish and seal, the levels of TC6 were too low to determine precise ER values; however, the ER value in penguin was 0.91 f 0.02, indicating a preference for the later eluting enantiomer (TC6-2). All samples were analyzed for the presence of some hepta- and octachlorobornenes, using the SE54 and the OV1701-BSCD columns. In Figure 7, we show SRM chromatograms 376+ 246+ and 410+ 280+ selective for TC3 and TC9, respectively. The two components were detected in the technical mixture (see Figure 7, panels a and c) and in smaller amounts in Baltic salmon (see Figure 7, panels e and g) but they were absent from seal and penguin. Apparently, the two bornenes show little accumulation. The faster metabolization of these compounds compared to that of some polychlorobornanes may be due to the presence of a C=C double bond. TC3 and TC9 were enantiomerically resolved on OV1701-BSCD. The chromatograms indicate racemic mixtures for TC3 (ER = 1.00 f 0.03) and TC9 (1.00 f 0.03) in the technical mixture (see Figure 7, panels b and d) but in Baltic salmon the enantiomers TC3-2 (ER = 0.3) and TC9-1 (ER = 1.4) are more abundant (see Figure 7, panels f and h). The enantiomer resolution is larger ( R values of 4.3 and 4.2 for TC3 and TC9, respectively) than that observed for the polychlorobornanes (Rvalues ranging from 0 to 1.3). Apparently, the presence of a C-C double
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Envlron. Sci. Technol.. Vol. 28, No. 1, 1994
125
70
c YJ
6oj
50-1
I(;, k,k, i 16100 .*,
l8lOO , L . ;TI& ,
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25 00
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TC5 TC6 TOX8
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Figure 6. SRM chromatograms 376+ 280+ of octachlorobornanesin technlcal toxaphene and in the aquatic samples, using the achlrai SE54 (left-side panels) and the chirai OV1701-BSCD HRGC column (right-side panels). (a and b) Technical toxaphene, (c and d) Baltic herrlng, (e and f) Arctic seal, and (g and h) Antarctic penguin. Note the separation of component TC6 into enantiomers on the chlral column; component TC5 and TOX8 not enantlomericaiiy resolved and partially coelutlng. -+
bond in the molecule aids in the chiral separation of the enantiomers. It is noteworthy that TC5, TC6, TOX8, and TOX9 do not respond to the ion transitions 376+ 246+ and 410+ 280+, because these compounds do not form RDA fragment ions via loss of C2HC13 from (M - HCl)*+parent ions. These components, however, were detected in SIM and SRM chromatograms with other transitions from the same parent ions (see Figures 5 and 6). Finally, in Figure 8, panels a-d, we show SRM chromatograms (342+ 2469 of technical toxaphene and Antarctic penguin analyzed on the achiral SE54 and the chiral OV1701-BSCDcolumn. This transition is selective for TC1, and the compound was detected in the technical mixture and in all the aquatic samples, with the highest levels in seal and penguin. Its highly selective detection by SRM is illustrated by comparing the chromatogram of the technical mixture in Figure 8a with the more complex ECNI chromatogram in Figure 3a. As illustrated in Figure 8, panels b and d, TC1 is enantiomerically resolved on the chiral column. The chromatograms indicate its presence as a racemic mixture (ER = 1.02 f 0.02) in the technical product (see Figure 8b), whereas in penguin a preference for the later eluting enantiomer TC1-2 is observed (ER = 0.74 f 0.03; see Figure 8d).
-
-+
-
Conclusions The enantiomer resolution of several polychlorobornanes and polychlorobornenes was accomplished using chiral HRGC, and the technique was applied to the analysis 126
Envlron. Scl. Technol., Vol. 28, No. 1, 1994
of a technical toxaphene mixture and environmental samples. Comparative analyses using both achiral and chiral HRGC yielded information not only on isomeric but also on enantiomeric composition. Analysis of technical toxaphene revealed all components, which were enantiomerically resolved, to be present as racemic or nearly racemic mixtures. I t is not known whether this finding is typical of toxaphene, or whether technical products from other sources have a different enantiomeric composition. The fact that the enantiomeric composition of TOX9, a principal environmental toxaphene component, is very similar in all aquatic species (ER = 0.98-1.34), points to more or less racemic toxaphene as a primary contaminant. E1 SRM was shown to be highly selective for the detection of some toxaphene compounds. The transitions selected (elimination of CzHzC12 and C2HCl3) seem unique to some bornanes and bornenes and are commonly not found with other chlorinated environmental contaminants. The technique showed better isomer selectivity than other mass spectrometric detection techniques (E1SIM, ECNI). Since the mass spectrometric properties of enantiomers are identical, enantiomers will show truly identical signal abundance in all SRM chromatograms (if not interfered with by other compounds), whereas those of isomers may differ significantly. The MS/MS technique may thus be of particular importance to chiral HRGC and may offer a way to distinguish between enantiomers and isomers. Hepta-, octa-, and nonachlorobornanes were detected in herring, in salmon, in seal from the Baltic and the Arctic,
Flgure 7. SRM chromatograms of hepta- (component TC3) and octachlorobornenes (component TC9) in (a-d) technical toxaphene and (e-h) Baltic salmon, using the achiral SE54 (left-side panels) and the chirai OV1701-BSCD HRGC column (right-side panels). (a, b, e, and f) SRM chromatograms 376+ 246+ showing elution of component TC3, and (c, d, g, and h) SRM chromatograms 410+ 280+ showing elution of componentTC9, respectlvely.Note the exceptlonally good enantiomeric resolution of components TC3 and TC9 on the chiral column. Sensitivity in chromatograms e-h is 15-50 X increased.
-
-
-
Figure 8. SRM chromatograms 342+ 246+ showing the elution of component TC1 (a heptachiorobornane), using the achiral SE54 (left-slde panels) and the chlral OV1701-BSCD HRGC column (right-side panels), in (a and b) technical toxaphene and (c and d) Antarctic penguin. Noto the enantlomerlc resolution of this component on the chlrai column.
and in penguin from the Antarctic. The isomer profiles were very similar with the same polychlorobornane congeners present, even in species from very distant locations. However, the isomer profiles differed significantly from those of technical toxaphene, thus confirming extensive alteration of the original mixture. Among the most accumulating compounds were TOX8 and TOX9, and among the least retained were compounds tentatively identified as polychlorobornenes and polychloroisocam-
phanes. TOX9 showed a very similar enantiomeric composition in all the species. Assuming that biological processes are often enantioselective, this indicates little biological degradation of this abundant compound. It cannot, however, rule out degradation by abiotic processes, such as by chemical and photochemical processes. Apart from the structures of TOX8 andTOX9, the exact structures of the other persistant aquatic components are still largely unknown. Some common features, however, Envhon. Sci. Technol., Vol. 28, No. 1, 1394
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are striking. It is noteworthy that most of these toxaphene components are recorded in SRM chromatograms which involvethe loss of C2H2C12, likely from RDA fragmentation of the six-membered carbon ring of the bornane structure. This indicates common substructural features and suggests that these components contain at least two Cl's at C-2/C-3 (or C-5/C-6), and possibly one each at all four locations, as is the case for TOX8 and TOX9. It would indicate that isomers without unchlorinated carbons at these positions are more resistant to metabolism. The use of MS/MS will aid in determining further structural details of compounds, such as the unknown TC compounds listed in this study. Acknowledgments
We thank C. Rappe and his staff at the Institute of Environmental Chemistry, University of Umefi, Umefi, Sweden, for the aquaticsamples. The receipt of the Arctic harp seal sample from M. Oehme, NILU, Lillestrerm, Norway, is gratefully acknowledged. We also thank M. Oehme and W. Vetter, Institute of Food Chemistry, University of Hohenheim, Stuttgart, Germany, for making available TOX8 and TOX9 isolated from harbor seal. Literature Cited
(1) Saleh,M. A. Rev. Environ. Contam. Toxicol. 1991,118,l-66. (2)Rapaport, R. A.; Eisenreich, S. J. Enuiron. Sci. Technol. 1988,22,931-941. (3) Musial, C. J.;Uthe, J. F. Znt. J. Enuiron. Anal. Chem. 1983, 14,117-124. (4) Muir, D. C. G.; Grift, N. P.; Ford, C. A.; Reiger, A.; Hendzel, M.; Lockhart, W.L. In Long Range Transport of Pesticides; Kurtz, D., Ed.; Lewis Publishers: Chelsea, MI, 1990; pp 329-346. (5)Parlar, H. Nachr. Chem. Tech. Lab. 1991,39,26-37. (6)Parlar, H. Znt. J. Environ. Anal. Chem. 1985,20,141-158. (7)Luckas, B.; Vetter, W.; Fischer, P.; Heidemann, G.; Plotz, J. Chemosphere 1990,21,13-19. (8) Stern, G.A.;Muir, D. C. G.;Ford,C.A.;Grift,N. P.;Dewailly, E.; Bidleman, T. F.; Walla, M. D. Environ. Sci. Technol. 1992,26,1838-1840. (9)Huhnerfuss, H.; Faller, J.; Konig, W. A.; Ludwig, P. Environ. Sci. Technol. 1992,26,2127-2133. (10)Buser, H.R.; Miiller, M. D.; Rappe, C. Environ. Sci. Technol. 1992,26,1533-1540.
128 Envlron. Scl. Technol., Vol. 28, No. 1, 1994
(11) Bidleman, T. F.; Patton, G. W.; Walla, M. D.; Hargrave, B. T.; Vass, W. P.; Erickson, P.;Fowler, B.; Scott, V.; Gregor, D. J. Arctic 1989,42,307-313. (12)Ribick, M. A.; Dubay, G. R.; Petty, J. D.; Stalling, D. L.; Schmitt, C. J. Environ. Sci. Technol. 1982,16,310-318. (13) Jansson, B.; Wideqvist, U. Int. J. Environ. Anal. Chem.
1983,13.309-321. (14) Vaz, 'R.; 'Blomkvist, G. Chemosphere 1986,14,223-231. (15) Swackhammer, D. L.; Charles, M. J.; Hites, R. A. Anal. Chem. 1987,59,913-917. (16)Van der Falk, F.; Wester, P. Chemosphere 1991,22,57-66. (17)Vetter, W.; Luckas, B.; Oehme, M. Chemosphere 1992,25, 1643-1652. (18) Buser, H. R.; Miiller, M. D. Anal. Chem. 1992,64,31683175. (19) Buser, H. R.; Miiller, M. D. Environ. Sci. Technol. 1993,27, 1211-1220. (20)Buser, H. R.; Oehme, M.; Vetter, W.; Luckas, B. Fresenius 2.Anal. Chem., in press. (21)Bartholom6, E.;Biekert, E.;Hellmann, H.; Ley, H.; Weigert, W. M.; Weise, E. UllmannsEncyklopiidie der Technischen Chemie,4th ed.; Verlag Chemie: Weinheim, Germany, 1981; Vol. 20,p 219. (22) Matsumura, F.; Howard, R. W.; Nelson, J. 0. Chemosphere 1975,3,271. (23)Turner, W. V.; Khalifa, S.; Casida, J. E. J.Agric. Food Chem. 1975,23,991. (24)Palmer, K. J.; Wong,R.Y.;Lundin,R. E.;Khalifa,S.;Casida, J. E. J. Am. Chem. SOC.1975,97,408. (25) Chandurkar, P. S.;Mataumura, F.; Ikeda, T. Chemosphere 1978,6,123-130. (26) Jansson, B.; Vaz, R.; Blomkvist, G.; Jenson, S.; Olsson, M. Chemosphere 1979,8,181-190. (27) Holmstead, R. L.; Khalifa, S.; Casida, J. E. J.Agric. Food Chem. 1974,22,939-944. (28) Gooch, J. W.; Mataumura, F. J.Agric. Food Chem. 1986, 33,844-848. (29) Saleh, M. A. J. Agric. Food Chem. 1983,31,748-751. (30) Seiber, J. N.; Landrum, P. F.; Madden, S. C.; Nugent, K. D.; Winterlin, W. L. J. Chromatogr. 1976, 114,361-368. Received for review April 13, 1993.Revised manuscript received August 12, 1993.Accepted October 5, 1993.' @Abstractpublished in Advance ACS Abstracts, November 15,
1993.
