Anal. Chem. 1994,66, 2155-2162
Identification of the (+)- and (-)-Enantiomers of Chiral Chlordane Compounds Using Chiral High-Performance Liquid Chromatography/Chiroptical Detection and Chiral High-Resolution Gas Chromatography/Mass Spectrometry Markus D. Muller’ and Hans-Rudolf Buser Swiss Federal Research Station. CH-8820 Wadenswil, Switzerland Synthetic racemic (f)-cis-chlordane, (f)-tmns-chlordane, (f)-chlordene, (&)-heptachlor, (f )-heptachlorepoxide, and (f )-oxychlordanewere analyzed by c h i d high-performance liquid chromatography (HPLC) using refractive index (RI) and chiroptical detection. chiroptical detection allowed the assignment of the (+)- and (-)-enantiomers of all compounds althoughsome were almost unresolved and showed no apparent enantiomer resolution by other detection means (RI). In this way, a single chiral HPLC column system with permethylated &cyclodextrin (PMCD) allowed the isolation of up to 100 pg of individual enantiomers at a time, or of nonracemic fractions enrichedwith one or the other enantiomer. Theseisolates were then used to identify the (+)-and (-)-enantiomers using chiral high-resolution gas chromatography (HRCC). Since the absolute configurations of the (+)- and (-)-enantiomers of these chiral chlordane compounds are known, it was possible to assign the exact structures to the enantiomers resolved and, retrospectively, to those detected at residue levels in aquatic vertebrate species and in human adipose tissue. The method was also applied to the isolation and identification of the a-hexachlorocyclohexaneenantiomers, and it should he valuable in other situations where chiral environmental contaminants are involved. Chlordane, an important chlorinated pesticide, consists of a complex mixture of various chemically similar compounds, predominantly hexa- to decachlorinated congeners and isomers;l*2it is now considered a possible human carcinogen.3 Several components of technical chlordane and some metabolites are persistent and ubiquitous environmental contaminants! A significantly changed isomer composition is generally observed in environmental and biological samples, with some minor components of technical chlordane showing much higher ab~ndance.~-~ These changes can be caused by abiotic processes in the environment, or by selective bioaccumulationor metabolism. Severalcompounds including the (1) Savage, E. P. Reu. Enuiron. Contam. Toxicol. 1989, 110, 117-128.
(2) Dearth. M.A.; Hitta, R. A. Enuiron. Sci. Technol. 1991,25, 245-254. (3) WHO/IARC. Occupational Exposures in Imecticide Application. of some PeJticides; IARC Monographs on the Evaluation of Carcinogenic Risks to Humans 53; World Healtb Organization, International Agency for Research on Cancer; Lyon, France, 1991; pp 115-177. (4) N0meir.A.A.; Hajja1.N. P. Rea Enuiron. Contom. Toxicol. 1987,ZOO, 1-22. ( 5 ) Norstrom. R. J.; Simon, M.; Muir, D. C. G.; Schweinsburg, R. E. Enuiron. ’ SCi. TCChd. 1988,22, 1063-1071. (6) Muir, D. C. G.; Norstrom, R. J.; Simon, M.Enuiron. Sei. Technol. 1988.22, 1071-1 079. (7) Kawano, M.;Inoue, T.;Hidaka, H.; Tatsuhwa,R. Chemosphere 1984,13, 95-100.
QOO3-27QQ194/Q366-2 155$O4.5O/Q 63 1994 American Chemical Society
main chlordane components cis- and trans-chlordane, heptachlor and chlordene, and the two major metabolites cisheptachlorepoxide (HEP) and oxychlordane (OXY)are chiral and thus exist as two optical isomers or enantiomers (for structures, see Chart 1). As previously pointed out, 8-10 biotic processes (uptake, metabolism, excretion) may be different for enantiomers whereas abiotic processes (chemical, photochemical, distribution, transport) will be the same. Previously, we reported on the application of chiral highresolution gas chromatography (HRGC) and mass spectrometry (MS)toward the enantioselective determinationof chiral chlordane compounds in a technical mixture, in ambient air, in a small number of aquatic vertebrate species, and in human adipose tissue.8-1° Interestingly, the samplesgenerallyshowed the presence of both enantiomers although with varying enantiomeric compositions. Whereas the technical mixture and ambient air showed enantiomericratios of approximately 1:1, these ratios were significantly changed in some biological samples with sometimes one or the other enantiomer being more abundant. At the time, an assignment of the absolute configuration to these enantiomers was not possible for lack of suitablereference compounds. However, for a more detailed understanding of the mechanisms involved in the transformation of these compounds, it is important to have the knowledge of the exact configuration of the enantiomers detected in environmental and biological samples. In this study we report on the application of chiral highperformance liquid chromatography (HPLC) in combination with chiroptical detection for the separation, isolation, and identification of small quantities of individual enantiomers from the synthetic racemic (f)compounds, or of fractions enriched with one or the other enantiomer. Through the use of chiroptical detection, the (+)- and the (-)-enantiomers of cis- and trans-chlordane, heptachlor, chlordene, HEP, and OXY, and of a-HCH were identified even for compounds where other detection means (refractive index, RI) showed no apparent enantiomer resolution. The isolated fractions were then used as qualitative standards to identify the (+)and the (-)-enantiomers in chiral HRGC analyses, using the same stationary phases and chiral selectors as in the previous investigations.8-1° In this way, the (+)- and (-)-enantiomers in these compounds were identified in a series of environmental (8) Buser, H. R.; MBllcr, M. D.; Rappc, C. Enuiron. Sci. Technol. 1992, 26, 1533-1540. (9) Buscr, H. R.; Miiller, M.D. Anal. Chem. 1992,64, 3168-3175. (10) Buser, H. R.; Miiller, M. D. Enuiron. Sci. Technol. 1993, 27, 1211-1220.
Ana!vtlcalChemk?by, Vol. 66,No. 13, July 1, 1994 2155
Chart 1. Absolute Conflguratlon d the (+)-ErrcmntkmcHI
of tho Chlofdam Compounds 1nv.rtlgatt.d. CI
CI
‘I’
(+)-chlordene
cI’
CI’
(+)-heptachlor
(+)-cis-chlordane
‘I’
(+)-trans-chlordane
CI
CI
f
CI
Ci
(+)-HEP
(+)-OXY
aAccordingto Mlyazakl et al.11q12 H-substituents not indicated. Chart 2. Structure8 of tha Two Enntknnn d a-HCH, Structure ol the A&al y-HCH.
CI
and the
CI
&;.
CI
.&.
: CI0”
I
i
!
I CI” I
a-HCH
Cl
a-HCH enantlomer II
enantlomer I
CI
CI
w ,’
pHCH (achlral)
Anassignment of the absolutestructvestothe (+)-and(-mntkmers of a-HCH Is currently not possible.
and biological samples retrospectively. Since the absolute configurations of these enantiomers are known from the work by Miyazaki et al. (refs 11 and 12; for the absolute stereochemistry of the compounds investigated, see Chart l), they can now be assigned to the enantiomers of these important environmental contaminantsin the various environmental and biological samples. Also included in the study was a-hexachlcrocyclohexane (a-HCH), another important environmental contaminant;this compound is chiral, as opposed to its achiral y-isomer (for structures, see Chart 2). EXPERIMENTAL SECTION Materials and Reference Compounds. Standard solutions of (f)-cis-chlordane, (*)-trans-chlordane, (&)-HEPand (&)OXY at concentrations of 10 ng/pL were obtained from Ehrenstorfer, Augsburg, FRG. A few milligrams of (*)-cischlordane, (f)-trans-chlordane, and cis- and trans-nonachlor were obtained from Chem Service, West Chester, PA, and (11) Miyazaki, A.; Hotta, T.; Marumo, S.; Sakai, M. J. Agdc. Food Chem. 1978, 26,915-911. (12) Miyazaki, A.; Sakai, M.; Marumo, S. J. Agric. Food Chem. 1980,28,1310-
1311.
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AM!~tkdChemIs&~, VOI. 66, NO. 13, Ju& 1, 1994
(*)-heptachlor from the Cantonal Laboratory, Zurich, Switzerland. All synthetic chiral compounds were racemic; they were isomerically pure (>98%). Small amounts of (*)HEP were prepared from (*)-heptachlor by reaction with Cr03-AcOH-H20l3 similarily, (&)-OXYwas prepared from trans-nonachlor via dehydrohalogenation to 2-chlorohep(f)-a-HCH and y H C H tachlor“ followed by epo~idati0n.l~ were from Riedel-de Haen (Seelze, Germany). Nonracemic HEP and OXY were prepared from enantiomerically enriched heptachlor and trans-chlordane by reaction with CrOg,lS respectively. Additionally, enantiomerically pure (+)- and (-)-limonene and enantiomerically enriched (+)- and (-)camphene were from Fluka (Buchs, Switzerland) and were used as optically active reference compounds for chiral HPLC. Chiral-HPLC and Fractionation of Racemic cbirlll Chlordane Compounds. The HPLC system consisted of a SpectraPhysics (San Jose, CA) Model 8800 pump delivering 0.7mL/ min of 80%methano1/20% 0.1%aqueous triethylamine acetate buffer (pH 4) with prior vacuum degassing. Samples (5-20 pL of 0.1-0.5% solutions in methanol) were injected via a Rheodyne (Cotati, CA) Model 7125 injector and a 20-pL loop, the amounts thus handled ranging from 5 to 100pg. The chiral HPLC column was a 200 X 4 mm column with immobilized PMCD on silica (7-pm particle size; MachereyNagel, Daren, FRG) and eluted cis- and trans-chlordane after 7-8 min. An ERMA (Tokyo, Japan) Model 7125A RI detector was used (sensitivity, 8 X IC5RI units f.s.) toestablish proper chromatographicconditions. Fraction collection was carried out while RI signals were observed and proper time lags (40 s) were taken into consideration to compensate for the delay between detection of the signal and elution of the compound at the outlet. From apparently unresolved peaks (peak width 40 s), cuts were taken approximately 10 s wide from the very front to the first inflexion point and from the second inflexion point to the very back of a peak. Theoretically (assuming Gaussian peak shape), the peak areas thus excised correspond to -12% each of the total peak area. For fully resolved components collection was almost quantitative.
-
(13) Singh, J. Bull. Enulron. Contam. Toxfcol. 1969, 4, 77-79. (14) Cochranc, W. P.; Forbes,M.; Chau, A. S. Y.J. Assoc. Off: AMI. Chem. 1970, 53,169-774.
(15) Schwcmmcr, B.; Cochranc, W. P.; Polen, P: B. Science 1970, 169, 1087.
Table 1. HRGC Cohmns uud column, polysiloxane, chirality stationary phase 1, chiral 2, chiral 3, chiral 4, achiral
PS086 BO86 OV1701 SE54
chiral selector, (contenta)
column material, dimensions
column tempb (rate)
PMCD (10%) BSCD (23%) PECD (30%) none
glass, 20-m, 0.30." i.d. glass, 16-m, 0.30-mm i.d. fused silica, 12-m, 0.25-mm i.d. fused silica, 25-m, 0.32-mm i.d.
100-120-250 OC (2 OC/min) 100-140-250 OC (3 OC/min) 100-140-250 OC (3 OC/min) 100-14G250 OC (3 OC/min)
a Concentration relative to polysiloxane stationary phase. 6 Injection, intermediate, and final hold temperatures; program rate between intermediate and final hold temperature listed; program rate between injection and intermediate temperature, 20 OC/min.
The polarimetric detector was an IBZ (Hannover, FRG) instrument operated with a 40-pL cell and a polychromatic halogen lamp with and without an interference filter of 589 nm (bandwidth 15 nm), respectively. Dextrorotation resulted in positive signals, levorotation in negative signals as recorded on a strip chart recorder (l-mV equivalenttoanoptical rotation of 1 mo at the instrument settings used). A lower flow rate of -0.5 mL/min was used for some samples to compensate for some loss in resolution from the larger dead volume of this system. Correct operation of the instrument was assured with (+)- and (-)-limonene, and with (+)- and (-)-camphene. Maximum sensitivity and linearity of detection were not determined since they are not relevant in the qualitative work reported. For GC/MS analysis, subsamples of the fractions were prepared by mixing 50 pL of effluent with -50 mg of Na2S04 and diluting with 500 pL of toluene. Aliquots of 0.5-1 pL were then injected for GC/MS. For total recovery the fractions were passed through a small column of silica topped with Na,S04 and then eluted with 5 mL of n-hexane or methylene chloride. Chiral HRGC/MS Analysis. A VG Tribrid doublefocusing magnetic sector hybrid mass spectrometer (VG Analytical, Manchester, England) was used for analyte detection and identification. The ion source was operated in the electron ionization mode (EI; 50 eV, 180 "C). Full-scan mass spectra (m/z 35-535, 1.16 s/scan, resolution M / A M = 500) were recorded for analyte identification in the HPLC fractions. Analyses were then repeated with selected-ion-monitoring (SIM) for optimal enantiomer/isomer separation using up to 10 ions simultaneously (0.50 s/scan; see Table I, ref IO). All samples were analyzed on an achiral SE54 and three chiral HRGC columns (see Table 1). The chiral columns were of the same type as in previous studies using PS086 or OV1701 as stationary phase and permethylated 8-cyclodextrin (PMCD), tert-butyldimethylsilyl-/3-CD(BSCD), or perethylated a-CD (PECD) as chiral selectors, and operated as previously described.8-lO Retention times were similar and elution orders identical to those previously observed. Enantiomeric excesses (ee) were defined as ee = 100(R - S ) / ( R S ) = 100 @I -pz)/@l p2) whereby R and S are the relative amounts of the dextro- and levorotatory enantiomers, andpl andp2 are the peak areas of the enantiomersdetermined in SIM analyses. Enantiomeric ratios (ER values) and enantiomer resolution (R) were as previously definedag The elution orders of enantiomers are specified as (+)/(-) or (-)/ (+), implying the (+)- or the (-)-enantiomers as earliereluting, respectively.
+
+
I
+4d 0
10 mln
Flguro 1. HPLC chromatograms(chiral immobilized PMCD column) of racemic chlordane compounds using (top, panels a-d) R I detection (sensitivity 2 X 10-5 R I unlts) and (bottom, panels e-h) chlroptlcai detection (sensitivity f40 mV) uslng polychromatic light of (a and e) ( f ) - ~ ~ n e , ( b a n d f ) ( f ~ ~ , ( c a n d g ) ( * ~ c M o r , and (d and h) (fkHEP. Note the presence of dextro- and levorotatory signalsfor heptachlorandHEPalthoughno visible enantiomer resolution was observed by RI detection. Note that (+)-trenschiordane, (+)ckhlordane, (+)-heptachlor, and (-)HEP are eariler-eiuted than the antipodes.
RESULTS AND DISCUSSION Separation and Isolation of Enantiomers of Chlordane Compounds using Chiral HPLC. The racemic compounds were analyzed by HPLC using a chiral PMCD column. When RI detection was used, varied enantiomer separation was observed with good resolution for some compounds but none for others; as expected, no enantiomer separation was observed using an achiral silica HPLC column. The compoundsshowing some enantiomer resolution were tram-chlordane (R > 1) and cis-chlordane (R 1);heptachlor, chlordene, HEP, OXY, and a-HCH remained practically unresolved (see Figure 1, panels a-d). Repeating these analyses using chiroptical detection indicated that (*)-cis- and (*)-tramchlordaneclearly showed positive signals (dextrorotation) for the earlier-eluted enantiomers and negative signals (levorotation) for the later-eluted enantiomers (see Figure 1, panels e and f). Interestingly, when chiroptical detection was used for (*)-heptachlor, (*)chlordene, (*)-HEP, (&)-OXY, and (&)- a-HCH, positive and negative signals were observed, although these compounds eluted practically unresolved, as indicated by the RI chromatograms; no chiroptical signals, however, were observed for achiral compounds such as cis- and trans-nonachlor, and y-HCH. For heptachlor and chlordene, a first positive deflection was followed by a negative deflection; for HEP, OXY, and a-HCH this was reversed (see Figure 1, panels g and h). The signals likely result from some enantiomer enrichment in the front and back of a peak and suggest some
-
AmtyticalChemistty, Vol. 66, No. 13, Ju& 1, 1994
2157
Table 2. EnanUomor Elutkn Orders ol Chlordane and Some Othw Compoundr Udng CMral HPLC and Chhal HRQC. HRGCC HPLCb PS086- E 0 8 6 OV1701 compounds PMCD PMCD BSCD PECD
-
Figure2. Theoreticalelutlonprofiles(Qausslan peak shapes)computed for closely eluted components (a! = 1.02) with addbe or subtractive response, as produced by RI and chiroptical detection, respectively, and for (a) eiutlon order (+)/(-) and (b) elution order (-)I(+).(Curves 1,2) Profiles for individual components with positive and negatfve responses,respectively,as producedfor enantiomers usingchiropticai detection; (Curve 3) profile for both components with subtractive response, as produced for racemates using chiroptical detection (summationof profiles 1and 2); (Curve 4) profile for both components with additiveresponse,as producedfor racematesusingR I (summation of absolute response of profiles 1 and 2). Note the reversed signal shape of profile 3 in panels a and b, from which the eiutlon order of the peaks can be deduced. Also note the reduced peak widths and amplitudes in profile 3 when compared to the others. Positive signals have upward, and negative slgnals, downward direction.
marginal enantiomer resolution with the (+)-enantiomers earlier-eluted for heptachlor and chlordene, and the (-)enantiomers earlier-eluted for HEP, OXY, and a-HCH. Apparently, the chiral HPLC system still shows some chiral recognition for these compounds although this was not detected when RI detection was used. For all the compounds studied, the sign (direction) of rotation of the chromatographicpeaks remained the same when polychromatic light or monochromatic light of 589 nm was used. However, the sensitivity (S/N ratio) was approximately 5 times lower when monochromatic light was used. Theoretical elution profiles computed for closely eluted components with additive or subtractive response such as produced by RI and chiroptical detection for enantiomers/ racemates,respectively, are shown in Figure 2. These profiles are very similar to those actually observed. Interestingly, the calculated peak widths for such marginally resolved enantiomers are narrower than for fully resolved enantiomerswhen subtractive (chiroptical) detection is used, due to signal attenuation/extinction in the center region. Comparable chromatograms were recently published in a study on the chiral separation of other c ~ m p o u n d s . ~ ~ J ~ Isolates collected from the very front and the very back of practically unresolved peaks, and analyzed by chiral HRGC, clearly showed the presence of nonracemic mixtures, enriched with one or the other enantiomer (see below). Enantiomer enrichment was sufficient to allow unambiguous assignment of the enantiomers and to establish the elution order of the (+)-and (-)-enantiomers in subsequent chiral HRGC analyses (see later). Reinjection of aliquots of some isolates on the chiral HPLC system confirmed an excess of a proper enantiomer, such as the (+)-enantiomer of heptachlor in the front-cut HPLC fraction (chromatograms not shown). In this way, isolates with microgram amounts of all chiral chlordane compounds and of a!-HCH enriched with one or (16) Mannschreck, A.; Zinncr, H.; Rtstet, N. Chimiu 1989, 13, 165-166. (17) Zukowski, J.; Tang, Y.; Bcrthod. A.; Armstrong, D. W. AMI. Chim. Acra 1992, 258,83-92.
2150
Adytic.qlChemIsby, VOI. 66, No. 13, July 1, 1994
(+) tram-chlordane (-)-tramchlordane (+)-cis-chlordane (-)-cis-chlordane (+)-heptachlor (-)-heptachlor (+)-chlordene (-)-chlordene (+)-HEP (-)-HEP (+)-OXY (-)-OXY (+)-a-HCH (-)-a-HCH (+)-camphene (-)-camphene
1
1
1
2 1 2 1 2
2
2
2 1 2 n.s. ns. n.s. ns. ns. ns. n.s. ns. 1 2 2
1
1
1
2 2 1
2 1
2 1
1
2
ns. n.s. 2 1
n.s. n.s. n.s. ns.
ns.d ns. 1 2 2 1 ns. n.s. 1 2 n.s. n.s. n.a.c n.a. n.a. n.a.
1 = First-eluted and 2 = second-eluted enantiomer. Chiral silicaPMCD HPLC column using chiroptical detection. C h i d HRGC columns using MS detection; for column dimensions and operating conditions, see text and Table 1 . dn.s. = no enantiomer separation observed. e n.a. = not analyzed.
70
a
+ /,
70
b
+ /.
Figure 3. E1 S I M chromatograms (m/z 373)showing elution of the (+)-and (-~nanuOmersof transand ckchlwdane isolated from the chlral HPLC column and analyzed using the chirai PS086PNICDHRGC cdumn: (a) (+b&ms-chbrdane (HPLC, fkst-eiuted), (b) (+)-ckchlordane (HPLC, flrst-eluted), (c) (-btren&lordane (HPLC, secondeluted), and (d) (-pchhlordane (HPLC, second-eluted). ER values were determined more precisely using the PSO8BBSCDcokrmn; see Figure 4. Note that enantiomer elutlon orders are Mentlcal to that by chiral HPLC.
the other enantiomer were obtained. The earlier-eluted enantiomers from the HPLC system (front-cuts) generally showed higher enantiomericenrichment than the later-eluted enantiomers. This is likely caused by unsymmetrical peak elution with tailing of the first- into the second-eluted enantiomer. In Table 2 we list the enantiomer elution orders observed by chiral HPLC of all compounds investigated. Chiral HRCC of the Enantiomer-Enriched Chlordane Isolates. Analysis of all HPLC fractions by achiral HRGC confirmed the presence of the proper analytes. The analyses were then repeated by using chiral HRGC. In Figure 3, panels a-d, we show SIM chromatograms (m/z 373) for the HPLC fractions of cis- and trans-chlordane, analyzed using the PS086-PMCD HRGC column. The chromatograms show these isolates enriched with one or the other enantiomer. For both compounds the (+)-enantiomer is earlier-eluted and the
+,
+,
, ,
,
,
24100
, , ,< , ,
26!00
,
,
, , ,
,
, , , , ,
24;OO
TI&
, , , , . T I h
26100
, , .
. , . ! lOlO0 . ..
8:'OO
. . , TIb5 r
10 0 , , ,
, , ,
lZL00
Qure 4. E1 SIM chromatograms(m/z 373) showing elution of the (+)-and (-)-enantiomers of trans- and ckhlordane isolatedfrom the chiral HPLC column and analyzed usingthe chirai PS08BBSCD HRGC column: (a) (+)-b.ans-chlordane (ER = 15.4), (b) (+)-ckchlordane (ER = 3.1),(c)(-)-transchkrdane(ER = O.O44),and(d)(-)-ckhkrdane
elution order [(+)/(-)I thus is the same as on thechiral HPLC system. The PS086-PMCD column shows poor enantiomer resolution, particularily for cis-chlordane, but good isomer resolution with no overlapping of enantiomers. Better enantiomer resolution and the same elution order were observed on PS086-BSCD (see Figure 4, panels a-d), but this column showed coelution of the (-)-enantiomer of trans-chlordane and the (+)-enantiomerof cis-chlordane. The chromatograms indicate enantiomeric excesses of =90 and 4 0 % for each isolate of trans- and cis-chlordane, respectively. The higher enantiomericenrichments for the isolates of tram-chlordane are expected from the higher enantiomer resolution for this compound by the chiral HPLC system.
~ ~ PI b 100
I
Figure 5. E1 SIM chromatograms (m/z 353, left-side panels; m/z 387, right-side panels)showing elution of the enantiomersof HEP (leftside panels)and OXY (right-aide panels) isolatedfrom the chhal HPLC column and analyzed using the chirai PSOBBBSCD HRGC column: (a) isolate enriched with (-)-HEP (HPLC, frontcut; ER = 0.321,(b) isolate enriched with (-)-OXY (HPLC; frontcut; ER = 0.29), (c) isolateenriched with (+)-HEP (HPLC, back-cut; ER = 1.31), and (d) isolate enriched with (+)-OXY (HPLC, back-cut; ER = 1.68). Note the reversed enantiomer elution order of the epoxides on the HRGC compared to the HPLC column.
In Figure 5 , panels a d , we show SIM chromatograms (m/z 353 and 387) for the HPLC fractions of HEP and OXY, analyzed using the PS086-BSCD column. For both compounds, the (+)-enantiomers are earlier-eluted and the
,
,
, ,-,
14100
,
,
'
, , ,
TIME
Figure 6. E1 SIM chromatograms (m/z 337, left-skle panels; m/z 353, rlght-side panels) showing elutbn and enantiomer separation of heptachlor(left-side~panels)and HEP (right+& panels), usingthe chirai OV1701-PECDHRGCcolumn: (a) isolateenriched with (+)-heptachlor (HPLC, frontcut; ER = 0.33),(b) its epoxldation product HEP showing a higher abundance of (+)-HEP (ER = 3.06, reciprocalvalue O S ) , (c) isolate enriched with (-)-heptachlor (HPLC; back-cut; ER = 1.23), and (d) and its epoxidation product HEP showing a hlgher abundance of (-)kEP(ER = 0.74, redpocaivaiue1.35). Notethereversedetmtbmer elutfonorderof heptachlorontheHRQCcomparedtotheHPLCcoiumn.
enantiomer elution order [(+)/(-)I thus is reversed from that on the chiral HPLC system. The chromatograms indicate enantiomeric excesses of 50 and 54% for the front-cuts [(-)enantiomers] and of 12 and 34% for the back-cuts [(+)enantiomers]of HEP and OXY, respectively. The values are lower than for trans-chlordane, but still sufficient for an unambiguous enantiomer assignment. In Figure 6, panels a and c, we show SIM chromatograms (m/z 337) for the HPLC fractions of heptachlor, analyzed using the OV1701-PECD column. On this column (-)heptachlor is earlier-eluted and the enantiomer elution order [(-)/(+)I thus is again reversed from that on the chiral HPLC system. The enantiomeric excesses of 50 and 10% for the (+)- and (-)-enantiomers, respectively, were still sufficient for an unambigous enantiomer assignment. Chlordene and a-HCH were similarily fractionized. The enantiomeric excesses were 20 and 8% for (-)- and (+)-a-HCH (see Figure 7), respectively, and 90 and 42% for (+)- and (-)-chlordene (data not shown), respectively. The elution orders were as listed in Table 2. Conversion of Nonracemic Heptachlorand Irsas-Chlordane into Epoxides. Heptachlor is converted into HEP by epoxidation with Cr03." Small aliquots (a few micrograms) of the HPLC fractions of heptachlor enriched with one or the other enantiomer were reacted. As illustrated by the SIM chromatogramsin Figure 6,panels bandd (m/z 373;0V1701PECD), the first-eluted enantiomer of HEP was formed from (+)-heptachlor (see Figure 6b), and the later-eluted enantiomer of HEP from (-)-heptachlor (see Figure 6d), and the reversed elution order of these enantiomersis thus confirmed. As expected, the ER values for HEP were approximately reciprocal to those of heptachlor. The data confirm the conversion of (+)-heptachlor into (+)-HEP, and of (-)heptachlor into (-)-HEP in a stereoselective way with conservation of the absolute configuration, as indicated by the reaction schemes in Scheme 1 and from the assignments made by Miyazaki et al.I2
Scheme 1. Fmatlon of (+)- and (-)-HEP from (+)- and (-)-Heptachlor, Re8pectlveiy, vla Ster.owkcttr. and Stw.orp.clllc EpoxMatlon
CI CQ
CI
(+)-heptachlor
!
80 70 60 50 40
30
b
(+)-HEP
CI
CI
Ci
CI
(-)-heptachlor
I-
Flgwe 7. E1 SIM chromatograms (m/z 219) showing elutlon and enantiomer separation of a-HCH, using a 20-m PSOBBPMCD HRW column: (a) isolate enriched with (-)-a-HCH (HPLC, front-cut; ER = 0.67) and (b) isolate enriched with (+)-a-HCH (HPLC, back-cut; ER = 1.17). Note the reversed enantiomer elution order on the HROC compared to the HPLC column.
trans-Chlordane is partially converted into OXY by reaction with CrO,.lS Racemic trans-chlordane (-1 mg) and small aliquots (a few micrograms) of the HPLC isolates enriched with one or the other enantiomer were reacted. As illustrated by the SIM chromatograms in Figure 8 (m/z 385; PS086-BSCD), the reaction of (& )-trans-chlordane gave (&)OXY (ER = 1.00,see Figure 8a), the reaction of (+)-transchlordane the first-eluted enantiomer of OXY (see Figure 8b), and the reaction of (-)-tram-chlordane the second-eluted enantiomer of OXY (see Figure 8c), respectively. The data thus confirm the conversion of (+)-trans-chlordane into (+)OXY,and of (-)-trans-chlordane into (-)-OXY, and are in agreement with the reaction scheme outlined in Scheme 2 and the assignments made by Miyazaki et al.I2 This reaction may proceed via dehydrogenation followed by epoxidation of the resulting C=C double bond. ChromatographicBehavior of Chlordane Enantiomers. In Table 2 we list theenantiomer elution orders for thecompounds investigated and the different chiral HRGC and the chiral 2160 Ana&ticalChem&tty, Vol. 66, No. 13, Ju& 1, 1994
C;
(-)-HE P
HPLC system used. For cis- and trans-chlordane the elution order was (+)/(-) on PSO86-PMCD as on the chiral HPLC system using this chiral selector, suggesting a similar mechanism of interaction in the gas-liquid as in the liquid-liquid phase for these two compounds. However, for a-HCH this was not the case, and for the other chlordane compounds it could not be confirmed because they were not resolved on PSO86-PMCD. For heptachlor, chlordene, HEP, and OXY resolved by chiral HRGC using other chiral selectors (BSCD or PECD), the elution orderswere reversed from that by HPLC using PMCD. For cis-chlordane, trans-chlordane, and HEP which are resolved by more than one chiral HRGC system, the same elution orders were observed for the different chiral selectors (PMCD, BSCD, or PECD). As expected, the same elution orders were observed for HRGC columns using the same chiral selector but different stationary phases (OV1701or PS086). Furthermore, chiral columns with the same stationary phase but differing amounts of a particular chiral selector (10 and 20% of PMCD on PS086) also showed the same elution orders, although column polarity and elution temperatures changed significantly. Presence of Chlordane and a-HCH Enantiomers in Environmental and Biological Samples. In previous papers8-10we reported on the enantiomeric composition of chlordane compounds in aquaticvertebrate speciesfrom the Baltic (fish, gray seal), Arctic (harp seal), and Antarctic (penguin), and in human adipose tissue. cis-Chlordane and trans-chlordane were detected at significant levels in fish (herring, salmon). The chromatograms in Figure 6, ref 8, and the enantiomer assignments now made identify (+)-cis-chlordane as being more abundant in herring and (-)-cis-chlordane as more abundant in salmon. Similarily, the results now indicate (-)trans-chlordane as being more abundant in herring and (+)trans-chlordane as more abundant in salmon. Heptachlor, and likely chlordene, and other major chlordane components, was absent in these species. This is not unexpected since heptachlor is rapidly converted into HEP or other metabolites by most organisms.’*
Schema 2. FonrrclHon d (+)- and (-)-OXY from (+)- a d (-)-tn?mChbrdme vla Stw-hrr
Epoxidakn
C@
CI
(+)-trans-chlordane
CI
Ci
(-)-trans- chlordane
!
,,,,,
, ,,,,,
,
,,,.,
.,,,,-,,,,,
,,,,,
. . , , . ~ , , ,, , , , ,
, , , , , L , , , ,, , ,,
,,
, , , , ,
1 ,
,,,,.
Flgure 8. E1 SIM chromatograms (m/z 387) showlng elution of OXY prepared vla epoxldatkn of "chlordane, using the PS088-BSCD HRQC cdwnn. (a) racemic OXY from racemic fms-chbrdane, (b) (+)OXY from (+)-hn&lordene (HPLC isolate, see Figure 3a), and (c) (+OXY from (-)tramchlordane (HPLC Isolate; see Figure 3c).
HEP is the major metabolite of heptachlor and is present in most biological samples including human tissue. Generally, the presence of both enantiomers was observed, although in varying enantiomeric ratios? The chromatograms in Figure 7, ref 9, and the assignments now made identify (-)-HEP as being more abundant in all aquatic species, particularily in seal; however, (+)-HEP was more abundant in human adipose tissue. Incubation of racemic heptachlor with rat liver homogenate (S9fraction) in a stereoselective reaction of the mixed-function oxidase system (MFO) yielded predominantly the earliereluted enantiomer of HEP (see chromatograms in Figure 3, ref lo), now identified as (+)-HEP. For the unreacted (18) Schmitt, C. J.; Zajicck, J. L.; Ribick, M. A. Arch. Enuiron. Conram. Toxicol.
1985, 14, 225-260.
(+)-OXY
CI
+, ,-
,
CI
ci
(-)-OXY
(remaining) heptachlor the chromatograms indicate a lower abundance of heptachlor-1, now identified as (-)-heptachlor. The results imply that (-)-heptachlor is reacting faster but the epoxidation product of its antipode [(+)-HEPI is more abundant. Presently, we have no explanation for this controversial finding. The actual metabolism of heptachlor may be more complicated than anticipated and may involve additional pathways and metabolites, as observed in another study.19 OXY is a major metabolite of octa- and nonachlordanes, and we reported the presence of both enantiomers in all the biological samples at surprisingly similar ratios (ER values 1.3-1.6).9 The assignments now made indicate a higher abundance of (+)-OXY.Previously, the formation of OXY from cis- and trans-chlordane in pigs was reported.15 The product isolated from trans-chlordane also showed dextrorotation with an [ a ] D value of 2.7O and thus an enantiomeric excess of (+)-OXY, whereas cis-chlordane gave optically inactive OXY. From this [ a ] D value and the [a],, value for (+)-OXYlisted by Miyazaki et al. (23O, see ref 12), theoptical purity is estimated at [~r]ob/[~r],axX 100 = 11.7%, corresponding to an ER value of 1.27, a value close to those and observed in our samples. As shown in Chart 1, (+)-OXY (+)-trans-chlordane are topographically equivalent at C1,and the above results may indicate that the (+)-enantiomer of trans-chlordane is metabolized somewhat faster. Data from enantioselective analyses of a-HCH in various biological samples using chiral HRGC were previously In these studies different CD derivatives were used as the chiral selector, but the exact elution order of the enantiomers as (+)/(-) was determined so far only by Faller et al.20for a column with 2,6-di-O-pentyl-3-O-butyryl-&CD; in the other studies the same elution order was assumed but not proven. As shown by the data listed in Table 2 we now confirm the same elution order for the PS086-PMCD column (19) Tashiro, S.;Matsumura, F. Arch. Emiron. Conram. Toxicol. 1978, 7 , 113127. (20) Faller, J.; Hiihnerfuss, H.; KOnig, W. A.; Krebbcr, R.; Ludwig, P.Enuiron. Sei. Technol. 1991, 265, 676478. (21) Miiller, M. D.; Schlabach, M.; Oehme, M. Enuiron. Sci. Technol. 1992, 26, 566- 569. (22) M b n e r , S.;Spraker, T. R.; Becker, P. R.; Ballschmiter, K. Chemosphere 1992, 24, 1171-1180.
Ana3/ticelChemist?y, Vd. 66, No. 13, JuEy 1, 1994
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and, thus, the assumption made by Miiller et al.21 Furthermore, the seal data reported by Miiller et a1.21 and Massner et a1.22with ER values > 1 suggest the same elution order on the OV1701-PMCD and the acetylated/pentylated &CD columns used in the latter studies. However, on an OV1701 column with permethylated y C D , the elution order was reversed. CONCLUSIONS Chiral HPLC with RI and chiroptical detection was used to analyze racemic chlordane compounds and a-HCH. A single chiral HPLC column with PMCD as chiral selector and chiropticaldetection identifiedthe levo- and dextrorotatory enantiomers even for compounds with just marginal enantiomer resolution and no resolution apparent by other detection means (RI detection). Collection of individual peaks or of narrow cuts of such apparently unresolved peaks yielded the more or less pure enantiomers or enantiomerically enriched (nonracemic) fractions. These fractions were then used for the unambiguous assignment of the enantiomers in subsequent analyses using chiral HRGC. In this way, it was possible to assign the (+)- and (-)-enantiomers of these compounds in various environmental biological samples, arid by using the same chiral selectors and stationary phases as in previous studies, retrospectively in such samples previously analyzed. The identification of these enantiomers is important for a full understanding of the environmental and biological behavior of chiral compounds. The nonracemic reference mixtures and the enantiomer enrichments obtained were fully satisfying for enantiomer assignment; enantiomers of higher purity are not required for this purpose. Similarily, pure enantiomers are dispensable for any quantitative studies since detector response of enantiomersis truly identical and can be calibrated by using the racemates.8 However, pure enantiomers may be desirable in model reactions to study the environmental and biological behavior of these compounds. Up to 100 pg of material was handled by the chiral HPLC system at a time. Although just a fraction was actually recovered from marginally resolved enantiomers, the rest of the material can also be recovered and reused in the separation process. This is important in situations where only small amounts of material are available. The amounts isolated were sufficient not only for HRGC analyses but also for further chemical reactions such as the conversion of enantiomerically enriched heptachlor and trans-chlordane to nonracemic HEP and OXY,respectively. In this way, the enantiomer assignments for these compounds were confirmed independently. (23) Testa, 9. Crundlagen der Organkchen Stereochemir; Verlag Chemic: Wcinhcim. FRG,1983. (24) Schurig, V.; Nowotny, H. P. Angew. Chem. 1990,102,969-986. (25) Apnstrong, D. W.; Li, W. Y.;Pitha, J. Anal. Chem. 1990,62,214-217. (26) Lipkowitz, K. B.; Baker, 9. Awl Chem. 1990,62, 774-777.
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The results are in contradiction to a previous assignment of heptachlor and HEP enantiomers, deduced from the reaction of heptachlor to HEP by the mixed-function oxidase system.'O This illustrates that for a detailed understanding of the metabolism of a compound multiple reaction pathways will have to be considered. The absolute configuration of the enantiomers of the chlordane compounds is known from the work by Miyazaki et al.,I1.12 but an assignment of the absolute configuration to (+)-and (-)-a-HCH is currently not possible. The (+)-series of enantiomers of the chlordane compounds except chlordene all are topographically equivalent at C1 (em-C1 in backward position, see Chart 1); even (+)-chlordene has the same configuration as (+)-heptachlor, consideringthe C = C double bond in ring B (note that the absolute configurations at C1 are R for (+)-cis- and (+)-trans-chlordane, (+)-HEP and (+)-OXY, but S for (+)-heptachlor; no asymmetric center for chlordene at CI). This implies common regiosterical requirements to yield dextro- or levorotation in these enantiomers, as known for other compounds such as steroids and particularily ketones (for prediction of optical rotation using sequenceandoctant rules, see ref 23). Furthermore, theelution orders of these enantiomers also show some common features in that (+)-cis- and (+)-trans-chlordane, and (+)-HEP and (+)-OXY, all are earlier-eluted on the chiral HRGC columns and thus show less interaction with the chiral selectors than the antipodes. Heptachlor and chlordene, which both have C = C double bonds in ring B, show reversed elution behaviors with the (-)-enantiomers earlier-eluted. Thus there appear to exist also common topographical requirements that govern interaction with the chiral selector and determine the elution orders of enantiomers. Although there were attempts to rationalize enantioselectivity in chiral HRGC with respect to thermodynamic^^^ and stereoselective interactions using nonracemic mixtures,25 prediction of elution orders and the assignment of absolute configurations have so far received little attention.26 The combination of chiral HPLC with chiroptical detection for the separation, identification and isolation, and chiral HRGC for subsequent enantioselectiveanalysisusing selective detection (MS), is a simple, yet powerful, tool and should prove valuable in other situations where chiral compounds are involved. ACKNOWLEDGMENT We thank Ciba-Geigy Ltd., Basle, Switzerland, and the Cantonal Laboratory, Zurich, Switzerland, for the loan of the chiroptical and the RI detectors. Received November 19, 1993. Accepted March 23, 1994.O Abstract published in Advance ACS Abstracts. May 15,
1994.
