Environ. Sci. Technol. 2001, 35, 960-965
Production of Toxaphene Enantiomers by Enantioselective HPLC after Isolation of the Compounds from an Anaerobically Degraded Technical Mixture WALTER VETTER* AND DOREEN KIRCHBERG Department of Food Chemistry, Friedrich-Schiller-University Jena, Dornburger Strasse 25, D-07743 Jena, Germany
Enantiomers of 12 chlorobornanes were separated on a chiral stationary HPLC phase. The investigated compounds included relevant chlorobornanes in technical toxaphene (Toxicant A and an unknown heptachlorobornane), anaerobically mediated media such as sediment, soil, and sewage sludge (B6-923, B7-1001), as well as eight persistent compounds of technical toxaphene (CTTs) frequently detected in biological samples (B7-1000, B71453, B8-1412, B8-1413 or P-26, B8-1414 or P-40, B8-1945 or P-41, B8-2229 or P-44, and B9-1679 or P-50). Sufficient amounts of these 12 CTTs were not commercially available and had to be produced in our lab. Eight CTTs were obtained from sewage sludge that was spiked with technical toxaphene and kept under anaerobic conditions for four weeks. The samples were extracted with hexane followed by RP-HPLC fractionation. The resulting toxaphene pattern was significantly simpler than that of the technical mixture. CTTs that showed intense fragmentation in GC/ ECNI-MS were preferably metabolized. Moreover, only one of the diastereomers that make Toxicant A (B8-806/B8809 or P-42a/b) resisted degradation in sewage sludge. We found that the persistent component of Toxicant A is 2,2,5endo,6-exo,8,9,9,10-octachlorobornane (B8-809 or P-42b). B9-1679 (P-50), B7-1453, and B8-1412 were earlier isolated from biological samples, and B7-1000 was isolated from naturally contaminated sediments. The fractions obtained after these procedures were suitable for enantioselective HPLC separations. The first eluting enantiomer was usually obtained as an enantiopure standard whereas the second eluting enantiomer also contained the other enantiomer. Attempts to determine the optical rotation with the help of a chiral HPLC detector failed. Elution orders of the enantiomers were established on three GC chiral stationary phases. Only the enantiomers of B7-1453 and B81945 (P-41) eluted in the same order from all CSPs while the others showed different enantiomer elution orders or were not resolved on one of the chiral GC stationary phases. The knowledge and consideration of these results is important for the interpretation of enantiomer ratios found in biological samples and comparison of literature data.
Introduction Enantioselective studies of chiral organochlorines (chloropesticides and atropisomeric PCBs) attract currently grow960
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ing interest in environmental chemistry (1, 2). In the past, different estrogenic activity of o,p′-DDT enantiomers and different insecticidal activity of enantiomers of chlordanerelated compounds have been reported (3, 4). Furthermore, investigation and evaluation of the enantiomer ratios of chiral organochlorines are suitable tools for elucidating the biodegradation pathways of these contaminants (2, 5). Application of enantioselective gas chromatography (eGC), i.e., the use of chiral stationary phases (CSP) based on modified cyclodextrins, allows the determination of chiral compounds enantioselectively at trace levels in environmental samples. Suitable CSPs for the enantioseparation of most of the chiral organochlorines are available (1, 2, 5). However, an objective interpretation of enantioselective data requires enantioenriched (or enantiopure) standards since reversals of the GC elution orders have been reported for organochlorine enantiomers (6, 7). Nonracemic standards can be produced with the help of enantioselective HPLC (eHPLC) (8-11). In the case of compounds of technical toxaphene (CTTs) this has not been achieved before. Toxaphene has been extensively used in diverse agricultural applications. It contains no principal component but consists of several hundred chlorinated bicyclic compounds which are mainly chiral (12, 13). Synthesis of single CTTs is not easy to achieve, and only limited amounts of relevant (racemic) standards are commerically available due to the work of Parlar and co-workers (14, 15). In contact with biota and also reducing media, the bulk of the CTTs can be metabolized, but the mechanisms leading to this change in composition are not fully understood. However, a toxaphene residue pattern that is weathered after significant biodegradation enables one to identify and quantify the most important CTTs on a congener-specific basis in various matrixes (13, 16). There is some evidence that such degradation occurs enantioselectively and that enantioselective investigation of CTTs in samples may help to elucidate the degradation pathways and interrelations among structurally related compounds. To support such studies, we attempted production and isolation of relevant CTT standards from anaerobically mediated toxaphene followed by separation of many environmentally relevant CTT enantiomers with the help of eHPLC.
Materials and Methods Reversed-Phase High Performance Liquid Chromatography (RP-HPLC). The HPLC system consisted of an SIL-10AD autosampler, an LC10AC pump (both Shimadzu, Jena, Germany), and a 201-202 fraction controller (Abimed-Gilson, Langenfeld, Germany). The flow of the mobile phase acetonitrile-water (86:14, v/v) was set at 1.0 mL/min. A Supelcosil LC-18-DB 250 mm × 4.6 mm column (Supelco, Bellefonte, USA) with 5 µm mesh size was used. Enantioselective High Performance Liquid Chromatography (eHPLC). The eHPLC system consisted of an LC6a pump (Shimazu, Jena, Germany) and a LiChroCart 250-4 (ChiraDex, Katalogue no. 51333) HPLC column (Merck, Darmstadt, Germany). Different mobile phases composed of methanol with 0-35% water (v/v) were used at flow rates of 0.9 mL/min. For selected experiments, an SPD-10a (Shimazu, Jena, Germany) UV detector (set at 210 nm) and/ or an OR-1590 chiral detector (Jasco, Gera, Germany) was used but failed to detect the CTTs. The UV detector was kept * Corresponding author phone: (+)3641 949 657; fax: (+)3641 949 652; e-mail:
[email protected]. 10.1021/es000174g CCC: $20.00
2001 American Chemical Society Published on Web 02/02/2001
in the system to record HPLC retention times with a HewlettPackard 3365 integrator. GC/ECD Parameters. All eHPLC fractions were analyzed on a dual GC/ECD system that consisted of a Hewlett-Packard 5890 gas chromatograph equipped with an HP 7673 auto sampler. A t-piece at the end of the split/splitless injector (splitless time 1.5 min) divided the carrier gas (Helium, 5.0 quality) flow onto two achiral columns (CP-Sil 2 and CP-Sil 8/20% C18, Varian/Chrompack, Middelburg, The Netherlands) and two ECDs (17). This system was used to determine the fractions with the respective target compound. A GC/ECD system (Hewlett-Packard 5890 Series II gas chromatograph equipped with a 63Ni electron capture detector) was used for studying the elution orders on 35% impure 6-O-tert-butyldimethylsilyl-2,3-di-O-methyl)-β-cyclodextrin diluted in OV-1701 (impure β-TBDM, BGB Analytik, Adliswil, Switzerland). The present badge of impure β-TBDM contained approximately 65% side products that have not been investigated in detail, yet. Only the impure and not pure, commercially available β-TBDM separates CTTs enantioselectively (18). Column parameters were as follows: 20 m length, 0.25 mm internal diameter, and 0.15 µm film thickness (18, 19). Furthermore, chemically bonded permethyl-β-cyclodextrin (CB-β-PMCD, ChirasilDex, Varian/ Chrompack, Middelburg, The Netherlands) was used with a length of 25 m, an internal diameter of 0.25 mm, and a film thickness of 0.1 µm. Samples were introduced with a cold injection system (Gerstel, Mu ¨ lheim, Germany): after 0.1 min at 72 °C, the injector temperature was raised at 12 °C/s to 250 °C. The ECD temperature was set at 270 °C. Helium was used as carrier gas. For analysis on the impure β-TBDM column, the following GC oven program was developed. After 2 min at 120 °C, the temperature was raised at 15 °C/min to 150 °C (0 min), 2 °C/min to 180 °C (80 min), and 20 °C/min to 220 °C (10 min). In the case of the CB-β-PMCD phase, the oven program started at 60 °C (2 min), followed by heating rates at 25 °C/min to 135 °C (20 min), 0.5 °C/min to 162 °C, and 25 °C/min to 220 °C (10 min). GC/ECNI-MS Parameters. Fractions with the target compounds as determined with the dual GC/ECD system (see above) were analyzed with a Hewlett-Packard 5890 series II/5989B GC/MS system in the ECNI-MS-SIM mode (20). Two chiral stationary phases were used: (i) 25% randomly tert-butyldimethylsilylated β-cyclodextrin (β-BSCD) diluted in PS086 (BGB Analytik, Adliswil, Switzerland). [column parameters were as follows: 30 m length, 0.25 mm internal diameter, and 0.20 µm film thickness (df) (6)] and (ii) impure β-TBDM (see above) (18, 19). In SIM mode we measured m/z 273, m/z 275, m/z 307, m/z 309, m/z 343, m/z 345, m/z 377, m/z 379, m/z 411, and m/z 413 to cover the range from penta- to nonachlorobornanes throughout the entire GC run. Toxaphene Standards and Analytical Procedure. One kilogram of Melipax powder (containing ∼10% technical toxaphene) was found in a garden shed in Jena (Germany) in 1998. About 100 g of Melipax powder was extracted with n-hexane in an ultrasonic bath and filtered through sodium sulfate. After evaporation of the solvent, technical toxaphene was obtained. Aged, reductive sewage sludge (liquid, solids < 15%) was obtained from the Jena municipal sewage plant. Several fractions of the 250 mL of sewage sludge were spiked with 200 µg of Melipax, respectively, and were kept in anaerobic milieu. After 4 weeks the samples were extracted twice with 100 mL of n-hexane. The n-hexane extracts were filtered through sodium sulfate, concentrated to approximately 2 mL. The extracts were passed through 8 g of activated silica in a 1 cm i.d. glass column (pre-washed with n-hexane). After elution with 48 mL of n-hexane, the CTTs were targeted into one fraction with 50 mL of ethyl acetate/cyclohexane (1:1, v:v). Several CTT fractions were combined and carefully evaporated to dryness. After dilution in approximately 1 mL
of the mobile phase (acetonitrile-water, 86:14, v:v), an aliquot of the sample (200 µL) was injected into the reversed-phase HPLC system. Fractions of 0.5 min (30 min: E2 75/6 min: 2.5 75/11 min: 0.05 75/20 minb 65/14 min: 2.5 75/9 min: 0.1 75/ not establishedc 75/ not establishedc 100/9 min: 0.3 75/7 min: 4
9-30 min 14- >30 min 8-17; >30 min 3-9 min 3-17 min 9-30 min 7-20 min 3-17 min 6-18 min 2-16 min 2-15 min 3-11 min
a Enantiomer distribution in fractions that showed maxima in the eHPLC runs was determined on β-BSCD (eGC), except for B8-1412 and B8-2229 which were enantioseparated on impure β-TBDM (eGC). E1 (i.e., ER > 20) or E2 (i.e., ER < 0.05) means pure enantiomer-1 or -2. Numbers (instead of E1 and E2) are the ratio of the first eluting peak over the second; in these cases only enantioenriched (and not enantiopure) standards were obtained. b Not enantioresolved by eGC; however, GC/ECD analysis provided two maxima which confirms the successful eHPLC enantiomer separation. c Not established due to impurities in chromatograms.
FIGURE 4. GC/ECNI-MS chromatograms of fractions (see Table 1) that contained enantiomer-1 (upper panels) and enantiomer-2 (lower panels) as obtained from eHPLC (ChiraDex). B9-1679, B6-923, B8-1413, and B7-1000 were analyzed on β-BSCD, and B8-1412 was analyzed on β-TBDM. pure B8-806 and a mixture of B8-806/B8-809 prove that the environmentally relevant compound in Toxicant A is B8809. The higher stability of B8-809 was also confirmed in blubber of marine mammals (data not shown) and is in agreement with results from molecular modeling (27). We also tried to separate the enantiomers of B7-515, but the isolate originally designated as B7-515 turned out to be another, unknown heptachlorobornane of the technical mixture (see Table 1). This CTT is most likely identical with one of the four heptachlorobornanes earlier detected in sediments from a coastal wedland (20). Our attempts to determine the optical rotation of pure enantiomers with the help of a chiral detector failed mainly since the enantiomers were not found in discrete sharp peaks but were distributed over a wide time range (Table 1). Therefore, the ChiraDex column was not suitable for solving this problem. Establishing the optical rotation of the enantiomers has to be performed by injection of pure enantiomers into a system consisting of a nonchiral stationary phase and a chiral detector (11). Nonchiral stationary HPLC phases give sharper peaks that may provide the necessary sensitivity for chiral detectors. However, initial experiments with B9-1679 using an RP-C18-HPLC column were not successful. 964
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The amounts of pure enantiomers produced so far ranged from >100 ng to >50 µg. Our results clearly confirm that eHPLC enables the production of pure enantiomers in order to (i) allow determination of the optical rotation of CTT enantiomers and (ii) gain amounts sufficient for toxicological investigations. A semipreparative HPLC column would simplify this by injection and enantioseparation of larger amounts of racemic CTTs. GC Elution Order of CTT Enantiomers on Different Chiral Stationary Phases. Enantiomer ratios are usually derived from the quotient of the (+) to the (-) enantiomer or, if the optical rotation is unknown, the quotient of the first to the second eluting enantiomer on a GC phase (1). In the case of the CTTs, the latter has to be done. From other chiral organochlorines it is known that enantiomers may elute in reversed order from two CSPs (1, 2, 5). To investigate this phenomenon, three GC-CSPs were tested with enantioenriched CTT solutions obtained from ChiraDex fractions (Table 2). All components except B6-923 were enantioseparated on at least two columns. However, many differences in the elution orders were observed. Only B7-1453 and B8-1945 eluted in the same order from the three GC columns and the
(Department of Food Chemistry, University of Jena, Germany), and to G. Schmidt (Jacso, Gera, Germany) for the loaning of the chiral detector. W.V. also thanks U. Berger (Promochem, Wesel, Germany) for the generous gift of a qualitative standard solution of B8-806.
Literature Cited
FIGURE 5. GC/ECNI-MS chromatogram of B6-923: (a) original fraction 20-21 min and (b) reinjected fraction 20-21 min.
TABLE 2. Elution Order of Toxaphene Enantiomers on Different Chiral Stationary GC Phasesa B6-923 B7-1000 B7-1001 B7-1453 B8-809 (P-42b) B8-1412 B8-1413 (P-26) B8-1414 (P-40) B8-1945 (P-41) B8-2229 (P-44) B9-1679 (P-50)
β-BSCD
impure β-TBDM
1b
c
1 2 1
2 1 2
1 2 1 2 2
2 1 2 1
CB-β-PMCD 2d 2 1 2 2 1 2 1 1
a β-BSCD ) tert-butyl-dimethylsilylated β-cyclodextrin; β-TBDM ) 6-O-tert-butyl-2,3-di-O-methyl-β-cyclodextrin; CB-β-PMCD ) chemically bonded permethyl-β-cyclodextrin. b 1,2 ) first and second eluting enantiomer from the ChiraDex HPLC column. c No enantiomer separation obtained. d Only partial resolution of the enantiomers.
eHPLC column whereas B7-1001 and B8-1414 eluted in the same order from the GC-CSPs, which was reversed from the ChiraDex (eHPLC) column (Table 2). All other components except B8-1412 showed different elution orders that may cause false interpretations of ERs. Our experiments confirmed that it is possible to obtain useful amounts of enantiopure CTT standards. Knowledge of the elution orders on GC-CSPs may help to establish uniform standard methods for the interpretation of data obtained from enantioselective studies. Furthermore, determination of the signs of optical rotation of pure enantiomers is an important tag for enantioselective analysis. For the longer term, it should be investigated whether enantiomers of toxaphene have different toxicological impact.
Acknowledgments We are grateful to B. Luckas for supporting our work, to E. Scholz for assistance in the sewage sludge experiments
(1) Vetter, W.; Schurig, V. J. Chromatogr. A 1997, 774, 143-175. (2) Kallenborn, R.; Hu¨hnerfuss, H. Chiral Environmental Pollutants; Springer-Verlag: Berlin, 2001. (3) McBlain, W. A. Life Sci. 1987, 40, 215-217. (4) Miyazaki, A.; Hotta, T.; Marumo, S.; Sakai, M. J. Agric. Food Chem. 1978, 26, 975-977. (5) Vetter, W. Food Rev. Int. In press. (6) Vetter, W.; Klobes, U.; Luckas, B.; Hottinger, G. Chromatographia 1997, 45, 255-262. (7) Oehme, M.; Mu ¨ ller, L.; Karlsson, H. J. Chromatogr. A 1997, 775, 275-285. (8) Buser, H.-R.; Mu ¨ ller, M. D. Anal. Chem. 1994, 66, 2155-2162. (9) Mo¨ller, K.; Bretzke, C.; Hu ¨ hnerfuss, H.; Kallenborn, R.; Kinkel, J. N.; Kopf, J.; Rimkus, G. Angew. Chem., Int. Ed. Engl. 1994, 33, 882-884. (10) Haglund, P. J. Chromatogr. A 1996, 724, 219-228. (11) Vetter, W.; Klobes, U.; Luckas, B.; Hottinger, G.; Schmidt, G. J. Assoc. Off. Anal. Chem. 1998, 81, 1245-1251. (12) Vetter, W. Chemosphere 1993, 26, 1079-1084. (13) Vetter, W.; Oehme, M. Toxaphene. Analysis and Environmental Fate of Congeners. In The Handbook of Environmental Chemistry, Volume 3, Part K: New Types of Persistent Halogenated Compounds; Paasivirta, J., Ed.; Springer-Verlag: Berlin, 2000; pp 237-287. (14) Burhenne, J.; Hainzl, D.; Xu, L.; Vieth, B.; Alder, L.; Parlar, H. Fresenius J. Anal. Chem. 1993, 346, 779-785. (15) Hainzl, D.; Burhenne, J.; Barlas, H.; Parlar, H. Fresenius J. Anal. Chem. 1995, 351, 271-285. (16) de Geus, H.-J.; Besseling, H.; Brouwer, A.; Klungsøyr, J.; McHugh, B.; Nixon, E.; Rimkus, G. G.; Wester, P. G.; de Boer, J. Environ. Health Persp. 1999, 107, 115-144. (17) Vetter, W.; Klobes, U.; Krock, B.; Luckas, B.; Glotz, D.; Scherer, G. Environ. Sci. Technol. 1997, 31, 3023-3028. (18) Klobes, U.; Vetter, W.; Luckas, B.; Hottinger, G. Chromatographia 1998, 47, 565-569. (19) Vetter, W.; Klobes, U.; Luckas, B.; Hottinger, G. J. Chromatogr. A 1999, 846, 375-381. (20) Vetter, W.; Maruya, K. Environ. Sci. Technol. 2000, 34, 16271635. (21) Krock, B.; Vetter, W.; Luckas, B.; Scherer, G. Chemosphere 1996, 33, 1005-1019. (22) Vetter, W.; Scholz, E.; Luckas, B.; Maruya, K. A. J. Agric. Food Chem. 2001, 49, in press. (23) Vetter, W.; Luckas, B.; Oehme, M. Chemosphere 1992, 25, 16431652. (24) Tribulovich, V. G.; Nikiforov, V. A.; Karavan, V. S.; Miltsov, S. A.; Bolshakov, S. Organohalogen Compd. 1994, 19, 97-101. (25) Buser, H.-R.; Haglund, P.; Mu ¨ ller, M. D.; Rappe, C. Organohalogen Compd. 1998, 35, 239-242. (26) Vetter, W.; Bartha, R.; Stern, G.; Tomy, G. Environ. Toxicol. Chem. 1999, 18, 2775-2781. (27) Vetter, W.; Scherer, G. Environ. Sci. Technol. 1999, 33, 34583461. (28) Vetter, W.; Pineiro Costas, N.; Bartha, R.; Gago Martı´nez, A.; Luckas, B. J. Chromatogr. A 2000, 886, 123-131. (29) Vetter, W.; Klobes, U.; Luckas, B. Chemosphere In press. (30) Jaus, A.; Oehme, M. Chromatographia 1999, 50, 299-304. (31) Stern, G. A.; Loewen, M. D.; Miskimmin, B. M.; Muir, D. C. G.; Westmore, J. B. Environ. Sci. Technol. 1996, 30, 2251-2258. (32) Turner, W. V.; Khalifa, S.; Casida, J. E. J. Agric. Food Chem. 1975, 23, 991-944.
Received for review August 3, 2000. Revised manuscript received December 7, 2000. Accepted December 7, 2000. ES000174G
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