Simultaneous Separation of Coplanar and Chiral Polychlorinated

Isomer-specific polychlorinated biphenyl determination in cetaceans from the mediterranean sea: Enantioselective occurrence of chiral polychlorinated ...
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Anal. Chem. 1999, 71, 70-77

Simultaneous Separation of Coplanar and Chiral Polychlorinated Biphenyls by Off-Line Pyrenyl-Silica Liquid Chromatography and Gas Chromatography. Enantiomeric Ratios of Chiral Congeners L. Ramos, L. M. Herna´ndez, and M. J. Gonza´lez*

Department of Instrumental Analysis and Environmental Chemistry, IQO, CSIC, Juan de la Cierva 3, 28006-Madrid, Spain

A method for the unambiguous determination of 41 key polychlorinated biphenyls (PCBs) (including coplanar and chiral congeners) and the enantiomeric ratio of chiral congeners is described. The method includes a fractionation step using a 2-(1-pyrenyl) ethyldimethylsilylated silica column for separating the polychlorinated biphenyls according to the number of chlorine atoms in the ortho positions. High-resolution gas chromatography with an electron capture detector and an achiral column was used to determine the PCB congener content in each fraction. The enantiomeric ratio of chiral congeners was calculated by high-resolution gas chromatography with a mass spectrometry detector using a chiral column. The method was found to be inexpensive, rapid, effective, and reliable under the operational conditions proposed. It eliminates the main coelution problems among the polychlorinated biphenyl congeners. It also makes it possible to determine the enantiomeric ratio of nine chiral congeners using monodimensional gas chromatography. The method was applied successfully to the analysis of the coplanar and atropisomeric polychlorinated biphenyl congeners in dolphin liver samples. The enantiomeric ratio of nine chiral congeners is also reported for the first time. Polychlorinated biphenyls (PCBs) have been used widely by industry worldwide since 1930. Although their use has been banned in many countries since the late 1970s, they still represent an important class of priority pollutants due to their persistence, toxicity, and bioaccumulation.1 Among the 209 theoretically possible PCB congeners, about 135 are present in technical mixtures and consequently can be found in environmental samples.2,3 Due to differences in their toxicity and biological activity, there is currently a great deal of interest in their unambiguous determination. Despite efforts to develop new stationary chromatographic phases, and the use of more sophisticated techniques, such as multidimensional gas (1) Gonza´lez, M. J.; Ferna´ndez, M. A.; Herna´ndez, L. M. Arch. Environ. Contam. Toxicol. 1991, 20, 343-348. (2) Larsen, B.; Bøwadt, S.; Tilio, R. Int. J. Environ. Anal. Chem. 1992, 47, 4768. (3) Ballschmiter, K.; Zell, M. Fresenius Z. Anal. Chem. 1980, 302, 20-31.

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chromatography (MDGC), two-dimensional gas chromatography, or high-speed gas chromatography,4-9 some important limitations still remain. A large number of stationary phases in high-resolution gas chromatography (HRGC), including long-chain alkyl polysiloxanes phases,10-13 liquid crystalline phases,14,15 and cyanopropyl2,16 and diphenyl groups in methyl polysiloxane copolymers,2,16-19 have been studied for separation of complex mixtures of PCBs. Larsen’s critical review in 199520 of the state of the art of HRGC separation of PCB congeners concluded that none of the 11 stationary phases commercially available, and for which there are published chromatograms and retention data for at least all PCB congeners occurring in significant concentrations in technical mixtures,2,11,16,19,21 were able to resolve all 209 congeners. Nor were they able to resolve the estimated 150 congeners present in technical mixtures, or the 36 priority congeners identified by McFarland and Clarke,22 (4) Kannan, N.; Petrick, G.; Schulz, D.; Duinker, J. Chemosphere 1991, 23, 1055-1076. (5) Haglund, P.; Asplund, L.; Ja¨rnberg, V.; Jansson, B. J. Chromatogr. 1990, 507, 389-398. (6) de Boer, J.; Dao, Q. T. J. High Resolut. Chromatogr. 1991, 14, 593-596. (7) Schomburg, G.; Husmann, H.; Hu ¨ binger. E. J. High Resolut. Chromatogr. 1985, 8, 395-399. (8) Kannan, N.; Petrick, G.; Schultz, B.; Duinker, J. C. J. Chromatogr. 1993, 642, 425-434. (9) Liu, Z.; Sirimane, S. R.; Patterson, D. G.; Needhem, L. L.; Phillips, J. B. Anal. Chem. 1994, 66, 3086-3092. (10) Fischer, R.; Wittlinger, R.; Ballschmiter, K. Fresenius J. Anal. Chem. 1992, 342, 421-425. (11) Fischer, R; Ballschmiter, K. Fresenius J. Anal. Chem. 1988, 332, 441-446. (12) Ballschmiter, K.; Mennel, A.; Buyten, J. Fresenius J. Anal. Chem. 1993, 346, 396-402. (13) Sissons, D.; Welti, D. J. Chromatogr. 1971, 60, 15. (14) Zielinski, W. L.; Miller, M. M.; Ulma, G.; Wasik, S. P. Anal. Chem. 1986, 58, 2692-2696. (15) de Boer, J.; Dao, Q. T.; van Dortmond, R. J. High Resolut. Chromatogr. 1992, 15, 249-254. (16) Bøwadt, S.; Skejø-Andressen, H.; Montanarella, L.; Larsen, B. Int. J. Environ. Anal. Chem. 1994, 56, 87-107. (17) Albro, P.; Corbet, J.; Schro¨eder, J. J. Chromatogr. 1981, 205, 103-111. (18) Bøwadt, S.; Larsen, B. J. High Resolut. Chromatogr. 1992, 15, 350-351. (19) Larsen, B.; Cont, M.; Montanerella, L.; Platzner, N. J. Chromatogr. A 1995, 115-129. (20) Larsen, B. J. High Resolut. Chromatogr. 1995, 18, 141-151. (21) Mullin, M.; Pochini, C.; McCrindle, S.; Romkes, M.; Safe, S.; Safe, L. Environ. Sci. Technol. 1984, 18, 468-476. (22) McFarland, V.; Clarke, J. Environ. Health Perspect. 1989, 81, 225-239. 10.1021/ac9806153 CCC: $18.00

© 1998 American Chemical Society Published on Web 11/21/1998

and most surprisingly, they were not even able to resolve the set of seven indicator congeners. Larsen20 concluded that no single commercially available capillary column was able to separate the priority PCBs from their sources of interferences. Despite the development of new stationary phases to improve the separation of key PCBs, the classic SE-54 column is considered to be the most reliable phase for HRGC techniques4 and is the most widely used for the analysis of PCBs. MDGC has been used successfully to clarify the identity of PCBs in industrial mixtures21,22 and in environmental samples6,23-25 and to resolve many of the questions relating to the relative amounts of PCBs. This technique has considerable potential but has not yet been fully refined to “heart-cut” the chromatographic peaks and to fully quantify each peak with the internal standard. The cost of MDGC, relative to a single-oven gas chromatograph, may also restrict the technique to a small number of laboratories for the time being. Other groups have chosen high-performance liquid chromatography and gas chromatography (HPLC-HRGC) for off-line coupling. In this technique, the purified extract is fractionated before the HRGC analysis. Four recent developments for fractionating PCBs have been described: (a) porous graphitic carbon,26 (b) C60/C70 fullerenes bound to polystyrene divinylbenzene,27 (c) C-18 dispersed PX-21 activated carbon,28 and (d) 2-(1-pyrenyl) ethyldimethylsilylated silica column (PYE).29 The four adsorbents have similar elution characteristics for PCBs, but PYE is more efficient and does not produce irreversible adsorption, it is possible to use a UV detector (because aromatic phase mobiles are not required) and isocratic elution, and a lower amount of solvent and time of analysis are required to obtain the PCB fractionation. The main disadvantage of this stationary phase is that samples must be almost completely devoid of lipids.30,31 In the early 1980s, the Bureau of Certificate References (BCR) group working on PCB analysis identified seven congeners which could be used in environmental monitoring programs on account of their high concentrations in technical mixtures and their recalcitrance (PCBs 28 (24-4 trichlorobiphenyl), 52 (25-25 tetrachlorobiphenyl), 101 (245-25 pentachlorobiphenyl), 118 (245-34 pentachlorobiphenyl), 138 (234-245 hexachlorobiphenyl), 153 (245245 hexachlorobiphenyl) and 180 (2345-245 heptachlorobiphenyl).3 This list has recently been extended to include 3 mono-ortho compounds (PCBs 105 (234-34 pentachlorobiphenyl), 114 (2345-4 pentachlorobiphenyl), and 156 (2345-34 hexachlorobiphenyl)) as well as PCBs 128 (234-234 hexachlorobiphenyl) and 170 (2345234 heptachlorobiphenyl).32 (23) Schultz, D. E.; Petrick, G.; Duinker, J. C. Environ. Sci. Technol. 1989, 32, 852-859. (24) Duinker, J. C.; Schultz, D. E.; Petrick, G. Anal. Chem. 1988, 60, 478-482. (25) de Boer, J.; Dao, Q. T. Int. J. Environ. Chem. 1991, 43, 245-251. (26) Creaser, C. S.; AlHaddad, A. Anal. Chem. 1989, 61, 1300-1302. (27) Stalling, D. L.; Guo, C. Y.; Saims, J. Chromatogr. Sci. 1993, 31, 265-278. (28) Feltz, K. P.; Tillitt, D. E.; Gale, R. W.; Peterman, P. H. Environ. Sci. Technol. 1995, 29, 709-718. (29) Haglund, P.; Asplund, L.; Ja¨rnberg, U.; Jansson, B. Chemosphere 1990, 20, 887-894. (30) Tuinstra, L. G. M.Th.; van Rhijn, J. A.; Roos, A. H.; Traag, W. A.; van Mazijk, R. J.; Kolkman, P. J. W. J. High Resolut. Chromatogr. 1990, 13, 797-802. (31) de Boer, J.; Stronck, C. J. N.; van der Valk, F.; Wester, P. G.; Daudt, M. J. M. Chemosphere 1992, 25, 1277-1283. (32) Wells, D. E.; Echarri, I. Int. J. Environ. Anal. Chem. 1992, 47, 75-97.

In recent years, attention has been focused on the toxicity of PCBs, especially of those congeners showing the same toxicity as polychlorinated dibenzo-p-dioxins and dibenzofurans. Thirteen PCBs, non-ortho (PCBs 77 (34-34 tetrachlorobiphenyl), 126 (34534 pentachlorobiphenyl) and 169 (345-345 hexachlorobiphenyl)), mono-ortho (PCBs 105 (234-34 pentachlorobiphenyl), 114 (2345-4 pentachlorobiphenyl), 118 (245-34 pentachlorobiphenyl), 123 (34524 pentachlorobiphenyl), 156 (2345-34 hexachlorobiphenyl), 157 (234-345 hexachlorobiphenyl), 167 (245-345 hexachlorobiphenyl), and 189 (2345-345 heptachlorobiphenyl)), and di-ortho substituted congeners (PCBs 170 (2345-234 heptachlorobiphenyl) and 180 (2345-245 heptachlorobiphenyl)), which are found to be inducers of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activity,33,34 exhibit high-affinity binding to the cytosolic receptor protein (Ah), and a toxic equivalent factor (TEF) has been assigned,35 belonging to the toxic PCB group. More recently still, attention has been focused on PCBs which display axial chirality in their nonplanar conformations.36 Nineteen out of 78 chiral PCBs, containing three or four chlorine atoms in the ortho positions, exist as stable enantiomers at ambient temperature. Among these 19 PCB congeners, at least 12 (PCBs 88 (2346-2 pentachlorobiphenyl), 84 (236-23 pentachlorobiphenyl), 91 (236-24 pentachlorobiphenyl), 95 (236-25 pentachlorobiphenyl), 131 (2346-23 hexachlorobiphenyl), 132 (234-236 hexachlorobiphenyl), 136 (236-236 hexachlorobiphenyl), 149 (236-245 hexachlorobiphenyl), 171 (2346-234 heptachlorobiphenyl), 174 (2345-236 heptachlorobiphenyl), 183 (2346-245 heptachlorobiphenyl), and 196 (2345-2346 octachlorobiphenyl)) are present in commercial PCB mixtures (Aroclor, Clophen, Phenochlor, and Kanechlor at 50 or 60% chlorination).3,24,37-39 Four of these congeners (PCBs 95, 132, 149, and 174) are relatively abundant in commercial mixtures, each accounting for over 2%. Chirality of biologically active compounds is of special importance due to the fact that most of these compounds are introduced into the environment as racemates and their uptake and metabolism by organisms may be selective for enantiomers.40,41 The enantiomeric ratio of chiral PCBs in animals which are in the upper trophic chain levels may give additional information on possible degradation pathways. In addition, it has been shown that the two atropisomers of PCB 88 (2346-2 pentachlorobiphenyl), 139 (2346-24 hexachlorobiphenyl), and 197 (2346-2346 octachlorobiphenyl) have different biological activities.42 Thus, the analysis of the chiral PCB congeners and the determination of their enantiomeric ratios in top predatory (33) Safe, S. CRC Crit. Rev. Toxicol. 1984, 13, 319-393. (34) Safe, S. Chemosphere 1987, 16, 791-802. (35) Alborgh, U. G.; Becking, G. C.; Birnbaum, L. S.; Brouwer, A.; Derks, H. J. G. M.; Feely, M.; Golor, G.; Hanberg, A.; Larsen, J. C.; Liem, A. K. D.; Schlatter, C.; Waern, F.; Younes, M.; Yrja¨nheikki, E. Chemosphere 1994, 28, 1049-1067. (36) Kaiser, K. L. E. Environ. Pollut. 1974, 7, 93-105. (37) Manchester-Nessvig, J. B.; Andren, A. W. Environ. Sci. Technol. 1989, 23, 1138-1148. (38) Driss, M. R.; Sabbah, S.; Bouguerra, M. L. Analusis 1989, 17, 252-258. (39) de Voogt, P.; Wells, D. E.; Reutergardh, L.; Brinkman, U. H. Th. Int. J. Environ. Anal. Chem. 1990, 40, 1-40. (40) Faller, J.; Hu ¨ hnerfuss, H.; Ko¨nig, W. A.; Krebber, R.; Ludwig, P. Environ. Sci. Technol. 1991, 25, 676-678. (41) Buser, H. R.; Mu ¨ ller, M. D.; Rappe, C. Environ. Sci. Technol. 1992, 26, 1533-1540. (42) Rodman, L. E.; Shedlofsky, S. I.; Mannschreck, A.; Pu ¨ ttmann, M.; Swim, A. T.; Robertson, L. W. Biochem. Pharmacol. 1991, 41, 915-922.

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animal species are currently of interest in order to assess the risk of exposure to PCBs. γ- and β-cyclodextrin as well as their mixtures have been successfully used as stationary phases in HRGC43-45 and as chiral selectors in cyclodextrin-modified micellar electrokinetic chromatography 46,47 for the enantiomeric separation of PCBs. Neither of these alternatives is able to separate the 19 chiral PCBs which are stable at room temperature. At present, MDGC using ChirasilDex as the chiral column in the achiral-chiral column combination is the only reliable methodology for the enantioselective analysis of chiral PCBs. The enantiomeric ratio of the two atropisomers of PCB 132 (234-236 hexachlorobiphenyl) in human breast milk and also those of PCB 95 (236-25 pentachlorobiphenyl) and 149 (236-245 hexachlorobiphenyl) in sediments and shark samples have been calculated by MDGC on Chirasil-Dex column.48-50 When a complete analysis of PCBs, including the determination of both the most toxic coplanar congeners and the enantiomeric ratio of those with chiral properties, is required, off-line HPLC(PYE)-HRGC with electron capture or mass spectrometry detection appears to be the most interesting, offering a highperformance, inexpensive, and simple alternative to MDGC. In this study, an off-line HPLC(PYE)-HRGC method is described for the unambiguous analytical determination of 41 interesting PCB congeners, including the most abundant, persistent, and toxic ones, and chiral congeners which are stable at room temperature. The enantiomeric ratio of nine chiral PCBs was also determined. The proposed methodology is a new application of HPLC(PYE) in PCB group separation prior to final determination by monodimensional HRGC/ECD. Particular attention has been paid to the separation of chiral PCBs from others, and their separation in the appropriate fraction to further determine the enantiomeric ratio of nine chiral PCBs (88, 91, 95, 132, 135, 136, 149, 174, and 196) by HRGC/LRMS with Chirasil-Dex stationary phase, without interference. In this study, the separation of the toxic non- and mono-ortho PCBs from PCB mixtures was improved with respect to that previously achieved by other research groups. Finally, the separation of other interesting PCBs, such as PCB 45, 88, 91, 114, 131, 136, 139, 175, and 178, from other coeluted PCBs is reported here for the first time. The method has been used successfully with dolphin liver samples from the Mediterranean. EXPERIMENTAL SECTION Chemicals. All solvent used were of high purity for pesticide residue analysis from Merck and Promochen (Wesel, Germany). (43) Schurig, V.; Glausch, A Naturwissenschaften 1993, 80, 468-469. (44) Ko¨nig, W. A.; Gehrcke, B.; Runge, T.; Wolf, C. J. High Resolut. Chromatogr. 1993, 16, 376-378. (45) Hardt, I. H.; Wolf, C.; Gehrcke, B.; Hochmuth, D. H.; Pfaffenberger, B.; Hu ¨ hnerfuss, B.; Ko¨nig, W. A. J. High Resolut. Chromatogr. 1984, 17, 859864. (46) Marina, M. L.; Benito, I.; Dı´ez-Masa, J. C.; Gonza´lez, M. J. J. Chromatogr. A 1996, 752, 265-270. (47) Marina, M. L.; Benito, I.; Dı´ez-Masa, J. C.; Gonza´lez, M. J. Chromatographia 1996, 42, 269-272. (48) Glausch, A.; Hahn, J.; Schurig, V. Chemosphere 1995, 30, 2079-2085. (49) Glausch, A.; Blanch, G. P.; Schurig, V. J. Chromatogr. A 1996, 723, 399404. (50) Blanch, G. P.; Glausch, A.; Schurig, V.; Serrano, R.; Gonzalez, M. J. J. High Resolut. Chromatogr. 1996, 19, 392-396.

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Table 1. PCB IUPAC Number, Structure, Number of Ortho Chlorines, Capacity Factor, Standard Deviation, and Coefficients of Variation (CV) on the Pyrenyl HPLC Column (250 × 0.46 mm i.d.) of the 41 PCBs Studied IUPAC no.

structure

no. of ortho Cl

capacity factor (k)

197 88 45 176 28 144 52 91 183 139 175 136 178 131 149 135 95 101 196 84 171

2346-2346 2346-2 236-2 2346-236 24-4 2346-25 25-25 236-24 2346-245 2346-24 2346-235 236-236 235-2356 2346-23 236-245 235-236 236-25 245-25 2345-2346 236-23 2346-234

4 3 3 4 1 3 2 3 3 3 4 4 3 3 3 3 3 2 3 3 3

0.320 0.398 0.409 0.429 0.432 0.476 0.486 0.491 0.492 0.503 0.510 0.531 0.524 0.533 0.544 0.554 0.556 0.565 0.590 0.608 0.637

153 174 132 110 129 180 138 118 123 114 194 170 167 105 156 157 189 77 126 169

245-245 2345-236 234-236 34-236 23-2345 2345-245 234-245 245-34 345-24 2345-4 2345-2345 2345-234 245-345 234-34 2345-34 234-345 2345-345 34-34 345-34 345-345

2 3 3 2 2 2 2 1 1 1 2 2 1 1 1 1 1 0 0 0

0.652 0.659 0.668 0.761 0.767 0.789 0.820 0.835 0.869 0.931 1.001 1.013 1.084 1.142 1.368 1.447 1.553 1.721 2.348 3.048

fraction I

II

III

IV

V

VI

SD/CV (%) 0.002/0.64 0.004/0.90 0.002/0.31 0.0007/0.12 0.002/0.38 0.013/2.6 0.002/0.30 0.001/0.21 0.02/3.4 0.004/0.61 0.001/0.22 0.001/0.22 0.002/0.28 0.003/0.51 0.0009/0.19 0.002/0.23 0.002/0.27 0.008/1.10 0.007/1.2 0.002/0.28 0.001/0.18 0.006/0.71 0.03/4.6 0.007/0.86 0.005/0.52 0.005/0.63 0.05/0.60 0.07/7.9 0.01/1.3 0.02/1.8 0.001/0.10 0.001/0.10 0.08/7.4 0.01/1.2 0.01/0.94 0.01/1.0 0.02/1.0 0.002/0.11 0.03/1.8 0.002/0.11 0.04/1.8 0.05/1.7

The 41 individual PCB congeners studied (Table 1) were purchase from Ehrenstorfer (Ausburg, Germany). Four working stock solutions were prepared from individual PCB standards. The first stock solution (COP-17) contained the most abundant and toxic congeners, PCBs 77 (34-34 tetrachlorobiphenyl), 101 (24525 pentachlorobiphenyl), 105 (234-34 pentachlorobiphenyl), 114 (2345-4 pentachlorobiphenyl), 118 (245-34 pentachlorobiphenyl), 123 (345-24 pentachlorobiphenyl), 126 (345-34 pentachlorobiphenyl), 138 (234-245 hexachlorobiphenyl), 153 (245-245 hexachlorobiphenyl), 156 (2345-34 hexachlorobiphenyl), 157 (234-345 hexachlorobiphenyl), 167 (245-345 hexachlorobiphenyl), 169 (345345 hexachlorobiphenyl), 170 (2345-234 heptachlorobiphenyl), 180 (2345-245 heptachlorobiphenyl), 189 (2345-345 heptachlorobiphenyl), and 194 (2345-2345 octachlorobiphenyl), at a concentration of 0.5 µg/mL each in n-C6H14. The second working stock solution (QUIR-19) contained the 19 chiral PCBs which are stable at room temperature (PCBs 45 (236-2 tetrachlorobiphenyl), 84 (236-23

pentachlorobiphenyl), 88 (2346-2 pentachlorobiphenyl), 91 (23624 pentachlorobiphenyl), 95 (236-25 pentachlorobiphenyl), 131 (2346-23 hexachlorobiphenyl), 132 (234-236 hexachlorobiphenyl), 135 (235-236 hexachlorobiphenyl), 136 (236-236 hexachlorobiphenyl), 139 (2346-24 hexachlorobiphenyl), 144 (2346-25 hexachlorobiphenyl), 149 (236-245 hexachlorobiphenyl), 171 (2346-234 heptachlorobiphenyl), 174 (2345-236 heptachlorobiphenyl), 175 (2346-235 heptachlorobiphenyl), 176 (2346-236 heptachlorobiphenyl), 183 (2346-245 heptachlorobiphenyl), 196 (2345-2346 octachlorobiphenyl), and 197 (2346-2346 octachlorobiphenyl)) at a concentration of 1 µg/mL each in n-C6H14. The third working stock solution (ARO-41) contained the 17 PCBs of stock solution COP17, plus the 19 PCBs of stock solution QUIR-19, plus PCBs 28 (24-4 trichlorobiphenyl), 52 (25-25 tetrachlorobiphenyl), 110 (34236 pentachlorobiphenyl), 129 (23-2345 hexachlorobiphenyl), and 178 (235-2356 heptachlorobiphenyl) at a concentration of 1 µg/ mL each in n-C6H14.The four working stock solution (QUIR-9) containing the nine chiral PCBs separated in their two atropisomers by Chirasil-Dex column (PCBs 84, 91, 95, 132, 135, 136, 149, 174, and 176) at a concentration of 1 µg/mL each in n-C6H14. Extraction and Cleanup. Extraction and cleanup followed a previously published semiautomatic procedure51 which is based on an adaptation of the method of Smith et al.52 This comprises a low-pressure chromatography system in which 0.5 g of dolphin liver samples, homogenated using a politron apparatus and dried with anhydrous sodium sulfate, was placed between two layers of anhydrous sodium sulfate on the top of a multilayer column containing neutral and base-modified silica gel. This column was connected to a second one filled with activated carbon (AmocoPX21) dispersed on glass fibers. The PCBs were eluted from the carbon column using cyclohexane/dichloromethane (80:20) and dichloromethane/toluene (80:20). The eluate was concentrated and transferred to a column containing silica gel impregnated with sulfuric acid and eluted with n-hexane to remove lipids. The eluent was evaporated to 1 mL under nitrogen before the final step of cleanup on a florisil column. Liquid Chromatography. The PCBs were separated in a Cosmosil 5-PYE column (2-(1-pyrenyl) ethyldimethylsilylated silica gel), 250 × 4.6 mm i.d., particle size 5 µm (Nacalai Tesque, Promochem GmbH) maintained at 25 °C. HPLC systems consisted of a Perkin-Elmer P10 pump, a Rheodyne 7125 injector equipped with a 20-µL loop, and a Hewlett-Packard HPLC series 1050 UV absorbance detector. The detector was operated at 225 nm. Hexane was used as mobile phase at a flow rate of 0.5 mL/min, and 20-ng portions of the PCB congeners were individually injected in the HPLC chromatographic system in order to calculate their capacity factors (k). Prior to the fractionation of the ARO-41 stock solution, 20-µL aliquots of the stock solutions COP-17 and QUIR-19 were injected in order to determine their chromatographic behavior and to test the chromatographic system. Six independent fractions were obtained as followed: The first 3.5 mL was discarded, and then 0.85, 0.45, 0.3, 0.5, 2.2, and 6 mL were collected to obtain FI to FVI, and subsequently analyzed by HRGC. The standard stock solution ARO-41 was injected four (51) Krokos, F.; Creaser, C. S.; Wright, C.; Startin, J. Organohalogen Compd. 1993, 11, 61-65. (52) Smith, L. M.; Stalling, D. L.; Johnson, J. L. Anal. Chem. 1984, 56, 18301836.

times in the HPLC(PYE) system. The six individual extracts obtained at each fractionation were concentrated as appropriate and subsequently injected in the HRGC/ECD chromatographic system to calculate the recoveries of the individual PCBs (Table 1). In all cases, the variation coefficients found were lower than 10%. HRGC/ECD. A Hewlett-Packard 5971 A gas chromatograph equipped with a 63Ni electron capture detector was used for the qualitative and quantitative analyses of the PCB congeners contained in each of the fractions collected from the PYE. The eluates of the different fractions were concentrated and taken up in a working solution containing PCBs 12 (34 dichlorobiphenyl) and 209 (23456-23459 decachlorobiphenyl) at a concentration of 1 ng/µL. These individual congeners were added just before chromatographic injection in order to correct injection errors and detector fluctuations. A 0.5-µL aliquot of this solution was injected into a 60-m BPX5 (5% phenyl 95% polysilphenylenesiloxane) fused-silica capillary column with 0.25-µm film thickness (SGE, Australia Pty. Ltd.). The column temperature was programmed from 60 (1 min) to 180 °C at 50 °C/min, then to 230 °C (40 mn) at 4 °C/min, and finally to 270 °C (10 min) at 4 °C/min. The injector and detector temperatures were 280 and 300 °C, respectively. Chromatographic data were obtained by the System Gold acquisition data system (Beckman, CA). The identification of the PCB congeners presented in each fraction was based on the knowledge of the composition of the standard stock solution injected, the retention order on the PYE and their retention time on GC relative to PCBs 12 and 209. The recoveries of the congeners in the different extracts were calculated by comparison of the individual peak area response of each congener with those corresponding to similar concentration of the stock standard solution. HRGC/LRMS. The determination of the enantiomeric ratio of nine chiral PCBs was performed by using a high-resolution gas chromatograph (HP5890 Series II) coupled to a low-resolution mass spectrometer working in the selected ion monitoring (SIM) mode (HP5971 A). The chiral stationary phase used was the Chirasil-Dex (30 m × 0.25 mm i.d., 0.2 µm of film thickness) supplied by Prof. V. Schurig (Tu¨bingen University, Germany). The column temperature was programmed from 80 (1 min) to 150 °C (10 min) at a rate of 20 °C/min, then to 160 °C (88 min) at a rate of 2 °C/min, and finally to 180 °C (100 min.) at a rate of 5 °C/ min. The eluent from the column was transferred to a quadrupole mass spectrometer with electronic impact ionization and subsequent ion detection. The interface temperature was 280 °C. The source temperature was 280 °C and the electron energy 70 eV. Two characteristic M/M + 2 ions of each PCB homologue (m/z ) 324/326 for the pentachloro-substituted congeners PCBs 84, 91, and 95; m/z ) 360/362 for the hexachloro-substituted congeners PCBs 132, 135, 136, and 149; and m/z 396/398 for the heptachloro-substituted congeners PCBs 174 and 176) were monitored for each analysis. Three different chromatographic windows were defined for each PCB homologue. Thus, pentachloro-, hexachloro- and heptachloro-substituted biphenyl congeners were monitored from 10 to 60 min, 60 to 100 min, and 100 to 140 min, Analytical Chemistry, Vol. 71, No. 1, January 1, 1999

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respectively. Quality control criteria were defined as simultaneous detection of a peak for both monitored ions within the expected retention time window for each congener and an ion intensity ratio of sample peaks within 20% of the mean values for calibration standards. RESULTS AND DISCUSSION The capacity factors (k) of the 41 individual PCBs investigated (including coplanar and chiral congeners) determined with HPLC(PYE) are given in Table 1. The chemical structures and the corresponding IUPAC numbers3 of the PCBs included in this work are also summarized in Table 1. The calculations of k were carried out by performing four different injections of the individual PCB congeners within a 3-day period. The CVs were below 4.6% in all cases except for PCBs 180 and 194, for which they were found to be 7.9 and 7.4%, respectively. Memory effects were not observed in any of the cases, and assays carried out 1 month later under similar experimental conditions gave the same results. In general, the elution order agrees with the results found by Haglund et al.5 using the PYE stationary phase, but some pairs that coeluted under his working conditions are better resolved in this study. This is the case for the pairs PCBs 144-52, 149-135, 95-101, 171-153, and 118-138. Probably the longer column length (25 cm instead the 15 cm used by Haglund5) or some differences in working temperature (not specified in Haglund’s5 published results) could explain these observed differences. Nevertheless, it is not easy to explain the differences observed in the elution pattern of the other PCBs. Thus, in the present study, PCB 144 eluted earlier than PCB 52; PCB 149 eluted earlier than PCB 135, and both congeners eluted before the PCBs 95-101 pair; PCB 171 eluted earlier than PCB 153 and the PCBs 118-138 pair before PCB 123. The capacity factors of PCB congeners 45, 88, 91, 114, 131, 136, 139, 175, and 178 are given in this study for the first time and consequently it is not possible to compare them with previous results. The representation of the calculated k for all the PCBs investigated with the total number and position of the chlorine atoms (number of chlorine atoms in the ortho position) (Figure 1) shows that the retention mechanism of the PCBs in this stationary phase is similar to that observed with active charcoal (Amoco-PX21)4 and very close to those observed with activated Florisil.53 Retention on the PYE stationary phase is highly influenced by solute planarity, and this phase has been shown to be excellent for isolating both coplanar non-ortho and mono-ortho PCBs from the bulk of PCBs in technical mixtures.5-29 Retention increased with the planarity of the molecule and with degree of chlorination. However, some important differences in retention have been observed, depending on the distribution pattern of chlorine atoms on the biphenyl ring, due to the fact that PCBs with similar molecule structure but with different π electron densities are separated. Initially, this stationary phase was developed to separate the toxic non-ortho PCBs (77, 126, and 169, possible with a carbon column) and the mono-ortho PCBs (not possible with a carbon column), and to improve the separation between the toxic and (53) Ramos, L.; Hernandez, L. M.; Gonzalez, M. J. J. Chromatogr. A 1997, 759, 127-137.

74 Analytical Chemistry, Vol. 71, No. 1, January 1, 1999

Figure 1. Plot of capacity factors (k) from Table 1 versus the number of chlorine atoms and the number of chlorine atoms in the ortho positions.

other coeluted PCBs in the 5% phenyl/95% methyl siloxane GC stationary phases (SE-54, CP-Sil-8, BP-5, DB-5 or similar).32,39,54 This column, therefore, has the potential to separate other key PCBs not previously studied and at the same time to separate chiral from coplanar PCBs. Figure 2 shows the HPLC chromatograms obtained when the CHIR-19 (Figure 2a), and the COP-17 (Figure 2b) standard solutions were injected into the HPLC(PYE). The k values of chiral PCBs were between 0.320 and 0.668, and they were well separated from the mono-ortho PCBs (k between 0.835 and 1.368), and both families were separated from the nonortho PCBs (k between 1.53 and 3.04). There are some overlaps between tri- and di-ortho PCBs, but they can be totally resolved by GC optimization. Among the 41 PCB congeners studied, those that may have coelution problems in HRGC with a DB-5 chromatographic column are6,15,24,25,39 PCBs 101-84, 77-110-136, 88-91, 144-135, 139-149-118123, 114-131, 153-132-105, and 126-129-178. The separation of some of them (PCBs 77-110, 118-149, 153-132-105, 126-129-178, and 84101) has been extensively studied and partially solved using both GC optimization with EC detection and/or MS detection (SIM mode and isotope dilution technique),23 as well as HPLC fractionation using PYE.32,55 The results obtained in Table 1 show the possibilities of the PYE column for resolving almost all the other PCB coelutions among the PCBs studied. PCBs 118, 123, 139, and 149. The coelutions that PCBs 149, 139, and 123 represent in the quantification of PCB 118 can be solved by optimization of the chromatographic variables in HRGC. The total elimination of the three PCBs (PCBs 123, 139, and 149) that coeluted with PCB 118 is always very difficult. But the results presented in Table 1 show that the four congeners can be totally resolved using PYE (kPCB 139 ) 0.503, kPCB 149 ) 0.542 and kPCB 118 ) 0.835, and kPCB 123 ) 0.869). (54) Kannan, N.; Petrick, G.; Schultz, D.; Duinker, J.; Boon, J.; van Arnhem, E.; Jansen, S. Chemosphere 1991, 23, 1055-1056. (55) Wells, D. E.; Echarri, I. Anal. Chim. Acta 1994, 286, 431-449.

Figure 3. HRGC/ECD chromatograms of the six fractions obtained after the HPLC(PYE) fractionation of the stock solution ARO-41 (fractions I-VI). Column: BPX-5, 60 m × 0.22 mm i.d. Chromatographic conditions are given in the Experimental Section. See Table 1 for structural identification of the PCB congeners.

Figure 2. HPLC(PYE) chromatograms of 20 µL of both stock solutions, CHIR-19 (a) and COP-17 (b), on a 250- × 4.6-mm-i.d. PYE column working at 25 °C. Mobile phase hexane at 0.5 mL/min. UV detection at λ ) 254 nm at 0.04 AUF.

PCB 77-110-136. The coelution of these three PCBs can cause serious problems for the quantification of PCB 77. This PCB is one of the most toxic ones, and it always has a very low concentration in technical and environmental samples. The separation of PCB 77 from its coelutants is very important to avoid any overestimation of its concentration. PYE is particularly suited to separating these three PCBs, which have chlorine atoms in the non-ortho- and two and four ortho-positions respectively, and consequently have very different retentions in the PYE column. Thus, kPCB 136 ) 0.531, kPCB 110 ) 0.761, and kPCB 77 ) 1.553. Pairs 88-91 and 144-135. The coelution of these two pairs of PCBs is very difficult to eliminate by the optimization of the chromatographic conditions, but they are completely separated using the PYE column: (kPCB 88 ) 0.382, kPCB 91 ) 0.491, kPCB 135 ) 0.554 and kPCB 144 ) 0.436 (Table 1). PCB 114-131. The PYE column appears to be particularly suited to separating chiral PCB 131 from mono-ortho PCB114. The difference between the value of k found for PCB 114 (0.931) and that obtained for PCB 131 (0.553) made fractiona-

tion with PYE the best way to separate these three PCB congeners. There is a wide variety of possibilities for PCB fractionation by HPLC(PYE), depending on the final objective of the analysis. As has been mentioned, six independent fractions were collected by PYE in this study. These fractions allowed a total separation of the most toxic congeners (non-ortho and mono-ortho from the bulk PCBs) and their unambiguous analysis by HRGC/ECD with a 5% methyl phenylsiloxane column. In addition, the proposed fractionation allowed a total separation of the coplanar from the chiral PCBs, and it is also possible to separate the chiral into three different fractions. These three fractions could later be injected in both chromatographic systems, HRGC/ECD with BPX-5 chromatographic column in order to perform the qualitative and quantitative analysis of the chiral PCBs, and HRGC/LRMS working in SIM mode to determine the enantiomeric ratio of the nine chiral PCBs which are separated into their two atropisomers using a Chirasil-Dex chromatographic column. GC chromatograms from the six fractions collected from the HPLC(PYE) analysis of the ARO-41 stock solution are shown in Figure 3. Fraction I (from k ) 0.235 to k ) 0.430) contained the PCBs 45, 88, 176, and 176 (four chiral PCBs); fraction II (from k ) 0.430 to k ) 0.540) contained PCBs 28, 52, 91, 131, 136, 139, 144, 175, 178, and 183 (seven chiral, two nontoxic coplanar, and PCB 178, which coeluted with PCB 126); fraction III (from k ) 0.540 to k ) 0.70) contained PCBs 84, 95, 101, 132, 135, 149, 153, 171, 174, and 196 (eight chiral and two coplanar di-ortho PCBs); fraction IV (from k ) 0.70 to k ) 0.830) contained di-ortho PCBs 110, 129, 180, and 138; fraction V (from k ) 0.830 to k ) 1.50) contained Analytical Chemistry, Vol. 71, No. 1, January 1, 1999

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Figure 5. HRGC/ECD chromatograms of the six fractions obtained after the HPLC(PYE) fractionation of a dolphin liver extract (fractions I-VI). Column: BPX-5, 60 m × 0.22 mm i.d × 0.25 µm film thickness. Chromatographic conditions are given in the Experimental Section. See Table 1 for structural identification of the PCB congeners. Figure 4. HRGC/LRMS(SIM) fragmentograms corresponding to (A) m/z 394 (hepta-CBs), (B) m/z 360 (hexa-CBs), (C) m/z 326 (pentaCBs), and (D) total trace ion mass chromatograms of the nine chiral PCBs from stock solution QUIR-9. GC column, Chirasil-Dex, 30 m × 0.22 mm i.d × 0.25 µm film thickness. Chromatographic conditions are given in the Experimental Section. See Table 1 for structural identification of the PCB congeners.

the mono-ortho PCBs 105, 114, 118, 123, 156, 157 and 167 and the di-ortho PCBs 170 and 194; and fraction VI contained the three non-ortho PCBs 77, 126, and 169 and the mono-ortho PCB 189 (from k ) 1.50 to k ) 3.50). Fractionation of the 41 PCB congeners prior to HRGC/ECD analysis made it possible to solve the most important coelution problems when monodimensional GC was used. Thus, PCBs 136, 110, and 77 were eluted in fractions II, IV, and VI, respectively; PCB 144 was eluted in fraction II, while PCB 135 was eluted in fraction III; PCBs 139, 149, and 118 were eluted in fractions II, III, and V, respectively; PCB 88 eluted in fraction I, while PCB 91 was eluted in fraction II; PCBs 178, 129, and 126 were eluted in fractions II, IV, and VI, respectively. Finally, PCBs 131 and 114 were eluted in fractions II and V, respectively. PCBs 118 and 123 were in fraction V, but they are separated by HRGC (Figure 3). The first three fractions containing the chiral PCBs were injected into the HRGC/LRMS(SIM) to calculate the enantiomeric ratio of chiral PCBs. Figure 4 shows the enantiomeric separation of nine chiral PCBs contained in the stock solution QUIR-9 (PCBs 88, 91, 95, 135, 136, 149, 132, 174, and 176) by HRGC using Chirasil-Dex as a stationary phase. LRMS in SIM mode 76 Analytical Chemistry, Vol. 71, No. 1, January 1, 1999

was chosen as the detection system in order to avoid any interference in enantiomeric ratio calculation of the atropisomers of PCBs. Until now, this calculation has only been possible using MDGC.49,50 The off-line HPLC(PYE)-HRGC/LRMS proposed in this paper is a very interesting alternative to the more expensive and sophisticated MDGC techniques used until now in this kind of study. The proposed methodology is more appropriate for routine work as it allows simultaneous analysis of the enantiomeric ratio for various PCBs in a maximum three chromatographic analyses, while a “heart-cut” run for each chiral PCB is needed when MDGC is used. Moreover, this method is more suitable when both enantiomeric ratio calculation of chiral PCBs and coplanar PCB determination are being carried out in a single sample. Not only is it possible to calculate the enantiomeric ratio of nine chiral PCBs, but the separation of key PCBs from interference is improved, and consequently so is the accuracy of PCB analysis. The method has been checked with a dolphin liver sample from the Mediterranean Sea, which exhibited both high PCB levels and a complex mixture of PCBs. The sample was treated according to the extraction and cleanup procedure described above. GC chromatograms from the six fractions collected from HPLC(PYE) analysis of the concentration sample extract are shown in Figure 5. Each PCB congener was included in the fraction expected. Thus, the four- and threeortho chlorinated PCB congeners were obtained in the three first fractions, and the di-, mono-, and non-ortho PCB congeners were mainly in fractions IV, V, and VI, respectively. The PCBs 77-110,

Gas chromatographic separation of the nine atropisomeric PCB congeners in their atropisomers was carried out later with the HRGC/LRMS system described above. Therefore, fractions I, II, and III were injected in a Chirasil-Dex column, and two characteristic ions were monitored. Figure 6 shows the fragmentograms obtained after the injection of fraction III, in which six of the nine separated enantiomer PCBs are obtained (PCBs 95, 84, 135, 149, 132, and 174). The chromatographic separation of the atropisomers of PCBs permits determination of the enatiomeric ratio of the mentioned PCBs as the proportion of the peak area of the first- to the second-eluted atropisomer peak. CONCLUSIONS Off-line HPLC(PYE)-HRGC/ECD/LRMS(SIM) has the potential to separate PCB chiral congeners (tri- and tetra-orthosubstituted) from “coplanar” PCBs and also to remove a number of ambiguities that can exist in the final determination of PCBs using monodimensional GC analysis. The PYE not only resolves coelution for PCBs which are difficult to eliminate by other methods, in a way that is simple and rapid, but it is also able to separate chiral PCBs from coplanar ones, except in a few cases. In addition, it is the most appropriate alternative to MDGC for the determination of chiral PCB enantiomeric ratio. Fractionation of a standard stock solution of 41 key PCBs into six fractions (according to their planarity and chlorination degree) takes less than 15 min using a PYE column. The subsequent determination of the PCBs present in the six fractions by GCECD allows the unambiguous determination of the 41 PCBs investigated. The method allows the enantiomeric ratio determination of the chiral PCBs present in the three first fractions by HRGC/LRMS(SIM) with a Chirasil-Dex stationary phase. The application of the method to real samples with PCB complex mixtures demonstrated that HPLC(PYE) fractionation of the PCB extracts before the HRGC analysis is a useful tool for the determination of the key PCB congeneres using a monodimensional HRGC/ECD, as well as the enantiomeric ratio of some chiral PCBs using an LRMS(SIM) detection system. Figure 6. HRGC/LRMS(SIM) fragmentograms of fraction III of a liver dolphin sample. (A) Window of penta-CBs (m/z 323.88/325.88, time from 10 to 60 min): (B) window of hexa-CBs (m/z 360/362, time from 60 to 100 min); (C) window of hepta-CBs (m/z 394/396, time from 100 to 140 min). See Table 1 for structural identification of the PCB congeners.

88-91, 144-135, 118-139-149, 131-114, and 126-129-178 were obtained in different fractions, as occurred when the standard stock solution ARO-41 was fractionated by HPLC(PYE) (Figure 3).

ACKNOWLEDGMENT The authors thank the CSIC and the Comunidad Auto´noma de Madrid for project 07M/0600/97. The authors also thank Prof. V. Schurig for kindly providing the Chirasil-Dex.

Received for review June 9, 1998. Accepted October 15, 1998. AC9806153

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