Anal. Chem. 1982, 54, 1798-1802
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weight hydrocarbon gases. If separation of argon/oxygen is not required, then single column operation (Porapak super Q)is possible with the split passing through the TCD being subsequently trapped for stable isotopic analysis. Determination of dissolved oxygen or easily oxidized components in interstitial fluids could be an additional application of this technique.
ACKNOWLEDGMENT I wish to thank R. 0. Barnes for generously making his samplers available, K. Beitz and H. Molge for equipment construction, and the captains and crews of R. V. Littorina and R. V. Valdiuia for ship support. The helpful discussions with E. Seibold and E. Suess and many other colleagues are also appreciated. LITERATURE CITED (1) Sayles, F. L.; Wilson, T. R. S.; Hume, D. N.; Mangelsdorf, P. C. Science 1973, 181, 154-156. (2) Sayles, F. L.; Wilson, T. R. S.; Hume, D. N.;Mangelsdorf, P. C. DeepSea Res. 1978, 23, 259-264. (3) Rudd, J. W. M.; Hamilton, R . D. Limnol. Oceanogr. 1975, 2 0 , 902-906. (4) Hesslein, R. H. Limnol. Oceanogr. 1978, 2 1 , 912-914.
(5) Lyons, W. B.; Gaudette, H. E.; Fogg, T. R. Abstracts of the 40th Annual Meeting of the American Society of Limnology and Oceanography, Michigan, 1977. (6) Kepkay, P. E.; Cooke, R. C.; Bowen, A. T. Geochim. Cosmochim. Acta 1981, 45, 1401-1409. (7) Barnes, R. 0. Deep Sea Res. 1973, 20, 1125-1128. (8) Swinnerton, J. W., Linnenbom, V. J.; Cheek, C. H. Anal. Chem. 1962, 3 4 , 483-485. (9) Weiss, R. F.; Craig, H. Deep-sea Res. 1973, 20, 291-303. (10) Weiss, R. F. Deep-sea Res. 1970, 17, 721-735. (1 1) Yamamoto, S.;Alcauskas, J. 8.; Crozler, T. E. J . Chem. Eng. Data 1978, 21, 78-80. (12) Mangelsdorf, P. C.; Wllson, T. R. S.; Daniell, E. Science 1989, 165, 17 1- 173. (13) Blschoff, J. L.; Greer, R. E.; Luistro, A. 0. Science 1970, 167, 1245-1246. (14) Grasshoff, K. "Methods of Seawater Analysis"; Verlag Chemie: Welnhelm, New York, 1976; 371 pp. (15) Inland Waters Directorate (Canada) Analytical Methods Manual, 1979, Ottawa, Envlronment Canada. (16) Cook, T. M.; Miles, D. L., Institute of Geological Sciences, Report 80/ 5, Natural Environment Research Council, London, 1980, 55 pp. (17) Hartmann, M.; Muller, P. J.; Suess, E.; von der Weiden, C. H. "Meteor" Forsch.-Ergebn., C 1973, 12, 74-86.
RECEIVED for review November 16,1981. Accepted May 17, 1982. This work was jointly funded by the Deutsche Forschungs Gemeinschaft and the Sonderforschungsbereiche 95.
Isolation and Identification of Benzene Metabolites in Vitro with Liquid Chromatography/Electrochemistry Daryl A. Roston' and Peter T. Kissinger" Department of Chemistry, Purdue University, West Lafayette, Indiana 47907
Microsomal preparatlons have been wldely used to study benzene metabolism and toxlclty. Such studles stem in part from flndings whlch suggest that the toxicity of benzene is due to the liver metabollsm of the compound. Thls study concerns the evaluatlon of ilquid chromatography/electrochemlstry (LCEC) for the determinatlon of mlcrosomai metaboiltes of benzene. Our approach Involves coupling the sensltivlty of LCEC with preconcentratlon of microsomal metabolites via solvent extraction of the incubatlon mixture. Absolute recoveries of -45 ng quantltles of hydroquinone, catechol, and phenol from mlcrosomai preparations are 18, 52, and 86 %, respectlveiy. Detectlon ilmlts observed for dlhydroxybenzene metabolites are 50.1 ng. The relative performance of UV, single-electrode, and dual-electrode detectors Is evaluated.
In vitro metabolism studies of xenobiotic compounds are often performed via microsomal incubations. The increased control of experimental conditions afforded by the use of subcellular fractions of tissue homogenates enhances the potential for elucidation of metabolic and toxicological questions. Because the absolute quantities of metabolites produced during microsomal incubations are generally extremely low, the determination of metabolites in complex preparations represents a particularly challenging analytical problem. The scope of microsomal studies is often a function of the selectivity and sensitivity of the analytical methodology employed. Present address: Department of Chemistry, Northern Illinois University, DeKalb, IL 60115. 0003-2700/82/0354- 1798$01.25/0
Liver microsomes have been extensively used to study benzene metabolism and toxicity ( 1 ) . Such studies stem in part from findings which suggest that the toxicity of benzene is due to the liver metabolism of the compound ( 2 ) . Often the major metabolite of benzene, phenol, is the only compound determined since it is the only metabolite present in quantities sufficient for detection. A variety of analytical methods have been used to study benzene-microsomal incubation mixtures, including colorimetry (3, 4), thin-layer chromatography ( 4 , 5 ) , and gas chromatography (6). (The cited references are representative of an extensive field.) Frequently, radiolabeled compounds are used in conjunction with one or more of the methods to improve capabilities. Recently, liquid chromatography with UV and/or scintillation counting detection has been used to study benzenemicrosomal incubation mixtures (5, 7, 8). Liquid chromatography/electrochemistry (LCEC) has been shown to be an extremely useful tool for studying biomedical problems involving the metabolism of aromatic amines and phenols (9, 10). The present report evaluates the use of LCEC for the isolation and identification of benzene microsomal metabolites. Our approach couples the preconcentration of neutral metabolites by solvent extraction with the low detection limits of LCEC to allow the determination of secondary as well as primary metabolites. Extraction efficiencies, hydrodynamic voltammetric data, and experiments employing a dual-electrode electrochemical detector are also reported.
EXPERIMENTAL SECTION The liquid chromatographic system was a Bioanalytical Systems LC-154. A Biophase CIScolumn (25 cm X 4.6 mm) was employed (Bioanalytical Systems Inc., West Lafayette, IN). Single electrode detection was achieved with a Apparatus.
0 1982 American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
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A = r n i x e d - f u n c t < o n oxidase 8 - G S H - S - a r y l transferase C .epoxide h y d r a s e
Benzene
Benzene Epoxide
\,
Phenol
Hydroquinone
Benzene Dihydrodiol
Catechol
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-02
Flgure 1.
Proposed metaibollc pathways for benzene.
Bioanalytical Systems LC-2A electrochemical detector equipped with a glassy carbon elect rode. Dual-electrde LCEC experiments were carried out with two modified BAS-LC-4A controllers. Technical details of this apparatus will be reported elsewhere (11). The dual-electrode detector cell has been previously described (12). An Altex Model 1/53analytical UV detector (254 nm) was also used. Reagents. Benzene and benzene derivatives were purchased from the following sources: benzene, catechol, and hydroquinone, J. T. Baker Chemical Co.; phenol, Mallinckrodt Inc. NADPH was purchased from P-L Biochemicals, Milwaukee, WI. All reagents were used without further purification. Procedures. Microsomal Incubations. Microsomes were prepared as previously reported (9). Benzene-microsomal incubations were performed ae follows: 400 p L of an NADPH-MgC1, solution was added to 2 mL of the microsomal-KC1-phosphate buffer suspension. The inixture was placed in the water bath for 5 min at 38 "C. A 40-pL portion of a benzene-acetone solution was then added to the incubation mixture. The incubation was allowed to proceed for 60 min. Concentrations in the incubation mixture were as followx NADPH, 0.7 mM; MgClz, 2.5 mM; benzene, 1.9 m M KC1,80 mM; pH 7.4 phosphate buffer, 21 mM; microsomal protein, 2.6 mg/mL. Microsomal protein concentrations were determined by the Lowry method (13). Benzene incubations were quenched by the addition of 2 mL of ethyl acetate to the incubation mixture, followed immediately by vortexing for 30 s. This step also served to extract neutral metabolites from the incubation mixture. 'The ethyl acetate had been deoxygenated before use by purging the solvent with nitrogen for several minutes. Benzene was added to the control incubation after quenching. After the initial extraction, 2 mg of ascorbic acid was added to the ethyl metate-microsomal mixture as an antioxidant. The mixture was then centrifuged at 2600 rpm for 10 min to facilitate separation of the organic and aqueous layers. After separation of the two layers, the extraction was repeated. Combined ethyl acetate llayers were evaporated under nitrogen at ambient temperature. The residue was reconstituted with 100 pL of 0.1 M pH 4 ammonium acetate buffer, which had been previously deoxygenated by purging with nitrogen. The reconstituted extract was centrifuged briefly before injection into the liquid chromatograph. Phenol incubations were performed in the same manner as the benzene incubations; however, a 30-min incubation period was used. The phenol concentration in the incubation mixture was 2.0 mM. Phenol incubations were quenched by the addition of 2 mL of cold methanol to the incubation mixture, followed immediately by vortexing. The quenched mixture was centrifuged at 2500 rpm for 10 min prior to injection into the liquid chromatograph. Recoveries were determined by adding known quantities of the compounds to blank microsomal preparations at 38 "C prior to the performance of the extraction procedure described above. Glassware used for pei*l'orming microsomal incubations and evaporating ethyl acetate was siliconized with Surfasil siliconizing fluid (Pierce Chemical Co., Rockford, IL). Liquid Chromatography. Pertinent experimental parameters were as follows: detector ]potential,+1.0 V vs. Ag/AgCl reference electrode, unless otherwise specified, flow rate, 1mL/min; injection volume, 20 pL. The mobile phase composition was 2% acetonitrile
06
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IO
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E,VOLTS
Flgure 2. Normalized hydrodynamic voltammograms for hydroquinone, catechol, and phenol.
I 2 nA
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