Vol. 30, Issue 3, 270-275, March 2002

Characterization by Liquid Chromatography-Nuclear Magnetic Resonance Spectroscopy and Liquid Chromatography-Mass Spectrometry of Two Coupled Oxidative-Conjugative Metabolic Pathways for 7-Ethoxycoumarin in Human Liver Microsomes Treated with Alamethicin

Michael B. Fisher,¹ David Jackson, Andreas Kaerner, Steven A. Wrighton, and Anthony G. Borel

Department of Drug Disposition (M.B.F., D.J., S.A.W., A.G.B.) and Pharmaceutical and Analytical Development Division (A.K.), Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana

The microsomal metabolism of 7-ethoxycoumarin (7-EC) was investigated using liquid chromatography (LC)-NMR and liquid chromatography-mass spectrometry (LC-MS) to characterize the coupling of oxidative-conjugative metabolism events. Within microsomes, cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs) are spatially disparate, each having surface and luminal localization, respectively. To optimize cofactor and substrate transit to UGT without compromising P450 activity, the pore-forming peptide alamethicin was used for microsomal perforation. Aqueous extracts of microsomal incubations containing NADPH and UDP-glucuronic acid were injected for LC-NMR and LC-MS analysis. The analytical complementarity of LC-NMR and LC-MS permitted the identification of four metabolites (M1 to M4). The metabolites M1 and M2 are novel microsomal metabolites for 7-EC, consistent with 3-hydroxylation and subsequent glucuronidation, respectively. Metabolites M3 and M4 were 7-hydroxycoumarin (7-HC) and 7-HC glucuronide, respectively. Viewed collectively, these results illustrate the utility of alamethicin in the examination of coupled oxidative-conjugative metabolism and the synergy of LC-NMR and LC-MS in metabolite identification.