Development and preliminary application of a high-performance liquid chromatographic assay for omeprazole metabolism in human liver microsomes

Abstract

A high-performance liquid chromatography assay was developed to measure the enzymatic activities of the 5-hydroxylation and sulphoxidation of omeprazole, a proton pump inhibitor, in human liver microsomes. The preliminary study was also undertaken to assess the assay's applicability for the enzyme kinetic analysis of omeprazole metabolism by human liver microsomes. The recovery of 5-hydroxyomeprazole, omeprazole sulphone and phenacetin (internal standard) after the precipitation of microsomal protein by acetonitrile was nearly complete. The intra-assay relative standard deviations (n = 6) were 8.2 and 12.8% for quantitation of the 5-hydroxylation and sulphoxidation activities of omeprazole, respectively. Eadie-Hofstee plots for the formation of 5-hydroxyomeprazole and omeprazole sulphone gave a biphasic relationship for all the microsomal samples studied (n = 6). The respective mean (+/- SD) high- and low-affinity component kinetic parameters for the 5-hydroxylation and sulphoxidation of omeprazole estimated by a two-enzyme kinetic analysis were: Km1 = 6.3 +/- 1.7 and 10.4 +/- 6.3 microM, Km2 = 183.2 +/- 180.4 and 671.2 +/- 639.4 microM, Vmax1 = 109.8 +/- 75.4 and 77.5 +/- 46.1 pmol mg-1 min-1, and Vmax2 = 163.3 +/- 94.1 and 318.3 +/- 163.3 pmol mg-1 min-1. The results suggest that the assay is reproducible, accurate and applicable for studying the metabolism of omeprazole in human liver microsomes.