HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ASSAY FOR HUMAN LIVER MICROSOMAL OMEPRAZOLE METABOLISM

Assays for the measurement of omeprazole metabolites in plasma and uri ne have been reported. but when applied to the determination of omepra zole metabolites formed by human liver microsomal incubations there we re obvious limitations in sensitivity. The present high-performance li quid chromatographic (HPLC) assay, which comprises extraction, evapora tion and reconstitution, several-fold more sensitive with a limit of d etection of approximately 2 pmol (2 nM in incubate) for omeprazole sul phone and 25 pmol (25 nM in incubate) for hydroxyomeprazole. Extractio n efficiency is essentially quantitative and is highly reproducible (c oefficient of variation = 2.1 % for both metabolites). The assay is li near over a wide range of concentrations and the formation of the meta bolites is linear with respect to both time (to 15 min) and protein co ncentration (to 1.5 mg/ml). Two minor metabolites, one of which was id entified tentatively as 5-O-desmethylomeprazole, were also formed by h uman liver microsomes and could be determined by this method. Prelimin ary studies of the formation of omeprazole sulphone and hydroxyomepraz ole showed that the formation kinetics in human liver microsomes were biphasic for both metabolites, suggesting that at least two different cytochrome P450 isoforms are involved in their formation.