Online hydrogen-deuterium exchange and a tandem-quadrupole time-of-flight mass spectrometer coupled with liquid chromatography for metabolite identification in drug metabolism

Abstract

A method of metabolite identification in drug discovery and development utilizing online hydrogen-deuterium (H-D) exchange and a tandem-quadrupole time-of-flight mass spectrometer coupled with liquid chromatography has been developed for the first time. For purposes of evaluating this method, nimodipine is incubated with rat, dog, monkey, and human hepatic microsomes over a 60-min period. This procedure involves the replacement of H2O with D2O in the mobile phase. The equilibration time required to change (switch) from H2O in the mobile phase into D2O is approximately 10 min. There is no significant difference between the retention times in H2O or D2O mobile phase. The results show that the separations of nimodipine and its metabolites and the subsequent H-D exchanges were achieved online. This leads to the identification of nimodipine metabolites formed in vitro. Five metabolites of nimodipine are identified and characterized. These metabolites are formed by dehydrogenation, demethylation of methoxy group, cleavage of the ester groups by hydrolysis or oxidation, and hydroxylation of methyl groups. The combination of the H-D exchange process and the assay system is found to be a powerful tool for the study of metabolite identification. The method appears to be highly desirable for identification of metabolites produced by dehydrogenation, oxidation, and dealkylation, which are discussed herein. This method also greatly enhances throughput, which in turn facilitates the ability to rapidly provide metabolite elucidation.