Usefulness of the hydrogen–deuterium exchange method in the study of drug metabolism using liquid chromatography-tandem mass spectrometry

Abstract

The usefulness of the hydrogen–deuterium (H-D) exchange method in the study of drug metabolism was investigated. Metabolite samples of denopamine and promethazine prepared in vitro were introduced to a triple stage quadrupole tandem mass spectrometer via a high performance liquid chromatography (HPLC) system using a deuterated mobile phase. Mass spectra by various ionization modes and collisionally induced dissociation (CID) mass spectra were obtained. A metabolite of denopamine was observed to have a shift of 7 mass units by the H-D exchange method, and this shift proved to be a glucuronidated metabolite. Discrimination between N- or S-oxide formation and hydroxylation observed in the metabolism of promethazine was also easily accomplished by this method. In this manner, the structures of the metabolites were elucidated unequivocally by determining the number of labile hydrogen atoms by the use of the H-D exchange method, since various reactions in drug metabolism are accompanied by an increase or a decrease in the number of labile hydrogen atoms. Additional information on the structures was obtained by analysis of the CID spectra of the molecular ion species. Thus, the combination of the H-D exchange method and tandem mass spectrometry was found to be very useful for the study of drug metabolism.