Liquid chromatography/quadrupole time‐of‐flight mass spectrometry in combination with online hydrogen/deuterium exchange technique for structural elucidation of phase I metabolites of iso‐phenylcyclopentylamine in rat bile

Abstract

MS/MS experiment and accurate mass measurement are powerful tools in metabolite identification. However, sometimes these data do not provide enough information to assign an unambiguous structure to a metabolite. In combination with MS techniques, hydrogen/deuterium (H/D) exchange can provide additional information for structural elucidation by determination of the number of exchangeable hydrogen atoms in a structure. In this study, the principal phase I metabolites of iso-phenylcyclopentylamine in rat bile were identified by high-performance liquid chromatography with electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS). Since N-oxidation may occur because of the existence of the primary amino group in the structure, it was difficult to differentiate the hydroxylated metabolites from N-oxides by ESI-Q-TOF-MS alone. Therefore, online H/D exchange technique was applied to solve this problem. Finally, 25 phase I metabolites were detected and structurally described, in which 11 were confirmed to be N-oxides. This study demonstrated the effectiveness of high-resolution mass spectrometry in combination with an online H/D exchange technique in rapid identification of drug metabolites, especially in discriminating hydroxylated metabolites from N-oxides.

Online Simultaneous Hydrogen/Deuterium Exchange of Multitarget Gas-Phase Molecules by Electrospray Ionization Mass Spectrometry Coupled with Gas Chromatography

“…This method can be implemented using both on-column and postcolumn techniques. On-column H/D exchange MS has been performed with a deuterated solvent as a mobile phase in liquid chromatography–mass spectrometry (LC–MS) − ,− or capillary electrophoresis mass spectrometry (CE-MS) . However, this method suffers from high consumption of an expensive deuterated solvent, such as D 2 O, and shifts in chromatographic retention times due to the different mobile phase–analyte–stationary phase interactions caused when two different mobile phases, such as H 2 O and D 2 O, are used.…”