Identification of imine or enamine drug metabolites using online hydrogen/deuterium exchange and exact mass measurements

Abstract

Drug metabolites that have imine or enamine partial structures cause extra mass-to-charge (m/z) increases in online hydrogen/deuterium exchange (HDX) in addition to hydroxyl or amine protons. Online HDX and exact mass measurement were used herein to characterize this extra increase property, and to further confirm proposed metabolite structures. Metabolites of two proprietary compounds as well as two commercially available compounds were analyzed using aqueous and HDX liquid chromatography coupled with an LTQ-Orbitrap. The exact mass measurements of both the precursor ions and product ions were acquired through data-dependent acquisition and compared with theoretical values of proposed fragment ions. Analysis of exact mass measurements of metabolite product ions under both normal aqueous and HDX conditions led to the identification of the isoxazole ring opening of compound C-1, and a double-bond formation on the methylpyrrolidine ring of compound C-2 during biotransformation. In both cases, imine or enamine structures formed in the metabolites caused extra m/z increases upon HDX that contributed confirmatory information to the structure identification. The compound 3,3-diphenyl-2-ethyl-1-pyrroline also demonstrated that the methylene protons adjacent to the imine were exchanged during online HDX. The exchangeability of methylene protons adjacent to imine or enamine moieties proved to be useful to narrow down or even pinpoint the metabolism sites of parent drugs when high-resolution exact mass measurement and online HDX were used.