Abstract
The metabolic fate of the emerging drug candidate S107, possessing the potential for misuse as performance‐enhancing agent in sports, was investigated by in vitro phase I and II experiments with human microsomal and S9 liver enzymes. The metabolites were identified by liquid chromatography‐mass spectrometry with electrospray ionisation in positive mode (LC‐ESI–MS/MS). Their collision‐induced dissociation behaviour was studied by high‐resolution/high accuracy Orbitrap MS^n^ analysis, supported by stable isotope labelling, H/D‐exchange experiments and density functional theory calculations. Monooxygenation accounted for the main phase I metabolic transformation due to N‐ and S‐oxidation of the 1,4‐benzothiazepine core, as substantiated by chemical synthesis, selective reduction methods and characteristic APCI in source fragmentation behaviour of the metabolites. Another dominant metabolic pathway was demethylation, yielding the N‐ and O‐demethylated metabolite, respectively. The latter was further conjugated by glucuronidation as well as sulfonation in subsequent phase II metabolic reactions, whereas the N‐demethylated metabolite was not amenable to conjugation. The active drug molecule itself was converted to two glucuronic acid conjugates, which are proposed to consist of two quaternary S107‐N^+^‐glucuronide isomers. All glucuronides were susceptible to enzymatic hydrolysis with β‐glucuronidase (Escherichia coli). A comprehensive LC‐ESI–MS(/MS)‐based detection method for urine was developed and its fitness for purpose was assessed. The assay can serve as a potential screening and/or confirmation method for S107 in clinical drug testing and doping control analysis in the future. Copyright © 2011 John Wiley & Sons, Ltd.