LAUSR

CYP-mediated fast metabolism may lead to poor bioavailability, fast drug clearance and significant drug interaction. Thus, metabolic stability screening in human liver microsomes (HLM) followed by metabolic soft-spot identification (MSSID) is routinely conducted in drug discovery. Liver microsomal incubations of testing compounds with fixed single or multiple incubation time(s) and quantitative and qualitative analysis of metabolites using high-resolution mass spectrometry are routinely employed in MSSID assays. The major objective of this study was to develop and validate a simple, effective, and high-throughput assay for determining metabolic soft-spots of testing compounds in liver microsomes using a single variable incubation time and LC/UV/MS. Model compounds (verapamil, dextromethorphan, buspirone, mirtazapine, saquinavir, midazolam, amodiaquine) were incubated at 3 or 5 µM with HLM for a single variable incubation time between 1 and 60 min based on predetermined metabolic stability data. As a result, disappearances of the parents were around 20–40%, and only one or a few primary metabolites were generated as major metabolite(s) without notable formation of secondary metabolites. The unique metabolite profiles generated from the optimal incubation conditions enabled LC/UV to perform direct quantitative estimation for identifying major metabolites. Consequently, structural characterization by LC/MS focused on one or a few major primary metabolite(s) rather than many metabolites including secondary metabolites. Furthermore, generic data-dependent acquisition methods were utilized to enable Q-TOF and Qtrap to continuously record full MS and MS/MS spectral data of major metabolites for post-acquisition data-mining and interpretation. Results from analyzing metabolic soft-spots of the seven model compounds demonstrated that the novel MSSID assay can substantially simplify metabolic soft-spot identification and is well suited for high-throughput analysis in lead optimization.