High-throughput quantitative and qualitative analysis of microsomal incubations by cocktail analysis with an ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometer system.

Abstract

Metabolite identification studies are very resource intensive and also are rarely performed in early discovery. Here, we report the validation of an ultraperformance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) platform for generating high-throughput stability data with structure elucidation in a single injection. Tandem mass spectrometry spectra were obtained for quantitative analysis using a generic information-dependent acquisition method from pooled microsomal samples incubated at low compound concentrations. A good correlation was observed between clearance determined using UPLC-HRMS and UPLC-triple-quadrupole analysis. Structural elucidation performed with MassMetaSite™ (Molecular Discovery, Perugia, Italy) software identified 85% of the major metabolites of eight marketed drugs and over 100 internal compounds under these conditions. For the first time, a high-throughput quantitative-qualitative workflow was established using a cocktail approach for sample analysis with UPLC-HRMS in order to enable metabolite identification in early discovery projects.