Analytical approaches to determine cytochrome P450 inhibitory potential of new chemical entities in drug discovery

The use of a cassette incubation of probe substrates with human liver microsomes (HLM) – also known as the ‘cocktail’ approach – is becoming a widely accepted approach to determine the interaction of new chemical entities (NCEs) with cytochrome P450 enzymes (CYP450) in early drug discovery. This article describes two LC–MS/MS-based analytical methods used at the high-throughput (HT) stage and late discovery (LD) stage for analysis of ‘cocktail’ incubates to analyze the probe metabolites 1′-hydroxymidazolam (CYP3A4), 4′-hydroxydiclofenac (CYP2C9), dextrorphan (CYP2D6), 1′-hydroxytacrine (CYP1A2) and 4′-hydroxymephenytoin (CYP2C19). The analytical methods are advantageous over currently reported methods due to their sensitivity, shorter analyses times (