In Vitro Trapping and Screening of Reactive Metabolites Using Liquid Chromatography-Mass Spectrometry

Metabolism catalyzed by the cytochrome P450 enzymes (CYPs) represents the most important clearance pathways for most drugs in humans. However, CYP-mediated metabolism can also lead to drug bioactivation resulting to formation of reactive metabolites that can potentially induce idiosyncratic toxicity by covalently binding to endogenous proteins and nucleic acids. Therefore, it has become imperative to implement strategies for screening and identifying bioactivation liability of drug candidates as an integrated approach to reduce the attrition rate in drug discovery and development. This chapter describes a detailed protocol for the in-vitro stable isotopic trapping and screening for reactive metabolites using common LC-MS/MS methodologies such as neutral loss scan and precursor ion scan.