Protocol to study in vitro drug metabolism and identify montelukast metabolites from purified enzymes and primary cell cultures by mass spectrometry

We present an optimized protocol set to study the production of drug metabolites in different in vitro systems. We detail the necessary steps to identify the metabolites of xenobiotics produced in different metabolic-competent systems, from purified enzymes to primary cell cultures. It is coupled to a high-resolution mass spectrometry analytical approach and can be adapted to study any xenobiotic. This protocol was optimized using montelukast, an antagonist of the cysteinyl leukotriene receptor 1, widely used for asthma management. For complete details on the use and execution of this protocol, please refer to Marques et al. (2022).1.