A Novel High Resolution Mass Spectrometry‐Based Analytical Strategy for Simultaneous Metabolite Profiling and Standard‐Free Metabolite Quantification of Artemisinin in Human Liver Microsomes and Plasma

Metabolite quantification without a radiolabeled analogue or the reference standard is challenging. This study presented a novel high resolution mass spectrometry (HRMS)‐based analytical strategy for simultaneous metabolite profiling and standard‐free metabolite quantification of drug candidates with limited restriction on structure. The model drug was artemisinin (ART), which is widely used in clinic to treat malaria. A major hydroxylated metabolite (M1) with minor isomer M2 was first found for ART in human liver microsomes using LC‐HRMS. Second, the MS response ratio (MRR) of the hydroxylation pathway in specific biological matrix was investigated using several probe substituents (midazolam, etc.). In contrast to varying (0.5–1.9‐fold difference) MS response of probe drugs and their metabolites at equimolar concentration, the MRR ratio of the hydroxylation pathway was relatively constant (∼0.6‐fold). Third, simulated calculation curves for M1 were obtained based on the calibration curves of ART and the MRR ratios of the hydroxylation pathway. Fourth, the present analytical strategy provided reliable (< 31.7% deviation from that obtained using a validated LC‐MS technique) quantitative data on enzyme kinetics and pharmacokinetics. As a result, M1 was found to be the predominant metabolite for ART (3.6‐fold of ART exposure) in human, and another unidentified hydroxylated metabolite M2 accounted for ∼40.0% of ART exposure. The results demonstrated that the new HRMS‐based analytical strategy along with the MRR ratio of a metabolic pathway evaluated by appropriate probe substituents can be a valuable tool for the simultaneous metabolite profiling and standard‐free metabolite quantification in early drug development.