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.