Metabolite identification of artemether by data-dependent accurate mass spectrometric analysis using an LTQ-Orbitrap hybrid mass spectrometer in combination with the online hydrogen/deuterium exchange technique

Abstract

Artemether (ARM), the O-methyl ether prodrug of dihydroartemisinin (DHA), is a first-line antimalarial drug used in areas of multi-drug resistance. Artemisinin drugs can be metabolized extensively in vivo and this seems related to their autoinduction pharmacokinetics. In the present study, the metabolite identification of ARM was performed by the generic data-dependent accurate mass spectrometric analysis, using high-resolution (HR) liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and tandem mass spectrometry (MS/MS) LTQ-Orbitrap hybrid mass spectrometer in conjunction with online hydrogen (H)/deuterium (D) exchange for rapid structural characterization. The LC separation was improved allowing the separation of ARM parent drugs and their metabolites from their diastereomers. A total of 77 phase I metabolites of ARM were identified in rat liver microsomal incubates and rat urine, including dihydroartemisinin and artemisinin. In rat bile, 12 phase II metabolites were found. Accurate mass data were obtained in both full scan and HR-MS/MS mode to support assignments of metabolite structures. Online H/D exchange LC/HR-ESI-MS experiments provided additional evidence in differentiating dihydroxylated deoxy-ARM from mono-hydroxylated ARM. The results showed the main phase I metabolites of artemether are hydroxylated, dehydro, demethylated and deoxy products, and they will undergo subsequent phase II glucuronidation processes. Most metabolites were reported for the first time. This study also demonstrated the effectiveness of high-resolution mass spectrometry in combination with an online H/D exchange LC/HR-MS(n) technique in rapid identification of drug metabolites.