Identification of the metabolites of neocnidilide in rat, monkey and human liver microsomes by liquid chromatography coupled to benchtop Orbitrap mass spectrometry

Neocnidilide, a bioactive component isolated from Angelica sinensis (Oliv.) Diels, displayed anti-inflammatory activity. The present work was performed to investigate in vitro metabolism of neocnidilide using liver microsomes. Neocnidilide (10 μM) was incubated with NADPH-supplemented rat monkey and human liver microsomes. To identify the reactive metabolites, glutathione (GSH) was included in the incubations. Liquid chromatography coupled to an Orbitrap mass spectrometer was used to detect and identify the metabolites. The structures of the metabolites were characterized by accurate masses and fragmentation patterns. A total of six hydroxylation metabolites and nine GSH conjugates were tentatively identified characterized. The metabolic pathways included hydroxylation, dehydrogenation and GSH conjugation. M6 was the major metabolite in human liver microsomes. CYP1A2 (25%), 2B6 (31.6%), 2C9 (10.5%) and 3A4 (18%) were the predominant isoenzymes governing its formation. This study provides valuable information on the in vitro metabolism of neocnidilide, which is indispensable for the further safety assessment of this compound.