Systematic Strategy for Metabolites of Amentoflavone <i>In Vivo</i> and <i>In Vitro</i> Based on UHPLC-Q-TOF-MS/MS Analysis

Amentoflavone, a biflavonoid occurring in many edible supplements, possesses some bioactivities, including antioxidant, anti-inflammation, antitumor, and neuroprotective activities. In the present study, an ultrahigh-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS/MS) method, combined with a three-step analytical strategy, was employed to identify metabolites <i>in vivo</i> (rat plasma, bile, urine, and feces) and <i>in vitro</i> (rat liver microsomes and rat intestine microsomes). A total of 39 metabolites in rats and nine metabolites in rat microsomes were elucidated by UHPLC-Q-TOF-MS/MS analysis, and the chemical structure of some isomers was further assigned by calculated Clog <i>P</i> values. Oxidation, internal hydrolysis, hydrogenation, methylation, sulfation, glucuronidation, glucosylation, O-aminomethylation, and degradation were the major metabolic pathways of amentoflavone. Noteworthy, O-aminomethylation and glucosylation could be considered as unique metabolic pathways of amentoflavone. This was the first report on metabolite identification of amentoflavone <i>in vivo</i> and <i>in vitro</i>, and the metabolic findings offer novel and valuable evidence for an in-depth understanding of the safety and efficacy of amentoflavone.