Metabolites identificaion of two bioactive constituents in Trollius ledebourii in rats using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry

Orientin and vitexin, 4'-hydroxyl-2-phenylchromen-4-one, are both major flavones derivatives found in Trollius ledebourii possessing definite pharmacological activities. In this study, in vitro metabolisms investigated on rat liver microsomes (RLMs) and in vivo metabolisms explored on Male Sprague Dawley rats of orientin and vitexin were tested, respectively. A systematic method based on ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was developed to characterize metabolites by means of electrospray ionization (ESI) mass spectrometry in positive ion mode. An on-line data acquisition method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) was developed to observe probable relevant metabolites. By comparison of chromatographic behaviors with reference substances, exact protonated ions, MS/MS fragment ions and relevant literature, a total of 12 metabolites of orientin and 23 metabolites of vitexin were detected, respectively, which suggested that orientin is more metabolically stable than vitexin. Oxidation, methylation, acetylation, reduction, loss of C6H10O5 and glucuronide conjugation were the major biotransformation routes of both of them in rats. More significant, glutamine conjugation, loss of CO and loss of CH2O were the unique metabolic pathways of vitexin compared with that of orientin for the first time. Besides, most metabolites were observed in rat urine and feces, implying that urine and feces were the active metabolic places for flavones. This is the first study on metabolisms of orientin and vitexin in vitro and in vivo simultaneously and the proposed metabolic pathways of them might provide further understanding of their pharmacological mechanisms and later study on their excretion.