Identification of in vivo and in vitro metabolites of 4,5-dimethoxycanthin-6-one by HPLC–Q-TOF-MS/MS

•A powerful HPLC–TOF/MS/MS method was employed to screen and identify the in vitro metabolites of 4,5-dimethoxycanthin-6-one and the in vivo metabolites in rat plasma and urine.•There were 8 phase I metabolites in rat and mouse liver microsomes, 7 phase I metabolites detected in dog and human liver microsomes.•3 phase I metabolites were detected in rat plasma and 5 phase I metabolites were identified in rat urine. No phase II metabolites were found in rat plasma and urine.