Abstract: AimTo identify the main metabolites of jatrorrhizine in rat urine. MethodsThe rat urine samples were collected 0-72 h after ig 12 mg·kg^-1 jatrorrhizine, then the samples were purified through C₁₈ solid-phase extraction cartridge. The purified samples were analyzed by combining liquid chromatography and tandem electrospray ionization ion trap mass spectrometry (LC-ESI/ITMSⁿ). Identification and structural elucidation of the metabolites were performed by comparing the changes in molecular masses, retention-times and full scan MSⁿ spectra with those of the parent drug. ResultsAt least seven phase I metabolites(such as de-methyl, de-hydrogen and hydroxyl metabolites) and eleven phase II metabolites (such as glucuronide conjugates and methyl-conjugates) were identified in rat urine. ConclusionThe developed LC-ESI/ITMSⁿ method is not only simple and rapid but also sensitive and specific for the identification of metabolites of jatrorrhizine in rat urine.