Abstract: AimTo estabilish a rapid and sensitive LC-ESI-ITMSⁿ method for the identification of ephedrine and its main metabolites in rat urine. MethodsAfter optimizing the detection condition of LC-ESI-ITMSⁿ chromatography and mass spectrometry by using a standard ephedrine, the ionization and cleavage rules of ephedrine in ESI-MS and ESI-MSⁿ modes were summarized, and then serving as the basis for the metabolite analysis of ephedrine in rat urine. Rat urine samples of 0-48 h were collected after ig 10 mg·kg^-1 ephedrine, then the samples were purified through C₁₈ solid-phase extraction cartridge. The purified samples were analyzed by LC-ESI-ITMSⁿ. ResultsThe structures of ephedrine metabolites were elucidated according to the changes of the molecular weights of the metabolites (ΔM) and their cleavage pattern in ESI-ITMSⁿ. As a result, three phase I metabolites and the parent drug ephedrine were identified existing in rat urine, but no phase II metabolites were found. ConclusionThe LC-ESI-ITMSⁿ method is rapid and highly sensitive and sepecific, it is suitable for the identification of ephedrine and its metabolites in rat urine.