Abstract: AimTo establish a rapid and sensitive LC-MSⁿ method for the identification of trigonelline and its main metabolites in rat urine. MethodsAfter optimizing the detection conditions of LC-MSⁿ chromatography and mass spectrometry using trigonelline, its ionization and cleavage in ESI-MS and ESI-MSⁿ modes were summarized, then serving as the basis for the metabolite analysis of trigonelline in rat urine. The 0-48 h urine samples of rats were collected after iv 8 mg·kg^-1 trigonelline, then, the samples were purified through C₁₈ solid-phase extraction cartridge. The purified samples were analyzed by LC-MSⁿ. ResultsThe structures of trigonelline 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, two phase I metabolites and the parent drug were identified existing in rat urine, and two phase II metabolites were identified. ConclusionThe LC-MSⁿ method is rapid and high sensitive and specific, it is suitable for the identification of trigonelline and its metabolites in rat urine.