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Alkaloids are the main bioactive ingredients derived from menispermi rhizoma (MR), which is often used in traditional Chinese medicine. However, simultaneous determination of different types of alkaloids in urine has not been reported. A sensitive, rapid, and efficient ultrahigh-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for rapid quantification of five major bioactive alkaloids (covering three different types) in rat urine has been established. Extraction of five alkaloids from rat urine was performed with acetonitrile precipitation. The separation was carried out on a C18 column, and the detection was performed with positive electrospray ionization (ESI) MS in multiple reaction monitoring (MRM) mode. The method was fully validated and successfully applied to a urinary excretion study of five alkaloids in rats after they were given an oral dose of MR extract. It is the first time that the urinary excretion profiles of dauricoside, acutumine, and acutumidine in rats has been reported. The results obtained lay the foundation for the clinical application and safety evaluation of MR. [ABSTRACT FROM AUTHOR]