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N6-[(S)-1- (phenyl)-propyl]-adenine riboside (YZG-331) is being developed as a novel sedative and hypnotic agent. The hydroxylated metabolites of YZG-331 have the same mass transition ion pair, making their determination in blood challenging. In this study, a rapid and sensitive liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of YZG-331 and its metabolites M1 (hydrolysis), M2 and M4 (hydrolysis and hydroxylation), M3, M5 and M6 (hydroxylation) in monkey blood. Propranolol was used as