Abstract: AimTo establish a LC/MS/MS method for determination of hydromorphone (HYD) in Beagle dog plasma. MethodsAfter incubation with β-glucuronidase for 16 h, an aliquot of 0.1 mL plasma was treated by liquid-liquid extraction. The analytes of interest were separated on a Zorbax SB C₈ column with the mobile phase consisting of methanol-water- formic acid (65∶35∶1). Atmospheric pressure chemical ionization source of MS was applied and operated in positive ion mode. ResultsThe linear calibration curve was obtained in the concentration range of 0.80-200.0 μg·L^-1. The lower limit of quantification was 0.80 μg·L^-1. The inter-day and intra-day precision (RSD) was below 6.0%, and the accuracy (RE) was within 1% calculated from QC samples. The method was used to determine the pharmacokinetic parameters of HYD after a single oral administration of 4 mg HYD sustained release tablets to Beagle dogs. ConclusionThe method was proved to be specific, sensitive, and suitable for the pharmacokinetic study of HYD sustained release formulation.