Objective To support the pharmacokinetic study of metformin (MET) hydroehloride/pioglitazone (PIO) hydroehloride tablet, an LC-MS/MS method was developed for the simultaneous determination of MET and PIO in Beagle dog plasma.Methods The analysis was performed on a FinniganTM TSQ Quantum Discovery MAXTM LC-MS/MS system.To increase reliability,moroxydine (IS-1) and rosiglitazone (IS-2) were used as internal standards for MET and PIO, respectively.Analytes were extracted from Beagle dog plasma by one-step protein precipitation using only 50 μl plasma.Chromatographic separation was performed on Phenomenex Synergi POLAR-RP 80A (250 mm×4.6 mm, 4 μm) column.The mobile phase consisted ofacetonitrile: water with 6 mM ammonium acetate and 0.1% formic acid (50∶50, v/v) at an isoeratic flow rate of 1.0 mL/min.The LC flow was split so that approximately 0.2 mL/min entered the mass spectrometer.The total chromatographic run time was only 8.0 min with the retention time of 3.0, 2.9, 6.1 and 4.9min for MET, IS-l, PIO and IS-2, respectively.The mass spectrometer was operated in positive selected reaction monitoring (SRM) mode with ESI source.The ion transitions monitored were m/z 130.0 → 60.1 for MET, m/z 172.0 → 113.0 for IS-1,m/z 356.8 → 133.9 for PIO and m/z 357.9 → 135.0 for 1S-2.The method was validated for specificity, linearity, sensitivity, accuracy,precision, stability, and recovery and matrix effect according to the FDA guideline for bioanalytical method validation.Results No endogenous components in plasma were found to interfere with MET and PIO determination.The lower limit of quantitation (LLOQ)values were 2 and 1 ng/mL for MET and PIO, respectively.The linear ranges were 2-I000 ng/mL for MET and 1-1000 ng/mL for PIO,respectively, with an average correlation coefficient of 0.999 using weighted (1/x) linear least-squares regression.Intra-day and inter-day precision (CV, %) ranged from 3.9% to 11.8% for MET and 2.9% to 11.3% for PIO, and accuracy within ±7.6% and ±12.7%from nominal for MET and PIO, respectively.The analytes were found to be stable for at least 60 days at-80℃, 24 h at room temperature, and stable for three freeze-thaw cycles.The processed extracts were stable for 72 h at 4℃.The recoveries were from 96.4% to 112.8% for MET and 102.1% to 104.5% for PlO, respectively.And the method was successfully applied to the pharmacokinetic study of compound mefformin and pioglitazone tablets in dogs.Conclusion An improved simultaneous determination method for MET and PIO by LC-MS/MS in dog plasma was developed and validated with good performance on specificity, linearity, accuracy, precision and robustness.The method has been successfully applied to the study of phatmacokinetics of compound MET and PIO tablets in dogs.And it will also be suitable for a reliable therapeutic drug monitoring in clinical application.