Abstract: The Ultron ES-OVM (150 mm×4.6 mm，5 μm) column was used as the chiral stationary phase to establish an HPLC method for the chiral separation of clopidogrel bisulfate enantiomers and its positional isomers.The process of enantioseparation was studied by stoichiometric displacement model for retention,thermodynamic parameters,enthalpy entropy-driven process and factors that affect the chromatographic behavior.The final chromatographic conditions were established as follows:a mixture of 0.02 mol/L KH₂PO₄-acetonitrile (80∶20) as the mobile phase with the flow rate of 0.8 mL/min,the detection wavelength of 220 nm,and the column temperature of 30 °C.The resolution data of clopidogrel bisulfate enantiomers and its positional isomers was more than 1.6.The limit of quantification (LOQ) of S-clopidogrel bisulfate,R-clopidogrel bisulfate and their clopidogrel bisulfate positional isomers were 0.115,0.102,0.118 and 0.106 mg/L;the linear ranges were 0.33-20.68,0.63-40.20,0.32-20.20 and 0.31-19.28 mg/L.According to general external standard method,the recoveries of clopidogrel bisulfate enantiomer and its positional isomers were in the range of 98.2%-101.7%,RSD＜2.0%.Method validation experiments show that this method was accurate and reliable to determine the content of clopidogrel bisulfate impurity isomers.