Abstract: The separation and determination of atracurium besilate and its three stereoisomerswere studied, Atracurium besilate and its related compounds ,and the three stereoisomers of.(±)atracurium besilate were fairly well separated with a nitrile- bonded phase column By acetonitrile-water-phosphoric acid-sulphuric acid(92 :8:0.4:0.03)mobile phase and methanol-sulphuric acid(1000 :1)mobile phase , respectively. The influence of the component concentrations of the mobilephase system on the retention and resolution was investigated. The results indicate that the methodgives good reproducibility and wide response linearity. The detection limit of atracurium besilate was10 ng.This method can be applied to the routine analysis and clinical determination of atracuriumbesilate。