Abstract: A new HPLC method for simultaneous determination of three isomers of atracuriumbesilate and its related impurities in tracrium injection(a preparation of atracurium besilate supplied byBurroughs Wellcome Co)is described on an ODS column with acetonitrile—tetrabutylammoniumchloride solution(14:86)as mobile phase. The influence of acetonitrile,tetrabutylammonium chlorideand pH on the chromatographic retention behavior of these compounds was also investigated.Gcodseparation and assay results have been obtained using the described method.