Abstract: A comparative study on the efficiency of separation of main co-existing impurities in HPLC analysis of vecuronium by methods given in the ministerial standards of China and in Europo-pharmacopoeia. Due to lack of vecuronium standard sample with known contents of main co-existing impurities, 5 portions of a vecuronium sample with significant amounts of the main co-existing impurities were destroyed and used as analytes in the testing. It was shown that effective separation of the main impurities was not attained by the present Chinese standards, even with optimization of the mixing ratio of the components and the pH condition of the mobile phase. However, by applying the condition of chromatographic separation given by the HPLC method of Europo-pharmacopoeia, good separation between the main peak of vecuronium and peaks of the main impurities, and by modifying the acidity of its mobile phase to pH 7.5, more effective separation was obtained.