OBJECTIVE To establish HPLC methods with different separation principles to analyze the relevant impurities in the APIs of bivalirudin from seven enterprises,to provide a basis for the comprehensive control of related substances of bivalirudin.METHODS Reversed-phase high performance liquid chromatography(RP-HPLC)was used to separate and analyze 11 kinds of impurities.Hydrophilic chromatography(HILIC)-HPLC was used to control four process impurities.Polymers were determined by size exclusion chromatography(SEC)-HPLC.RESULTS The established RP-HPLC could effectively separate the principal component and 11 impurities,the correction factors of 11 impurities were between 0.8-1.2,the detection concentration of bivalirudin was 0.1 μg·mL-1,and the detection limit was 0.004％.The established HILIC-HPLC could effectively separate the principal components and four process impurities,and the detection concentration of bivalirudin was 0.3 μg·mL-1,and the detection limit was 0.01％.Under SEC-HPLC conditions,the polymer and bivalirudin peaked sequentially,the resolution of the two was 2.9,the detection concentration of bivalirudin was 6 ng·mL-1,and the detection limit was 0.000 6％.Fifteen kinds of known impurities and polymers in 15 batches of samples from 7 enterprises were calculated by the self-control method of principal components,and the impurity contents from different enterprises had a certain correlation with their production processes.CONCLUSION The three different principles of the method have good specificity,high sensitivity,good durability,and reliable results,and can be used for quality control of substances related to bivalirudin.
维普
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