Determination of related substances in procaine benzylpenicillin by gradient elution of HPLC

Objective:To establish a method for the determination of related substances in procaine benzylpenicillin by using gradient elution of HPLC.Methods:C18 column was adopted;Mobile phase consisted of buffer (pH 7.0)-water-acetonitrile with gradient elution at a flow rate of 1.0 mL·min-1,and the detection wavelength was 225 nm.Results:4-Aminobenzoic acid,procaine,some impurities of benzylpenicillin and benzylpenicillin were obviously separated under the chromatographic system decribed above.Conclusion:The method is sensitive,accurate and specific.