Objective: To establish an HPLC method for simultaneous determination of the isomers 18α-diammonium glycyrrhizinate and 18β-diammonium glycyrrhizinate and detection of related substances in diammonium glycyrrhizinate injection. Methods: The analysis was performed on a Xbridge-C18 column with phosphate buffer (6.8 g of potassium dihydrogen phosphate and 1.7 g of potassium hydroxide, added 1 000 mL of water to dissolve, adjusted the pH value to 7.0 with potassium hydroxide test solution) as mobile phase A, acetonitrile as mobile phase B. Isocratic elution was used for content determination, and gradient elution was used for related substance detection. The flow rate was 1.0 mL·min-1. The column temperature was set at 30 ℃, the detection wavelength was 252 nm,and the injection volume was 20 μL. Results: The linear ranges of 18α-diammonium glycyrrhizinate and 18β-diammonium glycyrrhizinate were 34.64-103.9 and 15.20-45.59 μg·mL-1(r=0.999 9), respectively; the averagerecoveries(n=6) were 101.7% and 99.8% with RSDs of 1.6%, 2.0%, respectively. The contents of 18α-diammonium glycyrrhizinate and 18β-diammonium glycyrrhizinate, and total contentsin 5 batches of samples were 62.32%-76.31%, 16.03%-25.06%, and 87.01%-92.34%, respectively. With regard the detection of related substances, the main peaks of the two isomers and adjacent impurities couldbe effectively separated. The auxiliary materials did not interfere with the detection of related substances. The test solution was stable within 42 h; the linear range of 18α-diammonium glycyrrhizinate was 0.870 9-13.06 μg·mL-1 (r=0.999 9); the detection limits of 18α and 18β isomers were 3.528 and 3.148 ng, respectively; 24 impurities were detected in 5 batches of samples, the maximum single impurity content swere 1.63%-2.46%; and the total impurity contents were 6.47%-7.44%. Conclusion: This method is accurate, simple and reliable, and can be used to detect the content and related substances of diammonium glycyrrhizinate injection.
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