HPLC法测定甘草酸二铵注射液异构体含量及检测有关物质

Determination of diammonium glycyrrhizinate injection and detection of related substances by HPLC

目的:建立同时测定甘草酸二铵注射液中异构体18α-甘草酸二铵和18β-甘草酸二铵含量及检测有关物质的高效液相色谱方法。方法:采用Xbridge C_(18)色谱柱,以磷酸盐缓冲液(磷酸二氢钾6.8 g、氢氧化钾1.7 g,加水1000 mL溶解,用氢氧化钾试液调pH至7.0)为流动相A,乙腈为流动相B,含量测定采用等度洗脱,有关物质检测采用梯度洗脱,流速1.0 mL·min^(-1),柱温30℃,检测波长252 nm,进样量20μL。结果:18α-甘草酸二铵和18β-甘草酸二铵线性范围分别为34.64~103.9、15.20~45.59μg·mL^(-1)(r=0.9999);平均加样回收率(n=6)分别为101.7%、99.8%,RSD分别为1.6%、2.0%;5批样品中18α-甘草酸二铵和、18β-甘草酸二铵及其总含量分别为62.32%~76.31%、16.03%~25.06%、87.01%~92.34%。有关物质检测中,18α-甘草酸二铵和18β-甘草酸二铵两异构体峰与相邻杂质峰均能有效分离,辅料不干扰有关物质的测定,供试品溶液在42 h内稳定;18α-甘草酸二铵线性范围为0.8709~13.06μg·mL^(-1)(r=0.9999);18α、18β异构体检测限分别为3.528、3.148 ng;5批次样品共检出杂质24个,最大单个杂质含量为1.63%~2.46%;总杂质含量为6.47%~7.44%。结论:本方法准确、简便、可靠,可用于甘草酸二铵注射液的含量测定及有关物质检测。

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-C_(18)column with phosphate buffer(6.8 g of potassium dihydrogen phosphate and 1.7 g of potassium hydroxide,added 1000 mL of water to dissolve,adjusted the pH value to 7.0 with potassium hydroxide test solution)as mobile phase A,acetonitrile asmobile 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 20μL.Results:The linear ranges of 18α-diammonium glycyrrhizinate and 18β-diammonium glycyrrhizinate were 34.64-103.9 and 15.20-45.59μg·mL^(-1)(r=0.9999),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 0.8709-13.06μg·mL^(-1)(r=0.9999);the detection limits of 18αand 18βisomers 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.