HPLC法测定恩格列净片的有关物质

目的:建立恩格列净片有关物质测定的HPLC法。方法:采用辛烷基硅烷键合硅胶为填充剂的色谱柱(ZORBAX Rx-C8,250 mm×4.6 mm,5 μm),以pH 3.5的磷酸水溶液为流动相A,甲醇-乙腈(1:1)为流动相B,梯度洗脱,流速1.0 mL·min^-1,检测波长224 nm,柱温为25 ℃,进样量20 μL,对有关物质进行定性、定量分析。结果:在选定的色谱条件下,恩格列净与相邻杂质及各杂质之间的分离度均大于1.5;杂质IMPD(恩格列净还原产物)、杂质IMPC(恩格列净开环产物)、杂质EMGA(恩格列净中间体)、杂质IMPF(恩格列净缩合产物)、杂质IMPG(恩格列净脱葡萄糖醇产物)的定量限分别为28.30、53.07、73.60、65.00和395.57 ng,且在各自的线性范围内线性关系良好(r >0.999,n=5),平均回收率(n=9)分别为100.7%、102.9%、97.4%、102.1%和102.9%。对3批恩格列净片进行有关物质测定,结果总杂质量在0.05%~0.06%范围内。结论:经方法学验证,本法简便、灵敏,专属性好,可用于恩格列净片有关物质的测定。

Objective:To establish a method for the determination of the related substances of empagliflozin tablets.Methods:The analysis was carried out on a ZORBAX Rx-C8 chromatography column(250 mm×4.6 mm,5 μm).The mobile phase consisted of pH 3.5 phosphoric acid water solution and acetonitrile-methanol(1:1) with a gradient elution at a flow rate of 1.0 mL·min^-1 and the detection wavelength was 224 nm.The column temperature was 25 ℃,and the volume of injection was 20 μL.The related substances were qualified and quantified by the established method.Results:The resolutions were both greater than 1.5 among empagliflozin,adjacent impurities and other known impurities.The limits of impurity IMPD(empagliflozin reduction product),impurity IMPC(empagliflozin ring-opened product),impurity EMGA(empagliflozin intermediate),impurity IMPF(empagliflozin condensation product),impurity IMPG(empagliflozin deglucitol product)were 28.30,53.07,73.60,65.00,and 395.57 ng,respectively.The calibration curves of five known impurities were linear in the self-concentration range(r >0.999,n=5).The average recovery rates(n=9) were 100.7%,102.9%,97.4%,102.1% and 102.9%,respectively.The total impurities were 0.05%-0.06% in 3 batches of empagliflozin tablets.Conclusion:The established method is easy,sensitive and specific for the determination of the related substances in empagliflozin tablets according to methodology validation.