米拉贝隆有关物质的HPLC法测定

目的：建立测定米拉贝隆原料中有关物质的方法。方法：采用HPLC方法分离分析米拉贝隆的有关物质，使用Cosmosil C₁₈（250 mm×4.6 mm，5 μm）色谱柱；流动相A为10 mmol·L^-1庚烷磺酸钠+20 mmol·L^-1磷酸二氢钾溶液（用磷酸调至pH 3.0）-乙腈（90：10），流动相B为乙腈，梯度洗脱，流速1.0 mL·min^-1，检测波长220 nm，柱温35℃；采用流动相A为0.1%乙酸（用氨水调至pH 3.0）-乙腈（90：10），流动相B为乙腈，梯度洗脱，利用LC-MS/MS方法进行鉴别。结果：米拉贝隆与有关物质均能有效分离；LC-MS/MS鉴定了3种主要降解产物：杂质a（2-氨基噻唑-4-乙酸，化合物4）、杂质b（（R）-2-[[[2-（4-氨基苯基）乙基]氨基]甲基]苯甲醇，化合物8）和杂质c（2-（2-氨基噻唑-4-基）-N-[4-[2-（苯基乙基氨基）乙基]苯基]乙酰胺，化合物9）的定量限分别为7.02、7.32和9.18 ng，且在各自的线性范围内线性关系良好（r=1.000、0.999 0、1.000，n=5）；校正因子分别为1.4、1.6和1.3。结论：建立的方法可对米拉贝隆的有关物质进行定量测定。

Objective: To establish a method for the determination of the related substances in mirabegron. Methods: The related substances in mirabegron were separated and analyzed by RP-HPLC.A Cosmosil C₁₈ column was used with gradient elution at a flow rate of 1.0 mL·min^-1.The mobile phase consisted of phase A of 10 mmol·L^-1 heptane-1-sulfonic acid sodium salt and 20 mmol·L^-1 potassium dihydrogen phosphate solution (adjusted to pH 3.0 with phosphoric acid)-acetonitrile(90:10) and the mobile phase B of acetonitrile.The detection wavelength was 220 nm,and the column temperature was 35℃.The main impurities were identified using LC-MS/MS by the mobile phase A of 0.1% acetic acid(adjusted to pH 3.0 with ammonium hydroxide)-acetonitrile (90:10) and the mobile phase B of acetonitrile.Results: The related substances in mirabegron could be separated effectively with mirabegron.The main degradation products were identified using LC-MS/MS.The limits of quantification of impurity a(2-aminothiazol-4-acetic acid,compound 4),impurity b((R)α-[[[2-(4-aminophenyl) ethyl]amino]methyl]-benzenemethanol,compound 8) and impurity c(2-(2-aminothiazol-4-yl)-N-[4-[2-(phenylethylamino) ethl]phenyl]acetamide,compound 9) were 7.02,7.32 and 9.18 ng respectively.The calibration curves of the impurities were linear in the self-concentration range(r=1.000,0.999 0, 1.000;n=5).The calibration factors were 1.4,1.6 and 1.3.Conclusion: The established methods are suitable for qualitative determination of the related substances in mirabegron.