米拉贝隆有关物质的HPLC法测定

Determination of related substances in mirabegron by HPLC

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,5μm)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p H 3.0)-乙腈(90∶10),流动相B为乙腈,梯度洗脱,流速1.0 m L·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p H 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-l-sulfonic acid sodium salt and 20 retool. 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 separatedeffectively 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 ） - [ [ [ 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=l.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.