HPLC法测定苯磺酸左旋氨氯地平片有关物质及主要杂质鉴别

目的:建立苯磺酸左旋氨氯地平片有关物质测定方法,对主要杂质进行鉴别并采用杂质对照品测定校正因子。方法:采用HPLC方法分离分析苯磺酸左旋氨氯地平片有关物质,使用Agilent XDB C₁₈(250 mm×4.6 mm,5μm)色谱柱,流动相为甲醇-乙腈-0.7%三乙胺溶液(取三乙胺7.0 mL,加水至1000 mL,用磷酸调节pH至3.0±0.1)(35:15:50),流速1.0 mL·min^-1,检测波长238 nm,柱温30℃;采用2.3 g·L^-1醋酸铵-甲醇(40:60)为流动相,利用LC-MS/MS方法进行鉴别。结果:苯磺酸左旋氨氯地平与有关物质能有效分离;LC-MS/MS鉴定了5种主要杂质;杂质D(3-乙基-5-甲基-2-[(2-氨基乙氧基)甲基]-4-(2-氯苯基)-6-甲基吡啶-3,5-二羧酸)、杂质E(二乙基-2-[(2-氨基乙氧基)甲基]-4-(2-氯苯基)-6-甲基-1,4-二氢吡啶-3,5-二羧酸)、杂质F(二甲基-2-[(2-氨基乙氧基)甲基]-4-(2-氯苯基)-6-甲基-1,4-二氢吡啶-3,5-二羧酸)、氨氯地平乳糖加合物的定量限分别为9.3、10.4、5.1、19.1 ng,且在各自的线性范围内线性关系良好(r=1.000,n=5);平均回收率(n=9)分别为113.8%、101.6%、103.4%、107.9%;校正因子分别为2.0、1.1、1.0、3.0。结论:建立的方法可对苯磺酸左旋氨氯地平片有关物质进行定量测定。

Objective:To establish a method for the determination of the related substances of levamlodipine besylate tablets and to identify the main impurities as well as to determinate calibration factor by use of impurity references.Methods:The related substances in levamlodipine besylate tablets were separated and analyzed by RP-HPLC.The column was Agilent XDB C₁₈ (250 mm×4.6 mm,5 μm);the mobile phase was methanol-acetonitrile-0.7% triethylamine (added with 7.0 mL triethylamine and diluted with water to 1 000 mL,the solution adjusted with phosphoric acid to a pH of 3.0±0.1)(35:15:50)at a flow rate of 1.0 mL·min^-1; the detection wavelength was set at 238 nm; the column temperature was 30℃.The main impurities were identified using LC-MS/MS by 2.3 g·L^-1 ammonium acetate-methanol (40:60)as the mobile phase.Results:Levamlodipine besylate and related substances were effectively separated.Five impurities were identified using LC-MS/MS.The limits of quantification of impurity D (3-ethyl-5-methyl-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methylpyridine-3,5-dicarboxylate),impurity E (diethyl-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methy-1,4-dithydrolpyridine-3,5-dicarboxylate),impurity F (dimethyl-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methy-1,4-dithydrolpyridine-3,5-dicarboxylate),and amlodipine lactose adduct were 9.3 ng,10.4 ng,5.1 ng and 19.1 ng,respectively.The calibration curves of four known impurities were linear in the self-concentration range (r=1.000,n=5).The average recovery rates (n=9)were 113.8%,101.6%,103.4% and 107.9%;RSDs were 1.3%,2.2%,1.2% and 1.9%.The calibration factors were 2.0,1.1,1.0 and 3.0.Conclusion:The methods established are accurate and effective for qualitative determination of related substances in levamlodipine besylate tablets.