柱前衍生-高效液相色谱法检测安立生坦的手性拆分剂L-脯氨酸甲酯

采用4-氯-7-硝基苯-2-（嗯）-1,3-二唑（NBD-Cl）作为柱前衍生试剂,建立了柱前衍生化-高效液相色谱法（HPLC）结合荧光检测器测定安立生坦合成过程中使用的手性拆分剂L-脯氨酸甲酯的方法.考察了衍生反应温度、时间、衍生试剂加入量、缓冲液pH对L-脯氨酸甲酯衍生反应的影响.结果表明,衍生反应优化条件为：反应温度60℃,反应时间60 min,衍生试剂加入量为每毫升反应体系中加入0.3 mg NBD-Cl,衍生反应缓冲液pH为9.0.同时对此方法进行了方法学验证,L-脯氨酸甲酯在0.625～7.5 μg/mL范围内线性关系良好,r=0.999 4;检测限为2.5×10-3μg/mL,定量限为7.5×10-3μg/mL;平均回收率为91.9％～ 106.4％;重复性实验中RSD为0.33％;此方法耐用性良好,L-脯氨酸甲酯样品溶液及衍生产物溶液体系稳定.此方法灵敏、简便、专属性强,能对安立生坦原料药中残留的杂质L-脯氨酸甲酯进行检测及定量分析.

Pre-column derivatization-HPLC method for determination of L-proline methyl ester as chiral resolving agent of Ambrisentan

A pre-column derivatization high performance liquid chromatographic method with fluorescence detector for the determination of L-proline methyl ester as chiral resolving agent of Ambrisentan was developed by using 4-chloro-7-nitrobenzo-2-oax-1,3-diazole（NBD-Cl） as derivative reagent.Effects of temperature,time,NBD-Cl concentration,pH of borate buffer in the derivative reaction were investigated.The result showed that optimum condition of derivative reaction were as follows：0.3 mg/mL of NBD-Cl with borate buffer（pH =9.0） for 60 min at 60 ℃,the method was linear in the range of 0.625 ～7.5 μg/mL,the limit of detection was 2.5 × 10-3 μg/mL,the limit of quantitation was 7.5 × 10-3 μg/mL.The average recovery rate was 91.9％ ～ 106.4％ and RSD in the repeatability test was 0.33％.The ruggedness of the method was verified.The sample solvent was stable as well as the reaction system during detection.The results indicated the method showed the advantages of high sensitivity,convenience and good specificity,and could be applied in the determination of L-proline methyl ester in Ambrisentan.