N-苯甲酰基-O-苯甲酰基-L-酪氨酸及其对映异构体的手性高效液相色谱分析

Determination of N-benzoyl-O-benzoyl-L-tyrosine and its enantiomer by high performance liquid chromatography

建立了正相高效液相色谱法（NP-HPLC）测定中间体N-苯甲酰基-O-苯甲酰基-L-酪氨酸（L042）及其对映异构体N-苯甲酰基-O-苯甲酰基-D-酪氨酸（D042）含量的方法.采用CHIRALPAK AD-H手性柱,以环己烷-无水乙醇-三氟乙酸（50∶50∶0.2）为流动相,流速0.5mL·min-1,检测波长231nm,L042与其对映异构体获得良好的分离效果.在此条件下,L042、D042分别在浓度5.25～104.04μg·mL-1（n=6）,2.15～107.44μg·mL-1（n=7）范围内与峰面积呈良好的线性关系,相关系数均达到0.999 9;平均回收率分别为99.69%（RSD 1.31%）和101.12%（RSD 1.91%）;检出限分别为0.93和0.44μg·mL-1.建立了外标法测定L042含量的方法,对比了外标法及主成分自身对照法测定对映异构体D042的含量,选择节约检测成本的主成分自身对照法测定批量生产样品中的D042.该法准确、简便,适用于该中间体光学纯度的检测.

A normal phase high performance liquid chromatography（NP-HPLC）method for the determination of N-benzoyl-O-benzoyl-L-tyrosine（L042）and its enantiomer N-benzoyl-O-benzoyl-Dtyrosine（D042）is developed.Separation is achieved on a CHIRALPAK AD-H column using a cyclohexane∶absolute alcohol∶trifluoroacetic acid（50∶50∶0.2,V/V）mobile phase with the flow rate of 0.5mL·min-1.The UV detector wavelength is set at 231nm.The two enantiomers are reasonably separated.Under the condition,the standard curves are prepared with six calibrators over a concentration range of 5.25～104.04μg·mL-1 for L042and with seven calibrators over 2.15～107.44μg·mL-1 for D042.Regression equations reveal linear relationships（R =0.9999）between the peak area of each analyte and the concentration.The average recoveries are 99.69% with RSD 1.31% for L042and 101.12% with RSD1.91%for D042.The limits of detection are 0.93μg·mL-1 for L042and 0.44μg·mL-1 for D042.L042content is measured by external standard method.Compared with external standard method,the main component self-compare method has showed an advantage in terms of cost-saving feature.It has been successfully applied to analyze D042in a large number of samples.The method is accurate and simple,and is suitable for the optical purity control of the intermediate.