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目的:采用衍生化为辅助手段,建立高效液相色谱法测定缬沙坦起始物料L-缬氨酸甲酯盐酸盐中杂质L-异亮氨酸甲酯盐酸盐含量的方法。方法:采用岛津VP-ODS C18色谱柱(150 mm×4.6 mm,5μm);以乙酸钠缓冲液(pH 7.2)-甲醇-乙腈(20∶45∶30)为流动相,流速为1.0 mL.min-1;检测波长为262 nm;柱温为40℃。采用9-芴基甲酰氯为衍生化试剂,考察衍生化试剂浓度,反应静置时间,超声时间等衍生化条件,确定衍生化的方法并进行方法学验证。结果:衍生化反应条件为:衍生化试剂浓度10 mg.mL-1,反应静置时间20 min,超声10 s。经方法学验证,L-异亮氨酸甲酯盐酸盐在15～75μg.mL-1范围内线性良好,其平均回收率为92.943%,RSD为4.965%,溶液在5 h内稳定性。结论:该方法简便、快速、耐受性好,符合有关物质方法学验证要求,可用于杂质L-异亮氨酸甲酯盐酸盐的含量测定。
OBJECTIVE: To establish a HPLC method for the determination of L-isoleucine methyl ester hydrochloride in L-valine methyl ester hydrochloride, the starting material of valsartan, using derivatization as an auxiliary measure. METHODS: Shimadzu VP-ODS C18 column (150 mm × 4.6 mm, 5 μm) was used. The mobile phase was sodium acetate buffer (pH 7.2) -methanol-acetonitrile (20:45:30) with a flow rate of 1.0 mL. min-1; detection wavelength was 262 nm; column temperature was 40 ℃. Using 9-fluorenylformyl chloride as derivatization reagent, the derivatization conditions such as concentration of derivatization reagent, reaction standing time and ultrasonic time were investigated, and the derivatization method was determined and verified by methodology. Results: The derivatization conditions were as follows: the concentration of derivatization reagent was 10 mg.mL-1, the reaction was allowed to stand for 20 min and sonicated for 10 s. The method validation, L-isoleucine methyl ester hydrochloride in the range of 15 ~ 75μg.mL-1 good linearity, the average recovery was 92.943%, RSD was 4.965%, the solution within 5h stability. Conclusion: The method is simple, rapid and well tolerated. It meets the requirements of validation of the relevant material methodologies and can be used for the determination of impurity L-isoleucine methyl ester hydrochloride.