反相高效液相色谱法检测丁苯酞原料药中的杂质

Detection of the impurities in the raw material of 3-n-butylphthalide by a RP-HPLC method

目的:采用反相高效液相色谱法对丁苯酞原料药中的杂质进行分析。方法:采用Agilent ZORBAX plus苯基己基色谱柱(4.6 mm×250 mm,5μm),以乙腈-0.1%(v/v)乙酸水溶液为流动相,梯度洗脱,流速1.0 mL·min^-1,柱温30℃,检测波长227 nm,进样量50μL;溶剂为30%乙腈水溶液。结果:丁苯酞及其杂质分离良好;主成分及各杂质的定量下限均小于0.05μg·mL^-1,检测下限均小于0.03μg·mL^-1;在限度浓度的10%~400%范围内,各杂质的峰面积与浓度呈良好的线性关系;同一批次样品,重复测定6次,已知杂质M峰面积的RSD(n=6)为4.5%,最大未知单杂峰面积的RSD(n=6)为0,杂质总量的RSD(n=6)为3.9%。工艺验证3批样品中均只检测到了1个已知杂质即杂质E,且杂质E含量较低。结论:该方法专属性强,灵敏度高,重复性好,能严格、有效地控制丁苯酞原料药的质量。

Objective:To analyze impurities in the raw material of 3-n-butylphthalide using a reversed-phase high performance liquid chromatography(RP-HPLC).Methods:An Agilent ZORBAX plus Phenyl-Hexyl column(4.6 mm×250 mm,5μm)was used.The mobile phase consisted of acetonitrile and 0.1%(v/v)acetic acid aqueous solution with a gradient elution at the flow rate of 1.0 mL·min^-1,the column temperature was 30℃and the detection wavelength was 227 nm.The injection volume was 50μL and the dilution solvent was 30%(v/v)acetonitrile aqueous solution.Results:3-n-Butylphthalide and its impurities were adequately separated by the established RP-HPLC method.The lower limits of quantification of all 3-n-butylphthalide and its impurities were no more than 0.05μg·mL^-1,the lower limits of detection of all 3-n-butylphthalide and its impurities were no more than 0.03μg·mL^-1.The linear calibration curves of 13 impurities were obtained within the range of 10%to 400%of their limits.The repeatability RSDs(n=6)for impurity M,single impurity and total impurities were 4.5%,0%and 3.9%,respectively.Only a few amount of one known impurity(impurity E)were detected in 3 batches process validation samples.Conclusion:The established method is characterized as high specificity,high sensitivity and good repeatability.The quality of the raw material of 3-n-butylphthalide can be controlled by this method strictly and effectively.