高效液相色谱-电喷雾检测器测定熊去氧胆酸的有关物质

Determination of ursodeoxycholic acid by HPLC-CAD

目的:建立高效液相色谱-电喷雾检测器法测定熊去氧胆酸中的10个有关物质。方法:采用SHISEIDO Capcell PAK C18MGII(150 mm×4.6 mm,5μm)色谱柱,以0.1%甲酸-甲醇-乙腈(30∶45∶25)为流动相A,乙腈为流动相B,梯度洗脱,流速1.0 mL·min^(-1),柱温40℃,检测器温度35℃,采集频率5 Hz,过滤常数3.6 s。采用主成分自身对照法计算已知杂质和其它杂质的含量,并对建立的方法进行方法学验证。结果:熊去氧胆酸与各杂质分离度良好;熊去氧胆酸、杂质A、杂质B、杂质C、杂质E的质量浓度均在1.0~100.0μg·mL^(-1)范围内与峰面积呈良好的线性关系,定量限均为1.0μg·mL^(-1),检测限均为0.5μg·mL^(-1),杂质的平均回收率在99.3%~100.1%,RSD(n=9)不高于2.0%;供试品溶液在10℃条件下放置24 h内稳定;微调液相色谱参数后,对有关物质的检测结果无影响。3批样品有关物质结果显示,杂质A的含量均小于1.0%,其他单一杂质的含量均小于0.1%,杂质总量均小于1.5%。结论:本文所建立的HPLC-CAD法简便、灵敏、准确,可用于熊去氧胆酸的有关物质检测。

Objective: To establish an high performance liquid chromatography-charged aerosol detector method for the determination of the 10 related substances in ursodeoxycholic acid. Methods: A SHISEIDO Capcell PAK C_(18)MGII(150 mm×4.6 mm, 5 μm) column was adopted. The mobile phase A was composed of 0.1%formic acid-methanol-acetonitrile(30∶45∶25), and the mobile phase B was acetonitrile. The gradient elution was performed. The flow rate was 1.0 mL·min^(-1), the column temperature was 40 ℃, the detector temperature was 35 ℃, the sampling frequency was 5 Hz, and the filtration constant was 3.6 s. The contents of known impurities and other impurities were calculated by principal component self-comparison method, and the established method was validated. Results: Ursodeoxycholic acid was well separated from impurities. The mass concentrations of ursodeoxycholic acid, impurity A, impurity B, impurity C and impurity E all showed good linear relationship within the range of 1.0-100.0 μg·mL^(-1), the lower limit of quantification was 1.0 μg·mL^(-1), the lower limit of detection was 0.5 μg·mL^(-1), and the impurity recovery rates were between 99.3% and 100.1%, and the RSDs(n=9) of each recovery were less than 2.0%. The test solution was stable within 24 h after being placed at 10 ℃. Fine tuning of liquid chromatographic parameters had no effect on the detection results of related substances. The results of three batches of samples showed that the contents of impurity A were less than 1.0%, the contents of other single impurities were less than 0.1%, and the total amount of impurities were less than 1.5%. Conclusion: The established method is simple, sensitive and accurate, which can be used to determinate the related substances of ursodeoxycholic acid.