HPLC波长切换法结合化学计量学对不同厂家四黄止痢颗粒中5种成分的比较分析

Comparative analysis of 5 components in Sihuangzhili granules from different manufacturers by HPLC wavelength shift combined with chemometrics analysis

关键词:  四黄止痢颗粒  (R,S)-告依春  黄芩苷  盐酸小檗碱  甘草酸铵  大黄素  波长切换  化学计量学

Key words:  Sihuangzhili granules  (R,S)-goitrin  baicalin  berberine hydrochloride  ammonium glycyrrhizinate  emodin  wavelength shift  chemometrics analysis

摘要:

建立一种四黄止痢颗粒中(R,S)-告依春、黄芩苷、盐酸小檗碱、甘草酸铵、大黄素5个指标性成分的HPLC测定方法，同时结合化学计量学评价四黄止痢颗粒的质量。采用Inertsil ODS-3-C18（4.6x250 mm，5 μm）色谱柱，以乙腈（A）-0.3%三氟乙酸水溶液（B）为流动相，梯度洗脱，流速1mL·min-1，柱温30℃；采用波长切换检测（0~10 min，245 nm测定(R,S)-告依春；10~22 min， 278 nm测定黄芩苷；22~25 min，266 nm测定盐酸小檗碱；25~30 min，249 nm测定甘草酸铵；30~45 min，254 nm测定大黄素）。采用聚类分析、偏最小二乘判别分析等方法对不同厂家的四黄止痢颗粒进行质量评价。 (R,S)-告依春、黄芩苷、盐酸小檗碱、甘草酸铵、大黄素5个指标性成分在45 min内能够达到完全分离，进样浓度分别在0.071~0.71 μg·mL-1、14.03~140.3 μg·mL-1、3.11~31.1 μg·mL-1、0.116~1.16 μg·mL-1、0.0049~0.049 μg·mL-1范围内线性关系良好；平均加样回收率分别为102.7%、99.3%、101.1%、98.0%、97.3%。20批四黄止痢颗粒聚为4类；小檗碱、(R,S)-告依春和大黄素是影响不同厂家四黄止痢颗粒质量贡献较大的3种成分。该方法稳定性好，重复性好，精密度高，可用于四黄止痢颗粒中5种指标成分的同时测定。

Abstract:

An HPLC method was developed by using wavelength shift technique combined with chemometrics for simultaneous determination of the five components, including (R,S)-goitrin, baicalin, berberine hydrochloride, ammonium glycyrrhizinate and emodin in Sihuangzhili granules. The separation was performed on an Inertsil ODS-3-C18 (4.6x250 mm, 5 μm) column with mobile phase of acetonitrile (A)-0.3% trifluoroacetic acid solution (B) in gradient elution. The flow rate was 1mL ·min-1 and column temperature was 30℃.The detection wavelength was switched for (R,S)-goitrin at 245 nm within 0~10 min, baicalin at 278 nm within 10~22 min, berberine hydrochloride at 266 nm within 22~25 min, ammonium glycyrrhizinate at 249 nm within 25~30 min, and emodin at 254 nm within 30~45 min. In addition, cluster analysis and partial least square-discriminant analysis (PLS-DA) were utilized to evaluate the quality of Sihuangzhili granules from different manufacturers based on the contents of the five components.The results showed that the five index components could be completely separated within 45 min with good linear ranges of 0.071~0.71 μg·mL-1, 14.03~140.3 μg·mL-1, 3.11~31.1 μg·mL-1, 0.116~1.16 μg·mL-1, 0.0049~0.049 μg·mL-1 for (R,S)-goitrin, baicalin, berberine hydrochloride, ammonium glycyrrhizinate and emodin, respectively. The average recoveries were 102.7%, 99.3%, 101.1%, 98.0% and 97.3%, respectively. 20 batches of Sihuangzhili granules were clustered into four groups, and the results showed that berberine hydrochloride, (R,S)-goitrin and emodin, were major components that affected the quality of Sihuangzhili granules. The method is reproducible and stable, with high precision, which could be used for simultaneous determination of the five index componentsin Sihuangzhili granules.