基于HPLC指纹图谱和多成分同时测定的护肝剂质量评价研究

Quality evaluation of Liver-protection Agent based on HPLC fingerprint and simultaneous determinations of multiple components

目的建立护肝剂的HPLC指纹图谱,并对马钱素、芍药苷、野黄芩苷、黄芩苷、黄芩素、五味子甲素、五味子乙素、五味子丙素和熊果酸9种成分同时测定,为护肝剂的质量控制提供科学依据。方法采用安捷伦Zorbax SB-C18柱(150mm×4.6 mm,5μm)色谱柱;以乙腈-0.1%磷酸水溶液为流动相,梯度洗脱;体积流量1.0 mL/min;柱温30℃;紫外检测波长236、280、210 nm;进样量5μL;建立10批护肝剂HPLC指纹图谱共有模式,并采用化学计量学方法分析,同时对其9种有效成分进行定量测定。结果建立了护肝剂的HPLC指纹图谱,标识出39个共有峰,相似度均在0.9以上。系统聚类和主成分分析将样本大致分为4类。多成分定量分析条件通过方法学考察,平均加样回收率在95.13%～104.78%;10批护肝剂中含马钱素、芍药苷、野黄芩苷、黄芩苷、黄芩素、五味子甲素、五味子乙素、五味子丙素和熊果酸的平均质量浓度分别在216.3～223.0、126.1～137.1、144.7～149.0、1623.1～1992.7、481.9～520.0、14.9～18.7、33.8～37.0、2.9～3.7、39.7～43.6 mg/L。结论 HPLC指纹图谱结合多成分同时测定的方法,快速、简便、重复性好,可作为护肝剂的质量控制方法。

Objective To establish HPLC fingerprint strategy to simultaneously determine nine components in Liver-protection Agent, including loganin, paeonflorin, scutellarin, baicalin, baicalein, deoxyschizandrin, schisandrin, schizandrin C, and ursolic acid, and provide a scientific basis for the quality control of Liver-protection Agent and related medicinal preparations. Methods The analytical analysis was performed on an Agilent 1260-HPLC system equipped with a VWD detector and SB-C18 reversed phase column(Zorbax 150 mm × 4.6 mm, 5 μm). The analytes were eluted using a gradient mixture of two solvent: solvent A was distilled deionized water containing 0.1% ortho-phosphoric acid and solvent B was 100% acetonitrile. The mobile phase flow rate was 1.0 m L/min. The separation temperature was 30 ℃. The detection wavelengths were 236, 280, and 210 nm. The injection volume was 5 μL. Common patterns of HPLC fingerprints for 10 batches of Liver-protection Agent medicinal preparations were established, and chemometric analysis method was employed to analyze the hidden information. At the same time, methodological study was conducted for determinations of multiple components including loganin, paeonflorin, scutellarin, baicalin, baicalein, deoxyschizandrin, schisandrin, schizandrin C, and ursolic acid. Results The HPLC fingerprint strategy of Liver-protection Agent medicinal preparation had been set up, and 37 common peaks had been identified with the similarity of more than 0.9. Moreover, the samples were roughly divided into four categories by the methods of the systematic cluster analysis and principle component analysis. After validating the multiple component quantitative analysis condition through methodology, the average recovery rate was between 95.13% and 104.8%. The average mass concentration of loganin, paeonflorin, scutellarin, baicalin, baicalein, deoxyschizandrin, schisandrin, schizandrin C, and ursolic acid in ten batches of Liver-protection Agent was 216.3-223.0, 126.1-137.1, 144.7-149.0,1 623.1-1 992.7, 481.9-520.0, 14.9-18.7, 33.8-37.0, 2.9-3.7, and 39.7-43.6 mg/L, respectively. Conclusion The combination methods of HPLC fingerprint and simultaneous determinations of multiple components are rapid, simple, and reproducible, which can be adopted as one of the quality control methods for Liver-protection Agent and related medicinal preparations.