基于指纹图谱分析和多成分同时定量的护肝片质量评价研究

目的：建立护肝片的HPLC指纹图谱，并测定其主要成分的含量，为护肝片的质量控制提供可靠方法。方法：采用Agilent Zorbax SB-C₁₈色谱柱（250 mm&#215;4.6 mm，5 μm），以乙腈（A）-0.05%磷酸水溶液（B）为流动相，梯度洗脱（0~6 min，6% A；6~9 min，6% A→40% A；9~30 min，40% A→50% A；30~45 min，50% A；45~65 min，50% A→75% A），流速1.0 mL&#183;min^-1，检测波长250 nm，柱温30℃；建立护肝片HPLC指纹图谱，并对（R，S）-告依春、柴胡皂苷a、五味子醇甲、柴胡皂苷d、五味子酯甲、五味子甲素和五味子乙素的含量测定方法进行方法学考察。结果：在指纹图谱研究中，共确定护肝片HPLC指纹图谱20个共有峰，通过与混合对照品比较，指认其中7个指标成分，分别是（R，S）-告依春（1号峰）、柴胡皂苷a（3号峰）、五味子醇甲（5号峰）、柴胡皂苷d（6号峰）、五味子酯甲（8号峰）、五味子甲素（17号峰）和五味子乙素（20号峰），利用相似度软件对23批样品指纹图谱进行分析，各批样品相似度均在0.95以上。在建立的色谱条件下测定7个成分，分离度良好，方法精密度和重复性的RSD均<1.5%，供试品溶液在24 h内稳定，各成分具有良好的线性关系和较宽线性范围；6批护肝片中（R，S）-告依春、柴胡皂苷a、五味子醇甲、柴胡皂苷d、五味子酯甲、五味子甲素和五味子乙素质量分数分别为0.07~0.12、0.13~0.20、0.51~0.58、0.06~0.13、0.07~0.14、0.10~0.17和0.18~0.23 mg&#183;片^-1。结论：所建立的护肝片HPLC指纹图谱检测和定量测定分析方法快速、准确，可以有效地评价护肝片的质量。

Objective: To establish the HPLC fingerprint of Hugan tablets and determine the main components,so as to provide a reference for the quality control.Methods: The analysis was carried out on an Agilent Zorbax SB -C₁₈ column(250 mm&#215;4.6 mm,5 μm) with gradient elution of acetonitrile(A)-0.05% phosphoric acid aqueous solution(B)(0-6 min,6%A;6-9 min,6%A→40%A;9-30 min,40%A→50%A;30-45 min,50%A; 45-65 min,50%A→75%A) at a flow rate of 1.0 mL&#183;min^-1. The detection wavelength was 250 nm and the column temperature was 30℃. The HPLC fingerprint was established,and determination method of(R, S)-epigoitrin, saikosaponin a,schisandrin,saikosaponin d,schisantherin,deoxyschizandrin and schisandrin B was studied methodologically. Results: 20 common peaks were found in the fingerprint of Hugan tablets,7 of which were identified by comparison with the reference as(R, S)-epigoitrin(peak 1),saikosaponin a(peak 3),schisandrin(peak 5),saikosaponin d(peak 6),schisantherin(peak 8),deoxyschizandrin(peak 17) and schisandrin B(peak 20). The similarities of 23 batches of samples were above 0.95. The 7 compounds were well separated under the established chromatographic conditions. The RSD values for precision and repeatability tests were all less than 1.5% and the sample solution was stable during 24 h. All the compounds had a good linearity and wide linear range. The contents of(R, S)-epigoitrin,saikosaponin a,schisandrin,saikosaponin d,schisantherin,deoxyschizandrin and schisandrin B in 6 batches of Hugan tablets were 0.07-0.12 mg,0.13-0.20 mg,0.51-0.58 mg,0.06-0.13 mg,0.07-0.14 mg,0.10-0.17 mg tablet and 0.18-0.23 mg per tablet,respectively. Conclusion: The method is fast and accurate,and can be used for the quality control of Hugan tablets.