基于HPLC特征图谱及多成分同时定量的热炎宁颗粒质量评价研究

Quality evaluation of Reyanning granules based on HPLC characteristic chromatogram and simultaneous determination of multiple components

目的:建立热炎宁颗粒HPLC特征图谱和多成分同时定量的分析方法,为评价其质量提供依据。方法:采用Welch Ultimate Phenyl-Ether色谱柱(250 mm×4.6 mm,5μm),以乙腈(A)-0.15%磷酸水溶液(B)为流动相,梯度洗脱,流速为1.0 mL·min^(-1),柱温为30℃,检测波长为290 nm,进样量10μL。采用"中药色谱指纹图谱相似度评价系统(2012年版)"进行相似度评价,结合聚类分析(CA)、主成分分析(PCA)以及正交偏最小二乘-判别分析(OPLS-DA)对热炎宁颗粒进行质量评价。结果:建立了热炎宁颗粒的HPLC特征图谱,共标定了15个共有峰,并指认了其中6个共有峰,分别为单咖啡酰酒石酸、虎杖苷、野黄芩苷、菊苣酸、白藜芦醇、大黄素;16批样品相似度为0.957~0.992。CA、PCA将样品分为3类,结合OPLS-DA发现导致不同批次样品之间差异性的6个标志性化合物。定量分析的4个成分(虎杖苷、野黄芩苷、白藜芦醇、大黄素)分离度>1.5,精密度、稳定性(30 h)和重复性试验的RSD均<2.2%,平均加样回收率在96.4%~98.2%之间;16批热炎宁颗粒中虎杖苷、野黄芩苷、白藜芦醇、大黄素的含量范围分别为2.516~6.190、0.4546~1.950、0.2258~0.6297、0.1830~0.7135 mg·g^(-1)。结论:所建立的HPLC特征图谱及多成分同时定量的方法准确、重复性好,可为热炎宁颗粒的质量控制和评价提供依据。

Objective:To establish the HPLC characteristic chromatogram and simultaneous determination method of multi-components of Reyanning granules,so as to provide the basis for effective quality evaluation.Methods:The analysis was performed on Welch Ultimate Phenyl-Ether(250 mm×4.6 mm,5μm)column with mobile phase consisting of acetonitrile and 0.15%phosphoric acid at a flow rate of 1.0 mL·min^(-1)with gradient elution.The column temperature was 30℃.The detection wavelength was set at 290 nm,and the injection volume was 10μL.The similarity of Reyanning granules was evaluated by the Traditionai Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System(2012 edition).Then,the further quality evaluation of the drug was carried out by cluster analysis(CA),principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA).Results:The HPLC characteristic chromatogram of Reyanning granules was established and 15 common peaks were found,6 of them were indentified as monocaffeyltartaric acid,polydatin,scutellarin,chicoric acid,resveratrol and emodin,respectively.The similarities of 16 batches of samples were 0.957-0.992.The samples were classified into three categories by CA and PCA,and six components were main markers that caused differences in the different batches of samples by OPLS-DA.The resolution of 4 components of quantitative qnalysis(polydatin,scutellarin,resveratrol and emodin)was more than 1.5,the RSDs of precision,stability(30 h)and reproducibility tests were lower than 2.2%,and the average recovery rates of four compounds were between 96.4%and 98.2%.The contents of polydatin,scutellarin,resveratrol and emodin in 16 batches of Reyanning granules were 2.516-6.190,0.4546-1.950,0.2258-0.6297 and 0.1830-0.7135 mg·g^(-1),respectively.Conclusion:The method of HPLC characteristic chromatogram analysis and multi-index components determination was accurate and reproducible,which could be used for the quality control and evaluation of Reyanning granules.