丹参粉HPLC指纹图谱的建立及化学计量学方法评价

Establishment of HPLC fingerprint of Salvia miltiorrhiza powder and evaluation of chemometrics

目的:建立丹参粉高效液相(HPLC)指纹图谱,并结合多成分定量及化学计量学方法,对不同厂家丹参粉进行含量测定及质量评价。方法:采用HPLC法检测,色谱条件DiKMA C18色谱柱(4.6 mm×250 mm,5μm),乙腈-0.02%磷酸水溶液为流动相,梯度洗脱,流速为1.0 mL/min,检测波长275 nm。采用“中药色谱指纹图谱相似度评价系统(2012版)”计算相似度,SPSS 26.0进行系统聚类分析(HCA),SIMCA 14.1进行主成分分析(PCA)和偏最小二乘法判别分析(PLS-DA)。结果:共13个共有峰,相似度均在0.95以上,通过与对照品对比,共指认出4个成分:丹酚酸B(2号峰)、隐丹参酮(10号峰)、丹参酮Ⅰ(11号峰)、丹参酮ⅡA(13号峰);HCA及PCA结果显示,当平方欧氏距离为15时,23批样品可以聚为3类,S9单独为一类,S1、S4~S5聚为一类,S2~S3、S6~S8、S10~S23聚为一类;隐丹参酮对于样品在主成分1区分上荷载值较大,丹酚酸B对于样品在主成分2区分上荷载值较大;PLS-DA结果显示,4个成分中,丹酚酸B的VIP值>1.0。结论:建立丹参粉HPLC指纹图谱测定方法准确灵敏,稳定性和重复性好,化学计量法能够较好地辨识影响丹参粉质量的标志性成分,为丹参粉的质量研究提供参考。

Objective: The HPLC fingerprint of Salvia miltiorrhiza powder was established,and the content determination and quality evaluation of Salvia miltiorrhiza powder from different manufacturers were carried out with multicomponent quantitative and stoichiometry methods. Methods: HPLC method was adopted,the determination was performed on DiKMA C18 column(4.6 mm×250 mm,5 μm) with acetonitrile-0.02% phosphoric acid solution(gradient elution) as mobile phase. The flow rate was 1.0 mL/min. The detection wavelength was 275 nm. The similarity was evaluated by the ‘Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System(2012 version)’,hierarchical clustering analysis(HCA) was performed by SPSS 26.0,principal component analysis(PCA) and partial least squares discrimination analysis(PLS-DA) were performed by SIMCA 14.1. Results: There were 13 common peaks in HPLC fingerprints,the similarity was above 0.95,by comparison with the standard,four peaks were identified,salvianolic acid B(peak 2),cryptotanshinone(peak 10),tanshinone Ⅰ(peak 11),and tanshinone ⅡA(peak 13). The results of HCA and PCA showed that when the square Euclidean distance was 15,23 batches of samples could be clustered into three categories,S9 alone was one category,S1,S4~S5 were clustered into one category,S2~S3,S6~S8,S10~S23 were clustered into the other category. Loading score of PC1was higher for cryptotanshinone,while loading score of salvianolic acid B was higher for PC2. PLS-DA analysis showed that the VIP value of salvianolic acid B was greater than 1.0. Conclusion: The established fingerprint method for the determination of Salvia miltiorrhiza powder is accurate,sensitive,stable and reproducible,and chemometrics is good enough to identify the landmark components affecting the quality of Salvia miltiorrhiza powder,which provides reference for the quality research of Salvia miltiorrhiza powder.