目的 建立甜叶菊高效液相色谱（high performance liquid chromatography，HPLC）指纹图谱和分析其9种成分含量的一测多评（quantitative analysis of multicomponents by single-marker，QAMS）方法，实现对甜叶菊质量的系统评价。方法 采用HPLC法建立甜叶菊指纹图谱，运用相似度评价、聚类分析（cluster analysis，CA）、主成分分析（principal component analysis，PCA）和正交偏最小二乘-判别分析（orthogonal partial least squares discriminant analysis，OPLS-DA）等化学模式识别对指纹图谱进行分析；以新绿原酸和莱苞迪苷A（rebaudioside A）为内参物，通过建立其与绿原酸、异绿原酸A、异绿原酸C、莱苞迪苷D（rebaudioside D）、莱苞迪苷M（rebaudioside M）、莱苞迪苷C（rebaudioside C）、莱苞迪苷G（rebaudioside G）等9种成分的相对校正因子和相对保留时间，建立相应的QAMS分析方法，并与外标法（external standard method，ESM）测定结果进行比较。结果 15批甜叶菊HPLC指纹图谱中有12个共有峰，指认了其中9个特征色谱峰对应的化学成分，分别为新绿原酸、绿原酸、异绿原酸A、异绿原酸C、莱苞迪苷D、莱苞迪苷M、莱苞迪苷A、莱苞迪苷C、莱苞迪苷G。15批甜叶菊相似度为0.900～1.000，CA、PCA和OPLS-DA分析表明，甜叶菊供试品可分为2类，差异性成分为峰4、峰12（莱苞迪苷G）、峰9（莱苞迪苷M）、峰6（异绿原酸A）、峰7（异绿原酸C）和峰10（莱苞迪苷A）；建立了分析所指认的9种成分含量的QAMS方法，分析结果与ESM法测定的相应成分含量无显著差异。结论 所建立的HPLC指纹图谱和QAMS法操作简便、高效、经济、可靠，可为甜叶菊及其衍生产品的质量评价提供参考和依据。

Objective A method combining high performance liquid chromatography (HPLC) fingerprint with quantitative analysis of multi-components by single-marker (QAMS) was established to analyze the contents of nine components in Stevia rebaudiana Bertoni leaves (SrL) and realize the systematic evaluation of the quality of SrL samples. Methods The HPLC fingerprint of SrL samples was explored through HPLC analysis and comprehensively evaluated through a series of analytical methods including similarity evaluation, cluster analysis (CA), principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Neochlorogenic acid and Rebaudioside A were selected as internal reference standards for the exploration of the QAMS method, which was then applied to determine the content of 9 components in SrL samples including chlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, Rebaudioside D, Rebaudioside M, Rebaudioside C, Rebaudioside D and so on. Results There were 12 common peaks in the HPLC fingerprints of 15 batches of SrL samples and 9 peaks of them were confirmed, which were neochlorogenic acid, chlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, rebaudioside D, rebaudioside M, rebaudioside A, rebaudioside C and rebaudioside G, respectively. The similarity of 15 batches of SrL samples was 0.900—1.000. According to the further calculation of the fingerprints through CA, PCA and OPLS-DA, the tested SrL samples can be categorized into two groups. The differential components among the test SrL samples were identified as peak 4, peak 12 (rebaudioside G), peak 9 (rebaudioside M), peak 6 (isochlorogenic acid A), peak 7 (isochlorogenic acid C) and peak 10 (rebaudioside A), respectively. The QAMS method for quantitative analysis of the 9 confirmed components in SrL samples was established, and there was no significant difference between the analytical results from QAMS and ESM. Conclusion The established HPLC fingerprint and QAMS method were found to be simple, efficient, economical and reliable, which can provide a reference and basis for the quality evaluation of SrL samples and the derived products.

R286.2

国家自然科学基金资助项目(81973211/C0033375); 长沙市自然科学基金资助项目(kq2402180); 湖南中医药大学“十四五”重点学科-生物工程学科(校行发规字[2023] 2号)