佩兰的HPLC特征图谱建立及化学成分鉴定

HPLC Characteristic Chromatogram Establishment and Chemical Components ldentification of Eupatorium Fortunei

利用超高效液相色谱-四极杆-静电场轨道阱质谱(UHPLC-Q-Exactive Orbitrap MS)在正、负离子模式下采集总离子流图(TIC),根据分子精确质量数、二级谱图信息结合文献信息和化合物数据库,并进行对照品指认,对佩兰中的化学成分进行鉴定。采用Waters HSS T3(250 mm×4.6 mm,5.0μm)色谱柱,以乙腈-0.1%磷酸为流动相进行梯度洗脱,将采集的数据整理并导入“中药色谱指纹图谱相似度评价系统(2012版)”软件,建立23批佩兰的高效液相色谱(HPLC)特征图谱,对比正品与常见混伪品的特征图谱差异,以共有峰峰面积为变量采用聚类分析(HCA)进行验证。结果从佩兰中共鉴定34个化学成分,主要包括苯丙素类11个;吡咯里西啶类6个;核苷类3个;氨基酸类3个;黄酮类2个;其他糖苷、酸、碱、酮或醛类9个,通过对照品比对确认其中8个成分。23批佩兰药材的特征图谱共标定16个共有峰,相似度大于0.85,混伪品与佩兰对照特征图谱的相似度为0.452~0.746,HCA可明确对佩兰及其混伪品进行分类。所建方法的专属性、精密度、重复性、稳定性良好,成分鉴定结果可为完善佩兰药材质量控制标准提供参考,并能有效区分正品与混伪品。

A high performance liquid chromatography(HPLC)characteristic chromatogram analysis method of Eupatorium fortunei(E.fortunei)was established to differentiate it from its adulterants.Ul⁃trahigh performance liquid chromatography-Q-exactive orbitrap mass spectrometry(UHPLC-Q-Exac⁃tive Orbitrap MS)was operated under positive and negative ion mode to collect the data of total ions current(TIC).The ion source was a heated electrospray ionization(HESI)source.According to the accurate mass,fragment ions was combined with literature information,compound database and the reference substances were identified to conduct a preliminary identification of the chemical compo⁃nents in E.fortunei.The chromatographic separation was carried out on a Waters HSS T3(250 mm×4.6 mm,5.0μm)column,using acetonitrile-0.1%phosphoric acid as mobile phase by gradient elution.The collected HPLC data analyzed by a similar evaluation system for chromatographic finger⁃print of traditional Chinese medicine(version 2012,National Committee of Pharmacopoeia,China).HPLC charastersitic chromatograms of 23 batches of E.fortunei were established,facilitating a com⁃parative assessment between the characteristic chromatograms of genuine samples and its respective adulterants,and hierarchical cluster analysis(HCA)was used to verify the common peaks area as the variable.Results showed that a total of 34 chemical components were successfully identified within E.fortunei,including 11 phenylpropanoids,6 pyrrolizidine alkaloids,3 nucleosides,3 amino ac⁃ids,2 flavonoids,9 other glycoside,acid,alkali,ketone and aldehyde compounds,and the au⁃thenticity of 8 components were confirmed through comparison with the reference substance.There are 16 common peaks across the charastersitic chromatograms of 23 batches of E.fortunei and the similarities are greater than 0.85.The similarities between the chromatograms of the adulterants and the reference chromatogram of E.fortunei was 0.452-0.746.According to the area of common peaks,E.fortunei and its adulterants were clearly classified through hierarchical cluster analysis.The established HPLC characteristic chromatograms method of E.fortunei had good specificity,pre⁃cision,repeatability,stability,and components identification results not only serve as a valuable reference for quality control of E.fortunei but also prove highly effective in the reliable differentiation of E.fortunei and its adulterants,ensuring the quality and efficacy of E.fortunei is of significant im⁃portance.At the same time,this research also lays the foundation for further in-depth studies on the chemical composition and pharmacological effects of E.fortunei.