色谱 ›› 2021, Vol. 39 ›› Issue (6): 642-651.DOI: 10.3724/SP.J.1123.2020.08024

• 研究论文 • 上一篇    下一篇

非手性-手性色谱-预测多反应监测法分析中药毛前胡的化学成分

许霞, 李婷, 贾金茹, 汤慧婷, 李军, 赵云芳*(), 宋月林*()   

  1. 北京中医药大学, 中药学院中药现代研究中心, 北京 100029
  • 收稿日期:2020-08-22 出版日期:2021-06-08 发布日期:2021-04-13
  • 通讯作者: 赵云芳,宋月林
  • 作者简介:Tel:(010)64286100,E-mail: yunfang.zhao@163.com(赵云芳).
    * Tel:(010)64286100,E-mail: syltwc2005@163.com(宋月林);
  • 基金资助:
    国家重点研发计划(2018YFC1707300);国家自然科学基金项目(81773875);国家自然科学基金项目(81973444)

Analysis of chemical components of Chinese medicine Ligustici Radix by achiral-chiral liquid chromatography-predictive multiple reaction monitoring

XU Xia, LI Ting, JIA Jinru, TANG Huiting, LI Jun, ZHAO Yunfang*(), SONG Yuelin*()   

  1. Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China
  • Received:2020-08-22 Online:2021-06-08 Published:2021-04-13
  • Contact: ZHAO Yunfang,SONG Yuelin
  • Supported by:
    National Key Research & Development Program of China(2018YFC1707300);National Natural Science Foundation of China(81773875);National Natural Science Foundation of China(81973444)

摘要:

中药毛前胡为伞形科植物短片藁本Ligusticum brachylobum Franch.的干燥根,主要用于治疗风热咳嗽痰多、痰热喘满、咯痰黄稠等证,富含香豆素类化学成分,含有多组对映异构体和非对映异构体。为了深入研究毛前胡的化学成分组成,特别是对映异构体的组成,研究建立了非手性-手性色谱-预测多反应监测法(achiral-chiral-LC predictive MRM),同步实现毛前胡化学成分的化学选择性和立体选择性分离,以及高灵敏度定性分析。非手性色谱和手性液相色谱-串联质谱系统结合了RP-C18色谱柱的高效化学选择性分离能力以及手性色谱柱的立体选择性优势,有效避免了中心切割非手性-手性二维液相色谱构造复杂、重现性难以满足定量要求等缺陷。采用小内径核-壳型RP-C18色谱柱作为前端化学分离柱,实现结构类似香豆素的高效化学选择性分离;采用反相大内径AD-RH手性色谱柱,实现对映异构体的手性拆分;采用预测多反应监测模式,实现化学成分的高灵敏度检出;利用增强子离子扫描模式(EPI)采集各色谱峰的二级质谱信息,鉴定化学结构。通过定量离子对、定性离子对及两者的比值,判定是否为对映异构体。利用所构建的非手性-手性色谱耦联系统从毛前胡中共鉴定出60个化学成分,其中8对香豆素对映异构体得到了良好分离。本研究为毛前胡以及含有对映异构体中药的深入定性、定量分析提供可靠的方法。

关键词: 非手性-手性色谱, 预测多反应监测, 对映异构体拆分, 角型吡喃香豆素, 毛前胡

Abstract:

Ligustici Radix (Chinese name: maoqianhu) consists of the dried roots of Ligusticum brachylobum Franch., which is mainly distributed in the Yunnan and Sichuan provinces. This herbal medicine has been primarily used for the treatment of cough in traditional Chinese medicine. Ligustici Radix is rich in coumarin derivatives. Interestingly, enantiomers and diastereomers are widely used for these coumarins, thus posing a great challenge for in-depth chemical profile characterization. In the present study, a new analytical platform, achiral-chiral liquid chromatography-tandem mass spectrometry (achiral-chiral LC-MS/MS) was configured to profile the chemical composition of Ligustici Radix. Because achiral and chiral columns were serially coupled, especially enantiomers, both chemically and enantiomerically selective separations could be accomplished simultaneously. The newly configured achiral-chiral LC-MS/MS platform did not require any electronic valve; hence, it could overcome the drawbacks of heart-cutting achiral-chiral two-dimensional LC, i. e., sophisticated instrumentation and limited reproducibility due to the use of electronic valve(s) and the undesired retention time shift across different analytical runs. Some available candidates for chemically selective or enantiomerically selective separation were assayed; then, Capcell core RP-C18 column that was packed with core-shell type particles, and AD-RH column embedding amylose coated particles were employed the achiral and the chiral columns, respectively. The narrow-bore core-shell RP-C18 column served as the front tool to achieve efficient chemoselective separation of coumarin analogs, and enantioselective enantiomers were obtained by using a wide-bore AD-RH chiral column. The predictive multiple reaction monitoring (predictive MRM) mode allowed for the sensitive detection of potential components, and an enhanced product ion (EPI) scan, which was a unique function of Qtrap-MS, was programmed to record the MS2 spectra for all captured signals and thus aid structural annotation. Online energy-resolved mass spectrometry (online ER-MS) was introduced to pursue the suitable collision energy for each compound; in particular, inferior collision energy instead of the optimal one was utilized to suppress the response of the primary components such as praeruptorin A, B and pteryxin. The criteria to judge enantiomers or not included identical quantitative and qualitative precursor-to-product ion transitions, identical quantitative versus qualitative responses, and longer retention times from achiral-chiral LC over single-column achiral LC. As a result, a total of sixty components were observed and structurally identified. In particular, enantiomerically selective separations were achieved for eight enantiomers, cis-khellactone (CKL), qianhucoumarin G (QC-G), pteryxin (Pte), praeruptorin A (PA), cis-3'-isovaleryl-4'-acetylkhellactone (IAK), praeruptorin B (PB), praeruptorin E (PE), and cis-3',4'-diisovalerylkhellactone (DIK). Notably, none of the enantiomers were present as racemates; instead, the proportion of one enantiomer in each pair was greater than the other. Achiral-chiral LC-predictive MRM is a feasible choice for the quantitative and qualitative analyses of Ligustici Radix as well as other herbal medicines characterized by enantiomers and diastereomers.

Key words: achiral-chiral liquid chromatography (achiral-chiral LC), predictive multiple reaction monitoring (predictive MRM), enantioseparation, angular-type pyranocoumarins (APs), Ligustici Radix

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