Wu Lijuan, Wang Qian, Sun Runze, Liu Minghao, Jin Hui, Jia Jinru, Zhang Ke, Zhang Qian, Song Yuelin, Tu Pengfei, Zhao Yunfang
Modern Research Center for Traditional Chinese Medicine, Beijing Research Institute of Chinese Medicine, Beijing University of Chinese Medicine, Beijing 102401, China; School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102401, China.
SCIEX China, Beijing 100015, China.
J Chromatogr A. 2025 Aug 30;1757:466113. doi: 10.1016/j.chroma.2025.466113. Epub 2025 Jun 3.
Multi-components-targeted determination serves as the workhorse for quality evaluation of herbal medicines (HMs). However, this pipeline is extensively hampered by the lack of authentic compounds. Here, our efforts were devoted to circumventing this barrier by incorporating chromatographic fractionation that is exactly advantageous at chromatographing HMs extract into relatively simple mixtures and quantitative H NMR (qH-NMR) that enables direct determination of the content features in the resultant mixtures. Primary components-focused determination was conducted for Salvia miltiorrhiza, one of the most famous HMs, for applicability justification. The entire extract was fractionated into sixteen fractions by applying appropriate program to a home-made automated fraction collection device, and qH-NMR spectroscopy was deployed to identify and determine the contents of primary compounds, fifteen ones in total, in all fractions. After excluding multi-fraction distributions, thirteen fractions containing analytes-of-choice were mixed to act the pseudo-mixed standard solution role. Achiral and chiral columns were serially coupled to achieve satisfactory separation of fifteen targeted compounds, particularly methylenetanshinquinone vs. 1,2-dihydrotanshinone I. Tailored selected-reaction monitoring (tSRM) was applied to achieve content-extended measurement by specially assigning inferior collision energy to the abundant analytes. After method validation assays, achiral-chiral LC-tSRM was applied for simultaneous determination of nine phenolic acids along with six tanshinone analogues in S. miltiorrhiza roots collected from different habitats. As a result, habitat-dependent quantitative profiles occurred. Together, the incorporation of automated fractionation and qH-NMR is feasible for authentic compound-free quantitative analysis, and moreover, achiral-chiral LC-tSRM is eligible for content-extended quantitative measurement of HMs rich in isomers.
多成分靶向测定是中药材质量评价的主要方法。然而，由于缺乏正品化合物，该流程受到了极大的阻碍。在此，我们致力于通过结合色谱分离和定量核磁共振氢谱（qH-NMR）来克服这一障碍。色谱分离在将中药材提取物分离成相对简单的混合物方面具有优势，而qH-NMR能够直接测定所得混合物中的含量特征。以丹参为例进行了主要成分聚焦测定，以验证其适用性。通过将适当的程序应用于自制的自动馏分收集装置，将丹参全提取物分离成16个馏分，并采用qH-NMR光谱法鉴定和测定所有馏分中总共15种主要化合物的含量。在排除多馏分分布后，将13个含有目标分析物的馏分混合，充当伪混合标准溶液。将非手性柱和手性柱串联使用，以实现15种目标化合物的满意分离，特别是亚甲基丹参醌与1,2-二氢丹参酮I的分离。采用定制的选择反应监测（tSRM），通过为丰富的分析物专门设定较低的碰撞能量来实现含量扩展测量。经过方法验证试验后，采用非手性-手性液相色谱-选择反应监测（LC-tSRM）同时测定了来自不同产地的丹参根中9种酚酸和6种丹参酮类似物。结果显示了产地依赖性的定量特征。总之，结合自动馏分收集和qH-NMR可用于无正品化合物的定量分析，此外，非手性-手性LC-tSRM适用于对富含异构体的中药材进行含量扩展定量测量。
