Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai, 201203, China.
University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing, 100049, China.
Phytochem Anal. 2021 Apr;32(2):183-197. doi: 10.1002/pca.2927. Epub 2020 Mar 4.
Liuwei Dihuang Pills (concentrated pills, simplified as LWDHP), one of the most famous classic Chinese Patent Medicine (CPM), is produced by hundreds of pharmaceutical manufacturers with billions of Chinese yuan (CNY) in annual sales. However, current quality identification of LWDHP mainly relies on a thin-layer chromatography (TLC) method that is complicated and deficient.
The goal of this study is to simplify the identification process and provide a more comprehensive quality assessment method of LWDHP by developing an integrated strategy based on liquid chromatography coupled with mass spectrometry (LC-MS) and multivariate statistical analysis.
Ultra-high-performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UHPLC/QTOF-MS) was employed to perform qualitative analyses of a home-made LWDHP and to establish a stably characteristic compound library by analysis of batches of its component herbs. Then selective ion monitoring (SIM) of single MS was utilised to develop a rapid identification method based on the UHPLC/QTOF-MS analysis result. Multivariate statistical analysis was subsequently used for the quality assessment of different commercial samples.
Seventy-eight characteristic compounds were characterised, and 68 of them were recorded to establish a stably characteristic compound library. Thirty-one compounds were selected from the library for the establishment of SIM identification method. Good specificity, capability, and feasibility had been respectively verified by the analysis of blank sample, negative control (NC) preparation samples, home-made LWDHP sample, and commercial sample. Multivariate statistical analysis of 20 batches of commercial LWDHP samples revealed the quality consistency of the same vendor's product and quality difference between diverse vendors' products.
The SIM identification method by a single analysis could significantly simplify the identification process of LWDHP, and it was performed in a holistic mode for no less than two compounds of each component herb monitored. Moreover, it could also be combined with multivariate statistical analysis to conduct quality assessments of batches of samples. The integrated strategy used in the study of LWDHP could be applied for the identification of other CPM as well.
六味地黄丸（浓缩丸，简称 LWDHP）是最著名的经典中药（CPM）之一，由数百家制药厂生产，年销售额达数十亿元人民币。然而，目前 LWDHP 的质量鉴定主要依赖于薄层色谱（TLC）方法，该方法既复杂又不完善。
本研究旨在通过建立基于液相色谱-串联质谱（LC-MS）和多变量统计分析的综合策略，简化鉴定过程，并提供更全面的 LWDHP 质量评估方法。
采用超高效液相色谱-四极杆飞行时间质谱（UHPLC/QTOF-MS）对自制 LWDHP 进行定性分析，并通过对其成分草药的批次分析建立稳定的特征化合物库。然后，利用单 MS 的选择离子监测（SIM），基于 UHPLC/QTOF-MS 分析结果开发快速鉴定方法。随后，采用多变量统计分析对不同商业样品进行质量评估。
鉴定出 78 种特征化合物，其中 68 种被记录下来建立了稳定的特征化合物库。从库中选择 31 种化合物建立 SIM 鉴定方法。通过空白样品、阴性对照（NC）制备样品、自制 LWDHP 样品和商业样品的分析，分别验证了该方法的特异性、能力和可行性。对 20 批商业 LWDHP 样品进行多变量统计分析，揭示了同一供应商产品的质量一致性和不同供应商产品的质量差异。
单一分析的 SIM 鉴定方法可显著简化 LWDHP 的鉴定过程，并且以 holistic 模式对每个成分草药监测不少于两种化合物。此外，它还可以与多变量统计分析相结合，对批次样品进行质量评估。本研究中用于 LWDHP 鉴定的综合策略也可应用于其他 CPM 的鉴定。
