基于基于吸收的代谢物方法和使用极性切换UPLC-QQQ-MS/MS的快速定量发现质量标志物。
Discovery of quality markers of based on an absorption-based metabolite approach and rapid quantification using polarity-switching UPLC-QQQ-MS/MS.
作者信息
Tian Yifan, Liu Yuan, Kong Yuanlin, Li Limin, Yang Zhengming, Li Wenbin, Li Qiang, Ren Chaoqin, Sun Wenxia, Huang Yanfei
机构信息
Qinghai-Tibetan Plateau Ethnic Medicinal Resources Protection and Utilization Key Laboratory of National Ethnic Affairs Commission of the People's Republic of China, Southwest Minzu University, Chengdu, China.
Institute of Chinese Materia Medica Pharmacology and Toxicology, Sichuan Academy of Chinese Medicine Sciences, Chengdu, China.
出版信息
Front Pharmacol. 2024 Dec 6;15:1474768. doi: 10.3389/fphar.2024.1474768. eCollection 2024.
BACKGROUND
Regel (MQ) is a traditional Chinese medicine (TCM) used for clearing liver and lung heat in Tibetan medicine for over a thousand years. However, the lack of quality markers that correlate with pharmacological actions and absorption limits the safety and efficacy in its clinical application and on the drug market. Furthermore, a universal and rapid method for simultaneously determining quality markers in the MQ crude drug is still lacking.
PURPOSE
An absorption-based metabolite approach was used to discover and identify the quality markers of MQ. An efficient method based on polarity-switching ultra-performance liquid chromatography triple quadrupole mass spectrometry (UPLC-QQQ-MS/MS) was then established to determine the quality markers.
METHODS
The absorbed compounds and metabolites were first characterized using UPLC plus Q-Exactive Orbitrap tandem mass spectrometry (UPLC-Q-Exactive Orbitrap-MS) after giving oral MQ extract to rats. Subsequently, the absorbed compounds and precursors of metabolites correlating with the hepatocyte protection activity were screened as quality markers. Finally, a polarity-switching UPLC-QQQ-MS/MS method was developed for the quantitative analysis of the MQ crude drug, enabling the detection of quality markers in both negative and positive ion modes in a single run. The MS characteristics of target compounds were investigated to select appropriate product ions.
RESULTS
A total of 13 absorbed compounds and 30 metabolites were characterized. Among these, nine compounds, including five absorbed compounds and four precursors of metabolites intimately correlated with hepatocyte protection activity and absorption , were considered the quality markers of MQ in the current study. The quantification of quality markers was conducted using an Acquity UPLC HSS T3 (2.1 × 100 mm, 1.8 μm) column, the mobile phase consisting of acetonitrile and 0.1% formic acid solution (containing 10 mmol/L ammonium acetate). The validated UPLC-QQQ-MS/MS method was successfully applied to quantify the quality markers in the MQ crude drug.
CONCLUSION
We defined the quality markers and established a universal and rapid method for simultaneously determining the quality markers of MQ, which will be helpful for further investigation of the quality evaluation of MQ in clinical application and the drug market.
背景
热尔（MQ）是藏药中用于清肝肺热的一味传统中药，已有一千多年的应用历史。然而，缺乏与药理作用和吸收相关的质量标志物限制了其在临床应用和药品市场中的安全性和有效性。此外，仍缺乏一种通用且快速的同时测定MQ药材中质量标志物的方法。
目的
采用基于吸收的代谢物方法来发现和鉴定MQ的质量标志物。随后建立一种基于极性切换超高效液相色谱-三重四极杆质谱联用仪（UPLC-QQQ-MS/MS）的高效方法来测定质量标志物。
方法
给大鼠灌胃MQ提取物后，首先使用超高效液相色谱联用Q-Exactive Orbitrap串联质谱仪（UPLC-Q-Exactive Orbitrap-MS）对吸收的化合物和代谢物进行表征。随后，筛选与肝细胞保护活性相关的吸收化合物和代谢物前体作为质量标志物。最后，开发了一种极性切换UPLC-QQQ-MS/MS方法用于MQ药材的定量分析，能够在一次进样中同时在负离子和正离子模式下检测质量标志物。研究目标化合物的质谱特征以选择合适的产物离子。
结果
共鉴定出13种吸收化合物和30种代谢物。其中，9种化合物，包括5种吸收化合物和4种与肝细胞保护活性及吸收密切相关的代谢物前体，被认为是本研究中MQ的质量标志物。采用Acquity UPLC HSS T3（2.1×100 mm，1.8μm）色谱柱对质量标志物进行定量分析，流动相由乙腈和0.1%甲酸溶液（含10 mmol/L醋酸铵）组成。经过验证的UPLC-QQQ-MS/MS方法成功应用于定量分析MQ药材中的质量标志物。
结论
我们确定了质量标志物，并建立了一种通用且快速的同时测定MQ质量标志物的方法，这将有助于进一步研究MQ在临床应用和药品市场中的质量评价。
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