Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components
Summary
We describe a general protocol and systematic design that could be applied to separate and recognize complex components in alpine yarrow herb, Achillea millefolium L., a Chinese herbal medicine.
Abstract
Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) is the most widely used method in qualitative analysis of compounds. The method includes standardized and programmed protocols for sample pretreatment, MS tune, MS acquisition, and data processing. The sample pretreatments include collection, pulverization, solvent extraction, ultrasound, centrifugation, and filtration. Data post-processing was described in detail and includes data importing, self-established database construction, method establishment, data processing, and other manual operations. The above-ground part of the alpine yarrow herb, Achillea millefolium L., is used to treat inflammation, gastrointestinal disturbances, and pain and its 3-oxa-guaianolides could be useful leads for anti-inflammatory drug development. Three representative compounds in AML were identified, combining TOF-MS with a self-established database. Moreover, the differences from existing literature, liquid-phase parameter optimization, scan mode selection, ion source suitability, collision energy adjustment, isomer screening, method limitation, and possible solutions were discussed. This standardized analysis method is universal and can be applied to identify complex compounds in Chinese herbal medicine.
Introduction
Chinese medicine has accumulated the richest empirical knowledge in the world1. Qualitative analysis of chemical components in traditional Chinese herbal medicine has become a crucial topic in research2. Distinguishing chemical differences in Chinese herbal medicine is difficult because of category complexity and origin diversification3. The major compound types in Chinese herbal medicine include alkaloids, saponins, flavonoids, anthraquinones, terpenoids, coumarins, lignans, polysaccharides, polypeptides, and proteins1. However, the separation of compounds and identification of isomers hinder the development of qualitative research on Chinese herbal medicine.
The combination of ultra-high-performance liquid chromatography (UPLC) with suitable chromatography columns provides strong support for the separation of complex compounds in Chinese herbal medicine4. In recent years, high-resolution mass spectrometry has become increasingly popular in Chinese herbal medicine qualitative analysis. Commonly used high-resolution mass spectrometry methods include quadrupole time-of-flight mass spectrometry (Q-TOF-MS)5, orbitrap mass spectrometry (Orbitrap-MS)6, and Fourier-transform ion cyclotron mass spectrometry (FT-ICR-MS)7. FT-ICR-MS has the highest resolution but entails costly operation and maintenance costs8. Orbitrap-MS has advantages in detecting small molecular compounds, especially at molecular weights below 500 Da9. Q-TOF-MS is the most widely used method in qualitative analysis of serum pharmacochemistry10,11. Compared with the traditional network database or commercial database, joint analysis with a self-established database for data processing has become increasingly popular.
Alpine yarrow herb, Achillea millefolium L. (AML), a kind of Chinese herbal medicine, grows mainly in Xinjiang, Inner Mongolia, and the northeast areas of China12. The above-ground part of AML is widely used to treat inflammation, gastrointestinal disturbances, and pain, including rheumatalgia, toothache, and stomachache13. The 3-oxa-guaianolides from AML offer great potential as leads for anti-inflammatory drug development14. Current studies on chemical components in AML focus on sesquiterpenes, monoterpenes, flavonoids, and phenolic compounds15. However, for the identification of compounds in AML, no systematic qualitative induction scheme that could used for other Chinese herbal medicines is available. This study aims to provide a standardized identification of chemical components in Chinese herbal medicine by combining Q-TOF-MS and self-established database analysis.
Protocol
Representative Results
Discussion
High-resolution mass spectrometry combined with a self-established database offers a systematic qualitative technology to identify chemical components in Chinese herbal medicine. Unlike a commercial database, which contains common traditional Chinese medicine, a self-established database that uses compounds reported in the literature provides more accuracy in the analysis of rare or ethnic medicine16. Similar methods have been applied to other areas of research, including finished drug products<su…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was funded by China Postdoctoral Science Foundation (2022MD713780), Inheritance and Innovation Team of TCM Treatment of Immune Diseases, Chongqing Medical Scientific Research Project (Joint project of Chongqing Health Commission and Science and Technology Bureau) (2022DBXM007), and the Natural Science Foundation of Chongqing (cstc2018jcyjAX0370). A special project for performance incentive and guidance of Chongqing Scientific Research Institute (cstc2022jxjl120005, cstc2021jxjl130021).
Materials
| chloroform | Sinopharm Chemical ReagentCo., Ltd | CAS 67-66-3 | |
| ethyl acetate | ChuandongChemical | CAS 141-78-6 | |
| liquid chromatograph | Waters | ACQUITY Class 1 plus | |
| MassLynx | Waters | V4.2 | MS control software |
| Methanol | ChuandongChemical | CAS 67-56-1 | |
| n-butyl alcohol | ChuandongChemical | CAS 71-36-3 | |
| petroleum ether | ChuandongChemical | CAS 8032-32-4 | |
| Quadrupole time-of-flight mass spectrometry | Waters | SYNAPT XS | |
| UNIFI | Waters | Data analysis software |
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