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 identificati....

Protocol

1. Sample pretreatment

1. Collection of Chinese herbal medicine AML
   1. Plant alpine yarrow Herb, Achillea millefolium L. (AML) seeds in the ground in February. Collect the above-ground part of AML in July of the same year (Figure 1A).
      NOTE: AML used in this paper was collected in a mountainous area at an altitude of 400 m in Mianyang, Sichuan, China.
2. Drying treatment
   1. Wash all the collected AML in pure water to remove sediment and impurities. Dry the AML in an oven at 50 °C for 24 h (Figure 1B).

Results

Alpine yarrow herb was used as a model to display the representative result. As shown in Figure 4G, quercetin-3'-O-glucoside with m/z = 463.08935 transformed into an intermediate with m/z = 300.02828 via loss of a hexose molecule during the hydrolysis reaction. In another pathway, the break of the C-C bond in the flavonoid structure skeleton led to the formation of an intermediate with m/z = 223.06232, where hydroxymethyl and adjacent hydroxy in the hexose unit disappeared. Moreover, the cle.......

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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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