2D-HPLC-MS Technology Combined with Molecular Network for the Identification of Components in Tibetan Medicine Aconitum pendulum

Summary

This study utilizes two-dimensional high-performance liquid chromatography-mass spectrometry (2D-HPLC-MS) technology in conjunction with molecular networking to unravel the intricate chemical composition of the Tibetan medicinal plant Aconitum pendulum Busch (APB). The article provides a detailed protocol for the systematic exploration and identification of complex chemical components of herbal medicines.

Abstract

In this study, a comprehensive approach was employed, utilizing 2D-HPLC-MS technology in conjunction with the molecular network to unravel the intricate chemical composition of the Tibetan medicinal plant APB. Through the implementation of 2D-HPLC, enhanced separation of complex mixtures was achieved, enabling the isolation of individual compounds for subsequent analysis. The molecular network approach further aided in elucidating structural relationships among these compounds, contributing to the determination of potential bioactive molecules. This integrated strategy efficiently identified a wide array of chemical components present within the plant. The findings revealed a diverse spectrum of chemical constituents within APB, including alkaloids, among others. This research not only advances understanding of the phytochemical profile of this traditional Tibetan medicine but also provides valuable insights into its potential therapeutic properties. The integration of 2D-HPLC-MS and molecular network proves to be a powerful tool for systematically exploring and identifying complex chemical compositions in herbal medicines, paving the way for further research and development in the field of natural product discovery.

Introduction

Tibetan medicine is an integral part of traditional Chinese medicine, adhering to the principles of Tibetan medical practices, and is used for disease prevention and treatment¹. However, Tibetan herbal medicine contains complex plant chemical constituents characterized by significant fluctuations in content. Limited understanding of the fundamental bioactive elements has become a bottleneck in the modernization of Tibetan medicine². The application of liquid chromatography-mass spectrometry (LC-MS), combining the strong separation power of chromatography with the high sensitivity of mass spectrometry (MS), has been widel....

Protocol

1. Preparation

1. Sample preparation
   1. Use a 1/10,000 sensitivity balance to weigh 0.25 g (dry weight) of APB in a 3 mL microcentrifuge tube, add 2.5 mL of methanol, and then sonicate for 30 min (power 240 W, frequency 40 kHz).
   2. Perform centrifugation at 1.2 x g for 5 min, collect the supernatant, and filter through a membrane filter with a 0.22 µm pore size.
2. Preparation of experimental materials
   1. Prepare the two-dimensional mo.......

Discussion

The primary focus of this experiment was the optimization of a partial method within the framework of two-dimensional liquid phase separation. To achieve this, a novel at-column dilution modulator was seamlessly integrated into the two-dimensional liquid chromatography (2D-LC) system. This technical adjustment was of paramount importance as it significantly elevated the proficiency in profiling the chemical constituents present in Tibetan medicine known as APB. Incorporating the 2D-HPLC-MS technology enabled the comprehe.......

Materials

Name	Company	Catalog Number	Comments
Acetonitrile	Fisher chemical	F22M81203	Mobile phase
Aconitum pendulum	/	/	Herb medicine
Agilent 1290 Infinity (II) 2D-LC	Agilent Technologies	G2198-90001	Liquid chromatography
Disposable syringes	Chengdu Keen experimental equipment	/	1ml
EP tube	Chengdu Keen experimental equipment	/	3ml
Liquid phase injection bottle	Chengdu Keen experimental equipment	/	1.5ml
LTQ XL Mass Spectrometer	Thermo Fisher	LTQ21991	Mass Spectrometer
Microporous membranes	Chengdu Keen experimental equipment	/	0.22μm
Ultimate XB-C18,5 μm,2.1 x 200 mm	Welch	00201-31015	Reversed-phase column
Ultrasonic Cleaner	GT Sonic	UGT20DEC048Y	Ultrasonic Cleaner 240W 40KHz
XAmide,3 μm,100A	Dalian Mondi Technology	D2019110601	Hydrophilic column