Standardized Identification of Compound Structure in Tibetan Medicine Using Ion Trap Mass Spectrometry and Multiple-Stage Fragmentation Analysis

Summary

Here, we describe a general protocol and design that could be applied to identify trace amounts and minor constituents in the complex natural product formulations (matrixes) in Tibetan medicine.

Abstract

Tibetan medicines are complex and contain numerous unknown compounds, making in-depth research on their molecular structures crucial. Liquid chromatography-electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS) is commonly used to extract Tibetan medicine; however, many unpredictable unknown compounds remain after using the spectrum database. The present article developed a universal method for identifying components in Tibetan medicine using ion trap mass spectrometry (IT-MS). The method includes standardized and programmed protocols for sample preparation, MS setting, LC prerun, method establishment, MS acquisition, multiple-stage MS operation, and manual data analysis. Two representative compounds in the Tibetan medicine Abelmoschus manihot seeds were identified using multiple-stage fragmentation, with a detailed analysis of typical compound structures. In addition, the article discusses aspects such as ion mode selection, mobile phase adjustment, scanning range optimization, collision energy control, collision mode switchover, fragmentation factors, and limitations of the method. The developed standardized analysis method is universal and can be applied to unknown compounds in Tibetan medicine.

Introduction

The qualitative analysis of trace components in traditional Chinese medicine (TCM) has become a crucial topic in research. Due to the high numbers of compounds in TCM, it is difficult to isolate them for nuclear magnetic resonance spectrometer (NMR) or X-ray diffractometer (XRD) analysis, making mass spectrometry (MS)-based methods that only require low sample volumes increasingly popular. Additionally, liquid chromatography (LC) coupled with MS has been widely used in TCM research in recent years for the improved separation of complex samples and qualitative analysis of chemical compounds¹. One common method is liquid chromatography-electrospr....

Protocol

1. Sample preparation

1. Accurately weigh 1 g of the AMS sample, and place it in a conical flask with 30 mL of 80% methanol. Transfer the mixture to an ultrasound bath sonicator for 30 min of extraction at 25 °C. Centrifuge the sample at 14,000 x g for 5 min.
   NOTE: The frequency of the ultrasound bath sonicator is 40 KHz.
2. Prepare an injection syringe and a microporous membrane filter (0.22 μm, organic only). Filter the supernatant into a 2 mL sample bottle.

2. MS setting

1. Turn on the switch of the vacuum pump. Open the main valve of the argon cy....

Results

Cellobiose was used as a model to verify the feasibility of MSⁿ in positive ion mode. As shown in Figure 2A, the ESI-MS (positive ion mode) of cellobiose [C₁₂H₂₂O₁₁]⁺ produced the protonated molecule [M+H]⁺ at m/z 365. The product ion scan (CID-MS/MS) of [M+H]⁺ at m/z 365 resulted in the second fragment ion at m/z 305 (Figure 2B), which was further analyzed using MS³ and MS

Discussion

IT-MS and its MSⁿ technology offer a new approach to identifying the structure of trace TCM compounds. Unlike Q-TOF-MS, which could not deeply identify the fragment ions, IT-MS with MSⁿ technology excels due to its ability to isolate and accumulate ions. This article outlines a method for identifying trace compounds in Tibetan medicine using the IT-MS and MSⁿ technique. The method utilizes the n value in MSⁿ to determine the amount of fragment ion information provided. The cruc.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetonitrile	Thermo Scientific	CAS 75-05-8	LC-MS grade
Formic Acid	Knowles	CAS 64-18-6	HPLC grade
Linear ion trap mass spectrometer	Thermo Scientific	LTQ XL
liquid chromatograph	Thermo Scientific	U3000
LTQ Tune	Thermo Scientific	version 2.8.0	MS control software
Methanol	Thermo Scientific	CAS 67-56-1	LC-MS grade
Pure water	Thermo Scientific	CAS 7732-18-5	LC-MS grade
Xcalibur	Thermo Scientific	version 2.0	LC-IT-MS operational software