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-electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS), which is widely used in qualitative research on Tibetan medicine². With this method, complex components are enriched and separated in an LC column, and the mass-to-charge ratio (m/z) of the adduct ions is observed using an MS detector. Searching tandem MS (MS/MS or MS²) databases is currently the fastest approach for confident compound annotations in small molecule analysis using quadrupole time-of-flight (Q-TOF) MS and Orbitrap MS³. However, the poor quality of databases and the presence of various isomers hinder the identification of unknown compounds. In addition, the information provided by the MS/MS database is limited^4,5,6,7. It is significant to investigate the chemical compounds in each TCM using a general protocol that can be widely applied to other TCM.

IT-MS captures a wide range of ions by applying different radio frequency (RF) voltages to the ring electrodes⁸. IT-MS can perform time-series multiple-stage MS scans in diverse chronological orders, providing ingredient multiple-stage MS (MSⁿ) fragmentation, where n is the number of product ion stages⁹. Linear IT-MS is considered the best for structure identification as it can be used for sequential MSⁿ experiments¹⁰. Targeted ions can be isolated and accumulated in linear IT-MS¹. The MSⁿ (n ≥ 3) in IT-MS provides more fragment information than MS/MS in Q-TOF-MS. Since IT-MS cannot lock the target ion and its fragment ions, it is a powerful tool for the structure elucidation of unknown compounds, including isomers¹. MSⁿ technology has been widely applied to the structural analysis of unknown proteins, peptides, and polysaccharides^11,12. The abundance level of fragment ions in MSⁿ provides more molecular fragment information on targeted compounds in complex samples than MS/MS in Q-TOF-MS. Hence, applying MSⁿ technology to structural identification in TCM is essential.

Tibetan medicine is a significant component of TCM¹³, and these medicines are primarily derived from animals, plants, and minerals found in the plateau area¹⁴. The Tibetan medicine Abelmoschus manihot seeds (AMS) is the seed of Abelmoschus manihot (linn.) medicus. AMS is a traditional herbal medicine used to treat conditions such as atopic dermatitis, rheumatism, and leprosy. It contains chalcone, which possesses antibacterial, antifungal, anticancer, antioxidative, and anti-inflammatory effects¹⁵. In the present study, MSⁿ procedures were improved, and a detailed method was developed to identify compound structures in the Tibetan medicine AMS using IT-MS and MSⁿ. Certain MS parameters, including the ion mode, scanning range, and collision mode, were optimized to overcome problems in identifying trace compounds. This study aims to promote the standardized structure identification of trace compounds in TCM.

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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 cylinder and the partial pressure valve, and adjust the pressure to approximately 0.3 MPa. Open the nitrogen valve.
   NOTE: Wait for at least for 8 h to ensure a sufficient vacuum degree for the experimental conditions. Check that the gas pressure of argon and nitrogen is high enough before analysis.
2. Launch the MS control software. Click on Heated SEI Source in the software panel, and enter the MS parameters, including the heater temperature (350 °C), sheath gas flow rate (35 arb), aux gas flow rate (15 arb), spray voltage (3.8 KV for positive mode, −2.5 KV for negative mode), and capillary temperature (275 °C). Click on the Apply button to activate the ion source.

3. LC prerun, method establishment, and MS acquisition

1. Prepare mobile phase A and mobile phase B using 0.1% formic acid aqueous solution and pure acetonitrile, respectively. Degas them in an ultrasound bath sonicator for at least 15 min. Connect the solutions to the A and B fluid passages, respectively (Figure 1A). Prepare a methanol-water (1:9 v/v) solution, and then fill it into the cleanout fluid bottles of the pump and injector by hand.
   NOTE: The frequency of the ultrasound bath sonicator is 40 KHz.
2. Launch the LC-MS control software.
   1. Click on the Direct Control button to open the LC control panel. Open the purge valve in the counterclockwise direction on the pump module (Figure 1B).
   2. Click on the More Option button to open the pump setting, and set the purge parameters at 5 mLmin⁻¹ for 3 min. Click on the Purge button to start the bubble removal. Subsequently, close the purge valve.
3. Click on the Prime Syringe, Wash Buffer Loop, and Wash Needle Externally buttons to rinse the syringe for three cycles, the loop for one cycle, and the needle for one cycle, respectively. Place the sample bottle in the sampler (Figure 1C).
4. Click on the Instrument Setup button to open the method-editing window. Click on the New button to create a new LC-MS instrument method.
5. Establish a total run time for the LC method. Next, enter values to set the pressure limit, total flow rate, flow gradient, sample temperature, column temperature, and ready temperature delta in the method-editing window.
   NOTE: The default total flow rate of the mobile phase is constant at 0.3 mL/min with 50% A and 50% B and without column temperature in the absence of a chromatographic column. The default values of sample temperature and ready temperature delta are 15 °C and 0.1 °C, respectively. Other settings depend on the type of liquid chromatography column used.
6. Select the General MS or MSⁿ experiment type for the MS method. Enter values to configure the acquisition time, polarity, mass range, divert value number, and divert value duration. Click on the Save button to configure the settings as an instrument method.
   NOTE: The default settings without a chromatography column are as follows: acquisition time, 2 min; polarity, positive or negative; mass range, 100 to 1,200; divert value number, 2; and divert value duration, 1.99 min.

4. Operating multiple-stage mass spectrometry

1. Click on the Sequence Setup button to open the sequence table.
   1. In the table, enter the following information: sample type, file name, path, sample ID, instrument method, position, and injection volume.
   2. Click on the Save button to record the sequence table, and then click on the Start Analysis button to implement the settings and initiate the MS acquisition.
      NOTE: The default sample type is selected as unknown. The instrument method is the method saved in step 3.6. The sample bottle is placed in its unique location in the sample room. For example, RA1 is the first location in the first row of the red area in the sample room. The default injection volume is usually 2 μL, which depends on the concentration of the sample.
2. Double-click on the raw file in explorer to load the MS data into the data processing software. In the base peak chromatogram (BPI), select the area with the maximum area under the curve (AUC) by clicking and dragging the mouse. The corresponding MS spectra will be displayed in the same window.
3. Select a targeted ion for the next MS/MS analysis.
   1. Reopen the method-editing window. In the MSⁿ Setting table, set the m/z of the targeted ion to one decimal place in the Parent Mass column.
   2. Select Collision mode, and enter the collision energy (CE) value. Set the MS/MS scan range. Click on the Save button to record the MS method, and enter a new file name in the sequence table. Click on the Start button to initiate the MS/MS acquisition.
      NOTE: The MS/MS scan range was 40%-130% of the targeted parent ion. The default CE value in collision-induced dissociation (CID) mode is 35%.
4. Double-click on the raw file in explorer to load the MS/MS raw file into the data processing software.
   1. Identify the strongest fragment ion in the MS/MS spectrum, and enter its m/z value into the MSⁿ method list. In the MSⁿ Setting table, set the MS³ parameters, including collision mode, CE value, and scan range.
   2. Click on the Save button to record the MS method, and enter a new file name in the sequence table. Click on the Start button to initiate the MS³ acquisition.
5. Double-click on the raw file in explorer to load the MS³ raw file into the data processing software. Repeat step 4.4 to obtain the MS⁴ spectrum.
6. Complete the MSⁿ experiment when no stable fragment ions are observed in the spectrum.

5. Manual MSⁿ data analysis

1. Double-click on the raw files to open all the mass spectra from MS to MSⁿ. Manually calculate the m/z difference values between the ion and the corresponding fragment ions.
   NOTE: For example, the m/z difference value between the ion (m/z 617.25) and the corresponding fragment ions (m/z 571.28) was 45.97 in MS/MS, the m/z difference value between the ion (m/z 571.28) and corresponding fragment ions (m/z 525.38) was 45.90 in MS³, and the m/z difference values between the ion (m/z 525.38) and the corresponding fragment ions (m/z 344.93 and 273.16) were 180.45 and 252.22 in MS⁴, respectively.
2. Manually draw the "core" structure according to MS⁴ results (the last level of MSⁿ). Manually derive the original structure using functional groups or molecular segments based on the m/z difference value. Manually draw the molecular cleavage paths according to each molecular structure in MSⁿ. Examples of manual molecular derivation are detailed in the representative results section.

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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

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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...

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Materials

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