High-resolution Tandem Mass Spectrometry for Studying Chemical Constituents of Gynura bicolor DC

Abstract

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). Ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) has gradually become a leading technology for the identification of TCM ingredients. Gynura bicolor DC. (BFH), a perennial stemless herb used for medicine and food in China has medicinal effects such as clearing heat, moistening the lung, relieving cough, dispersing stasis, and relieving swelling. Polyphenols and flavonoids contain numerous isomers, which hinder the identification of the complex compounds in BFH. This paper presents a systematic protocol for studying chemical constituents of BFH based on solvent extraction and integrated data via UPLC-Q-TOF-MS.

The method described here includes systematic protocols for sample pretreatment, MS calibration, MS acquisition, data processing, and analysis of results. Sample pretreatment includes collection, cleaning, drying, crushing, and extraction. MS calibration consists of multipoint and single-point correction. Data processing includes data importing, method establishment, analysis processing, and result presentation. Representative results of the typical fragmentation pattern of phenolic acids, esters, and glycosides in Gynura bicolor DC. (BFH) are presented in this paper. In addition, organic solvent selection, extraction, data integration, collision energy selection, and method improvement are discussed in detail. This universal protocol can be widely used to identify complex compounds in TCM.

Introduction

Traditional Chinese medicine (TCM) has been clinically practiced in China for thousands of years, and it plays a vital role in maintaining the health of Chinese people¹. The composition of TCM is diverse and complex, and TCM has been widely reported in many qualitative studies focusing on the chemical composition². The chemical components in TCM can be roughly divided into the following categories such as alkaloids, organic acids, phenylpropanoids, coumarins, lignans, quinones, flavonoids, terpenoids, triterpenoid saponins, steroid saponins, cardiac glycosides, and tannins³. Given the large numbers of unknown components and indistinguishable isomers in TCM, the separation and analysis of the desired chemical components are important subjects for the fundamental research of TCM⁴.

Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) has been applied to analyze substances in traditional Chinese medicine (TCM), which can be separated by ultra-high-performance liquid chromatography^5,6. The high resolution of MS can provide extensive ion information, which is used for database analysis with error less than 5 ppm⁷. After turning on the collision energy, the secondary MS mode can obtain secondary fragment ions, whose intensity and number are affected by the magnitude of energy⁸.

Gynura bicolor DC. (BFH), a perennial stemless herb widely used for medicine and food (Figure 1A), is a rare and endangered plant unique to China⁹. BFH has abundant anthocyanins, polyphenols, flavonoids, and strong antioxidant capacity¹⁰. BFH has medicinal effects including clearing heat, cooling blood, moistening the lung, relieving cough, dispersing stasis, relieving swelling, relieving summer heat, and eliminating heat. Few studies have focused on the chemical composition of BFH¹¹. Polyphenols and flavonoids contain numerous isomers, which make the identification of the complex compounds in BFH difficult. A universal method for the identification of chemical components must be developed, which can be applied to all kinds of TCM. This study aimed to report a systematic protocol for studying chemical constituents of BFH based on solvent extraction and integrated data via UPLC-Q-TOF-MS.

Protocol

1. Sample pretreatment

1. Wash the whole herb of BFH in pure water until there are no visible deposits and impurities. Place the clean BFH in a dish and then place it in the oven (Figure 1B). Set the oven to 50 °C for 24 h.
   NOTE: The entire plant of BFH was collected in Sichuan Province, China.
2. Crush dried BFH in a high-speed multifunctional crusher. Transfer the coarse powder into a 50-mesh sieve (Figure 1C). Collect the fine powder in an airtight bag, and store it in a drying tower.
3. Accurately weigh six BFH samples of 0.25 g each, and place them in six conical flasks. Add 30 mL of organic solvent (50% ethanol-water, 70% ethanol-water, chloroform, petroleum ether, ethyl acetate, and n-butyl alcohol) to each conical bottle (Figure 1D).
4. 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. Prepare an injection syringe and a 0.22 μm organic microporous membrane filter. Filter all the supernatants into 2 mL sample bottles.

2. MS calibration

1. Using a 1,000 µL range pipette, transfer 500 µL of 0.1 M sodium hydroxide solution to a 100 mL volumetric flask and then transfer 450 µL of deionized or ultrapure water to the 100 mL volumetric flask (Figure 2).
2. Using a 200 µL range pipette, add 50 µL of formic acid to the 100 mL volumetric flask. Make a total volume of 100 mL with 90:10 2-propanol:water, and sonicate the solution for 5 min. Label the flask as 0.5 mM sodium formate solution in 90:10 2-propanol:water and store it in the refrigerator.
   NOTE: The sodium formate solution expires in 1 week at room temperature.
3. Launch the UPLC-Q-TOF-MS control software and then open the MS tune window. In the Sample Flow Control panel, set 25 μL/min in the Infusion Flow Rate option and select C in the Reservoir option. Wait about 1 min for the automatic purge to the end.
4. Click on the Flow button to start delivering sodium formate solution. Click on the Positive button to switch to the positive ion mode, and click on the Sensitivity button to switch to the sensitivity mode.
   NOTE: Proceed to the next step until the characteristic peaks of sodium formate appear on the real-time spectrum window.
5. In the UPLC-Q-TOF-MS control software, open the MS Console window. In the left position, select SYNAPT XS and Intellistart options in sequence.
6. Tick the Create Calibration, and click on the Start button. Click on the Next button, and select Calibration Profile in the drop-down menu. Click on the Next button, and select the Sensitivity mode in Positive polarity. Click on the Next, Tune Page, Next, and Start buttons in turn.
7. Switch to the MS tune window, and click the Negative button to switch to the negative ion mode. Go back to the MS Console window.
8. Tick the Create Calibration, and click on the Start button. Click on the Next button, and select Calibration Profile in the dropdown menu. Click on the Next button, and select the Sensitivity mode in Negative polarity. Click on the Next, Tune Page, Next, and Start buttons in turn.
9. In the LockSpray Flow Control panel, set a 50 μL/min flow rate and click on the Flow button to let the LE solution enter the mass spectrometer. Switch to the MS tune window, tick the LockSpray Source Setup, and click on the Start button to begin calibration.

3. MS acquisition

1. Switch to the main interface, and right-click on the Inlet File column to open the liquid chromatography method.
2. Click on the Inlet button and then set parameters including Run time and Gradient. Click on the Autosampler button and then set parameters including Run time, Column, and Sample. Click the Save button.
3. Right-click on the MS Method column to open the mass spectrometry method.
4. Click on the Information button and then set parameters including End Time, Polarity, Analyser Mode, Low Mass, High Mass, and Ramp Transfer Collision Energy. Click the OK button and then the Save button.
5. Fill in the sequence table including file name, vial, and vol. Click on the Save button.
6. Select the row in the sequence table for the sample you want to run. Click on the Run button, and click on the OK button in the window that just pops up.

4. Data processing

1. Launch the Data analysis software. In the My Work column, click on the Import MassLynx Data button to open a new window.
2. Click on the plus sign button, and check all the five raw files from the positive mode. Enter a group name, and click on the Create UNIFI Sample Set button to start data import (Figure 3A).
3. Double-click on the Analysis Method file in the positive mode. Click on the Processing label, and click on the Target by Mass button. Enter the number of 5 ppm into the input box after Target match tolerance and Fragment match tolerance.
4. Click on the Home button, and click on the Adducts button. Select the rows of +H and +Na, and click on the right arrow-shaped button. Click on the Save button (Figure 3B).
5. Double-click on the Analysis Method file in the negative mode. Click on the Processing label, and click on the Target by Mass button. Enter the number of 5ppm into the input box after Target match tolerance and Fragment match tolerance.
6. Click on the Home button and then click on the Adducts button. Select the rows of -H and +HCOO and click on the right arrow-shaped button. Click on the Save button.
7. In the My Work column, click on the Analysis button to open a new window. Choose the data just imported and the method just created and then click on the Next button. Enter the filename. Select the folder where the analysis data are stored and then click on the Finish button
8. Click on the Process button and wait for a few minutes until the results show up (Figure 3C).
9. Copy the result table into a blank form (Figure 3D).

5. Result analysis

1. Select a compound in the result table. The corresponding secondary mass spectrum will be displayed in the lower right corner (Figure 4A).
2. Click on the table-shaped button, and select the High Energy Fragments option to display the table of secondary fragment ions (Figure 4B).
3. Select a secondary fragment ion in the table. Move the mouse over the ion peak corresponding to the secondary mass spectrum, and the corresponding structural fracture will be displayed on the screen (Figure 4C).
   NOTE: Manually assess the reasonability of the cracking given by the software, and distinguish isomers based on the secondary fragment ions.
4. Manually draw the fragmentation pattern in the drawing software.
   NOTE: Examples of fragmentation patterns are described in detail in the representative results section.

Representative Results

The chemical composition identification of BFH was used as a model to display the representative results. Base peak chromatograms of solvent-extracted Gynura bicolor DC. are shown in Supplemental File 1-Supplemental Figure S1-S6, and the observed retention time (RT), component name, formula, mass-to-charge ratio (m/z), and mass error are listed in Tables S1-S6. In the ZBTK, all 35 kinds of compounds were identified from separated peaks in UPLC-TOF-MS. As shown in Supplemental File 1-Supplemental Table S1, the main chemicals contained acid (quinic acid, DL-malic acid, citric acid, o-coumaric acid, and 3-O-trans-coumaroylquinic acid), glycosides (gentiopicrin, picroside II, suffruticoside J, copoline, and borneol-2-O-β-D-apiofuranosyl-(1→6)-β-D-glucoside), and esters (bilobalide, trimethyl citrate, and trans-O-glucosyl-methyl-trans-cinnamate). In the ZBTK-CT, 25 kinds of extra compounds were detected (Supplemental File 1-Supplemental Table S2), and 45 kinds of total compounds were observed.

When chloroform was used as an extractant, all 50 compounds were found. Among them, the 28 kinds of extra compounds included coronaric acid, sanleng acid, agrimol A, and methyl artemisate (Supplemental File 1-Supplemental Table S3). When petroleum ether was used as an extractant, 44 kinds of total compounds were observed. We found 17 kinds of extra compounds including notopterol, onjisaponin, and cnideol B (Supplemental File 1-Supplemental Table S4). A total of 65 compounds were obtained in solvent using ethyl acetate as an extractant. Compared with the previous four extractives, 37 extra compounds were noted such as daphnetin, acetovanillone B, and stigmasterol-3-O-β-D-glucoside (Supplemental File 1-Supplemental Table S5). In the butyl alcohol extractive, we found 67 kinds of total compounds, which contained 26 kinds of extra compounds including rengyoside C, chromones, aturametelin F, and deacetylmatricarin-8-O-β-D-glucopyranoside (Supplemental File 1-Supplemental Table S6). In summary, we found 168 compounds based on all the compound results of six extractants.

To deeply understand the cleavage pathway of compound types in UPLC-TOF-MS, we selected three compounds as examples. For acids, 3-O-trans-coumaroylquinic acid with m/z = 337.09222 could lose a C₇H₁₀O₅ or C₉H₈O₂ group via the hydrolysis reaction to form intermediates with m/z = 163.04116 or 199.05557, respectively (Figure 4D). For esters, trans-O-glucosyl-methyl-trans-cinnamate with m/z = 385.11308 could be converted into intermediate with m/z = 153.07485 or 135.04679 via C-C cleavage at different sites (Figure 4E). For glycosides, citrusin C with m/z = 385.11308 transformed into intermediate with m/z = 272.09174 via C-C bond cleavage in the aliphatic chain, whereas the loss of the C₇H₆O₂ group resulted in intermediate with m/z = 113.02534 (Figure 4F). In another pathway, the breakage of the C-O-C bond on the benzene ring led to the formation of an intermediate with m/z = 163.11245, while the hexose unit disappeared.

The representative results of molecular docking revealed 2D and 3D structures in Figure 4G,H, respectively. Gene ontology enrichment analysis showed the results of gene function in terms of biological process (BP), cellular component (CC), and molecular function (MF; Figure 4I). The Kyoto Encyclopedia of Genes and Genomes pathway annotation analysis showed the pathway results of differentially expressed genes, which could be used to further understand the function of genes (Figure 4J). The detailed results of molecular docking are shown in Supplemental File 1-Supplemental Table S7 and Supplemental File 1-Supplemental Figure S7-S9.

Figure 1: Pretreatment method of traditional Chinese medicine. (A) Gynura bicolor DC. (B) Cleaning and drying. (C) Pulverization. (D) Organic solvent addition. (E) Ultrasound extraction. (F) Centrifugation of mixture. (G) Filtration of supernatant. Please click here to view a larger version of this figure.

Figure 2: Operation procedure of UPLC-Q-TOF-MS/MS. (A) Sodium formate preparation. (B) Mass axis calibration. (C) MS tune. (D) Sample placement. (E) Mass spectrum acquisition. (F) Mass spectrum display. Please click here to view a larger version of this figure.

Figure 3: Data processing and integration. (A) Data conversion. (B) Method modification. (C) Data processing. (D) Statistical analysis of results. Please click here to view a larger version of this figure.

Figure 4: Deep result analysis. (A) Secondary mass spectrum. (B) Secondary fragment ion display. (C) Fracture site display. (D) fragmentation pattern 1. (E) Fragmentation pattern 2. (F) Fragmentation pattern 3. (G) Demonstration of the active ingredient action target. (H) 3D structure of the active ingredient action target. (I) GO enrichment analysis. (J) KEGG pathway annotation analysis. Please click here to view a larger version of this figure.

Supplemental File 1: Identification of compounds, molecular docking results, base peak chromatograms, and network analysis. Please click here to download this File.

Discussion

Besides water decoction¹², organic solvent extraction is another common method of TCM pretreatment¹³. According to the principle of similar phase dissolution, numerous components have been extracted by the combination of various organic solvents¹⁴. Ultrasonic-assisted extraction is one of the main methods used to obtain components in TCM¹⁵. Supercritical carbon dioxide extraction is good for extracting certain kinds of substances such as lignans¹⁶. Moreover, microwave-assisted extraction in ionic liquids (ILs) was investigated as an alternative to conventional organic solvent extractions¹⁷. However, the precise control of microwave energy is difficult to achieve, which may cause secondary reactions such as etherification dehydration and esterification dehydration.

Optimal settings of collision energy can yield additional second-order MS fragment information. Information from tandem MS, i.e., MS^E mode, was provided by two parallel alternating scans using either low energy to obtain molecular ion information or high energy to obtain full-scan accurate mass fragment ions¹⁸. During the setting of high-energy parameters, the range of 10-35 V is usually used in ramp transfer collision energy to enhance precursor fragmentation efficiency¹⁹.

The vast amount of raw MS data was generated from high-resolution MS. Raw MS data, which included molecular ion peaks in the MS spectrum and fragment ion peaks in the MS/MS spectrum, must be thoroughly processed and integrated. MS data analysis software aimed to analyze all metabolites in a biological sample comprehensively²⁰. The screening of sulfated metabolites was based on the data-dependent acquisition of full MS scans and multidimensional metabolite data²¹. On the basis of evolving technology, artificial intelligence prediction will be a feasible development direction for MS data analysis.

The majority of compounds can be identified by this method. However, the limited secondary fragments cannot fully identify all isomers including flavonoids and tannins. To achieve structure identification, researchers can employ tandem mass spectrometry to provide further information on fragment ions, which help in the identification of compound structures⁸. Given the limitation of electrospray ionization ion or atmosphere pressure chemical ionization source, high-resolution MS is not suitable for macromolecule analysis. Matrix-assisted laser desorption ionization can promote the ionization of large molecular compounds, such as proteins²². In summary, UPLC-Q-TOF-MS/MS is one of the most widely applicable techniques for the identification of TCM compounds. It has the potential to be applied to other fields, including clinical medicine and energy chemistry.

Materials

Name	Company	Catalog Number	Comments
chloroform	Sinopharm Chemical ReagentCo., Ltd	CAS 67-66-3
ethanol	ChuandongChemical	CAS 64-17-5
ethyl acetate	ChuandongChemical	CAS 141-78-6
liquid chromatograph	Waters	ACQUITY Class 1 plus
MassLynx	Waters	V4.2	MS control software
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