This work was funded by the National Natural Science Foundation of China (82104881), Inheritance and Innovation Team of TCM Treatment of Immune Diseases, Chongqing Medical Scientific Research Project (Joint project of Chongqing Health Commission and Science and Technology Bureau) (2022DBXM007), A special project for performance incentive and guidance of Chongqing Scientific Research Institute (cstc2022jxjl120005), A special project for Chongqing Postdoctoral Science Foundation (2022CQBSHTB3035), Senior Medical Talents Program of Chongqing for Yong and Middle-aged, the Program for Scientific Institutions of Chongqing (independent research project No.2022GDRC015).

1. Sample pretreatment
<ol>
	<li>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 &#176;C for 24 h. 
	NOTE: The entire plant of BFH was collected in Sichuan Province, China.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Transfer the mixture to an ultrasound bath sonicator for 30 min of extraction at 25 &#176;C. Centrifuge the sample at 14,000 x g for 5 min. Prepare an injection syringe and a 0.22 &#956;m organic microporous membrane filter. Filter all the supernatants into 2 mL sample bottles.</li>
</ol>
2. MS calibration
<ol>
	<li>Using a 1,000 &#181;L range pipette, transfer 500 &#181;L of 0.1 M sodium hydroxide solution to a 100 mL volumetric flask and then transfer 450 &#181;L of deionized or ultrapure water to the 100 mL volumetric flask (Figure 2).</li>
	<li>Using a 200 &#181;L range pipette, add 50 &#181;L 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.</li>
	<li>Launch the UPLC-Q-TOF-MS control software and then open the MS tune window. In the Sample Flow Control panel, set 25 &#956;L/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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>In the LockSpray Flow Control panel, set a 50 &#956;L/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.</li>
</ol>
3. MS acquisition
<ol>
	<li>Switch to the main interface, and right-click on the Inlet File column to open the liquid chromatography method.</li>
	<li>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.</li>
	<li>Right-click on the MS Method column to open the mass spectrometry method.</li>
	<li>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.</li>
	<li>Fill in the sequence table including file name, vial, and vol. Click on the Save button.</li>
	<li>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.</li>
</ol>
4. Data processing
<ol>
	<li>Launch the Data analysis software. In the My Work column, click on the Import MassLynx Data button to open a new window.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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</li>
	<li>Click on the Process button and wait for a few minutes until the results show up (Figure 3C).</li>
	<li>Copy the result table into a blank form (Figure 3D).</li>
</ol>
5. Result analysis
<ol>
	<li>Select a compound in the result table. The corresponding secondary mass spectrum will be displayed in the lower right corner (Figure 4A).</li>
	<li>Click on the table-shaped button, and select the High Energy Fragments option to display the table of secondary fragment ions (Figure 4B).</li>
	<li>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.</li>
	<li>Manually draw the fragmentation pattern in the drawing software. 
	NOTE: Examples of fragmentation patterns are described in detail in the representative results section.</li>
</ol>

Identifying Chemical Composition of &lt;em&gt;Gynura bicolor&lt;/em&gt; by UPLC-TOF Mass Spectrometry

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Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+chinese+medicine+and+separation+science.">Traditional chinese medicine and separation science.</a> J Sep Sci. 31 (11), 2113-2137 (2008).</li><li>Yuan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+classification+and+identification+of+complex+chemical+compositions+in+traditional+chinese+medicine+based+on+uplc-q-tof/ms+coupled+with+data+processing+techniques+using+the+kudiezi+injection+as+an+example.">Rapid classification and identification of complex chemical compositions in traditional chinese medicine based on uplc-q-tof/ms coupled with data processing techniques using the kudiezi injection as an example.</a> Anal Methods. 7 (12), 5210-5217 (2015).</li><li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hplc+coupled+with+chemical+fingerprinting+for+multi-pattern+recognition+for+identifying+the+authenticity+of+clematidis+armandii+caulis.">Hplc coupled with chemical fingerprinting for multi-pattern recognition for identifying the authenticity of clematidis armandii caulis.</a> JoVE. 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The authors declare no competing financial interests.

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 substan...

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 chromatography5,6. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>chloroform</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>CAS 67-66-3</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>ChuandongChemical</td>
			<td>CAS 64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>ethyl acetate</td>
			<td>ChuandongChemical</td>
			<td>CAS 141-78-6</td>
			<td></td>
		</tr>
		<tr>
			<td>liquid chromatograph</td>
			<td>Waters</td>
			<td>ACQUITY Class 1 plus</td>
			<td></td>
		</tr>
		<tr>
			<td>MassLynx</td>
			<td>Waters</td>
			<td>V4.2</td>
			<td>MS control software</td>
		</tr>
		<tr>
			<td>n-butyl alcohol</td>
			<td>ChuandongChemical</td>
			<td>CAS 71-36-3</td>
			<td></td>
		</tr>
		<tr>
			<td>petroleum ether</td>
			<td>ChuandongChemical</td>
			<td>CAS 8032-32-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Quadrupole time-of-flight mass spectrometry</td>
			<td>Waters</td>
			<td>SYNAPT XS</td>
			<td></td>
		</tr>
		<tr>
			<td>UNIFI</td>
			<td>Waters</td>
			<td></td>
			<td>Data analysis software</td>
		</tr>
	</tbody>
</table>

high-resolution tandem mass spectrometry for studying chemical constituents of gynura bicolor dc

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.

Preparation of &lt;em&gt;Gynura bicolor DC&lt;/em&gt; samples for High-Resolution Tandem Mass Spectrometry

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

Begin by sample pretreatment. 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 at 50 degrees Celsius for 24 hours.Crush the dried BFH in a high-speed multifunctional crusher. Transfer the coarse powder into a 50-mesh sieve. Collect the fine powder in an airtight bag and store it in a drying tower.Then accurately weigh six BFH samples of 0.25 grams each and place them in six conical flasks. Add 30 milliliters of organic solvent to each conical flask. Transfer the mixture to an ultrasound bath sonicator for 30 minutes at 25 degrees Celsius.Next, centrifuge the sample at 14, 000g for five minutes. Using an injection syringe and the 0.22-micrometer organic microporous membrane filter, filter all the supernatants into two-milliliter sample bottles.

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-Supp...

Read this Scientific Article Protocol about High-resolution Tandem Mass Spectrometry for Studying Chemical Constituents of Gynura bicolor DC at JoVE.com

We describe a general protocol that integrates high-resolution mass spectrometry analysis and molecular docking in traditional Chinese medicine research.

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). ...

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296 Views.  Chongqing Hospital of Traditional Chinese Medicine. Begin by sample pretreatment. 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 at 50 degrees Celsius for 24 hours.Crush the dried BFH in a high-speed multifunctional crusher. Transfer the coarse powder into a 50-mesh sieve. Collect the fine powder in an airtight bag and store it in a drying tower.Then accurately weigh six BFH samples of 0.25 grams each and place them in six co...

Video: Preparation of Gynura bicolor DC samples for High-Resolution Tandem Mass Spectrometry

Calibration and Data Acquisition on High-Resolution Tandem Mass Spectrometer for Chemical Composition Analysis

To begin, prepare 0.5 millimolar sodium formate solution. Once the UPLCQOFMS control software is launched, open the MS tune window in the sample flow control panel set 25 microliters per minute in the infusion flow rate option, select C in the reservoir option, and wait about one minute for the automatic purge to end. Next click flow 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. Then open the MS console window, select synapse access and IntelliStart options in sequence. Click on create calibration, and click on the start button.Click on the next button and select calibration profile in the dropdown menu. After clicking next, select the sensitivity mode in positive polarity. Then sequentially click on the next, tune page, next, and start buttons.Switch to the MS tune window and click the negative button to switch to the negative ion mode. In the MS console window, tick create calibration, then click start. Click on next.Select calibration profile and select the sensitivity mode in negative polarity. Again, click on next, tune page, next, and start buttons. Next in the lock spray flow control panel, set the flow rate to 50 microliters per minute and click on the flow button to allow the LE solution to enter the mass spectrometer.Switch to the MS tune window. Tick the lock spray source setup, and click on the start button to begin calibration. For MS acquisition, switch to the main interface and right click on the inlet file column to open the liquid chromatography method.Click on the inlet button and set parameters such as runtime and gradient. Then click the auto sampler button and set parameters including runtime, column, and sample, and click on save. Next, right click on the MS method column to open the mass spectrometry method.Under information set parameters including end time, polarity, analyzer mode, low mass, high mass, and ramp transfer collision energy. Click okay and then save. Then fill in the sequence table with the file name vial and volume.Save the sequence, select the sample to run from the sequence table and initiate the run.

Processing and Analysis of Mass Spectrometry Data for Chemical Composition Analysis of Gynura bicolor DC

After launching the data analysis software, go to the my work column and click on import mass links data to open a new window. Click on the plus sign and select all five raw files from the positive mode acquired by high resolution mass spectrum metric analysis. Enter a group name and click on create unify sample set to start data import.Double click on the analysis method file in the positive mode. Click on processing, followed by the target by mass button. Enter 5PPM for target match tolerance and fragment match tolerance.Click on home, and then adducts. Select the rows of protylated and acetylated. Click on the right arrow-shaped button, and then save.In the my work column, click on analysis. Choose the imported data and method, and then click on next. Enter the file name, select the folder for storing the analysis data, and click finish.Then, click on the process button. Copy the result table into a blank form for analysis. Next, select a compound in the result table.The corresponding secondary mass spectrum will appear in the lower right corner. Click on the table-shaped button, and select the high energy fragments option to display the table of secondary fragment ions. Select a secondary fragment ion in the table.Move the cursor over the ion peak corresponding to the secondary mass spectrum, and the corresponding structural fracture will be displayed. Manually draw the fragmentation pattern in a drawing software. The chemical composition identification of BFH revealed 168 compounds based on the compound results of six extractants.To understand the cleavage pathway in UPLC-TOF-MS for acids, 3-O-trans-coumaroylquinic acid was demonstrated. Cleavage via hydrolysis reaction led to the formation of intermediates. For esters, trans-O-glucosyl-methyl-trans-cinnamate showed conversion into intermediates via carbon-carbon cleavage.For glycosides cintrucin C transformed into an intermediate via carbon-carbon bond cleavage in the alphalytic chain, or via the breakage of the carbon oxygen carbon bond on the benzene ring. Gene ontology enrichment analysis showed the probable gene functions of the identified compounds. Also, the KEGG pathway annotation analysis showed the pathways for differentially expressed genes.