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 Gynura bicolor 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><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.

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

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

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

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). ...

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467 Views.  Chongqing Hospital of Traditional Chinese Medicine. Begin met de voorbehandeling van het monster. Was het hele kruid BFH in zuiver water totdat er geen zichtbare afzettingen en onzuiverheden meer zijn. Leg de schone BFH in een schaaltje en zet deze vervolgens 24 uur in de oven op 50 graden Celsius.Plet de gedroogde BFH in een multifunctionele breker met hoge snelheid. Breng het grove poeder over in een zeef met 50 mazen. Vang het fijne poeder op in een luchtdichte zak en bewaar het in een droogtoren.

Video: Voorbereiding van Gynura bicolor DC-monsters voor tandem-massaspectrometrie met hoge resolutie

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

Monitoring Plant Hormones During Stress Responses

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. Among the most severe stresses are insect herbivory, pathogen infection, and drought stress. For each of these stresses a specific set of hormones and/or combinations thereof are known to fine-tune the responses, thereby ensuring the plant's survival. The major hormones involved in the regulation of these responses are jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). To better understand the role of individual hormones as well as their potential interaction during these responses it is necessary to monitor changes in their abundance in a temporal as well as in a spatial fashion. For the easy, sensitive, and reproducible quantification of these and other signaling compounds we developed a method based on vapor phase extraction and gas chromatography/mass spectrometry (GC/MS) analysis (1, 2, 3, 4). After extracting these compounds from the plant tissue by acidic aqueous 1-propanol mixed with dichloromethane the carboxylic acid-containing compounds are methylated, volatilized under heat, and collected on a polymeric absorbent. After elution into a sample vial the analytes are separated by gas chromatography and detected by chemical ionization mass spectrometry. The use of appropriate internal standards then allows for the simple quantification by relating the peak areas of analyte and internal standard.

A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

Biology

Early Detection of Cyanobacterial Blooms and Associated Cyanotoxins using Fast Detection Strategy

Fast detection of cyanobacteria and cyanotoxins is achieved using a Fast Detection Strategy (FDS). Only 24 h are needed to unravel the presence of cyanobacteria and related cyanotoxins in water samples and in an organic matrix, such as bivalve extracts. FDS combines remote/proximal sensing techniques with analytical/bioinformatics analyses. Sampling spots are chosen through multi-disciplinary, multi-scale, and multi-parametric monitoring in a three-dimensional physical space, including remote sensing. Microscopic observation and taxonomic analysis of the samples are performed in the laboratory setting, which allows for the identification of cyanobacterial species. Samples are then extracted with organic solvents and processed with LC-MS/MS. Data obtained by MS/MS are analyzed using a bioinformatic approach using the online platform Global Natural Products Social (GNPS) to create a network of molecules. These networks are analyzed to detect and identify toxins, comparing data of the fragmentation spectra obtained by mass spectrometry with the GNPS library. This allows for the detection of known toxins and unknown analogues that appear related in the same molecular network.

A fast-multidisciplinarystrategy for early detection of cyanobacterial blooms and associated cyanotoxins is described here. It allows for the detection of cyanobacteria and related cyanotoxins in water samples and in organic matrices, such as bivalve samples, in 24 h.

Fast detection of cyanobacteria and cyanotoxins is achieved using a Fast Detection Strategy (FDS). Only 24 h are needed to unravel the presence of ...

Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, penicillin, paclitaxel, rapamycin, or artemisinin, commonly used in medical practice, have been first identified and isolated from natural products. However, the identification and isolation of biologically active specialized metabolites from natural sources is a challenging and time-consuming process. Traditionally, individual metabolites are isolated and purified from complex mixtures, following the extraction of biomass. Subsequently, the isolated molecules are tested in functional assays to verify their biological activity. Here we present the use of cellular membrane affinity chromatography (CMAC) columns to identify biologically active compounds directly from complex mixtures. CMAC columns allow for the identification of compounds interacting with immobilized functional transmembrane proteins (TMPs) embedded in their native phospholipid bilayer environment. This is a targeted approach, which requires knowing the TMP whose activity one intends to modulate with the newly identified small molecule drug candidate. In this protocol, we present an approach to prepare CMAC columns with immobilized tropomyosin kinase receptor B (TrkB), which has emerged as a viable target for drug discovery for numerous nervous system disorders. In this article, we provide a detailed protocol to assemble the CMAC column with immobilized TrkB receptors using neuroblastoma cell lines overexpressing TrkB receptors. We further present the approach to investigate the functionality of the column and its use in the identification of specialized plant metabolites interacting with TrkB receptors.

The protocol describes the preparation of cell membrane affinity chromatography (CMAC) columns with immobilized cell membrane fragments containing functional transmembrane tropomyosin kinase receptor B proteins. The use of CMAC columns in the identification of specialized plant metabolites interacting with these receptors and present in complex natural mixtures is also explained.

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, ...

Biochemistry

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLC&#946; also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity ...

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

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.

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.

Tibetan medicines are complex and contain numerous unknown compounds, making in-depth research on their molecular structures crucial. Liquid ...

Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) is the most widely used method in qualitative analysis of compounds. The method includes standardized and programmed protocols for sample pretreatment, MS tune, MS acquisition, and data processing. The sample pretreatments include collection, pulverization, solvent extraction, ultrasound, centrifugation, and filtration. Data post-processing was described in detail and includes data importing, self-established database construction, method establishment, data processing, and other manual operations. The above-ground part of the alpine yarrow herb, Achillea millefolium L., is used to treat inflammation, gastrointestinal disturbances, and pain and its 3-oxa-guaianolides could be useful leads for anti-inflammatory drug development. Three representative compounds in AML were identified, combining TOF-MS with a self-established database. Moreover, the differences from existing literature, liquid-phase parameter optimization, scan mode selection, ion source suitability, collision energy adjustment, isomer screening, method limitation, and possible solutions were discussed. This standardized analysis method is universal and can be applied to identify complex compounds in Chinese herbal medicine.

We describe a general protocol and systematic design that could be applied to separate and recognize complex components in alpine yarrow herb, Achillea millefolium L., a Chinese herbal medicine.

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography ...

Classification of Tropane Alkaloids from Plant Metabolites Using LC-MS/MS

Applications of Liquid-Chromatography Tandem Mass Spectrometry in Natural Products Research: Tropane Alkaloids as a Case Study

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and can yield promising leads for resistant or emerging diseases. The challenge, however, is twofold: not only must researchers find natural products and elucidate their structures, but they must also identify what is worth isolating and assaying (and what is already known - a process known as dereplication). With the advent of modern analytical instrumentation, the pace of natural product discovery and dereplication has accelerated. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become an especially valuable technique for identifying and classifying chemical structures. Tropane alkaloids (TAs) are plant-derived compounds of great medicinal and toxicological significance. In this study, we developed an LC-MS/MS-based screening workflow utilizing the multiple MS/MS configurations available on a triple-quadrupole (QQQ) mass spectrometer to annotate and classify TA structures based on their distinct fragmentation patterns. By using a combination of data-dependent (DD) product ion scans, precursor ion scans (PrIS), and neutral loss scans (NLS), we applied this method to TA-rich extracts of the nightshades Datura stramonium and Datura metel. This method is rapid, sensitive, and was successfully employed for both preliminary dereplication of complex TA-containing samples and for the discovery of a novel candidate for isolation, purification (and eventual bioassay).

Author Spotlight: Discovering New Alkaloids in Plants with Advanced Mass Spectrometry Techniques

We present a method for rapid mass spectrometry (MS)/mass spectrometry (MS)-based annotation and classification of tropane alkaloids, useful for both preliminary dereplication of tropane-containing samples and discovery of novel alkaloids for isolation.

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and ...

Primary Organ Identification in Medicinal Plants Using 13C6&#45;Glucose Labeling

13C6&#45;Glucose Labeling Associated with LC&#45;MS&#58; Identification of Plant Primary Organs in Secondary Metabolite Synthesis

This paper presents a novel and efficient method for certifying primary organs involved in secondary metabolite synthesis. As the most important secondary metabolite in Parispolyphylla var. yunnanensis (Franch.) Hand. -Mzt. (PPY), Paris saponin (PS) has a variety of pharmacological activities and PPY is in increasing demand. This study established leaf, rhizome, and stem-vascular-bundle 13C6-Glucose feeding and non-feeding four treatments to precisely certify the primary organs involved in Paris saponins VII (PS VII) synthesis. By combining liquid chromatography-mass spectrometry (LC-MS), the 13C/12C ratios of leaf, rhizome, stem, and root in different treatments were quickly and accurately calculated, and four types of PS isotopic ion peak(M&#8722;) ratios were found: (M+1) &#8722;/M&#8722;, (M+2) &#8722;/M&#8722;, (M+3) &#8722;/M&#8722; and (M+4) &#8722;/M&#8722;. The results showed that the ratio of 13C/12C in the rhizomes of the stem-vascular-bundle and rhizome feeding treatments was significantly higher than that in the non-feeding treatment. Compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the leaves increased significantly under leaf and stem-vascular-bundle feeding treatments. Simultaneously, compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the leaves under rhizome treatment showed no significant difference. Furthermore, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the stem, root, and rhizome showed no differences among the four treatments. Compared to the non-feeding treatment, the ratio of the Paris saponin II (PS II) molecule (M+2) &#8722;/M&#8722; in leaves under leaf feeding treatment showed no significant difference, and the (M+3) &#8722;/M&#8722; ratio of PS II molecules in leaves under leaf feeding treatment were lower. The data confirmed that the primary organ for the synthesizing of PS VII is the leaves. It lays the foundation for future identification of the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants.

Author Spotlight: Plant Primary Organs Profiling Using 13C6&#45;Glucose Labeling and LC&#45;MS

The developed method of 13C6-Glucose labeling combined with liquid chromatography high-resolution mass spectrometry is versatile and lays the foundation for future studies on the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants, as well as the comprehensive utilization of these secondary metabolites.

This paper presents a novel and efficient method for certifying primary organs involved in secondary metabolite synthesis. As the most important secondary ...

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

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