Name: 2d-hplc-ms technology combined with molecular network for the identification of components in tibetan medicine aconitum pendulum
Uploaded: 2023-12-08T14:50:00.000+00:00
Duration: 7 min 50 s
Description: Watch this Scientific Journal Video about Author Spotlight: Integrating 2D-HPLC-MS and Molecular Networking in Natural Medicine Analysis at JoVE.com

This work was funded by National Natural Science Foundation of China (82130113), the National Natural Science Foundation of China (82204765), the Nature Science Foundation of Sichuan (2022NSFSC1470), Sichuan Provincial Postdoctoral Special Funding Project (TB2023020) and the Xinglin Scholars Research Promotion Program of Chengdu University of Traditional Chinese Medicine (BSH2021030). These funds provide support in terms of experimental equipment, experimental materials, and publication fees.

1. Preparation
<ol>
	<li>Sample preparation
	<ol>
		<li>Use a 1/10,000 sensitivity balance to weigh 0.25 g (dry weight) of APB in a 3 mL microcentrifuge tube, add 2.5 mL of methanol, and then sonicate for 30 min (power 240 W, frequency 40 kHz).</li>
		<li>Perform centrifugation at 1.2 x g for 5 min, collect the supernatant, and filter through a membrane filter with a 0.22 &#181;m pore size.</li>
	</ol>
	</li>
	<li>Preparation of experimental materials
	<ol>
		<li>Prepare the two-dimensional mobile phase, use acetonitrile as the organic phase B phase, and configure 0.1% formic acid ultrapure water as the aqueous phase A.</li>
		<li>Perform two-phase filtration (0.22 &#181;m) and sonicate using an ultrasound machine (40 kHz) for 15 min. Purge the replaced mobile phase to remove the bubbles. In this experiment, the C18 column was used as the 1D column, the hydrophilic column as the 2D column, and the column was mounted to the instrument.</li>
	</ol>
	</li>
	<li>Adjustment of the appropriate shunt ratio
	<ol>
		<li>Connect the 2D-LC instrument out line through a tee to the mass inlet and the other end of the tee to a shunted line. Adjust the flow rate to a suitable value to ensure that the flow rate into the mass spectrum is 0.3-0.5 mL/min. 
		&#8203;NOTE: Since the second dimension of the 2D-LC is usually set above 2 mL/min, this flow rate is too large for MS, so it is necessary to perform a split.</li>
	</ol>
	</li>
</ol>
2. 2D-LC operation
<ol>
	<li>Double-click the Instrument 1 Online icon, and the chemical workstation will automatically communicate with the 1260 LC and enter the workstation.</li>
	<li>For 2D-LC method parameter setting, select the Method and Run Control screen from the View menu to invoke the desired interface, which will normally be entered by default. Click on the injector module, right-click Method, set the injection volume, flow rate and mobile phase time gradient of the 1D pump.</li>
	<li>Enter the sample run time under Stop Time. Click on the main menu Instrument and click 2D-LC Method on the drop-down menu. Select Comprehensive in 2D-LC Mode. Enter 2 min at Modulation time, 1.9 min at 2D Gradient Stop time. Set Flow Settings to 2 mL. Edit 2D-Gradient and set to wavelengths. After editing the method, select Save Method As on the Method menu to name the new method, and then click OK. 
	&#8203;NOTE: The external transfer pump requires manual setting of the flow rate.</li>
</ol>
3. MS operation
<ol>
	<li>Turn ON the switch of the vacuum pump. Open the argon cylinder main valve and the pressure divider valve and adjust the pressure to about 0.3 MPa. Open the nitrogen valve.</li>
	<li>Wait at least 8 h to ensure adequate vacuum for experimental conditions. Check that the air pressure of argon and nitrogen is high enough before analysis.</li>
	<li>To launch the MS control software, click on the Heating SEI Source in the software panel and enter the MS parameters, including heater temperature (350 &#176;C), sheath flow rate (35 arb), auxiliary air flow rate (15 arb), spray voltage (3.8 KV in positive mode, -2.5 KV in negative mode), and capillary temperature (275 &#176;C). Click the Apply button to activate the ion source.</li>
	<li>To set up the MS method, enter values to configure acquisition time, polarity, mass range, transfer value number, transfer value duration, and more. Set MS Run Time (min): 93.00. Set up ScanEvent Details: ITMS + c norm o (100.0-1200.0), CV=0.0v. ITMS + c norm Dep MS/MS, most intense ion from (1): Activation Type: CID, Min. Signal Required: 500, Isolation Width: 2.00, Normalized Coll. Energy: 35.0, Default Charge State:3, Activation Q: 0.250, ActivationTime: 93.000. To set data-dependent settings, use separate polarity settings as disabled, Neutral loss in top:3, Product in top: 3. Click Save to configure the settings as the instrument method. 
	NOTE: The end of run time is consistent with the 2D-LC.</li>
	<li>Click the Sequence Setup button to open the sequence table. Enter the sample type, file name, path, sample ID, instrument method, location, injection amount, and other information in the form.</li>
	<li>Click the Save button to record the sequence listing, then click the Start Analysis button to set up and start MS acquisition. Click Run Control on the 2D-LC instrument and select Run Method. At the same time, the MS instrument also clicks the Run button. 
	&#8203;NOTE: Because the 2D-LC and MS are not controlled by the same software in this experiment, they need to be set up on both instruments separately.</li>
</ol>
4. Molecular network operation
<ol>
	<li>Data preparation: Export 2D-HPLC-MS raw mass spectral data and convert secondary mass spectral data into mzXML or mzML data via ProteoWizard&#39;s Msconvert. (http://proteowizard.sourceforge.net/).</li>
	<li>To upload data, download the WinSCP software on the official website and upload mzML data to the account created by GNPS via FTP protocol. Once the installation is complete, launch WinSCP.</li>
	<li>Connect to GNPS FTP server: In the WinSCP interface, on Session configuration page, fill in the following connection information: File protocol: Select FTP. For Hostname: Enter massive.ucsd.edu., for port number: Keep the default 22.</li>
	<li>Enter the account number and password. After filling in the above information, click the Login button to establish a connection to the GNPS FTP server.</li>
	<li>Create molecular networks by opening a web browser and visiting the GNPS website (https://gnps.ucsd.edu/). New users need to sign up for an account and then log in.</li>
	<li>On the main interface of the GNPS website, click the Tasks tab in the top navigation bar and select Create A New Task from the drop-down menu. On the task creation page, click the Add Files button, then select and upload the data file. (e.g., mzML format).</li>
	<li>In the Task Parameters tab, set the various parameters that generate the MN. These parameters include peak extraction algorithm, peak overtravel value, similarity calculation method, etc. Make the appropriate parameter settings as needed. Click the Launch Analysis button at the bottom of the page to run the task.</li>
	<li>After the task runs, find the created task in the task list and click the Service Name to view the analysis results. The website provides MN diagrams, substitutes, symbiotic networks, and other related information.</li>
</ol>

Analyzing Tibetan Medicinal Plant APB Using 2D-HPLC-MS with Molecular Networking

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The authors declare no competing financial interests.

The primary focus of this experiment was the optimization of a partial method within the framework of two-dimensional liquid phase separation. To achieve this, a novel at-column dilution modulator was seamlessly integrated into the two-dimensional liquid chromatography (2D-LC) system. This technical adjustment was of paramount importance as it significantly elevated the proficiency in profiling the chemical constituents present in Tibetan medicine known as APB. Incorporating the 2D-HPLC-MS technology enabled the comprehensive examination of the intricate composition of APB. By leveraging the merits of two-dimensional separation techniques, we achieved notable enhancements in both resolution and sensitivity. This translated to the successful identification and quantification of a more extensive spectrum of compounds.
In addition, troubleshooting of this technology is also worth discussing. When the peak shape of the liquid phase is abnormal, the main consideration is whether there are bubbles in the flow path and whether the mobile phase ratio is normal. The solution is to degas the mobile phase and optimize the mobile phase ratio. When the liquid phase pressure is abnormal, blockage and leakage are mainly considered. The solution is to replace or clean the clogged parts and reinforce the leaking parts.
This method, however, has certain limitations that deserve attention. It may not be ideally suited for the analysis of volatile and oil components due to the specific properties of these compounds. Despite these constraints, the implementation of two-dimensional separation techniques represents a significant advancement in our analytical capabilities. Not only does it find relevance in Tibetan medicine, but it also exhibits the potential for broader applications in fields such as pharmaceuticals, natural products, and environmental analysis.
MN proved to be a valuable tool for identifying and annotating the chemical components present in APB. This article only lists some of the identified components, and the unidentified components can continue to be analyzed and identified by classical methods. By constructing networks based on spectral similarity, MNs establish connections between related compounds and facilitate their structural elucidation.
The results obtained from this study contribute to our understanding of the chemical composition of APB and its potential therapeutic effects. Aconitine and hypaconitine have analgesic, cardiotonic, and antitumor effects20,21. 14-Benzoylaconine has anti-inflammatory and analgesic effects22. 14-O-acetylneoline has anti-tumor properties19. Further investigations are warranted to explore the pharmacological properties and potential synergistic interactions among these components.
In conclusion, the combined use of 2D-HPLC-MS technology and MN proved to be an effective approach for the identification and characterization of chemical components in APB. Research into APB is ongoing, and there may be other unidentified compounds that merit future investigation. This study provides a foundation for further research on the therapeutic efficacy and safety of this traditional Tibetan medicine, as well as its potential applications in modern healthcare practices.

Tibetan medicine is an integral part of traditional Chinese medicine, adhering to the principles of Tibetan medical practices, and is used for disease prevention and treatment1. However, Tibetan herbal medicine contains complex plant chemical constituents characterized by significant fluctuations in content. Limited understanding of the fundamental bioactive elements has become a bottleneck in the modernization of Tibetan medicine2. The application of liquid chromatography-mass spectrometry (LC-MS), combining the strong separation power of chromatography with the high sensitivity of mass spectrometry (MS), has been widely used in natural medicine analysis3. However, due to the limitation of a single separation mechanism, components with highly similar structures tend to coelute in one-dimensional liquid chromatography separation. In subsequent mass spectrometric analysis, the low-abundance co-eluted components are difficult to detect due to ion suppression caused by high-abundance components4.
2D-HPLC, which stands for two-dimensional high-performance liquid chromatography, represents a novel chromatographic method that harmoniously combines different separation mechanisms using a pair of columns. This includes the fusion or alternation of normal-phase chromatography with reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography with reversed-phase liquid chromatography5. By merging these complementary chromatographic properties, the enhanced separation capability is achieved, effectively addressing the challenges caused by complex sample matrices6. Furthermore, by coupling two-dimensional chromatography with MS, the powerful separation ability of 2D-HPLC and the high sensitivity detection capability of MS can be fully integrated, providing support for the study of complex drug systems and their fundamental constituents7,8,9.
Tibetan medicine commonly features a multifaceted array of components and functionalities, where active ingredients typically exist in intricate compositions and at minimal concentrations. By integrating 2D-LC as a robust separation system and MS as an exceedingly sensitive detector, the challenges posed by intricate samples in terms of separation and identification can be more effectively addressed10,11,12. This amalgamation substantially contributes to advancing the exploration of the chemical composition of Tibetan medicine.
Regardless of whether it is traditional LC-MS or 2D-LC-MS, a vast amount of information can be obtained. However, extracting structural information of complex system components from this massive amount of information has always been a significant challenge. Therefore, researchers have developed various methods for screening and mining MS data. Global Natural Products Social Molecular Networking (GNPS) is an MS/MS data organization and visualization platform where 2D-LC-MS mass spectrometry data can be uploaded13. Each spectrum is considered as a vector and compared to all other spectra using cosine similarity. When the similarity between two spectra exceeds a threshold, they are connected in a molecular network (MN). This can be used for the rapid identification of known compounds and the determination of various unknown natural products14.
Tibetan medicine usually has multiple functions, but its complex composition and significant concentration differences make it difficult to effectively elucidate the relationship between function and material basis15. An in-depth study of Tibetan medicine requires the systematic characterization of as many components as possible. In the framework of this study, we intend to use APB in Tibetan medicine as the research object to demonstrate the strategy and process of systematically studying the complex chemical components of Tibetan medicine using 2D-HPLC-MS technology and MN technology. In the construction of the 2D chromatographic system, we combined reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography with significant separation mechanisms to achieve more effective separation of the complex components of APB16. In addition, to overcome solvent incompatibility between the two dimensions, an at-column dilution modulation mode was adopted. By combining the powerful separation capability of 2D-LC with the high sensitivity detection capability of MS, the spectral information of the complex components in APB is obtained more effectively and comprehensively. Furthermore, through the network and visualization of massive spectral information by MN technology, the components of APB are systematically analyzed. The strategy and process demonstrated in this study are expected to be applied to the study of other Tibetan medicines, promoting research on the material basis of Tibetan medicine, which is of great significance for advancing Tibetan medicine resources and improving quality control standards for Tibetan herbs17. The overall experimental process is shown in Figure 1.
In the experiment presented here, a new at-column dilution modulator was introduced into the Agilent two-dimensional liquid chromatography (2D-LC) system18. By adding an independent delivery pump, the flow path of the analysis was changed, resulting in a high orthogonality of the 2D-LC analysis. The coupling and switching between the two dimensions are accomplished by two six-port valves, as shown in Figure 2. When one sample loop is filled in the first dimension, another sample loop is analyzed in the second dimension. This means that the filling time of the 1D loop and the running time of the 2D are equal. This requires the fast gradient generated by the Binary pump in the second dimension. No peaks are lost when the entire effluent is analyzed. This is particularly helpful for the analysis of unknown samples. It results in a large number of 2D chromatograms that need to be combined for data analysis.

<table><tbody><tr><td>Acetonitrile</td><td>Fisher chemical</td><td>F22M81203</td><td>Mobile phase</td></tr><tr><td>Aconitum pendulum</td><td>/</td><td>/</td><td>Herb medicine</td></tr><tr><td>Agilent 1290 Infinity (II) 2D-LC&amp;nbsp;</td><td>Agilent Technologies</td><td>G2198-90001</td><td>Liquid chromatography</td></tr><tr><td>Disposable syringes</td><td>Chengdu Keen experimental equipment</td><td>/</td><td>1ml</td></tr><tr><td>EP tube</td><td>Chengdu Keen experimental equipment</td><td>/</td><td>3ml</td></tr><tr><td>Liquid phase injection bottle</td><td>Chengdu Keen experimental equipment</td><td>/</td><td>1.5ml</td></tr><tr><td>LTQ XL Mass Spectrometer</td><td>Thermo Fisher</td><td>LTQ21991</td><td>Mass Spectrometer</td></tr><tr><td>Microporous membranes&amp;nbsp;</td><td>Chengdu Keen experimental equipment</td><td>/</td><td>0.22&amp;mu;m</td></tr><tr><td>Ultimate XB-C18,5 &amp;mu;m,2.1 x 200 mm</td><td>Welch</td><td>00201-31015</td><td>Reversed-phase column</td></tr><tr><td>Ultrasonic Cleaner</td><td>GT Sonic</td><td>UGT20DEC048Y</td><td>Ultrasonic Cleaner 240W 40KHz</td></tr><tr><td>XAmide,3 &amp;mu;m,100A</td><td>Dalian Mondi Technology</td><td>D2019110601</td><td>Hydrophilic column</td></tr></tbody></table>

2d-hplc-ms technology combined with molecular network for the identification of components in tibetan medicine aconitum pendulum

In this study, a comprehensive approach was employed, utilizing 2D-HPLC-MS technology in conjunction with the molecular network to unravel the intricate chemical composition of the Tibetan medicinal plant APB. Through the implementation of 2D-HPLC, enhanced separation of complex mixtures was achieved, enabling the isolation of individual compounds for subsequent analysis. The molecular network approach further aided in elucidating structural relationships among these compounds, contributing to the determination of potential bioactive molecules. This integrated strategy efficiently identified a wide array of chemical components present within the plant. The findings revealed a diverse spectrum of chemical constituents within APB, including alkaloids, among others. This research not only advances understanding of the phytochemical profile of this traditional Tibetan medicine but also provides valuable insights into its potential therapeutic properties. The integration of 2D-HPLC-MS and molecular network proves to be a powerful tool for systematically exploring and identifying complex chemical compositions in herbal medicines, paving the way for further research and development in the field of natural product discovery.

APB was utilized as a model organism to validate the feasibility of the 2D-HPLC-MS technology in conjunction with the MN method. By importing the MS raw data into the MN with the parameters set to default, an MN was generated. MN is a visual computational strategy that visualizes all molecular ions detected in a complete LC-MS-MS experiment and the chemical relationships between these molecular ions13.
MN is based on the secondary mass spectrum fragments formed by the compound after entering MS-MS; compounds with similar structures produce similar MS-MS ion fragments under the same conditions, and the similarity of these mass spectral data is calculated by computer algorithms (expressed by the cosine value 0-1, the greater the similarity, the greater the cosine value, that is, if two MS-MS mass spectra are completely uncorrelated, the cosine value is 0)14. If the two mass spectra MS-MS are completely consistent, and the cosine value is 1, integrate these mass spectra into a visual network map according to the size of the similarity. In this MN diagram, each node represents the MS-MS mass spectrum of a compound, and the connection between nodes represents the correlation between the MS-MS maps of the two compounds, indicating that they are structurally similar or represent a uniform type of compound.
Alkaloids are the active ingredient and the main ingredient in the APB19. As shown in Figure 3 and Figure 4, four of the alkaloid components identified by the MN are aconitine, 14-benzoylaconine, 14-O-acetylneoline, and hypaconitine. The cosine values of these four compounds are 0.95, 0.95, 0.93, and 0.82, respectively, so the data is reliable. These four compounds have the same parent nucleus. Aconitine, 14-benzoylaconine, and hypaconitine are similar, and only the substituents are different (Figure 3 and Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66239/66239fig01.jpg" /> 
Figure 1: Identifying unknown compound structures in Tibetan medicine using 2D-HPLC-MS analysis. This is a simple experimental operation process to visualize the protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66239/66239fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66239/66239fig02.jpg" /> 
Figure 2: Flow path of the two-dimensional liquid phase. This has been referenced in the manufacturer&#39;s instructions in 2D-LC valve with loop. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66239/66239fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66239/66239fig03.jpg" /> 
Figure 3: Four chemical components identified by molecular networks. Using the protocol described here, four compounds were annotated in the generated network. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66239/66239fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66239/66239fig04.jpg" /> 
Figure 4: MS-MS spectra of four chemical components. Raw secondary mass spectra are provided for the four compounds. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66239/66239fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">Identification</td>
			<td colspan="2">Molecular 
			formula</td>
			<td>tR(min)</td>
			<td>[M+H]+ 
			Measured 
			(m/z) &#160;</td>
			<td>[M+H]+ 
			Lib</td>
			<td colspan="2">MSE fragmentation</td>
			<td>Cosine</td>
			<td colspan="2">Type</td>
		</tr>
		<tr>
			<td rowspan="2">1</td>
			<td colspan="2" rowspan="2">14-Benzoylaconine</td>
			<td colspan="2" rowspan="2">C32H45NO10</td>
			<td rowspan="2">21.67</td>
			<td rowspan="2">604.61</td>
			<td rowspan="2">604.31</td>
			<td colspan="2">554.6304</td>
			<td rowspan="2">0.95</td>
			<td colspan="2" rowspan="2">Alkaloids Terpenoids</td>
		</tr>
		<tr>
			<td colspan="2">328.6808</td>
		</tr>
		<tr>
			<td rowspan="2">2</td>
			<td colspan="2" rowspan="2">Aconitine</td>
			<td colspan="2" rowspan="2">C34H47NO11</td>
			<td rowspan="2">25.54</td>
			<td rowspan="2">646.58</td>
			<td rowspan="2">646.32</td>
			<td colspan="2">614.5858</td>
			<td rowspan="2">0.95</td>
			<td colspan="2" rowspan="2">Alkaloids Terpenoids</td>
		</tr>
		<tr>
			<td colspan="2">586.5756</td>
		</tr>
		<tr>
			<td rowspan="3">3</td>
			<td colspan="2" rowspan="3">Hypaconitine</td>
			<td colspan="2" rowspan="3">C33H45NO10</td>
			<td rowspan="3">31.63</td>
			<td rowspan="3">618.66</td>
			<td rowspan="3">616.31</td>
			<td colspan="2">525.7743</td>
			<td rowspan="3">0.82</td>
			<td colspan="2" rowspan="3">Alkaloids Terpenoids</td>
		</tr>
		<tr>
			<td colspan="2">499.9276</td>
		</tr>
		<tr>
			<td colspan="2">452.734</td>
		</tr>
		<tr>
			<td rowspan="2">4</td>
			<td colspan="2" rowspan="2">14-O-acetylneoline</td>
			<td colspan="2" rowspan="2">C26H41NO7</td>
			<td rowspan="2">9.82</td>
			<td rowspan="2">480.56</td>
			<td rowspan="2">480.3</td>
			<td colspan="2">462.5589</td>
			<td rowspan="2">0.93</td>
			<td colspan="2" rowspan="2">Alkaloids Terpenoids</td>
		</tr>
		<tr>
			<td colspan="2">398.4953</td>
		</tr>
	</tbody>
</table>
Table 1: Identification of the chemical constituents in APB by 2D-HPLC-MS Technology Combined with Molecular Network. Detailed data for the four compounds is provided here.

Watch this Scientific Journal Video about Author Spotlight: Integrating 2D-HPLC-MS and Molecular Networking in Natural Medicine Analysis at JoVE.com

Author Spotlight: Integrating 2D-HPLC-MS and Molecular Networking in Natural Medicine Analysis

This study utilizes two-dimensional high-performance liquid chromatography-mass spectrometry (2D-HPLC-MS) technology in conjunction with molecular networking to unravel the intricate chemical composition of the Tibetan medicinal plant Aconitum pendulum Busch (APB). The article provides a detailed protocol for the systematic exploration and identification of complex chemical components of herbal medicines.

2D-HPLC-MS Technology Combined with Molecular Network for the Identification of Components in Tibetan Medicine Aconitum pendulum

In this study, a comprehensive approach was employed, utilizing 2D-HPLC-MS technology in conjunction with the molecular network to unravel the intricate ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

416 Views.  Chengdu University of Traditional Chinese Medicine. In deze studie hebben we een alomvattende aanpak toegepast met behulp van 2D-HPLC-MS-technologie in combinatie met moleculaire netwerken om de ingewikkelde chemische samenstelling van de Tibetaanse medicinale plant APB te ontrafelen. We willen een efficiëntere methode ontwikkelen om de chemische samenstelling van natuurlijke geneesmiddelen te identificeren. De grootste uitdaging van de huidige experimenten is dat de sectie massaspectrometrie niet is uitgerust met massaspectrometrie met hoge ...

Video: Auteur in de schijnwerpers: integratie van 2D-HPLC-MS en moleculaire netwerken in de analyse van natuurlijke geneeskunde

Network Analysis of the Default Mode Network Using Functional Connectivity MRI in Temporal Lobe Epilepsy

Functional connectivity MRI (fcMRI) is an fMRI method that examines the connectivity of different brain areas based on the correlation of BOLD signal fluctuations over time. Temporal Lobe Epilepsy (TLE) is the most common type of adult epilepsy and involves multiple brain networks. The default mode network (DMN) is involved in conscious, resting state cognition and is thought to be affected in TLE where seizures cause impairment of consciousness. The DMN in epilepsy was examined using seed based fcMRI. The anterior and posterior hubs of the DMN were used as seeds in this analysis. The results show a disconnection between the anterior and posterior hubs of the DMN in TLE during the basal state. In addition, increased DMN connectivity to other brain regions in left TLE along with decreased connectivity in right TLE is revealed. The analysis demonstrates how seed-based fcMRI can be used to probe cerebral networks in brain disorders such as TLE.

The Default Mode Network (DMN) in Temporal Lobe Epilepsy (TLE) is analyzed in the resting state of the brain using seed-based functional connectivity MRI (fcMRI).

Functional connectivity MRI (fcMRI) is an fMRI method that examines the connectivity of different brain areas based on the correlation of BOLD signal ...

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 ...

Chemistry

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 ...

Identifying Chemical Composition of Gynura bicolor by UPLC-TOF Mass Spectrometry

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 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.

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

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

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Surfactant Depletion Combined with Injurious Ventilation Results in a Reproducible Model of the Acute Respiratory Distress Syndrome (ARDS)

Various animal models exist to study the complex pathomechanisms of the acute respiratory distress syndrome (ARDS). These models include pulmo-arterial infusion of oleic acid, infusion of endotoxins or bacteria, cecal ligation and puncture, various pneumonia models, lung ischemia/reperfusion models and, of course, surfactant depletion models, among others. Surfactant depletion produces a rapid, reproducible deterioration of pulmonary gas exchange and hemodynamics and can be induced in anesthetized pigs using repeated lung lavages with 0.9% saline (35 mL/kg body weight, 37 &#176;C). The surfactant depletion model supports investigations with standard respiratory and hemodynamic monitoring with clinically applied devices. But the model suffers from a relatively high recruitability and ventilation with high airway pressures can immediately reduce the severity of the injury by reopening atelectatic lung areas. Thus, this model is not suitable for investigations of ventilator regimes that use high airway pressures. A combination of surfactant depletion and injurious ventilation with high tidal volume/low positive end-expiratory pressure (high Tv/low PEEP) to cause ventilator induced lung injury (VILI) will reduce the recruitability of the resulting lung injury. The advantages of a timely induction and the possibility to perform experimental research in a setting comparable to an intensive care unit are preserved.

Surfactant Depletion Combined with Injurious Ventilation Results in a Reproducible Model of the Acute Respiratory Distress Syndrome ARDS

A combination of surfactant washout using 0.9% saline (35 mL/kg body weight, 37 &#176;C) and high tidal volume ventilation with low PEEP to cause moderate ventilator induced lung injury (VILI) results in experimental acute respiratory distress syndrome (ARDS). This method provides a model of lung injury with low/limited recruitability to study the effect of various ventilation strategies for extended periods.

Various animal models exist to study the complex pathomechanisms of the acute respiratory distress syndrome (ARDS). These models include pulmo-arterial ...

A Surgical Model of Heart Failure with Preserved Ejection Fraction in Tibetan Minipigs

More than half of heart failure (HF) cases are classified as heart failure with preserved ejection fraction (HFpEF) worldwide. Large animal models are limited for investigating the fundamental mechanisms of HFpEF and identifying potential therapeutic targets. This work provides a detailed description of the surgical procedure of descending aortic constriction (DAC) in Tibetan minipigs to establish a large animal model of HFpEF. This model used a precisely controlled constriction of the descending aorta to induce chronic pressure overload in the left ventricle. Echocardiography was used to evaluate the morphological and functional changes in the heart. After 12 weeks of DAC stress, the ventricular septum was hypertrophic, but the thickness of the posterior wall was significantly reduced, accompanied by dilation of the left ventricle. However, the LV ejection fraction of the model hearts was maintained at &gt;50% during the 12-week period. Furthermore, the DAC model displayed cardiac damage, including fibrosis, inflammation, and cardiomyocyte hypertrophy. Heart failure marker levels were significantly elevated in the DAC group. This DAC-induced HFpEF in minipigs is a powerful tool for investigating molecular mechanisms of this disease and for preclinical testing.

The present protocol describes a step-by-step procedure to establish a minipig model of heart failure with preserved ejection fraction using descending aortic constriction. The methods for evaluating cardiac morphology, histology, and function of this disease model are also presented.

More than half of heart failure (HF) cases are classified as heart failure with preserved ejection fraction (HFpEF) worldwide. Large animal models are ...

Bridging the Technology Divide in the COVID-19 Era: Using Virtual Outreach to Expose Middle and High School Students to Imaging Technology

Increasing the diversity of students choosing careers in science, technology, engineering, and mathematics (STEM) fields is an area of intense focus across the USA, especially in kindergarten through 12th grade (K-12)-focused pipeline programs in medical schools. A diverse STEM workforce contributes to better problem-solving and equity in health care. Two of the many major barriers for rural students are the lack of sufficient STEM role models and limited access to technology in the classroom. Medical schools often serve as an important resource for students in the local community who can easily gain access to STEM professionals and modern technology through on-campus, sponsored events and STEM outreach to the local classrooms. However, underrepresented minority (URM) students often live in socioeconomically distressed parts of rural states such as Arkansas, where access to STEM role models and technology is limited. Virtual learning in the COVID-19 era has proven that the imaging technology resources of a medical school can be harnessed to reach a wider audience, especially students living in rural areas far from the medical school campus.

This article presents an overview of how synchronous web-based virtual outreach can be used to expose 6th-12th grade students to advanced imaging technologies such as ultrasound, computerized tomography, and electroencephalography. The paper discusses the methods and equipment needed to livestream integrated educational sessions for effective student engagement in STEM.

Increasing the diversity of students choosing careers in science, technology, engineering, and mathematics (STEM) fields is an area of intense focus ...

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

The composition of the Tibetan medicine Rhodiola granules (RG) is complex, and the overall quality of RG is difficult to determine. Therefore, establishing a method to determine the multi-component in vitro dissolution of RG is of great significance for quality control. This study uses the second paddle method of the fourth general rule 0931 from the Chinese Pharmacopoeia (2020 edition), compliant with apparatus 2 of the United States Pharmacopeia (USP). The dissolution apparatus was set to a rotation speed of 100 rpm with ultrapure water as the dissolution medium. A sample volume of 1 mL was collected at each timepoint. Furthermore, the cumulative dissolution of gallic acid, salidroside, and ethyl gallic acid in RG at different time points was determined by high-performance liquid chromatography (HPLC). Finally, the dissolution curves were drawn, and the curves were fitted to the GompertzMod, the Gompertz, the Logistic, and the Weibull equations. The results showed that the cumulative dissolution of gallic acid in RG was over 80% at 1 min, the cumulative dissolution of salidroside and ethyl gallic acid was over 65% at 5 min, and the cumulative dissolution of each index component decreased after 30 min. The curve fitting demonstrated that the GompertzMod equation was the best-fitting model for each index component of RG. In conclusion, the dissolution test method described in this protocol is simple, accurate, and reliable. It can characterize the dissolution behavior of the index components in RG in vitro, which provides a methodological reference for quality control of RG and quality evaluation of other ethnic compounds.

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

Here, we test the dissolution of Rhodiola granules (RG) in vitro, draw dissolution curves of salidroside, gallic acid, and ethyl gallate in ultrapure water, and fit the curves to different mathematical models. This protocol provides information and guidance for in vivo bioequivalence and in vivo-in vitro correlation studies of RG.

2D-HPLC-MS Technology Combined with Molecular Network for the Identification of Components in Tibetan Medicine Aconitum pendulum

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