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 comprehe...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#160;</td>
			<td>Chengdu Keen experimental equipment</td>
			<td>/</td>
			<td>0.22&#956;m</td>
		</tr>
		<tr>
			<td>Ultimate XB-C18,5 &#956;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 &#956;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.

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

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

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 c...

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

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.

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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305 Views.  Chengdu University of Traditional Chinese Medicine. 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. 