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

In this study, we employed a comprehensive approach utilizing 2D-HPLC-MS technology in conjunction with molecular networking to reveal the intricate chemical composition of the Tibetan medicinal plant APB. We aim to establish a more efficient method for identifying the chemical composition of natural medicines. The main challenge of the current experiments is that the mass spectrometry section is not equipped with the high resolution mass spectrometry, resulting in the detection of fewer components.

2D-LC-MS enables more accurate identification and quantification of compound complex mixtures based on sample separation combined with molecular network technology, it can better process complex data information and improve the efficient and the accuracy of analysis. Our laboratory, we are focused applying existing technologies to get the separation of natural medicinal chemical components. Begin sample preparation by weighing the Aconitum pendulum bush or APB.

Using a one by 10, 000 sensitivity balance, weigh 0.25 grams of dry APB in a three milliliter microcentrifuge tube. Add 2.5 milliliters of methanol to it, and then sonicate for 30 minutes at a power of 240 watts and a frequency of 40 kilohertz. Centrifuge the sonicated sample at 1.2G for five minutes, and collect the supernatant.

Filter it through a 0.22 micron membrane filter. Next, prepare the two dimensional mobile phase by using acetonitrile as the organic phase, or phase B, and ultra-pure water containing 0.1%formic acid as the aqueous phase, or phase A.After performing two phase filtration through the 0.22 micron filter, subject the mobile phases to ultrasonic sonication at 40 kilohertz for 15 minutes. Purge the replaced mobile phase to remove the bubbles.

Select a C18 column as the 1D column and a hydrophilic column as the 2D column, and mount them to the instrument. To adjust the appropriate shunt ratio, connect the 2D-LC instrument outline through a T to the mass inlet, and the other end of the T to a shunted line. Adjust the flow rate to a suitable value to ensure the appropriate flow rate for the mass spectrum.

To begin the 2D-Liquid Chromatography or 2D-LC, double click the instrument one online icon, and the chemical workstation will automatically communicate with the 1260LC and enter the workstation. For the 2D-LC method parameter setting, click on the injector module, right click Method, and set the injection volume flow rate and mobile phase time gradient to the 1D pump. Enter the sample runtime under Stop Time.

Click on the main menu instrument and click 2D-LC Method on the dropdown menu. Select Comprehensive in 2D-LC Mode. Enter two minutes at modulation time and 1.9 minutes at 2D gradient stop time.

Set flow settings to two milliliters. Edit 2D gradient and set wavelengths. After editing the method, select Save Method As on the method menu to name the new method, and click OK.To begin, 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 megapascals.

To set up the MS method, enter the runtime in minutes to 93.00, and enter values to configure acquisition time, polarity, mass range, transfer value number, transfer value duration, and more. Set up scan event details according to the values displayed on the screen. To set data-dependent settings, use separate polarity settings as disabled.

Set neutral loss within top to three, and product mass list within top to three. Save to configure the settings as the instrument method. 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. Save the information to record the sequence listing. Then click the Start Analysis button to set up and start MS acquisition.

To begin the data preparation, export 2D-HPLC-MS raw mass spectral data. Next, to connect to the GNPS FTP server, in the WIN SCP interface on the Session Configuration page, fill in the connection information. After filling in the information, click the Start button to establish a connection to the GNPS FTP server.

Create molecular networks by opening a web browser and visiting the GNPS website. New users need to sign up for an account and then log in. On the main interface of the GNPS website, click Create Molecular Network under Data Analysis.

On the task creation page, click the Select Input Files button, then select and upload the data file. In the Workflow Option tabs, set the various parameters that generate the molecular network or MN.These parameters include the peak extraction algorithm, peak over travel value, similarity calculation method, and so on. After making the appropriate parameter settings as needed, click the Submit button at the bottom of the page to run the task.

After the task runs, find the created tasks in the Jobs tab and click the workflow name to view the analysis results. The website provides MN diagrams, substitutes, symbiotic networks, and other related information. Four of the alkaloid components in the APB samples identified by the MN were Aconitine, 14-Benzoylaconine, 14-Oecetalnelene, and Hypoconatine.

The four compounds had the same parent nucleus. Aconitine, 14-Benzoylaconine, 14-Benzoylaconine, and Hypoconatine were similar, and only the substituents were different.