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

This method can help researchers analyze the structures of Tibetan medicine compounds, especially the tree's components. It can provide insight into the qualitative study of compound's content in natural medicine. The main advantage of this technique is that the accuracy of the compound structure obtained in this case is higher than that of the database.

This technique can be used in the structure analysis of metabolic intermediates in bladder or urine. To begin, the Tibetan medicine Abelmoschus manihot's seeds sample preparation. Accurately weigh one gram of AMS and place it in a conical flask containing 30 milliliters of 80%methanol.

Using an ultrasound bath sonicator at 40 kilohertz, perform extraction on the mixture for 30 minutes at 25 degrees Celsius. Then, centrifuge the sample at 14, 000g for five minutes. Prepare an injection syringe and an organic 0.22 micron microporous membrane filter, and filter the supernatant into a two-milliliter sample bottle.

After turning on the switch of the vacuum pump, open the main valve of the argon cylinder along with the partial pressure valve and adjust the pressure to approximately 0.3 megapascals. Then, open the nitrogen valve. Launch the mass spectrometry, or MS, control software.

Click on Heated ESI Source in the software panel and set the MS parameters, including heater temperature, gas flow rates, spray voltage for positive and negative modes, and capillary temperature. Click on the Apply button to activate the ion source for liquid chromatography or LC.Prepare mobile phases A and B using 0.1%formic acid in an aqueous solution in pure acetonitrile respectively. Degas them in an ultrasound bath sonicator at 40 kilohertz for at least 15 minutes before connecting the solutions to the A and B fluid passages respectively.

Prepare a 1:9 volume by volume methanol water solution, then manually fill it into the clean-out fluid bottles of the pump and injector. After launching the LC-MS control software, click Direct Control to open the LC control panel. Open the purge valve in the counterclockwise direction on the pump module.

Click on More Option to open the pump setting and set the purge parameters at five milliliters per minute for three minutes. Click on Purge to start the bubble removal. Once done, close the purge valve.

Then, click on Prime Syringe to rinse the syringe for three cycles, Wash Buffer Loop to rinse the loop for one cycle, and Wash Needle Externally to wash the needle for one cycle. Place the sample bottle in the sampler. Click on Instrument Setup to open the Method Editing Window and click on New to create a new LC-MS instrument method.

Establish a total runtime for the method and set the pressure limit. Total flow rate, flow gradient, sample temperature, column temperature, and ready temperature delta in the Method Editing Window. For the MS method, select the General MS or MSn experiment type.

Enter values of the acquisition time, polarity, mass range, divert value number, and duration. Click on Save to configure the settings as an instrument method. Click on Sequence Setup to open the sequence table, wherein enter the information about sample type, file, name, path, sample ID, instrument method, position, and injection volume.

Record the sequence table by clicking Save, then implement the settings and initiate the MS acquisition. To load the MS data into the data processing software, double-click on the raw file in Explorer. In the base peak chromatogram BPI, select the maximum area under the curve or AUC by clicking and dragging the mouse.

The corresponding MS spectrum will be displayed in the same window. Select a targeted ion for the next MS/MS analysis. Reopen the Method Editing Window, and in the MSn setting table, set the m/z of the targeted ion to one decimal place in the Parent Mass column.

Next, select collision mode and enter the collision energy, or CE value. Set the MS/MS scan range. Click on Save to record the MS method and enter a new file name in the sequence table.

Click on the Start button to initiate the MS/MS acquisition. Once the acquisition is complete, double-click on the raw file in Explorer to load the MS/MS raw file into the data processing software. Identify the strongest fragment ion in the spectrum and enter its m/z value in the MSn method list.

In the MSn setting table, set the MS3 parameters, including collision mode, CE value, and scan range. Again, click Save to record the MS method and enter a new file name in the sequence table. Click Start to initiate the MS3 acquisition.

As demonstrated before, double-click on the raw file in the Explorer to load the MS3 raw file into the data processing software, and repeat the steps to obtain the MS4 spectrum. To analyze the data, double-click on the raw files to open all the mass spectra from MS to MSn. Manually calculate the m/z difference values between the ion and the corresponding fragment ions.

Then, manually draw the core structure according to the MS4 results and derive the original structure using functional groups or molecular segments based on the m/z difference value. Manually draw the molecular cleavage paths according to each molecular structure in MSn. The ESI-MS of model carbohydrate cellobiose produced the proteinated molecule M H at m/z 365 in positive mode.

The product ion scan, or CID MS/MS resulted in a second fragment ion at m/z 305. Further, MS3 and MS4 analysis sequentially resulted in the third and fourth fragment ions at m/z 254 and m/z 185, respectively. The MS/MS analysis revealed the sequence of ion fragmentations, namely ring opening hydrolysis, CC cleavage, and dehydration.

So, this method was found suitable for carbohydrates. The preliminary qualitative analysis of AMS using LC Q-TOF MS revealed the presence of numerous unknown compounds. The negative MSn analysis of the M H ion at m/z 617 produced a second fragment ion at m/z 571.

The MS3 analysis of this fragment ion produced a third fragment ion at m/z 525 by the loss of hydroxyethyl as methanol corresponding to 32 dalton and hydroxyl as water, which is 18 dalton. The MS4 analysis produced fourth fragment ions at m/z 344 and 273. Manual identification of the core structure revealed the molecular structure of the compound at m/z 617 and its cleavage paths in MSn.

The product ion scan of the M H ion of another unknown compound was performed, and its molecular structure and cleavage mechanism were also revealed. After the vacuum pump is turned on, wait for at least five hours to ensure sufficient vacuum degree for the experimental conditions. Statistic analysis can be used to collate the fragments having the same or similar mass-to-charge ratio.

With mass spectrometry solution increasing, this technique could help researchers obtain newer compounds in natural medicines.