Name: Technical Approach for Structural Analysis of an Unknown Compound in Huoxiang Zhengqi Oral Liquid based on Linear Ion Trap Mass Spectrometry (Video) | JoVE Journal
Uploaded: 2026-04-03T14:13:00.000+00:00
Duration: 7 min 45 s
Description: Learn multi-stage mass spectrometry fragmentation for structural elucidation of unknown compounds in Huoxiang Zhengqi oral liquid. This linear ion trap method enables deeper fragmentation analysis than traditional approaches for Chinese herbal medicines.

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5 minutes

This work was funded by a special project for performance incentive and guidance of Chongqing Scientific Research Institute (cstc2022jxjl120005). Science and technology research project of Chongqing Municipal Education Commission (KJZD-K202315102). Chongqing Medical Scientific Research Project (Joint project of Chongqing Health Commission and Science and Technology Bureau (2022DBXM007). Hospital Special of Xinglin Scholar of Chengdu University of TCM (YYZX202160).

1. Sample pretreatment
<ol>
	<li>Open the packaging of the commercial HXZQ. Accurately transfer 0.1 mL of HXZQ into a 2 mL sample bottle, then add 0.9 mL of water. Shake the solution until it is thoroughly mixed.</li>
	<li>Prepare an injection syringe (1 mL) and a microporous membrane filter (0.22 &#956;m). Filter the solution into a new 2 mL sample bottle. 
	NOTE: Take appropriate personal precautions during experimental procedures.</li>
</ol>
2. Ultra-performance liquid chromatography (UPLC) preparation
<ol>
	<li>Double-click on the Xcalibur software. Click Ready to Download, then click the Direct Control button. Click on the Pump Module column in the pop-up window, set %B to 50, %C to 0, and %D to 0 (Figure 1).</li>
	<li>Click on the Motor button to switch it to the On state. Click on the More Options button, set the Flow to 5 [mL/min] and the Time to 180 [s] in the pop-up window. Click on the Purge button, then click on the OK button in the pop-up window. 
	NOTE: The mobile phase is constant at 0.3 mL/min with 50% A (0.1% formic acid solution) and 50% B (acetonitrile) without column temperature in the absence of a chromatographic column. The default injection volume is usually 1 &#956;L.</li>
</ol>
3. MS preparation
<ol>
	<li>Return to the software main window, click on the Sequence Setup View button. Click on the Open button to import the already edited template, and right-click on the Method Name and click on the Open File button to open the method file.</li>
	<li>In the pop-up Instrument Setup window, set the First Mass (m/z) to 100 and the Last Mass (m/z) to 1200. Click on the Save button to save the method. 
	NOTE: The default MS conditions included ion source temperature at 350 &#176;C, initial MS range during 80 &#8211; 1200 Da, collision mode at collision-induced dissociation (CID), and collision energy at 35.</li>
</ol>
4. Full MS test
<ol>
	<li>Click on the Run sequence button, then click on the OK button in the pop-up window. Wait for the sample injection to be completed (Figure 2). 
	NOTE: Before the test, please place the 2 mL sample bottle in the sample tray of the instrument.</li>
	<li>Click on the Roadmap View button, and click on the Qual Browser icon to open the Qual Browser window. Click on the Open button, select the Data File with .raw format in the folder, and double-click on it to open the file.</li>
	<li>Right-click on the Chromatogram Window, then click on the Ranges button. In the Scan filter section, select ESI Full MS. In the Plot type section, choose TIC. Click on the OK button, and then the window will display the total ion chromatogram of the sample.</li>
	<li>Click on the Pushpin button in the upper right corner of the mass spectrum window.</li>
	<li>In the chromatogram window, click and slide to select a Time Region with the strongest relative abundance. The mass spectrum window will display the corresponding mass spectrum ions. Record the m/z values for the next level of mass spectrometry.</li>
</ol>
5. MS/MS test
<ol>
	<li>Return to the Instrument Setup window. In the Parent Mass column of the n=2 row, enter the m/z value of the compound recorded in the previous step. Click on the Save button to save the method.</li>
	<li>Return to the software window. Click the Sequence Setup View button, modify the File name, and then click the Save button to save the sequence.</li>
	<li>Click on the Run sequence button, then click on the OK button in the pop-up window. Wait for the sample injection to be completed.</li>
	<li>Return to the Qual Browser window. Click on the Open button, select the Data File with .raw format in the folder, and double-click on it to open the file</li>
	<li>Right-click on the Chromatogram Window, then click on the Ranges button. In the Scan filter section, select ESI Full MS. In the Plot type section, choose TIC. Click on the OK button, and then the window will display the total ion chromatogram of the sample.</li>
	<li>Click on the Pushpin button in the upper right corner of the mass spectrum window.</li>
	<li>In the chromatogram window, click and slide to select a Time Region with the strongest relative abundance. The mass spectrum window will display the corresponding mass spectrum ions. Record the m/z values for the next level of mass spectrometry.</li>
</ol>
6. MSn test
<ol>
	<li>Return to the instrument setup window. In the Parent Mass column of the n=3 row, enter the m/z value of the compound recorded in the previous step. Click on the Save button to save the method.</li>
	<li>Repeat steps 4.2 to 4.5 to complete the sample injection and data viewing. Stop the MSn analysis once no stable fragment ions are observed.</li>
</ol>
7. Parameter optimization
<ol>
	<li>Return to the instrument setup window. In the act type column, click CID and then select PQD or ETD to change collision mode.</li>
	<li>In the normalized collision energy column, click 35 and then set to 20 or 50 to change collision energy. 
	NOTE: Combine all chemical residues and solvents into the organic waste container.</li>
</ol>
8. Result analysis
<ol>
	<li>Manually draw the parent ion and fragmentation ion in the drawing software, including parent ion structure, compound name, and mass-to-charge ratio (m/z) value.</li>
	<li>For example, for the unknown compound with m/z=623.21, observe the resulting fragmentation. Here, the fragment intermediate had an m/z=461.15 at MS/MS. The mass difference was 162.06 Da, corresponding to a hexose structure.</li>
	<li>Analyze this further, the fragment intermediate with m/z=461.15 fragmented again to form neobyakangelicol with m/z=315.09 at MS3. The mass difference was 146.06 Da, corresponding to a deoxyhexamethylose structure. That is, the intermediate with m/z=461.15 had one more deoxyhexamethylose unit than neobyakangelicol.</li>
	<li>In the structure of neobyakangelicol, analyze the bonding position. Here, the exposed hydroxyl group was the most likely position to be connected to a deoxyhexamethylose. Similarly, the hydroxyl group at C1 on the deoxyhexamethylose unit was the most likely to be connected to a hexose. Finally, obtain the structure of the unknown compound with m/z=623.21.</li>
</ol>

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The authors declare no competing financial interests.

The combination of LIT-MS and its MSn fragmentation technology provides a method for identifying unknown compounds in CHCs. Unlike traditional tandem MS modes in Orbitrap and Q-TOF MS, linear ion trap can specifically capture target ions, effectively avoiding interference from co-eluting ions23. This method achieves molecular-level precision, providing relatively accurate chemical structural information, thereby partially addressing the challenge of qualifying unknown compounds due to d...

Chinese herbal compounds (CHCs), as invaluable assets of traditional Chinese medicine (TCM), have accrued millennia of validated clinical experience1. They fulfill an irreplaceable role in disease prevention, therapeutic intervention, and rehabilitation2. Through multi-herb synergism, CHCs treat the human body holistically, operationalizing the core tenets of holism and syndrome differentiation-based therapy3. Within contemporary medical frameworks, CHCs garner global recognition for their polypharmacological mechanisms engaging multi-target pathways4, with broad applications spanning digestive disorders, respiratory infections, and immunomodulation5. Huoxiang Zhengqi oral liquid (HXZQ) epitomizes classical CHC formulations6. Its composition integrates multiple medicinal herbs such as patchouli, perilla leaf, angelica dahurica, atractylodes, poria, and dried tangerine peel. It is renowned for its ability to confer diaphoretic, heat-clearing, dampness-resolving, and spleen-stomach tonifying properties7. HXZQ is clinically deployed against dampness-induced influenza-like syndromes, epigastric distension, emesis, and diarrheal conditions8. Technological advancements have shifted CHC research from phenomenological observation to molecular, mechanistic, and compound-level investigations9, such as utilizing proteomics and metabolomics techniques to identify the active components, thereby establishing scientific foundations for TCM globalization. Consequently, rigorous analysis of HXZQ&#8217;s chemical constituents and pharmacodynamics not only advances CHC standardization but also catalyzes innovative drug discovery10.
The HXZQ is a multi-herb formulation whose inherent chemical complexity arises from its composite botanical constituents11. Characterized by a diverse array of phytochemical classes&#8212;including volatile oils, coumarins, lignans, polysaccharides, and alkaloids&#8212;HXZQ contains both well-characterized bioactive compounds and a substantial pool of structurally unannotated constituents12. Dynamic fluctuations in key components (e.g., volatile oils, flavonoids, alkaloids) can occur due to variations in extraction protocols and storage conditions, underscoring the critical need for systematic chemical profiling6. Within the framework of traditional Chinese medicine (TCM) modernization, in-depth compositional analysis of classical formulations like HXZQ not only elucidates the material basis underlying their therapeutic efficacy but also provides empirical support for quality control, standardized manufacturing, and adverse event monitoring10. While high-abundance compounds in HXZQ have been extensively documented, a significant portion of its chemical component remains uncharacterized13. The structural diversity of its constituents, combined with the low abundance of many potentially bioactive molecules, poses formidable challenges to comprehensive identification using conventional analytical techniques such as chromatography and spectroscopy alone14. Notably, isomerism is prevalent across multiple phytochemical classes in HXZQ, including coumarins, lignans, and polysaccharides, further complicating structural differentiation15. Additional barriers to accurate compound annotation include low analyte abundance and matrix interference effects. Collectively, these factors highlight a key frontier in HXZQ research: the development of robust analytical strategies to achieve precise, high-coverage identification of its full chemical complement.
Contemporary mass spectrometry (MS) platforms encounter inherent limitations when characterizing complex matrices, including ion co-elution artifacts and incomplete spectral database coverage16. In this context, tandem mass spectrometry (MS/MS) and multi-stage mass spectrometry (MSn) have emerged as indispensable analytical strategies for de novo structural elucidation of unknown compounds17. While conventional high-resolution MS systems such as quadrupole time-of-flight (Q-TOF) MS and Orbitrap MS generate high-quality MS/MS fragment data, their utility is constrained to single-stage fragmentation events. Despite this limitation, these platforms deliver rich structural insights with exceptional sensitivity and resolution, particularly when analyzing complex mixtures18. In contrast, linear ion trap (LIT) MS employs a multi-stage collision-induced dissociation (CID) mode that enables sequential, iterative fragmentation of molecular ions. This unique capability allows for stepwise dissection of compound skeletons and functional groups, facilitating unambiguous qualitative identification of structurally diverse unknown analytes19. To address the unmet need for comprehensive characterization of complex CHC matrices, this study presents an LIT-MS-based analytical workflow tailored for unknown compound identification. Leveraging the LIT&#39;s high ion capture efficiency and rapid scanning speed, this approach enhances the throughput and accuracy of de novo structural annotation. Application of this workflow to HXZQ aims to: (1) complement existing methodologies for profiling uncharacterized chemical constituents in HXZQ; (2) establish a technical framework to support standardization research for other CHC formulations; and (3) accelerate the translation of TCM from empirical practice to evidence-based, precision medicine.

<table><tbody><tr><td>Acetonitrile</td><td>Thermo Scientific</td><td>CAS 75-05-8</td><td>Liquid state</td></tr><tr><td>Formic acid</td><td>Thermo Scientific</td><td>CAS 64-18-6</td><td>Liquid state</td></tr><tr><td>Huoxiang Zhengqi Oral Liquid</td><td>Chongqing Taiji Industry (Group) Co., Ltd.</td><td>State Drug Standard Code Z50020409</td><td>Object of study</td></tr><tr><td>Linear ion trap mass spectrometer</td><td>Thermo Scientific</td><td>LTQ XL</td><td>IT-MS instrument</td></tr><tr><td>liquid chromatograph</td><td>Thermo Scientific</td><td>U3000</td><td>UPLC instrument</td></tr><tr><td>Xcalibur</td><td>Thermo Scientific</td><td>version 2.0</td><td>UPLC-IT-MS operational software</td></tr></tbody></table>

technical approach for structural analysis of an unknown compound in huoxiang zhengqi oral liquid based on linear ion trap mass spectrometry

Technical Approach for Structural Analysis of an Unknown Compound in Huoxiang Zhengqi Oral Liquid based on Linear Ion Trap Mass Spectrometry

Chinese herbal compounds (CHCs) play an irreplaceable role in Chinese medicine, and identifying their complex constituents has been a significant research focus in recent years. Huoxiang Zhengqi oral liquid is a classic Chinese patent medicine whose chemical composition requires further investigation at the molecular level. Traditional mass spectrometers, such as time-of-flight and Orbitrap, typically only provide secondary fragmentation information. Based on a linear ion trap mass spectrometer, compounds could be broken down more thoroughly, thereby obtaining deeper fragment information. This paper develops a structural exploration technique for unknown compounds in CHCs, encompassing sample pretreatment, ultra-performance liquid chromatography preparation, mass spectrometry preparation, full-spectrum testing, secondary mass spectrometry testing, multi-level mass spectrometry testing, and result analysis. Representative results demonstrate the derivation process of the compound structure. We discuss factors influencing the experimental technique, such as isomers, polyhydroxy compounds, and instrument resolution. Based on the derivation of the microscopic molecular structure of unknown compounds through multistage mass spectrometry fragmentation, the established experimental method is versatile and applicable for structural characterization of bioactive small molecules in traditional Chinese medicine and their linkage to pharmacological mechanisms.

We suggested that all the m/z information needed to be collated first, and then the mass difference value between the parent ion and the fragment ion was calculated. Found the compound that was reported in the database or literature, and then deduced the unknown compound structure reversely based on this known structure.
All detected compounds and their corresponding fragment ions were presented by m/z values. A subset of these fragment ions could be matched to authenticated reference standard...

Here, we show a standard protocol that combines multi-stage mass spectrometry trees with a fragmentation process based on Huoxiang Zhengqi oral liquid.

Chinese herbal compounds (CHCs) play an irreplaceable role in Chinese medicine, and identifying their complex constituents has been a significant research ...

A multi-stage mass spectrometry fragmentation method was developed to identify and characterize complex constituents in Huoxiang Zhengqi oral liquid. Traditional tandem mass spectrometry cannot resolve unknown compound structures. Multi-stage mass spectrometry provides deeper fragmentation for comprehensive structural elucidation.For ultra-performance liquid chromatography preparation, start by double clicking on the Xcalibur software to open it. Click Ready to Download, then click Direct Control. In the pop-up window, click the Pump Module column and set percentage B to 50, percentage C to zero and percentage D to zero.Click the Motor button to switch it to the on state. Click more options, set the flow to five milliliters per minute and the time to 180 seconds in the pop-up window. Click Purge, then click Execute despite warning in the pop-up window.Return to the main software window and click Sequence Setup View. Click Open to import the edited template. Right click on method name and click Open File to open the method file.In the software main window, set the first mass to 100 mass to charge ratio and the last mass to 1200 mass to charge ratio. Click Save to save the method. Click Run Sequence, select Standby in the after sequence set system, then click Okay in the pop-up window.Wait for the sample injection to be completed. Click Roadmap View, then click the Qual Browser icon to open the Qual Browser window. Click Open, select the data file with RAW format and double click to open it.Right click on the chromatogram window and click Ranges. In the scan filter section, select ESI full MS.And in the plot type section, choose TIC, then click Okay. Observe the total ion chromatogram displayed.Click the push pin button in the mass spectrum window. In the chromatogram window, click and slide to select a time region with the strongest relative abundance. Observe the corresponding mass spectrum ions and record the mass to charge ratio values.Open the instrument setup window. Locate the parent mass column of the N equals two row and enter the previously recorded mass to charge ratio value. Click Save to save the method.Return to the sequence setup view in the software window, modify the file name and click Save to save the sequence. Click Run Sequence, then click Okay in the pop-up window. Wait for the sample injection to be completed.Go to the Qual Browser window, click Open, select the RAW data file and double click to open it. Right click on the chromatogram window and click Ranges. In the scan filter section, select ESI full MS.In the plot type section, choose TIC and click Okay to display the chromatogram.In the mass spectrum window, click the push pin button. Select a time region with the strongest relative abundance and observe the mass spectrum ions. Record the mass to charge ratio values for the next level of mass spectrometry.In the instrument setup window, locate the parent mass column of the N equals three row and enter the previously recorded mass to charge ratio value. Click Save to save the method. As previously demonstrated, repeat the data viewing procedure to complete the sample injection and analysis.After opening the raw data file, click the push pin button in the mass spectrum window and observe the changing fragment ion peaks. In the instrument setup window, go to the Act Type column and click CID. Then select PQD or ETD to change the collision mode.In the Normalized Collision Energy column, click 35 and change it to 50 to adjust the collision energy. Draw the parent ion and fragmentation ions in drawing software, including the parent ion structure, compound name, and mass to charge ratio value. As an example, identify the fragment at mass to charge ratio 461.15, and examine the precursor ion with mass to charge ratio 623.21 in the tandem mass spectrometry spectrum.Calculate the mass difference. Analyze the further fragmentation of the intermediate ion with mass to charge ratio 461.15. To generate a product ion with mass to charge ratio 315.09 in the MS cubed, calculate the mass difference based on the bonding position and linkage analyses of all the fragments.Deduce the final structure of the unknown compound with a mass to charge ratio of 623.21. The unknown compound with a mass to charge ratio of 623.21 lost one hexose unit and yielded a fragment ion with a mass to charge ratio of 461.15. Tertiary mass spectrometry fragmentation of the intermediate produced neobyakangelicol with a mass to charge ratio of 315.09 after the loss of one rhamnose unit.Quadruple mass spectrometry of neobyakangelicol produced a fragment ion with a mass to charge ratio of 135.09. The structure of the unknown compound was deduced from the multi-stage fragmentation pattern. The unknown compounds with mass to charge ratios of 545.41 and 365.12 were deduced using the same fragmentation method.This protocol enables detailed analysis and structural characterization of more unknown compounds in medicinal herbs and Chinese patent medicines. A clear understanding of the core structure of the main components is essential for accurate fragmentation and analysis using this protocol. This procedure enables the development of a multi-stage mass spectrometry fragmentation database for improved compound identification and comparative analysis.

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Learn multi-stage mass spectrometry fragmentation for structural elucidation of unknown compounds in Huoxiang Zhengqi oral liquid. This linear ion trap method enables deeper fragmentation analysis than traditional approaches for Chinese herbal medicines.

Technical Approach for Structural Analysis of an Unknown Compound in Huoxiang Zhengqi Oral Liquid based on Linear Ion Trap Mass Spectrometry (Video) | JoVE Journal

Technical Approach for Structural Analysis of an Unknown Compound in Huoxiang Zhengqi Oral Liquid based on Linear Ion Trap Mass Spectrometry

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