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

Summary

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

Abstract

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.

Introduction

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’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—including volatile oils, coumarins, lignans, polysaccharides, and alkaloids—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'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.

Protocol

1. Sample pretreatment

1. 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.
2. Prepare an injection syringe (1 mL) and a microporous membrane filter (0.22 μm). Filter the solution into a new 2 mL sample bottle.
   NOTE: Take appropriate personal precautions during experimental procedures.

2. Ultra-performance liquid chromatography (UPLC) preparation

1. 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).
2. 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 μL.

3. MS preparation

1. 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.
2. 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 °C, initial MS range during 80 – 1200 Da, collision mode at collision-induced dissociation (CID), and collision energy at 35.

4. Full MS test

1. 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.
2. 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.
3. 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.
4. Click on the Pushpin button in the upper right corner of the mass spectrum window.
5. 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.

5. MS/MS test

1. 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.
2. 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.
3. 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.
4. 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
5. 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.
6. Click on the Pushpin button in the upper right corner of the mass spectrum window.
7. 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.

6. MSⁿ test

1. 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.
2. 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.

7. Parameter optimization

1. Return to the instrument setup window. In the act type column, click CID and then select PQD or ETD to change collision mode.
2. 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.

8. Result analysis

1. 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.
2. 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.
3. 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.
4. 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.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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