Summary

Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components

Published: November 03, 2023
doi:

Summary

We describe a general protocol and systematic design that could be applied to separate and recognize complex components in alpine yarrow herb, Achillea millefolium L., a Chinese herbal medicine.

Abstract

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) is the most widely used method in qualitative analysis of compounds. The method includes standardized and programmed protocols for sample pretreatment, MS tune, MS acquisition, and data processing. The sample pretreatments include collection, pulverization, solvent extraction, ultrasound, centrifugation, and filtration. Data post-processing was described in detail and includes data importing, self-established database construction, method establishment, data processing, and other manual operations. The above-ground part of the alpine yarrow herb, Achillea millefolium L., is used to treat inflammation, gastrointestinal disturbances, and pain and its 3-oxa-guaianolides could be useful leads for anti-inflammatory drug development. Three representative compounds in AML were identified, combining TOF-MS with a self-established database. Moreover, the differences from existing literature, liquid-phase parameter optimization, scan mode selection, ion source suitability, collision energy adjustment, isomer screening, method limitation, and possible solutions were discussed. This standardized analysis method is universal and can be applied to identify complex compounds in Chinese herbal medicine.

Introduction

Chinese medicine has accumulated the richest empirical knowledge in the world1. Qualitative analysis of chemical components in traditional Chinese herbal medicine has become a crucial topic in research2. Distinguishing chemical differences in Chinese herbal medicine is difficult because of category complexity and origin diversification3. The major compound types in Chinese herbal medicine include alkaloids, saponins, flavonoids, anthraquinones, terpenoids, coumarins, lignans, polysaccharides, polypeptides, and proteins1. However, the separation of compounds and identification of isomers hinder the development of qualitative research on Chinese herbal medicine.

The combination of ultra-high-performance liquid chromatography (UPLC) with suitable chromatography columns provides strong support for the separation of complex compounds in Chinese herbal medicine4. In recent years, high-resolution mass spectrometry has become increasingly popular in Chinese herbal medicine qualitative analysis. Commonly used high-resolution mass spectrometry methods include quadrupole time-of-flight mass spectrometry (Q-TOF-MS)5, orbitrap mass spectrometry (Orbitrap-MS)6, and Fourier-transform ion cyclotron mass spectrometry (FT-ICR-MS)7. FT-ICR-MS has the highest resolution but entails costly operation and maintenance costs8. Orbitrap-MS has advantages in detecting small molecular compounds, especially at molecular weights below 500 Da9. Q-TOF-MS is the most widely used method in qualitative analysis of serum pharmacochemistry10,11. Compared with the traditional network database or commercial database, joint analysis with a self-established database for data processing has become increasingly popular.

Alpine yarrow herb, Achillea millefolium L. (AML), a kind of Chinese herbal medicine, grows mainly in Xinjiang, Inner Mongolia, and the northeast areas of China12. The above-ground part of AML is widely used to treat inflammation, gastrointestinal disturbances, and pain, including rheumatalgia, toothache, and stomachache13. The 3-oxa-guaianolides from AML offer great potential as leads for anti-inflammatory drug development14. Current studies on chemical components in AML focus on sesquiterpenes, monoterpenes, flavonoids, and phenolic compounds15. However, for the identification of compounds in AML, no systematic qualitative induction scheme that could used for other Chinese herbal medicines is available. This study aims to provide a standardized identification of chemical components in Chinese herbal medicine by combining Q-TOF-MS and self-established database analysis.

Protocol

1. Sample pretreatment

  1. Collection of Chinese herbal medicine AML
    1. Plant alpine yarrow Herb, Achillea millefolium L. (AML) seeds in the ground in February. Collect the above-ground part of AML in July of the same year (Figure 1A).
      NOTE: AML used in this paper was collected in a mountainous area at an altitude of 400 m in Mianyang, Sichuan, China.
  2. Drying treatment
    1. Wash all the collected AML in pure water to remove sediment and impurities. Dry the AML in an oven at 50 °C for 24 h (Figure 1B).
  3. Powder preparation
    1. After drying, crush AML into a coarse powder using a high-speed multifunctional crusher. Pass the powder through a 50-mesh sieve (Figure 1C).
  4. Solvent extraction
    1. Place 1 g of accurately weighed AML sample in a conical flask with 30 mL of a 75% ethanol-water solution (Figure 2A).
    2. Extract the mixture in an ultrasound bath sonicator for 30 min at 25 °C (Figure 2B).
    3. Centrifuge the sample at 14,000 × g for 5 min (Figure 2C).
    4. Fit an injection syringe with a microporous membrane filter (0.22 μm, organic only) and filter the supernatant into a 2 mL sample bottle (Figure 2D).

2. MS tune

  1. Launch the LC-MS control software (Figure 3A). Open the MS tune module and purge the 1 ng/µL leucine encephalin (LE) solution two times.
  2. In the LockSpray Flow Control panel, set a 50 μL/min flow rate and click on the Flow button to let the LE solution enter the mass spectrometer (Figure 3B).
  3. Click the LockSpray Source Setup button to complete the MS tune in positive mode (Figure 3C). Click on the negative icon to switch the ion mode. Click the LockSpray Source Setup button to complete the MS tune in negative mode.

3. MS acquisition

  1. Set a sequence table, including file name, MS method, inlet file, vial, and volume. Click on the Save button to record the sequence table.
  2. Click on the Run button and Start button in sequence (Figure 3D). Select the Acquire sample data only option. Click on the OK button to start data acquisition.
  3. Click on the Chromatogram button to open the real-time total ion chromatogram (TIC) window (Figure 3E). Click on the Display button and TIC button in sequence. Select the BPI Chromatogram option, then click on the OK button to display the base peak chromatogram (BPI) window (Figure 3F).

4. Data processing

  1. Launch the data analysis software.
  2. Click on the My Work button and Import MassLynx Data button in turn to enter the child window (Figure 4A). Select the raw data files and enter the sample set name, then click on the Create UNIFI Sample Set button to import the raw data of MS spectra.
    NOTE: Make sure the positive and negative raw data are imported separately.
  3. Database establishment
    1. Click on the Administration button in the initial window (Figure 4B). Click on the Import library items button. Select the database template file in .xlsx format with all separated compound structures in .mol format in the same folder.
    2. Enter a name for the database. Click on the Verify button to make sure all the compounds are displayed. Click on the Import button to finish the build of a self-established database.
      ​NOTE: All the compounds in independent .mol format files that need to be imported into the database are prepared based on the literature references. Compound structure files are drawn by oneself using drawing software.
  4. Click on the Create analysis method button to open a child window. Click on the Generate a process only method button to establish a data processing method.
  5. Data analysis
    1. Click on the Create analysis button to open a child window.
    2. Click on the Create analysis from existing data button, then select the imported data and established method.
    3. Click on the Process button to start a long data calculation (Figure 4D).
    4. Click on the Investigate button to switch to the TIC window.
    5. Click on the Select traces button and select the TOF MSE BPI. Click on the Replace all button to view the BPI.
  6. Right-click and select the Add column option | display compound information, including compound name, natural mass, observed m/z, mass error, observed retention time (RT), detector counts, response, adducts, alternate assignments, and total number of fragments found (Figure 4E).
  7. Select the High-energy fragments option to display the secondary mass spectrum fragments of the selected compound (Figure 4F).
  8. Manually draw the molecular cleavage paths according to each secondary fragment (Figure 4G).
    NOTE: Examples are described in detail in the representative results section.

Representative Results

Alpine yarrow herb was used as a model to display the representative result. As shown in Figure 4G, quercetin-3'-O-glucoside with m/z = 463.08935 transformed into an intermediate with m/z = 300.02828 via loss of a hexose molecule during the hydrolysis reaction. In another pathway, the break of the C-C bond in the flavonoid structure skeleton led to the formation of an intermediate with m/z = 223.06232, where hydroxymethyl and adjacent hydroxy in the hexose unit disappeared. Moreover, the cleavage of the hexose skeleton resulted in an intermediate with m/z = 71.01498. For phenolic acids, 3,5-dicaffeoylquinic acid with m/z = 515.11923 could lose a C9H6O3 or C18H12O7 group via hydrolysis reaction to form intermediates with m/z = 353.08783 or 173.04579, respectively (Figure 4H). For flavonoids, luteolin could be converted into an intermediate with m/z = 133.03002 via C-O-C and C-C cleavage (Figure 4I).

Figure 1
Figure 1: Identification of chemical components in Chinese herbal medicine AML using Q-TOF-MS and statistical analysis. (A) Collection of Chinese herbal medicine. (B) Drying treatment. (C) Powder preparation. (D) Mass spectrometry analysis. (E) Data processing. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Extraction method of Chinese herbal medicine AML. (A) Organic solvent addition. (B) Ultrasound extraction. (C) Centrifugation of mixture. (D) Filtration of supernatant. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Operation procedure of Q-TOF-MS. (A) Instrument accuracy calibration. (B) Standard solution preparation. (C) MS tuning (D) Sample injection. (E) TIC spectrum display. (F) BPI spectrum display. (G) Raw MS spectra of quercetin-3'-O-glucoside. (H) Raw MS/MS spectra of quercetin-3'-O-glucoside. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Data analysis process. (A) Data importing. (B) Self-established database construction. (C) Method establishment. (D) Data processing. (E) Compound information. (F) Secondary mass spectrum fragment. (G) Fragmentation pattern of glycosides. (H) Fragmentation pattern of phenolic acids. (I) Fragmentation pattern of flavonoids. Please click here to view a larger version of this figure.

Discussion

High-resolution mass spectrometry combined with a self-established database offers a systematic qualitative technology to identify chemical components in Chinese herbal medicine. Unlike a commercial database, which contains common traditional Chinese medicine, a self-established database that uses compounds reported in the literature provides more accuracy in the analysis of rare or ethnic medicine16. Similar methods have been applied to other areas of research, including finished drug products17, dyes18, and plastic packaging19. Compared with the popular commercial database matching, the ability to identify compounds in unusual traditional Chinese medicine is significantly improved. The three identified compounds are consistent with the reported literature, but we will provide more information on secondary fragment ions and fragmentation patterns.

In TOF-MS, the negative ion mode is suitable for the identification of most types of compounds, such as flavonoids and anthraquinones. In the negative ion mode, the charged ion detected by mass spectrometry was the compound that lost a proton or added a formate ion. Alkaloids are suitable for detection in positive ion mode and form a positive charge by losing an electron, while polysaccharides are more likely to appear in positive ion mode with a proton or sodium ion. In our experiment, the electrospray ionization MS source is used for the targeted detection of polar and moderately polar compounds. For compounds of weak polarity, the atmospheric pressure chemical ionization MS source must be used. For large molecular compounds (m/z > 5,000), matrix-assisted laser desorption ionization MS source is the most suitable method to use. Although the self-established database can increase the accuracy of MS data, the attribution of isomers remains a common problem in high-resolution mass spectrometry.

The self-established database can be used to characterize the compounds specifically, but various isomers exist20. For example, a large number of flavonoid isomers are present21, which makes identifying the exact structures of the compounds difficult. Therefore, the secondary mass spectrum fragment information needs to be combined to derive an accurate judgment22. In secondary mass spectrometry data, the ion fragments of unknown compounds need to be investigated in the site during bond cleavage. As needed, we can increase the collision energy value to obtain more secondary fragments.

Although the UPLC-Q-TOF-MS has many advantages for compound identification compared with other analysis methods, some limitations remain. First, the separation of complex samples in the UPLC module is important. Better separation can reduce the co-efflux of compounds, which affects the identification of secondary fragments. Better separation of chemical compounds requires suitable liquid phase conditions, including a special liquid chromatography column, mobile phase with additives, and optimized gradient elution. Second, manual confirmation of secondary fragments is still required for isomer recognition. In recent years, multistage mass spectrometry based on ion trap mass spectrometry has been able to distinguish isomers23. In summary, UPLC-Q-TOF-MS technology is widely used in fields such as pharmacy, environmental chemistry, and material science, and it has the potential to gradually develop into a basic qualitative approach.

Declarações

The authors have nothing to disclose.

Acknowledgements

This work was funded by China Postdoctoral Science Foundation (2022MD713780), Inheritance and Innovation Team of TCM Treatment of Immune Diseases, Chongqing Medical Scientific Research Project (Joint project of Chongqing Health Commission and Science and Technology Bureau) (2022DBXM007), and the Natural Science Foundation of Chongqing (cstc2018jcyjAX0370). A special project for performance incentive and guidance of Chongqing Scientific Research Institute (cstc2022jxjl120005, cstc2021jxjl130021).

Materials

chloroform Sinopharm Chemical ReagentCo., Ltd CAS 67-66-3
ethyl acetate ChuandongChemical CAS 141-78-6
liquid chromatograph Waters ACQUITY Class 1 plus
MassLynx Waters V4.2 MS control software
Methanol ChuandongChemical CAS 67-56-1
n-butyl alcohol ChuandongChemical CAS 71-36-3
petroleum ether ChuandongChemical CAS 8032-32-4
Quadrupole time-of-flight mass spectrometry Waters SYNAPT XS
UNIFI Waters Data analysis software

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Gao, Y., Li, M., Jiang, X., Yang, F., Zhou, C., Zhang, B., Liu, D. Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components. J. Vis. Exp. (201), e66091, doi:10.3791/66091 (2023).

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