This study utilizes two-dimensional high-performance liquid chromatography-mass spectrometry (2D-HPLC-MS) technology in conjunction with molecular networking to unravel the intricate chemical composition of the Tibetan medicinal plant Aconitum pendulum Busch (APB). The article provides a detailed protocol for the systematic exploration and identification of complex chemical components of herbal medicines.
In this study, a comprehensive approach was employed, utilizing 2D-HPLC-MS technology in conjunction with the molecular network to unravel the intricate chemical composition of the Tibetan medicinal plant APB. Through the implementation of 2D-HPLC, enhanced separation of complex mixtures was achieved, enabling the isolation of individual compounds for subsequent analysis. The molecular network approach further aided in elucidating structural relationships among these compounds, contributing to the determination of potential bioactive molecules. This integrated strategy efficiently identified a wide array of chemical components present within the plant. The findings revealed a diverse spectrum of chemical constituents within APB, including alkaloids, among others. This research not only advances understanding of the phytochemical profile of this traditional Tibetan medicine but also provides valuable insights into its potential therapeutic properties. The integration of 2D-HPLC-MS and molecular network proves to be a powerful tool for systematically exploring and identifying complex chemical compositions in herbal medicines, paving the way for further research and development in the field of natural product discovery.
Tibetan medicine is an integral part of traditional Chinese medicine, adhering to the principles of Tibetan medical practices, and is used for disease prevention and treatment1. However, Tibetan herbal medicine contains complex plant chemical constituents characterized by significant fluctuations in content. Limited understanding of the fundamental bioactive elements has become a bottleneck in the modernization of Tibetan medicine2. The application of liquid chromatography-mass spectrometry (LC-MS), combining the strong separation power of chromatography with the high sensitivity of mass spectrometry (MS), has been widely used in natural medicine analysis3. However, due to the limitation of a single separation mechanism, components with highly similar structures tend to coelute in one-dimensional liquid chromatography separation. In subsequent mass spectrometric analysis, the low-abundance co-eluted components are difficult to detect due to ion suppression caused by high-abundance components4.
2D-HPLC, which stands for two-dimensional high-performance liquid chromatography, represents a novel chromatographic method that harmoniously combines different separation mechanisms using a pair of columns. This includes the fusion or alternation of normal-phase chromatography with reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography with reversed-phase liquid chromatography5. By merging these complementary chromatographic properties, the enhanced separation capability is achieved, effectively addressing the challenges caused by complex sample matrices6. Furthermore, by coupling two-dimensional chromatography with MS, the powerful separation ability of 2D-HPLC and the high sensitivity detection capability of MS can be fully integrated, providing support for the study of complex drug systems and their fundamental constituents7,8,9.
Tibetan medicine commonly features a multifaceted array of components and functionalities, where active ingredients typically exist in intricate compositions and at minimal concentrations. By integrating 2D-LC as a robust separation system and MS as an exceedingly sensitive detector, the challenges posed by intricate samples in terms of separation and identification can be more effectively addressed10,11,12. This amalgamation substantially contributes to advancing the exploration of the chemical composition of Tibetan medicine.
Regardless of whether it is traditional LC-MS or 2D-LC-MS, a vast amount of information can be obtained. However, extracting structural information of complex system components from this massive amount of information has always been a significant challenge. Therefore, researchers have developed various methods for screening and mining MS data. Global Natural Products Social Molecular Networking (GNPS) is an MS/MS data organization and visualization platform where 2D-LC-MS mass spectrometry data can be uploaded13. Each spectrum is considered as a vector and compared to all other spectra using cosine similarity. When the similarity between two spectra exceeds a threshold, they are connected in a molecular network (MN). This can be used for the rapid identification of known compounds and the determination of various unknown natural products14.
Tibetan medicine usually has multiple functions, but its complex composition and significant concentration differences make it difficult to effectively elucidate the relationship between function and material basis15. An in-depth study of Tibetan medicine requires the systematic characterization of as many components as possible. In the framework of this study, we intend to use APB in Tibetan medicine as the research object to demonstrate the strategy and process of systematically studying the complex chemical components of Tibetan medicine using 2D-HPLC-MS technology and MN technology. In the construction of the 2D chromatographic system, we combined reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography with significant separation mechanisms to achieve more effective separation of the complex components of APB16. In addition, to overcome solvent incompatibility between the two dimensions, an at-column dilution modulation mode was adopted. By combining the powerful separation capability of 2D-LC with the high sensitivity detection capability of MS, the spectral information of the complex components in APB is obtained more effectively and comprehensively. Furthermore, through the network and visualization of massive spectral information by MN technology, the components of APB are systematically analyzed. The strategy and process demonstrated in this study are expected to be applied to the study of other Tibetan medicines, promoting research on the material basis of Tibetan medicine, which is of great significance for advancing Tibetan medicine resources and improving quality control standards for Tibetan herbs17. The overall experimental process is shown in Figure 1.
In the experiment presented here, a new at-column dilution modulator was introduced into the Agilent two-dimensional liquid chromatography (2D-LC) system18. By adding an independent delivery pump, the flow path of the analysis was changed, resulting in a high orthogonality of the 2D-LC analysis. The coupling and switching between the two dimensions are accomplished by two six-port valves, as shown in Figure 2. When one sample loop is filled in the first dimension, another sample loop is analyzed in the second dimension. This means that the filling time of the 1D loop and the running time of the 2D are equal. This requires the fast gradient generated by the Binary pump in the second dimension. No peaks are lost when the entire effluent is analyzed. This is particularly helpful for the analysis of unknown samples. It results in a large number of 2D chromatograms that need to be combined for data analysis.
1. Preparation
2. 2D-LC operation
3. MS operation
4. Molecular network operation
APB was utilized as a model organism to validate the feasibility of the 2D-HPLC-MS technology in conjunction with the MN method. By importing the MS raw data into the MN with the parameters set to default, an MN was generated. MN is a visual computational strategy that visualizes all molecular ions detected in a complete LC-MS-MS experiment and the chemical relationships between these molecular ions13.
MN is based on the secondary mass spectrum fragments formed by the compound after entering MS-MS; compounds with similar structures produce similar MS-MS ion fragments under the same conditions, and the similarity of these mass spectral data is calculated by computer algorithms (expressed by the cosine value 0-1, the greater the similarity, the greater the cosine value, that is, if two MS-MS mass spectra are completely uncorrelated, the cosine value is 0)14. If the two mass spectra MS-MS are completely consistent, and the cosine value is 1, integrate these mass spectra into a visual network map according to the size of the similarity. In this MN diagram, each node represents the MS-MS mass spectrum of a compound, and the connection between nodes represents the correlation between the MS-MS maps of the two compounds, indicating that they are structurally similar or represent a uniform type of compound.
Alkaloids are the active ingredient and the main ingredient in the APB19. As shown in Figure 3 and Figure 4, four of the alkaloid components identified by the MN are aconitine, 14-benzoylaconine, 14-O-acetylneoline, and hypaconitine. The cosine values of these four compounds are 0.95, 0.95, 0.93, and 0.82, respectively, so the data is reliable. These four compounds have the same parent nucleus. Aconitine, 14-benzoylaconine, and hypaconitine are similar, and only the substituents are different (Figure 3 and Figure 4).
Figure 1: Identifying unknown compound structures in Tibetan medicine using 2D-HPLC-MS analysis. This is a simple experimental operation process to visualize the protocol. Please click here to view a larger version of this figure.
Figure 2: Flow path of the two-dimensional liquid phase. This has been referenced in the manufacturer's instructions in 2D-LC valve with loop. Please click here to view a larger version of this figure.
Figure 3: Four chemical components identified by molecular networks. Using the protocol described here, four compounds were annotated in the generated network. Please click here to view a larger version of this figure.
Figure 4: MS-MS spectra of four chemical components. Raw secondary mass spectra are provided for the four compounds. Please click here to view a larger version of this figure.
Identification | Molecular formula |
tR(min) | [M+H]+ Measured (m/z) |
[M+H]+ Lib |
MSE fragmentation | Cosine | Typ | |||||
1 | 14-Benzoylaconine | C32H45NO10 | 21.67 | 604.61 | 604.31 | 554.6304 | 0.95 | Alkaloids Terpenoids | ||||
328.6808 | ||||||||||||
2 | Aconitine | C34H47NO11 | 25.54 | 646.58 | 646.32 | 614.5858 | 0.95 | Alkaloids Terpenoids | ||||
586.5756 | ||||||||||||
3 | Hypaconitine | C33H45NO10 | 31.63 | 618.66 | 616.31 | 525.7743 | 0.82 | Alkaloids Terpenoids | ||||
499.9276 | ||||||||||||
452.734 | ||||||||||||
4 | 14-O-acetylneoline | C26H41NO7 | 9.82 | 480.56 | 480.3 | 462.5589 | 0.93 | Alkaloids Terpenoids | ||||
398.4953 |
Table 1: Identification of the chemical constituents in APB by 2D-HPLC-MS Technology Combined with Molecular Network. Detailed data for the four compounds is provided here.
The primary focus of this experiment was the optimization of a partial method within the framework of two-dimensional liquid phase separation. To achieve this, a novel at-column dilution modulator was seamlessly integrated into the two-dimensional liquid chromatography (2D-LC) system. This technical adjustment was of paramount importance as it significantly elevated the proficiency in profiling the chemical constituents present in Tibetan medicine known as APB. Incorporating the 2D-HPLC-MS technology enabled the comprehensive examination of the intricate composition of APB. By leveraging the merits of two-dimensional separation techniques, we achieved notable enhancements in both resolution and sensitivity. This translated to the successful identification and quantification of a more extensive spectrum of compounds.
In addition, troubleshooting of this technology is also worth discussing. When the peak shape of the liquid phase is abnormal, the main consideration is whether there are bubbles in the flow path and whether the mobile phase ratio is normal. The solution is to degas the mobile phase and optimize the mobile phase ratio. When the liquid phase pressure is abnormal, blockage and leakage are mainly considered. The solution is to replace or clean the clogged parts and reinforce the leaking parts.
This method, however, has certain limitations that deserve attention. It may not be ideally suited for the analysis of volatile and oil components due to the specific properties of these compounds. Despite these constraints, the implementation of two-dimensional separation techniques represents a significant advancement in our analytical capabilities. Not only does it find relevance in Tibetan medicine, but it also exhibits the potential for broader applications in fields such as pharmaceuticals, natural products, and environmental analysis.
MN proved to be a valuable tool for identifying and annotating the chemical components present in APB. This article only lists some of the identified components, and the unidentified components can continue to be analyzed and identified by classical methods. By constructing networks based on spectral similarity, MNs establish connections between related compounds and facilitate their structural elucidation.
The results obtained from this study contribute to our understanding of the chemical composition of APB and its potential therapeutic effects. Aconitine and hypaconitine have analgesic, cardiotonic, and antitumor effects20,21. 14-Benzoylaconine has anti-inflammatory and analgesic effects22. 14-O-acetylneoline has anti-tumor properties19. Further investigations are warranted to explore the pharmacological properties and potential synergistic interactions among these components.
In conclusion, the combined use of 2D-HPLC-MS technology and MN proved to be an effective approach for the identification and characterization of chemical components in APB. Research into APB is ongoing, and there may be other unidentified compounds that merit future investigation. This study provides a foundation for further research on the therapeutic efficacy and safety of this traditional Tibetan medicine, as well as its potential applications in modern healthcare practices.
The authors have nothing to disclose.
This work was funded by National Natural Science Foundation of China (82130113), the National Natural Science Foundation of China (82204765), the Nature Science Foundation of Sichuan (2022NSFSC1470), Sichuan Provincial Postdoctoral Special Funding Project (TB2023020) and the Xinglin Scholars Research Promotion Program of Chengdu University of Traditional Chinese Medicine (BSH2021030). These funds provide support in terms of experimental equipment, experimental materials, and publication fees.
Acetonitrile | Fisher chemical | F22M81203 | Mobile phase |
Aconitum pendulum | / | / | Herb medicine |
Agilent 1290 Infinity (II) 2D-LC | Agilent Technologies | G2198-90001 | Liquid chromatography |
Disposable syringes | Chengdu Keen experimental equipment | / | 1ml |
EP tube | Chengdu Keen experimental equipment | / | 3ml |
Liquid phase injection bottle | Chengdu Keen experimental equipment | / | 1.5ml |
LTQ XL Mass Spectrometer | Thermo Fisher | LTQ21991 | Mass Spectrometer |
Microporous membranes | Chengdu Keen experimental equipment | / | 0.22μm |
Ultimate XB-C18,5 μm,2.1 x 200 mm | Welch | 00201-31015 | Reversed-phase column |
Ultrasonic Cleaner | GT Sonic | UGT20DEC048Y | Ultrasonic Cleaner 240W 40KHz |
XAmide,3 μm,100A | Dalian Mondi Technology | D2019110601 | Hydrophilic column |