With the rapid development of science and technology, exploring new analytical techniques and establishing new strategies for analyzing Chinese medicine components are important tools to promote the analytical research of complex components of Chinese medicine. In particular, the development of chromatography-mass spectrometry techniques and data processing strategies can greatly facilitate the rapid, high-throughput characterization and accurate quantification of complex components of Chinese medicines. This paper will discuss the progress of new techniques and strategies for analyzing Chinese medicine components by mass spectrometry.
chromatography-mass spectrometry
Chromatography-mass spectrometry (LC-MS) is a fast, efficient, highly sensitive, and selective technique that can provide a wealth of structural information and improve the efficiency of identification and characterization of trace components in complex systems is now widely used in the analysis and identification of Chinese medicine components. In particular, liquid chromatography-mass spectrometry (LC-MS) has evolved from high-performance liquid chromatography (HPLC) to ultra-high-performance liquid chromatography (UPLC)/ultra-high-pressure liquid chromatography (U HPLC) and from one-dimensional liquid chromatography (1D-LC) to two-dimensional liquid chromatography (2D-LC). Liquid chromatography (2D-LC). The mass spectrometry component is mostly high-resolution mass spectrometry, such as time-of-flight mass spectrometry (TOF), quadrupole-time of flight mass spectrometry (Q-TOF), Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR) and orbital ion trap mass spectrometry (Orbitrap). Ultra Performance Liquid Chromatography (UPLC) is more efficient and faster than High-Performance Liquid Chromatography (HPLC). When combined with High-Resolution Mass Spectrometry (HRMS), it has significant advantages in analyzing and identifying Chinese medicine components. Two-dimensional liquid chromatography is particularly suitable for analyzing complex samples due to its high peak capacity (theoretical peak capacity of up to 10,000). By maximizing the orthogonality between the first and second-dimensional liquid chromatography, the distribution of peaks can be expanded to analyze and identify a wider range of components. Two modes of analysis are available: full 2D (LC × LC) and multiple heart-cutting (HMC). With the increasing pressure tolerance of UHPLC systems and the maturation of precision flow rate and multi-channel injection valve switching automation, UHPLC has become popular and is used in conjunction with HMS for the analysis and identification of Chinese medicine components. Gas chromatography-mass spectrometry (GC-MS) is an important tool for analyzing and identifying volatile oil components in Chinese medicine, with NIST and Wiley standard spectral libraries providing mass spectral information for rapid and accurate identification. The development of GC from one-dimensional (GC) to two-dimensional (GC × GC) has significantly increased the peak capacity and enhanced the separation of components. GC-MS techniques have evolved from GC-quadrupole mass spectrometry (GC-MS) to GC-triple quadrupole mass spectrometry (GC-MS/MS) and GC-time-of-flight mass spectrometry (GC-MS/MS). -Time of flight mass spectrometry (GC-TOF/MS) has improved the quality and accuracy of mass spectrometry data, as well as the sensitivity of detection. Sample pre-treatment techniques for GC-MS have evolved from direct sample injection and headspace injection to headspace solid phase microextraction (HS-SPME) and single drop liquid-liquid microextraction (SDME). These new pre-treatment techniques integrate extraction, enrichment, and injection steps to improve analytical efficiency and expand the applications of GC-MS. Chromatography-mass spectrometry is one of the essential techniques for analyzing and identifying the components of traditional Chinese medicines. Only by choosing the appropriate analytical technique according to the structural characteristics and physicochemical properties of the components of traditional Chinese medicines can the analysis and identification of complex components of traditional Chinese medicines be achieved accurately.
Mass spectrometry techniques and strategies for the identification of Chinese medicinal ingredients
Mass spectrometry techniques are improving in terms of resolution, accuracy, and sensitivity. In particular, new multifunctional hybridization mass spectrometry techniques offer a variety of scanning modes and fragmentation methods for the analysis and identification of complex components while providing high-resolution, accurate, and wide-coverage mass spectrometry data to support the identification of components with high quality. At the same time, the analysis and utilization of these multidimensional data have become a new constraint. Therefore, new techniques and strategies of mass spectrometry for identifying TCM components have become a hot research topic.
1. Ion drip mass spectrometry (IM-MS) is a technique that combines ion drip spectroscopy with mass spectrometry. It can identify components of similar shape and size and is particularly suitable for analyzing isomers and structurally similar components. Using ion flowmetry after chromatographic separation can greatly improve the analysis and identification of components.
2. The setting of mass spectrometry acquisition parameters for identifying and characterizing herbal components relies on high-quality mass spectrometry data. High-resolution mass spectrometry, such as time-of-flight mass spectrometry (TOF), quadrupole-time-of-flight mass spectrometry (Q-TOF), Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR), and electrostatic field orbital ion trap mass spectrometry (Orbitrap), are the mainstream instruments for the identification and characterization of TCM components. In addition to the differences in instrument hardware and software, using the FT-ICR and Orbitrap instruments can be a significant advantage. In addition to the differences in instrument hardware and software, the settings of the acquisition rate, resolution, precursor ion mass window, fragmentation voltage, and collision energy can also directly affect the mass spectrometry data and, thus, the identification and characterization of the Chinese medicine components. The acquisition rate measures the number of spectra acquired per unit time of the mass spectrometer. The identification of unknown components of TCM requires the selection of non-targeted modes. Therefore, acquiring as many spectra as possible to obtain complete mass spectral information for component identification is essential. Time-of-flight mass spectrometry is the fastest mass spectrometer available and, when coupled with ultra-performance liquid chromatography, is suitable for discovering the components of complex systems. Generally, QTOF can obtain 2 to 100 high-resolution full-scan spectra per second, which is sufficient for analyzing TCM components. However, TOF technology cannot provide secondary mass spectrometry information by itself, making it difficult to identify TCM components accurately. For mass analyzers such as TOF, the acquisition rate of the instrument does not affect the resolution but does affect the sensitivity. As a result, several major instrument manufacturers have worked to design and improve the hardware or software configuration of TOFs to combine high sensitivity and wide dynamic range with high acquisition rates. For example, the Xevo G2-S QTOF from Waters uses a patented stacked ring assembly to focus and guide ions at the ion source, significantly improving ion transfer efficiency and increasing the sensitivity of the mass spectrometer by more than 25 times; the disadvantage of the TOF is that it does not have cascade capability and can only provide secondary mass spectrometry data even when coupled with a quadrupole. Therefore, TOF has limited application when multiple levels of mass spectrometry are required to infer or confirm the structure of Chinese medicine components and must be used in conjunction with other tandem mass spectrometry. The resolution of high-resolution mass spectrometers is usually between 1 and 250,000 (FWHM). For the identification of TCM components, resolutions in tens of thousands to hundreds of thousands are generally adequate for the analysis. In recent years, some ultra-high resolution mass spectrometers have been introduced by major instrument manufacturers, such as Thermo’s Orbitrap Fusion mass spectrometer with a resolution of 450,000 (FWHM) and Bruker’s Fourier Transform Ion Cyclotron Resonance mass spectrometer (FT-ICR) with a resolution of 1 million (FWHM). FT-ICR technology is unique in its high sensitivity, ultra-high resolution, ultra-high mass accuracy, and multi-stage mass spectrometry capabilities for identifying and characterizing complex components in Chinese medicine. The width of the precursor ion mass window depends on the sensitivity and selectivity of the data acquisition. A narrower mass window improves resolution and selectivity but filters out most of the precursor ions, which may result in information loss. Therefore, a wider precursor ion mass window (1 to 3 u) is more suitable for analyzing and identifying TCM components. The choice of fragmentation voltage and collision energy directly affects the fragmentation information of the secondary mass spectra. Setting multiple collision energy fragments to obtain fragmentation information of the analyte at each collision energy and matching it with the mass spectral database can help to identify as many TCM components as possible. In addition, many mass spectrometers currently support fast positive and negative polarity switching, which can be used to obtain high-quality mass spectral data and fragmentation information of unknown components with different charge distribution and ionization characteristics.
3. The full scan mode is the most common and simplest mode of mass spectrometer acquisition for primary mass spectrometry data, allowing the acquisition of excimer ion and molecular mass information. Acquiring secondary mass spectrometry data usually requires a choice of acquisition modes, and different instrument manufacturers have their proprietary technologies for high-resolution mass spectrometers. Currently, the main modes of secondary mass spectrometry data acquisition for high-resolution mass spectrometry include data-dependent acquisition (DDA) and data-independent acquisition (DIA). In DDA, the mass spectrometer first performs a full scan. Then it selects precursor ions that meet certain criteria to trigger secondary fragmentation, including abundance, charge, dynamic exclusion, background deduction, etc. DDA mode reduces the presence of interfering ions by pre-screening the precursor ions, resulting in high-quality data for fragmented ions. It is currently the most commonly used acquisition mode. However, DDA as a selective acquisition mode has low coverage, and in general, ions with higher intensities are more likely to be selected as targets for secondary mass spectrometry acquisition, thus risking the loss of valuable ions of key components when they do not meet the screening criteria or are co-flowed with many higher intensity ions. It is often difficult to obtain high-quality fragment ion information for many trace components in Chinese medicine, which can be difficult to identify. The DIA model does not pre-screen the parent ions, which theoretically enables comprehensive fragment information to be obtained for all ions. Some strategies for DIA have been developed, such as the full information tandem mass spectrometry (MSE) technique (developed by Waters) and the SWAT H technique (developed by AB SCIEX in collaboration with ETH Zurich). DIA data processing software is still in the developmental stage compared to the many sophisticated analytical tools available for DDA. The DDA and DIA models have both advantages and disadvantages for the analysis and identification of complex components in Chinese medicine. The combination of the two has yielded good results.
4. Mass spectrometry data processing strategies are rich in structural information. They have been proven effective in identifying TCM components by summarising the structure-related patterns of compounds and establishing databases. Researchers have explored and summarized compounds’ mass spectrometric cleavage patterns and integrated various data processing and analysis methods to develop a variety of commercial mass spectrometric data processing strategies. Commonly used strategies include background subtraction (BS), mass loss filtering (MDF), daughter ion filtering (PIL), neutral loss filtering (NLF), and principal component analysis (PCA). An identification model that integrates multiple mass spectrometry data can fully explore similar unknown components and has the advantage of being comprehensive and systematic. The disadvantage is that a large amount of mass spectrometry data identified needs to be analyzed for identification on a case-by-case basis, which is slow. Therefore, the combination of the mass mentioned above spectrometry data processing strategies with cheminformatics and computational science has led to several more selective and efficient data processing strategies for complex components of TCM, including mass spectral tree similarity filter technique (MTSF) based on template compounds, fragmentation trees (MTSF) based on fragmentation fingerprints to identify unknown compounds de novo, and the identification of unknown compounds based on fragmentation trees (MTSF). “The MTSF technique, the fragmentation trees (FTs) strategy, the molecular networking (MN) strategy based on secondary fragment similarity scores, and the molecular descriptor strategy for compound prediction. Mass spectral tree similarity filtering (M TSF) calculates the similarity of an unknown compound to a template (known) compound using the primary high-resolution mass spectral data of the compound as the “trunk” and the multi-level mass spectral data as the “branches.” The fragmentation tree (FTs) strategy provides a rational reconstruction of the parent ion structure by combining fragment ions to characterize the structure of the unknown component from the bottom up. A representative compound identification software based on the fragment tree strategy is SIRIUS, currently updated to version 4.0 (SIRIUS 4), which enables a wide range of structure predictions for small molecules such as drugs, natural products, and metabolites. The Global Natural Products Social Network (GNPS) platform, which builds molecular networks (MNs) based on secondary fragment similarity to uncover unknown components, has received considerable attention since its inception in 2014.
Conclusion and outlook
The flourishing development of modern analytical and testing technologies and the emergence of research strategies have provided strong technical support for breaking through the bottleneck in studying traditional Chinese medicines’ material basis and mechanism of action. Emerging technologies such as multidimensional chromatography, ion drip mass spectrometry, and mass spectrometry imaging provide a wide scope for research on TCM. Different research methods, ideas, and solutions will form a more scientific, feasible, and effective research system for TCM.
Post time: Dec-17-2022
