A mass spectrometry imaging method for analyzing chemical components and spatial distribution of dried traditional Chinese medicine decoction pieces by using imprinting

Abstract

The application provides a mass spectrum imaging method for analyzing chemical components and spatial distribution of dried traditional Chinese medicine decoction pieces by using imprinting method, comprising: applying pressure to the traditional Chinese medicine decoction pieces to be tested to obtain a relatively flat cross section; establishing a "sandwich" structure, and transferring the components of the cross section of the traditional Chinese medicine decoction pieces to a transfer film by using imprinting method; performing ionization scanning on the transfer film in a mass spectrometer by using mass spectrum imaging detection method to obtain mass spectrum imaging data of the chemical components in the transfer film of the traditional Chinese medicine decoction pieces to be tested; performing data processing on the mass spectrum imaging data to identify the chemical components in the sample to be tested; and analyzing the spatial distribution rule of metabolites in the traditional Chinese medicine decoction pieces to be tested. The application can overcome the difficulty that the dried traditional Chinese medicine decoction pieces are hard and have uneven surfaces, and are difficult to be prepared into imaging samples, fill the gap of the mass spectrum imaging method for dried traditional Chinese medicine decoction pieces, and has the advantages of simple operation, high sensitivity, wide dynamic range, and multiple types of component coverage.

Description

Technical Field

[0001] This invention belongs to the field of mass spectrometry imaging technology, and relates to a mass spectrometry imaging method for analyzing the chemical composition and spatial distribution of dried Chinese herbal medicine slices using the imprinting method. Background Technology

[0002] As raw materials for direct preparation and formulation, processed Chinese medicinal herbs are a crucial link in ensuring the safety and efficacy of clinical use of traditional Chinese medicine (TCM). Studying the chemical composition of processed Chinese medicinal herbs is of great significance for understanding the growth and development of TCM, elucidating the pharmacodynamic material basis and pharmacological mechanisms of action. Because metabolites undergo complex interactions during the growth and storage of TCM, it is necessary to explore the spatiotemporal information of TCM and comprehensively understand the spatial distribution of metabolites in processed Chinese medicinal herb samples.

[0003] Compared to classical histochemical methods, mass spectrometry imaging technology eliminates the need for sample fragmentation, providing additional spatial information for metabolite studies and enabling visualization of traditional Chinese medicine (TCM) metabolites. Furthermore, mass spectrometry is a label-free technique that can be used without knowledge of the analytes, simultaneously detecting hundreds of compounds. Compared to fluorescence microscopy and in situ hybridization, it offers advantages such as high information throughput and fast analysis speed. Air-assisted desorption / electrospray ionization mass spectrometry (AFADESI-MSI) is a highly sensitive, high-coverage, and highly specific mass spectrometry imaging method. This method maintains tissue integrity and performs well at low m / z values, providing a powerful tool for the visualization analysis of TCM metabolites. The advent of AFADESI-MSI technology has significantly advanced research progress in areas such as the distribution of TCM components, quality markers of TCM, and the pharmacological, toxicological, and migration pathways of TCM metabolites.

[0004] However, most Chinese herbal medicine slices are processed and dried, making it difficult to prepare samples using traditional frozen sectioning methods. Furthermore, their morphology and physical properties vary greatly, and the irregular surface of the slices leads to drastic changes in signal intensity, making direct imaging methods unsuitable. These sample preparation challenges significantly hinder the mass spectrometry imaging analysis of Chinese herbal medicine slices compared to that of ordinary plant and animal tissues. Summary of the Invention

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] Step S1: Apply pressure to the Chinese herbal medicine slices to be tested to obtain a relatively flat cross-section;

[0007] Step S2: Establish a "sandwich" structure and use the imprinting method to transfer the cross-sectional components of the Chinese herbal medicine slices onto the transfer film;

[0008] Step S3: Use mass spectrometry imaging detection method to perform ionization scanning on the above-mentioned transfer film in a mass spectrometer to obtain mass spectrometry imaging data of chemical components in the transfer film of the Chinese herbal medicine to be tested.

[0009] Step S4: Process the mass spectrometry imaging data to identify the chemical components in the sample to be tested;

[0010] Step S5: Analyze the spatial distribution pattern of metabolites in the Chinese herbal medicine pieces to be tested.

[0011] Preferably, the specific method of step S1 is as follows:

[0012] Select Chinese herbal medicine slices with relatively flat cross-sections and thin thickness, apply 0.5 tons of pressure using a manual tablet press for 10 seconds to obtain Chinese herbal medicine slices with flat cross-sections to be tested.

[0013] Preferably, the transfer method in step S2 is as follows:

[0014] Spray 300-500 μL of 80% acetonitrile solution onto the transfer membrane (nylon microporous filter membrane). Place the Chinese herbal medicine sample to be tested on the moistened transfer membrane. Cover both sides of the transfer membrane-Chinese herbal medicine sample pair with absorbent paper, rubber plate, and sponge paper in sequence. Apply a pressure of 0.3-0.5 tons using a manual tablet press for 5-10 seconds. Finally, stick the transfer membrane onto the glass slide with double-sided tape.

[0015] Preferably, the determination method in step S3 is as follows:

[0016] Analysis was performed using an AFADESI-MSI platform and a Q-Orbitrap mass spectrometer (Q Exactive, ThermoScientific, USA). The spray solvent was 80% acetonitrile solution, with a solvent flow rate of 5 μL / min and a pump flow rate of 45 L / min. The spray distance from the section was 0.5–0.8 mm, the distance between the sample surface and the sprayer was 2–4 mm, and the distance between the sprayer and the ion delivery tube was 2–4 mm. Positive ion scanning was performed in Full Scan mode, with an MS resolution of 70,000, a mass range of 70–1000 Da, automatic gain control (AGC) target of 3E6, a maximum injection time of 200 ms, and a capillary temperature of 350 °C.

[0017] Preferably, the data analysis method in step S4 is as follows:

[0018] The raw data (.raw format) was converted to .imzML format using imzMLConverter software, and then imported into the Cardinal software package for background subtraction, peak alignment, and peak filtering to obtain mass spectrometry images of the metabolites. The data in the mass spectrometry images were compared with high-resolution mass spectrometry data from the literature and the SmetDB database to identify the chemical components in the sample.

[0019] Preferably, the analysis method in step S5 is as follows:

[0020] The spatial distribution patterns of metabolites in the tested Chinese herbal medicine were analyzed by comparing the mass spectrometry images with the original cross-sectional photos of the Chinese herbal medicine and the photos of the transfer film.

[0021] The beneficial effects of this invention are as follows:

[0022] This invention employs a mass spectrometry imaging method for analyzing the chemical composition and spatial distribution of dried Chinese herbal medicine slices using the imprinting method. This method overcomes the difficulties in preparing imaging samples due to the dryness, hardness, and uneven surface of the Chinese herbal medicine slices, thus filling the gap in mass spectrometry imaging methods for dried Chinese herbal medicine slices. The method is simple to operate, highly sensitive, has a wide dynamic range, and covers a wide variety of components, exhibiting significant advantages. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The image shows the "sandwich" structure of the imprinting sample preparation in Example 1.

[0025] Figure 2 The image shows the total ion flow chromatogram of the licorice blot in positive ion mode in Example 1.

[0026] Figure 3 The diagram shows the relationship between the licorice imprint mass spectrometry imaging results and the original herbal slices in Example 1. A: Licorice slices; B: Licorice imprint; C: Spatial shrinkage centroid clustering diagram of licorice imprint imaging.

[0027] Figure 4 The image shown is a mass spectrometry image of the positive ion mode licorice imprint metabolite in Example 1.

[0028] Figure 5The total ion chromatograms of licorice imprints on different transfer membranes in positive ion mode are shown in Comparative Example 1: A. Polytetrafluoroethylene microporous membrane (pore size 0.45 μm); B. Nylon 66 microporous membrane (pore size 0.45 μm); C. Nylon 66 microporous membrane (pore size 0.45 μm).

[0029] Figure 6 The following are mass spectrometry images of licorice metabolites from different transfer membranes in positive ion mode, as shown in Comparative Example 1: A1 Polytetrafluoroethylene microporous membrane (pore size 0.45 μm); A2 Nylon 66 microporous membrane (pore size 0.45 μm); A3 Nylon 66 microporous membrane (pore size 0.45 μm); B1 m / z 189.1344; B2 m / z 337.1706; B3 m / z 266.1750; B4 m / z 491.2984; B5 m / z 381.0782; B6 m / z 251.1596.

[0030] Figure 7 The following are mass spectrometry images of licorice imprint metabolites in different transfer spraying solvents in positive ion mode, as shown in Comparative Example 2: A1 80% acetonitrile; A2 50% acetonitrile; A3 80% methanol; A4 methanol; A5 acetonitrile; B1 m / z 188.0764; B2 m / z 80.0348; B3 m / z 162.0759; B4 m / z 175.1188. Detailed Implementation

[0031] The invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the invention are all common knowledge and general knowledge in the art, and the invention does not have any particular limitations.

[0032] This invention provides a mass spectrometry imaging method for analyzing the chemical composition and spatial distribution of dried Chinese herbal medicine slices using the imprinting method. The method includes: applying pressure to the Chinese herbal medicine slices to obtain a relatively flat cross-section; establishing a "sandwich" structure and transferring the cross-sectional components of the Chinese herbal medicine slices onto a transfer membrane using the imprinting method; performing ionization scanning on the transfer membrane in a mass spectrometer using a mass spectrometry imaging detection method to obtain mass spectrometry imaging data of the chemical components in the transfer membrane of the Chinese herbal medicine slices; processing the mass spectrometry imaging data to identify the chemical components in the sample; and analyzing the spatial distribution patterns of metabolites in the Chinese herbal medicine slices. This invention overcomes the difficulties of preparing imaging samples due to the dryness, hardness, and uneven surface of Chinese herbal medicine slices, filling the gap in mass spectrometry imaging methods for dried Chinese herbal medicine slices. The method is simple to operate, highly sensitive, has a wide dynamic range, and covers a wide variety of components, exhibiting significant advantages.

[0033] Embodiments of the present invention relate to a mass spectrometry imaging method for analyzing the chemical composition and spatial distribution of licorice slices using the blot method, comprising the following steps:

[0034] Step S1: Apply pressure to the licorice slices to be tested to obtain a relatively flat cross-section;

[0035] Step S2: Establish the "sandwich" structure of licorice slices and use the imprinting method to transfer the cross-sectional components of licorice to the transfer film;

[0036] Step S3: Use mass spectrometry imaging detection method to perform ionization scanning on the above-mentioned transfer film in a mass spectrometer to obtain mass spectrometry imaging data of the chemical components in the transfer film of the licorice slices to be tested.

[0037] Step S4: Process the mass spectrometry imaging data to identify the chemical components in the licorice slices to be tested;

[0038] Step S5: Analyze the spatial distribution pattern of metabolites in licorice slices.

[0039] The specific steps are as follows:

[0040] Step S1: Apply pressure to the licorice slices to be tested to obtain a relatively flat cross-section;

[0041] Because traditional Chinese medicine (TCM) slices have a certain thickness, unsuitable pressure may damage the transfer film or cause the TCM tissue to detach and adhere to the transfer film. Excessive pressure or prolonged pressure may also cause component diffusion, reducing the imaging accuracy of the transfer. This invention selects licorice slices with a thickness of less than 3 mm and a smooth cut surface without obvious curvature. A manual tablet press (MC-12, Changsha Miqi Instrument Equipment Co., Ltd.) is used to apply pressure for 5–10 seconds at a pressure of 0.3–0.5 tons. This yields licorice samples with a smoother cut surface and no significant change in the morphology of the cut tissue, which are then photographed and recorded.

[0042] Step S2: Establish the "sandwich" structure of licorice slices and use the imprinting method to transfer the cross-sectional components of licorice to the transfer film;

[0043] 300–500 μL of 80% acetonitrile solution was sprayed onto a transfer membrane (nylon 66 microporous filter membrane, 50 mm in diameter, 0.22 μm in pore size, purchased from Tianjin Jinteng Experimental Equipment Co., Ltd.) to moisten the membrane without obvious water stains. The licorice root slices to be tested were placed on the moistened transfer membrane. Absorbent paper, a rubber sheet, and sponge paper were then sequentially placed on both sides of the transfer membrane-herb sample pair to obtain a "sandwich" structure for sample preparation using the imprint method. See details in [link to documentation]. Figure 1 Apply 0.3–0.5 tons of pressure using a manual tablet press for 5–10 seconds. Remove the transfer film, inspect it for integrity and clarity, and take a photograph. Adhere the transfer film to a glass slide using double-sided tape to obtain the licorice slice imprint sample.

[0044] Step S3: Use mass spectrometry imaging detection method to perform ionization scanning on the above-mentioned transfer film in a mass spectrometer to obtain mass spectrometry imaging data of the chemical components in the licorice transfer film to be tested.

[0045] Licorice blot samples were analyzed using an AFADESI-MSI platform and a Q-Orbitrap mass spectrometer (Q Exactive, Thermo Scientific, USA). Mass spectrometry imaging was performed with continuous scanning in the x-direction at a constant velocity of 0.3 mm / s and a stepping interval of 0.15 mm in the y-direction. The spray solvent was 80% acetonitrile solution at a flow rate of 5 μL / min, the pump flow rate was 45 L / min, the spray distance from the slice was 0.7 mm, the distance between the sample surface and the sprayer was 3 mm, and the distance between the sprayer and the ion delivery tube was 3 mm. Positive ion scanning was performed in Full Scan mode with an MS resolution of 70,000, a mass range of 70–1000 Da, an automatic gain control (AGC) target of 3E6, a maximum injection time of 200 ms, and a capillary temperature of 350 °C.

[0046] Step S4: Process the mass spectrometry imaging data to identify the chemical components in licorice;

[0047] The mass spectrometry imaging data of the measured licorice blot samples were processed. The raw data (.raw format) was converted to .imzML format files and imported into the Cardinal software package for background subtraction, peak alignment, and peak filtering to obtain the mass spectrometry image of the licorice blot. Figure 2 The total ion chromatogram of licorice blot is shown. Data from the mass spectrometry images were compared with high-resolution mass spectrometry data from licorice-related literature and the SmetDB database to identify the chemical components of the licorice decoction pieces. Specific identification results are shown in Table 1.

[0048] Table 1. Identification of chemical components in licorice blot mass spectrometry imaging.

[0049]

[0050]

[0051] Step S5: Analyze the spatial distribution pattern of metabolites in licorice slices.

[0052] Further spatial shrinkage centroid clustering analysis was conducted to obtain the partitioning information of the licorice imprint. This clustering diagram is a visualization of the overall expression level clustering results, which effectively presents the outlines of different regions in the licorice imprint. The clustering diagram of the licorice imprint was compared with the original cross-sectional photograph of licorice slices and the photograph of the licorice imprint. Figure 3 The results showed that the overall distribution of licorice components was closely related to its tissue morphology and structure.

[0053] Figure 4 Mass spectrometry images of some chemical components in licorice are shown. Amino acids are widely distributed throughout the cross-section, with relatively lower concentrations in the cork layer. Flavonoids and coumarins are present in higher concentrations in licorice, mostly concentrated in the cork layer. The distribution of these components exhibits clear tissue specificity.

[0054] Comparative Example 1:

[0055] The transfer membrane described in step S2 is optimized. The transfer membranes to be optimized include: polytetrafluoroethylene microporous filter membrane (50 mm in diameter, 0.22 μm in pore size, Hangzhou Xinxing Co., Ltd.); nylon 66 microporous filter membrane (50 mm in diameter, 0.22 μm in pore size, purchased from Tianjin Jinteng Experimental Equipment Co., Ltd.); and nylon 66 microporous filter membrane (50 mm in diameter, 0.45 μm in pore size, purchased from Tianjin Jinteng Experimental Equipment Co., Ltd.).

[0056] The same mass spectrometry imaging method as in Example 1 was used, the difference being that in step S2, the three types of transfer membranes were selected for imaging. The specific experimental steps were as follows: the three types of transfer membranes were cut to appropriate sizes, moistened with 500 μL of 80% acetonitrile, and then imprinted onto the same licorice slices under the same pressure (0.5 tons, applied for 10 s). The mass spectrometry imaging results showed that, compared with the polytetrafluoroethylene microporous membrane, the nylon 66 microporous membrane showed a better correspondence between mass spectrometry imaging and the licorice tissue structure, with no significant component migration. The nylon 66 microporous membrane could better display the true component distribution of licorice. Compared with the 0.45 μm nylon 66 microporous membrane, the 0.22 μm nylon 66 microporous membrane collected a richer variety of chemical components, such as... Figure 5 , Figure 6 Therefore, the nylon 66 microporous filter membrane (50 mm in diameter, 0.22 μm in pore size, purchased from Tianjin Jinteng Experimental Equipment Co., Ltd.) was the final transfer membrane selected for the in-situ spectral imaging method.

[0057] Comparative Example 2:

[0058] The solvents used for spraying the transfer film and transferring auxiliary components in step S2 of the imprinting method are optimized. The solvents to be optimized include: acetonitrile, methanol, 80% acetonitrile, 80% methanol, and 50% acetonitrile.

[0059] The same mass spectrometry imaging method as in Example 1 was used, the difference being that in step S2, the five spray solvents mentioned above were selected to spray and wet the transfer membrane. The specific experimental steps were as follows: the transfer membrane (nylon 66 microporous filter membrane, 50 mm in diameter, 0.22 μm in pore size, purchased from Tianjin Jinteng Experimental Equipment Co., Ltd.) was cut into five pieces. Simultaneously, the five transfer membranes were wetted with 500 μL of acetonitrile, methanol, 80% acetonitrile, 80% methanol, and 50% acetonitrile, respectively. The same pressure (0.5 tons, applied for 10 s) was used to transfer the same licorice slice using the imprinting method. The mass spectrometry imaging results showed that 80% acetonitrile was used as the spray solvent, resulting in the best component transfer results. Figure 7 As shown.

[0060] In summary, the method described in this invention successfully solves the sample preparation difficulties caused by the drying and unevenness of Chinese herbal medicine slices through the form of imprinting. It can perform mass spectrometry imaging on dried licorice slices, and the imaging results are highly consistent with the tissue structure of licorice, with obvious tissue specificity of components.

[0061] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A mass spectrometry imaging method for analyzing the chemical composition and spatial distribution of dried Chinese herbal medicine slices using the imprinting method, characterized in that, Includes the following steps: Step S1: Apply pressure to the Chinese herbal medicine slices to be tested to obtain a relatively flat cross-section; Step S2: Establish a "sandwich" structure and use the imprinting method to transfer the cross-sectional components of the Chinese herbal medicine slices to be tested onto the transfer film; The specific transfer method in step S2 is as follows: spray 300~500 μL of 80% acetonitrile solution onto the transfer film, place the Chinese herbal medicine pieces to be tested on the moistened transfer film, cover the two sides of the transfer film-Chinese herbal medicine sample pair with absorbent paper, rubber plate and sponge paper in sequence, apply a pressure of 0.3~0.5 tons for 5~10 s, and finally stick the transfer film onto the glass slide; Step S3: Use mass spectrometry imaging detection method to perform ionization scanning on the transfer film in a mass spectrometer to obtain mass spectrometry imaging data of the chemical components of the Chinese herbal medicine slices to be tested on the transfer film; Step S4: Process the mass spectrometry imaging data to identify the chemical components in the Chinese herbal medicine pieces to be tested; Step S5: Analyze the spatial distribution pattern of metabolites in the Chinese herbal medicine pieces to be tested.

2. The mass spectrometry imaging method for analyzing the chemical composition and spatial distribution of dried Chinese herbal medicine slices using the imprinting method according to claim 1, characterized in that, Step S1 includes: selecting Chinese herbal medicine slices with relatively flat cross-sections and thin thickness, applying a pressure of 0.3 to 0.5 tons to them for 5 to 10 seconds to obtain Chinese herbal medicine slices with flat cross-sections to be tested.

3. The mass spectrometry imaging method for analyzing the chemical composition and spatial distribution of dried Chinese herbal medicine slices using the imprinting method according to claim 1, characterized in that, In step S2, the transfer membrane is a nylon 66 microporous filter membrane.

4. The mass spectrometry imaging method for analyzing the chemical composition and spatial distribution of dried Chinese herbal medicine slices using the imprinting method according to claim 3, characterized in that, In step S2, 300~500 μL of 80% acetonitrile solution is sprayed onto the nylon 66 microporous filter membrane to make the transfer membrane wet and free of obvious water stains; And / or, The nylon 66 microporous filter membrane has a diameter of 50 mm and a pore size of 0.22 μm.

5. The mass spectrometry imaging method for analyzing the chemical composition and spatial distribution of dried Chinese herbal medicine slices using the imprinting method according to claim 1, characterized in that, In step S3, the mass spectrometer parameters are set as follows: The mass spectrometer used was a Q-Orbitrap mass spectrometer, and the platform used was an AFADESI-MSI platform. The spray solvent was 80% acetonitrile solution, the solvent flow rate was 5 μL / min, the pump flow rate was 45 L / min, the distance between the spray and the slice was 0.5~0.8 mm, the distance between the sample surface and the sprayer was 2~4 mm, and the distance between the sprayer and the ion delivery tube was 2~4 mm.

6. The mass spectrometry imaging method for analyzing the chemical composition and spatial distribution of dried Chinese herbal medicine slices using the imprinting method according to claim 1, characterized in that, In step S3, the parameters for the ionization scan are set as follows: Positive ion scanning, Full Scan mode, MS resolution of 70,000, mass range of 70 ~ 1000 Da, automatic gain control target of 3E6, maximum injection time of 200 ms, capillary temperature of 350 ℃.

7. The mass spectrometry imaging method for analyzing the chemical composition and spatial distribution of dried Chinese herbal medicine slices using the imprinting method according to claim 1, characterized in that, In step S4, the data processing software includes: imzMLConverter and Cardinal software packages; And / or, In step S4, data preprocessing includes: The raw data was converted into .imzML format files using imzMLConverter software, and then imported into the Cardinal software package for background subtraction, peak alignment, and peak filtering to obtain mass spectrometry images of metabolites.

8. The mass spectrometry imaging method for analyzing the chemical composition and spatial distribution of dried Chinese herbal medicine slices using the imprinting method according to claim 7, characterized in that, In step S4, the data in the mass spectrometry image are compared with the high-resolution mass spectrometry data in the literature and the SmetDB database to identify the chemical components in the Chinese herbal medicine slices to be tested.

9. The mass spectrometry imaging method for analyzing the chemical composition and spatial distribution of dried Chinese herbal medicine slices using the imprinting method according to claim 1, characterized in that, Step S5 includes: The spatial distribution patterns of metabolites in the tested Chinese herbal medicine were analyzed by comparing the mass spectrometry images with the original cross-sectional photos of the Chinese herbal medicine and the photos of the transfer film.

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