Method for mass spectrometry imaging detection of compounds containing ortho-diphenolic hydroxyl functionality in tissues

Abstract

The application provides a mass spectrometry imaging detection method for a compound containing an ortho-diphenol hydroxyl functional group in a tissue and application thereof, and belongs to the technical field of mass spectrometry detection. The method at least comprises: synthesizing a derivatization reagent in a solution, spraying the derivatization reagent solution to the surface of a prepared tissue section to obtain a derivatization product of the compound containing the ortho-diphenol hydroxyl functional group; and performing matrix spraying on the tissue section after derivatization, and then performing MALDI mass spectrometry imaging analysis. The application can effectively improve the detection sensitivity of the compound containing the ortho-diphenol hydroxyl functional group in the tissue, realizes the visual analysis of the compound containing the ortho-diphenol hydroxyl functional group in a biological tissue, such as an endogenous metabolite and an exogenous drug, and therefore has good practical popularization and application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of mass spectrometry, and relates to a MALDI mass spectrometry imaging method for high-sensitivity detection of compounds containing ortho-diphenol hydroxyl functional groups in biological tissues in a positive ion mode and application thereof TECHNICAL BACKGROUND

[0002] Phenolic hydroxyl is one of the most common functional groups in small molecule drugs, and traditional Chinese medicine active ingredients such as flavonoids and polyphenols mostly contain multiple phenolic hydroxyl groups. A large number of in vivo and in vitro studies in recent years have shown that these compounds have strong antioxidant activity, and due to their other pharmacological effects and very small toxic and side effects, they show strong development potential as antioxidant drugs.

[0003] For example, chlorogenic acid polyphenol is known as a natural antioxidant, and some antioxidant health products mainly contain chlorogenic acid. Researchers have found that chlorogenic acid can reduce the cell apoptosis of retinal nerve cells caused by hypoxia or excessive carbon monoxide. Since chlorogenic acid can reduce retinal cell death, chlorogenic acid has the effect of preventing diseases caused by retinal degeneration. Quercetin is a representative of flavonoids, which has been shown to have the ability to promote hippocampal precursor cell proliferation and neurogenesis. For another example, danshensu is a water-soluble active ingredient in Danshen, which has obvious pharmacological activities, including myocardial protection, thrombosis inhibition, blood lipid reduction, uric acid reduction, neuroprotection, prevention and treatment of liver fibrosis, and anti-tumor, anti-inflammatory and immune enhancement. Danshensu isopropyl ester is an effective component screened from the numerous metabolites of compound Danshen, which has significant anti-hypoxia and anti-ischemia effects. These compounds containing phenolic hydroxyl functional groups generate relatively stable semiquinone free radicals by reacting with oxygen free radicals through phenolic hydroxyl groups, thereby playing an antioxidant role. The structure-activity relationship study on this mechanism shows that the antioxidant activity of ortho-phenolic hydroxyl structure is the strongest. The ortho-phenolic hydroxyl group can be stabilized by forming intramolecular hydrogen bonds after being abstracted to generate free radicals.

[0004] Another important endogenous small molecule containing ortho-diphenol hydroxyl groups is monoamine neurotransmitter, which is an endogenous central neurotransmitter generated by hydroxylation of aromatic amino acids in neuron cell bodies, and contains ortho-diphenol hydroxyl groups in the molecular structure. It is transported to the nerve ending along the microtubule or microfilament, and is stored in the vesicle of the nerve ending. When the neuron is excited, the nerve impulse is transmitted to the nerve ending, the vesicle is broken, and it is released into the synaptic cleft. Most of them diffuse to the postsynaptic membrane and bind to the receptors there, producing biological effects of intercellular transmission of nerve information.

[0005] Current tissue homogenate followed by LC-MS analysis has confirmed the distribution of these pharmacodynamic compounds and endogenous neurotransmitters in cerebrospinal fluid and brain tissue. Currently, most of our knowledge of the tissue localization of these pharmacodynamic compounds and endogenous neurotransmitters comes from whole-body autoradiography or LC-MS analysis after tissue homogenization. However, most pharmacodynamic compounds and endogenous neurotransmitters are unevenly distributed in tissues, and characterizing the tissue distribution of pharmacodynamic compounds is critical to drug development.

[0006] Matrix-assisted laser desorption / ionization (MALDI) mass spectrometry imaging technology can generate pixelated mass spectrometry data with near-cellular resolution and provide spatial mapping of metabolites and drugs according to XY positions on tissue sections, and has been used to measure and visualize the tissue spatial distribution of metabolites and drugs. However, the reactivity of hydroxyl groups is low, and it is still challenging to perform mass spectrometry imaging analysis on such low-abundance and / or difficult-to-ionize small molecule drugs in tissue samples. The introduction of specific recognition groups and mass spectrometry sensitization groups through chemical derivatization can achieve recognition of o-diphenol hydroxyl compounds and effectively improve the sensitivity of mass spectrometry detection. There is no commercial derivatization reagent for in situ mass spectrometry imaging analysis of o-diphenol hydroxyl compounds. Kaya et al. synthesized a pyridinium-containing boronic acid molecule, 4-(N-Methyl)pyridinium Boronic Acid, which derivatized o-diphenol hydroxyl-containing monoamine neurotransmitters through boronate formation (boronic acid-diol reaction). The specific synthesis steps are as follows: first, under an inert nitrogen atmosphere, 155 mg of 4-pyridyl boronic acid pinacol ester (0.81 mmol) was dissolved in 15 mL of anhydrous acetonitrile; 0.26 mL of methyl iodide (4.1 mmol) was added, and the solution was heated to 80°C for 6 h. The acetonitrile was removed under reduced pressure. The yellow intermediate was dissolved in 10 mL of water, 1M hydrochloric acid (2 mL) was added, and stirred at room temperature for 1 h. After 1 h, the water phase was washed with tetrahydrofuran to remove pinacol. The synthesis of this derivatization reagent molecule has many steps and takes a long time, and after synthesis, 4-(N-Methyl)pyridinium Boronic Acid needs to be separated and purified from the synthesis system for derivatization. SUMMARY

[0007] In view of the problems in the prior art, the present application aims to provide a mass spectrometry imaging detection method for compounds containing o-diphenol hydroxyl functional groups in tissues. The present application synthesizes a derivatization reagent, which can be applied to MALDI mass spectrometry imaging of in situ derivatization of compounds containing o-diphenol hydroxyl groups, significantly improves the detection sensitivity of compounds containing o-diphenol hydroxyl groups in tissues, and realizes the visual analysis of endogenous metabolites and exogenous drugs containing o-diphenol hydroxyl functional groups in biological tissues. The synthesis method of the derivatization reagent: the reaction of the oxonium ion of pyrylium salt with the primary amine of aminomethylphenylboronic acid hydrochloride to synthesize N-alkyl pyridinium derivative pyridinium-methyl phenylboronic acid is carried out in a methanol aqueous solution, the reaction is completed immediately, heating and long-term stirring are not required, and only a small amount of organic base is added as an oxidizing agent. Moreover, the synthesized pyridinium-methyl phenylboronic acid does not need to be separated and purified from the synthesis solution system, and the synthesis solution system can be directly sprayed on the surface of a tissue section for mass spectrometry imaging detection of compounds containing o-diphenol hydroxyl functional groups.

[0008] The technical scheme of the present application is as follows:

[0009] In the first part of the present application, a mass spectrometry imaging detection method for compounds containing o-diphenol hydroxyl functional groups in tissues is provided, and the method at least comprises:

[0010] (a) generating N-alkyl pyridinium derivative pyridinium-methyl phenylboronic acid by the reaction of the oxonium ion of pyrylium salt with the primary amine of aminomethylphenylboronic acid hydrochloride in a solution;

[0011] (b) spraying the derivatization reagent on the surface of a tissue section to perform a derivatization reaction, forming a borate ester five-membered ring by the o-diphenol hydroxyl group of the compound containing the o-diphenol hydroxyl functional group and the boronic acid, transferring the permanent positive charge of pyridinium-methyl phenylboronic acid to the compound containing the o-diphenol hydroxyl functional group, and obtaining a derivatization product of the compound containing the o-diphenol hydroxyl functional group;

[0012] (c) performing matrix spraying on the tissue section after the above derivatization treatment to obtain a tissue section sample that can be used for mass spectrometry imaging;

[0013] (d) collecting the mass spectrum signal of the derivatization product by using a laser desorption ion source (MALDI), qualitatively analyzing the compound containing the o-diphenol hydroxyl functional group in the tissue, and extracting the mass-to-charge ratio information of the derivatization product to obtain a mass spectrometry imaging graph.

[0014] As a further scheme of the present application: the synthesis of the derivatization reagent in the solution is mixing a pyrylium salt solution and an aminomethylphenylboronic acid hydrochloride solution, adding an organic base catalyst, and fully mixing to obtain a derivatization reagent pyridinium-methyl phenylboronic acid solution;

[0015] As a further scheme of the present application: R1 to R5 of the pyrylium salt can be one, two or three of hydrogen and substituent methyl, phenyl, benzyl, and the number of substituents can be one, two, three, four or five, and the anion is BF4;

[0016] As a further scheme of the present application: the aminomethylphenylboronic acid hydrochloride can be one of 3-aminomethylphenylboronic acid hydrochloride and 4-aminomethylphenylboronic acid hydrochloride;

[0017] As a further scheme of the present application: the concentration of the pyrylium salt solution and the aminomethylphenylboronic acid hydrochloride solution is respectively 10-50 mM, preferably 20-30 mM, more preferably 25 mM and 10-50 mM, preferably 20-30 mM, more preferably 25 mM; the molar ratio of the pyrylium salt and the aminomethylphenylboronic acid hydrochloride in the mixed solution synthesis system is 10:1-1:1, preferably 8:1-2:1, more preferably 2:1;

[0018] As a further scheme of the present application: the solvent of the pyrylium salt and the aminomethylphenylboronic acid hydrochloride is a methanol aqueous solution, preferably, the volume concentration of methanol in the aqueous solution is 25-100%, further preferably 50-75% of methanol;

[0019] As a further scheme of the present application: the organic base catalyst is one or both of triethylamine and N, N-diisopropyl ethylamine;

[0020] As a further scheme of the present application: the volume ratio of the organic base to the solution synthesis system is 1:1000-4:1000;

[0021] As a further scheme of the present application: the spraying amount of the derivatization reagent pyridinium-methylphenylboronic acid solution on one side surface of the tissue section is 100-500 nmol / cm 2 , preferably 150-300 nmol / cm 2 ;

[0022] As a further scheme of the present application: the matrix is α-cyano-4-hydroxycinnamic acid (CHCA) with a mass concentration of 7-10 mg / mL, the solvent is 40-60% acetonitrile aqueous solution containing 0.1-0.2% trifluoroacetic acid; the spraying amount on one side surface of the tissue section is 0.07-0.35 mg / cm 2 , preferably 0.15-0.25 mg / cm 2 ;

[0023] As a further scheme of the present application: the tissue section is selected to be an animal tissue section, including a mouse brain tissue section or a rat brain tissue section;

[0024] As a further scheme of the present application: the compound containing ortho-diphenol hydroxyl functional group is one or more than two of endogenous metabolites and exogenous drugs containing ortho-diphenol hydroxyl functional group in the tissue, including one or more than two of catechol neurotransmitters and phenolic acids, flavonoid drugs.

[0025] The beneficial technical effects of one or more of the above technical solutions are:

[0026] (1) The synthesis of the derivatization reagent used in the above technical solution is completed in a solution system, which is simple to operate without heating and incubation. The synthesized derivatization reagent does not need to be separated and prepared, and the mixed solution of the derivatization reagent can be used for derivatization.

[0027] (2) The derivatization condition is mild, and the reaction step is simple. The reaction occurs under very mild conditions and rapidly at ambient temperature and pressure without any stirring or agitation, which is suitable for in situ derivatization analysis of the compound containing ortho-diphenol hydroxyl functional group in the tissue, avoiding the delocalization of the target compound and the dissolution of the tissue.

[0028] (3) In the above technical method, the permanent positive charge is introduced into the compound containing ortho-diphenol hydroxyl functional group by derivatization treatment, which improves the detection sensitivity of the compound containing ortho-diphenol hydroxyl functional group in MALDI mass spectrometry, and realizes in situ MALDI mass spectrometry imaging analysis of the compound containing ortho-diphenol hydroxyl functional group in the tissue.

[0029] (4) The above technical solution effectively realizes the simultaneous mass spectrometry imaging visualization analysis of the compound containing ortho-diphenol hydroxyl functional group such as endogenous metabolites and exogenous drugs under positive ion conditions, and has good practical application value.

[0030] The present application can effectively improve the detection sensitivity of the compound containing ortho-diphenol hydroxyl functional group in the tissue, realize the visualization analysis of the compound containing ortho-diphenol hydroxyl functional group such as endogenous metabolites and exogenous drugs in biological tissue, and therefore has good practical popularization and application value.

[0031] Drawings of the specification

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows

[0033] Figure 1 is the mass spectrometry characterization graph of 2,4,6-trimethylpyridinium-4-methylphenylboronic acid in the embodiment 1 of the present application;

[0034] Figure 2 is the derivatization reaction formula of dopamine (A), danshensu isopropyl ester (B), quercetin (C) and chlorogenic acid (D) in the embodiment 2 of the present application;

[0035] Figure 3a is the MALDI mass spectrometry data of the dopamine (A), danshensu isopropyl ester (B), quercetin (C) and chlorogenic acid (D) derivatization products on ITO slides in Example 2 of the present application;

[0036] Figure 3 b is the MALDI mass spectrometry data of dopamine (A), danshensu isopropyl ester (B), quercetin (C) and chlorogenic acid (D) on ITO slides in Example 3 of the present application;

[0037] Figure 4 is the mass spectrometry imaging map of endogenous dopamine in mouse brain tissue sections in Example 4 of the present application;

[0038] Figure 5 is the mass spectrometry imaging map of danshensu isopropyl ester on the surface of mouse brain tissue sections in Example 5 of the present application;

[0039] Figure 6 is the mass spectrometry imaging map of quercetin on the surface of mouse brain tissue sections in Example 6 of the present application;

[0040] Figure 7 is the mass spectrometry imaging map of chlorogenic acid on the surface of mouse brain tissue sections in Example 7 of the present application; DETAILED DESCRIPTION

[0041] Example 1: Synthesis of 2,4,6-trimethylpyridinium-4-methylphenylboronic acid (1) Precisely weigh 21 mg of 2,4,6-trimethylpyridinium tetrafluoroborate, add 2 mL of methanol: water (50:50, v / v) solution, mix well by vortex, and ultrasonic for 5 minutes to obtain a 2,4,6-trimethylpyridinium tetrafluoroborate (50 mM) solution, which is ready for use;

[0042] (2) Precisely weigh 37.2 mg of 4-aminomethylphenylboronic acid hydrochloride, add 4 mL of methanol: water (50:50, v / v) solution, mix well by vortex, and ultrasonic for 5 minutes to obtain a 4-aminomethylphenylboronic acid hydrochloride (50 mM) solution, which is ready for use;

[0043] (3) Take 2,4,6-trimethylpyrylium hydrochloride solution and 4-aminomethylphenylboronic acid hydrochloride solution each 1 mL, add 2 μL of triethylamine, vortex mix, to obtain 2,4,6-trimethylpyridinium-4-methylphenylboronic acid solution; high-performance preparative liquid chromatography separation, column: Agilent Poroshell 120 EC-C18 (100 mm x 3.0 mm, 2.7 μm), mobile phase A water (containing 0.5% formic acid by volume), mobile phase B acetonitrile (containing 0.5% formic acid by volume), elution gradient 0-8 min, 2% B-100% B, collect the chromatographic peak at retention time 1.75 min, to obtain 2,4,6-trimethylpyridinium-4-methylphenylboronic acid peak, freeze-dried into powder, ready for use; the product is characterized by LC-ESI-QTOF-MS and MALDI-TOF-MS, as shown in Figure 1 As described above, the m / z of 2,4,6-trimethylpyridinium-4-methylphenylboronic acid is 256.1531, which is consistent with the theoretical ratio and mode. The fragment ion of m / z 135.0627 is generated by losing trimethylpyridinium from the ion of m / z 256.1531.

[0044] Example 2: Derivatization of dopamine, danshensu isopropyl ester, quercetin and chlorogenic acid and MALDI mass spectrometric analysis

[0045] (1) Precisely weigh 21 mg of 2,4,6-trimethylpyrylium tetrafluoroborate, add 2 mL of methanol: water (50:50, v / v) solution, vortex mix, ultrasonic for 5 minutes, to obtain 2,4,6-trimethylpyrylium tetrafluoroborate (50 mM) solution, ready for use;

[0046] (2) Precisely weigh 37.2 mg of 4-aminomethylphenylboronic acid hydrochloride, add 4 mL of methanol: water (50:50, v / v) solution, vortex mix, ultrasonic for 5 minutes, to obtain 4-aminomethylphenylboronic acid hydrochloride (50 mM) solution, ready for use;

[0047] (3) Take 2,4,6-trimethylpyrylium hydrochloride solution and 4-aminomethylphenylboronic acid hydrochloride solution each 1 mL, add 2 μL of triethylamine, vortex mix, to obtain 2,4,6-trimethylpyridinium-4-methylphenylboronic acid solution, ready for use;

[0048] (4) Precisely weigh 35 mg of CHCA, add 5 mL of acetonitrile: water (60:40, v / v) solution containing 0.2% trifluoroacetic acid by volume concentration, vortex mix, ultrasonic for 5 minutes, to obtain CHCA (7 mg / mL) solution, ready for use;

[0049] (5) Precisely weigh 1 mg of dopamine, danshensu isopropyl ester, quercetin and chlorogenic acid standard substance respectively, and prepare standard substance solution by dissolving them in 50% methanol water solution respectively, with a concentration of 1 mg / mL, for standby use.

[0050] (6) Drop 1 uL of dopamine, danshensu isopropyl ester, quercetin and chlorogenic acid standard substance solution (1 mg / mL) respectively on the surface of ITO glass slide about 3 mm 2 , with an interval of 1 mm, and place them in a vacuum dryer for drying at room temperature for 15 minutes;

[0051] (7) Use the Bruker full-automatic matrix spraying instrument ImagePrep, with a spraying chamber area of about 100 cm 2 , place the ITO glass slide containing the standard substance drying point on one of the surfaces in the middle of the spraying chamber, and spray the 2,4,6-trimethylpyridinium 4-methylphenyl borate synthesis solution obtained in step (3) into the spraying chamber, with a total of 1 mL of solution, and a total of 12 times of spraying, so that the final spraying amount of 2,4,6-trimethylpyridinium 4-methylphenyl borate solution in the synthesis solution system is 500 nmol / cm 2 , and naturally dry at room temperature after each layer of spraying, and after the last layer of spraying, place the ITO glass slide in a vacuum dryer for drying at room temperature for 15 minutes;

[0052] (8) Use the Bruker full-automatic matrix spraying instrument ImagePrep, with a spraying chamber area of about 100 cm 2 , place the ITO glass slide containing the standard substance drying point on one of the surfaces in the middle of the spraying chamber, and spray the CHCA / CHCA solution prepared in step (4) into the spraying chamber, with a total of 5 mL of solution, and a total of 20 times of spraying, so that the final spraying amount of CHCA is 0.35 mg / cm 2 , and naturally dry at room temperature after each layer of spraying, and after the last layer of spraying, place the ITO glass slide containing the standard substance drying point on one of the surfaces in a vacuum dryer for drying at room temperature for 15 minutes;

[0053] (9) Use the Bruker UltraFlex III MALDI-TOF / TOF type mass spectrometer in positive ion, reflection mode, with a mass-to-charge ratio detection range of 140-1000, to perform mass spectrum analysis on the ITO glass slide containing the standard substance drying point on one of the surfaces;

[0054] (10) According to the derivatization reaction formula of dopamine, danshensu isopropyl ester, quercetin and chlorogenic acid ( Figure 2 ), use the Bruker DataAnalysis to extract the mass spectrum signal of the derivatization product, with m / z of 373.21, 460.23, 574.22 and 522.17 respectively. The results are as follows Figure 3As shown in Figure a, it can be seen that when 2,4,6-trimethylpyridinium-3-methyl phenylboronic acid synthesis solution is used as a derivatization reagent and CHCA is used as a matrix, mass spectrometric signals of the derivatization products of dopamine, danshensu isopropyl ester, quercetin and chlorogenic acid can be obtained.

[0055] Example 3: MALDI mass spectrometric analysis of dopamine, danshensu isopropyl ester, quercetin and chlorogenic acid

[0056] (1) 35 mg of CHCA was precisely weighed and dissolved in 5 mL of acetonitrile: water (60:40, v / v) solution containing 0.2% trifluoroacetic acid by volume concentration, vortexed and ultrasonicated for 5 minutes to obtain a CHCA (7 mg / mL) solution, which was ready for use;

[0057] (2) 1 mg of dopamine, danshensu isopropyl ester, quercetin and chlorogenic acid standard substances were precisely weighed respectively and dissolved in 50% methanol by volume concentration to obtain standard substance solutions with a concentration of 1 mg / mL, which were ready for use.

[0058] (3) 1 uL of the standard substance solutions (1 mg / mL) of dopamine, danshensu isopropyl ester, quercetin and chlorogenic acid was added respectively to the surface of an ITO glass slide about 3 mm 2 apart, and placed in a vacuum desiccator for drying at room temperature for 15 minutes;

[0059] (4) A Bruker full-automatic matrix spraying instrument ImagePrep was used, the area of the spraying chamber was about 100 cm 2 , the ITO glass slide containing the standard substance drying points on one surface was placed in the middle of the spraying chamber with the surface containing the standard substance drying points facing upwards, and the CHCA solution prepared in step (1) was sprayed into the spraying chamber, a total of 5 mL of solution was used, and the spraying was performed 20 times, the final spraying amount of CHCA was 0.35 mg / cm 2 , and after each layer was sprayed, it was naturally dried at room temperature, and after the last layer was sprayed, the ITO glass slide containing the standard substance drying points on one surface was placed in a vacuum desiccator for drying at room temperature for 15 minutes;

[0060] (5) A Bruker UltraFlex III MALDI-TOF / TOF mass spectrometer was used to perform mass spectrometric analysis on the surface of the ITO glass slide containing the standard substance drying points in one surface in positive ion and reflection mode, with a mass-to-charge ratio detection range of 140-1000;

[0061] (10) According to the accurate molecular weights of dopamine, danshensu isopropyl ester, quercetin and chlorogenic acid, which are 153., 240., 302. and respectively, the mass spectrometric signals of the derivatization products were extracted by using Bruker DataAnalysis. The results are shown in Figure a. Figure 3b, no peaks of derivatives of dopamine, danshensu isopropyl ester, quercetin and chlorogenic acid (m / z 373.21, 460.23, 574.22 and 522.17) and no response signals of ion peaks and water loss peaks of dopamine, danshensu isopropyl ester, quercetin and chlorogenic acid were observed without using derivatization reagents.

[0062] Example 4: Derivatization of endogenous dopamine in mouse brain tissue and MALDI mass spectrometry imaging analysis

[0063] (1) The frozen mouse brain tissue was taken and coronal sections of the brain tissue with a thickness of 14 μm were prepared using a microtome;

[0064] (2) The brain tissue sections were transferred to an indium tin oxide (ITO) glass slide and placed in a vacuum desiccator for drying at room temperature for 15 minutes;

[0065] (3) 21 mg of 2,4,6-trimethylpyrylium tetrafluoroborate was precisely weighed, 2 mL of a methanol: water (50:50, v / v) solution was added, vortexed and mixed, and ultrasonicated for 5 minutes to obtain a 2,4,6-trimethylpyrylium tetrafluoroborate (50 mM) solution, which was prepared for use;

[0066] (4) 18.6 mg of 4-aminomethylphenylboronic acid hydrochloride was precisely weighed, 4 mL of a methanol: water (50:50, v / v) solution was added, vortexed and mixed, and ultrasonicated for 5 minutes to obtain a 4-aminomethylphenylboronic acid hydrochloride (25 mM) solution, which was prepared for use;

[0067] (5) 1 mL of the 2,4,6-trimethylpyrylium hydrochloride solution and 1 mL of the 4-aminomethylphenylboronic acid hydrochloride solution were mixed, 2 μL of triethylamine was added, vortexed and mixed, and a 2,4,6-trimethylpyridinium 4-methylphenylboronic acid solution was obtained, which was prepared for use;

[0068] (6) 35 mg of CHCA was precisely weighed, 5 mL of an acetonitrile: water (60:40, v / v) solution containing 0.2% (v / v) trifluoroacetic acid was added, vortexed and mixed, and ultrasonicated for 5 minutes to obtain a CHCA (7 mg / mL) solution, which was prepared for use;

[0069] (7) The ITO glass slide containing the dried spot of the standard was placed in the middle of the spraying chamber of a Bruker automatic matrix spraying instrument ImagePrep with a spraying chamber area of about 100 cm2, the 2,4,6-trimethylpyridinium 4-methylphenylboronic acid solution obtained in step (5) was sprayed into the spraying chamber, a total of 1 mL of the solution was used, and the spraying was performed 12 times, so that the final spraying amount of the 2,4,6-trimethylpyridinium 4-methylphenylboronic acid solution in the synthesis system was 250 nmol / cm2. 2After each layer of spraying, allow it to air dry at room temperature. After the last layer of spraying, place the ITO glass slide in a vacuum desiccator and dry it at room temperature for 15 minutes.

[0070] (8) Use the Brook ImagePrep fully automatic matrix sprayer, with a spray chamber area of ​​approximately 100 cm². 2 Place the ITO slide with the side containing the standard drying point facing upwards in the center of the spray chamber. Spray the CHCA solution prepared in step (6) into the spray chamber, using a total of 3 mL of solution, in 12 sprays, with a final spray amount of 0.21 mg / cm³. 2 After each layer of spraying, allow it to air dry at room temperature. After the last layer of spraying, place the ITO glass slide with the side containing the drying point of the standard sample facing up in a vacuum desiccator and dry at room temperature for 15 minutes.

[0071] (9) Using a Bruker UltraFlexⅢ MALDI-TOF / TOF mass spectrometer, in positive ion and reflection mode, with a mass-to-charge ratio detection range of 140-1000, mass spectrometry analysis was performed on the surface of mouse brain tissue slices attached to ITO slides.

[0072] (10) The mass spectrometry signal (m / z 373.21) of the dopamine derivatization product was extracted using Bruker DataAnalysis. The results are as follows: Figure 4 As shown, when 2,4,6-trimethylpyridinium-3-methylphenylboronic acid synthesis solution is used as the derivatization reagent and CHCA is used as the matrix, mass spectrometry signals (m / z 373.21) of endogenous dopamine derivatization products in mouse brain tissue slices can be obtained. The dopamine derivative signal is stronger in the striatal dopaminergic neuron region of the mouse, while no dopamine derivative signal is found in the cortical region without dopaminergic neurons.

[0073] Example 5: Mass spectrometry imaging analysis of tanshinone isopropyl derivatives on the surface of mouse brain tissue sections

[0074] (1) Frozen mouse brain tissue was taken and coronal sections of brain tissue with a thickness of 14 μm were prepared using a slicer;

[0075] (2) Transfer the brain tissue slices onto indium tin oxide (ITO) slides and dry them in a vacuum desiccator at room temperature for 15 minutes;

[0076] (3) Accurately weigh 12.8 mg of 2,4-diphenylpyranium tetrafluoroborate, add 2 mL of methanol-water (50:50, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain 2,4-dimethylpyranium tetrafluoroborate (20 mM) solution for later use;

[0077] (4) precisely weigh 14.9 mg of 4-aminomethylphenylboronic acid hydrochloride, add 4 mL of methanol: water (50:50, v / v) solution, vortex mix, and ultrasonic for 5 minutes to obtain a 4-aminomethylphenylboronic acid hydrochloride (20 mM) solution, ready for use;

[0078] (5) mix 1 mL of 2,4-dimethylpyrylium tetrafluoroborate solution and 1 mL of 4-aminomethylphenylboronic acid hydrochloride solution, add 2 μL of triethylamine, vortex mix to obtain a derivatization reagent 2,4-diphenylpyridinium-4-methylphenylboronic acid solution, ready for use;

[0079] (6) precisely weigh 35 mg of CHCA, add 5 mL of acetonitrile: water (60:40, v / v) solution containing 0.2% trifluoroacetic acid by volume concentration, vortex mix, and ultrasonic for 5 minutes to obtain a CHCA (7 mg / mL) solution, ready for use;

[0080] (7) precisely weigh the danshensu isopropyl ester standard, and prepare a series of concentration standard solution with 50% methanol aqueous solution, with concentrations of 1.08, 0.27, and 0.055 mg / mL, ready for use;

[0081] (8) use dry drop method to drop 0.2 uL of the danshensu isopropyl ester series concentration standard solution on the surface of the same mouse brain tissue section, with each standard solution dot occupying an area of 3 mm 2 , and with a 1 mm interval, and place in a vacuum desiccator for room temperature drying for 15 minutes;

[0082] (9) use the Bruker full-automatic matrix spray instrument ImagePrep with a spray chamber area of about 100 cm 2 , place the ITO glass slide with the standard drying dot on one of the surfaces upward in the middle of the spray chamber, spray the 2,4,6-trimethylpyridinium 4-methylphenylboronic acid synthesis solution obtained in step (5) into the spray chamber, use a total of 1 mL of the solution, and spray in 12 times, with a final spraying amount of 2,4,6-trimethylpyridinium 4-methylphenylboronic acid solution in the synthesis solution system of 200 nmol / cm 2 , and after each layer of spraying, naturally dry at room temperature, and after the last layer of spraying, place the ITO glass slide in a vacuum desiccator for room temperature drying for 15 minutes;

[0083] (10) use the Bruker full-automatic matrix spray instrument ImagePrep with a spray chamber area of about 100 cm 2 , place the ITO glass slide with the standard drying dot on one of the surfaces upward in the middle of the spray chamber, spray the CHCA solution prepared in step (6) into the spray chamber, use a total of 1 mL of the solution, and spray in 4 times, with a final spraying amount of 0.07 mg / cm 2After each layer of spraying, allow it to air dry at room temperature. After the last layer of spraying, place the ITO glass slide with the side containing the drying point of the standard sample facing up in a vacuum desiccator and dry at room temperature for 15 minutes.

[0084] (11) Using a Bruker UltraFlexⅢ MALDI-TOF / TOF mass spectrometer, in positive ion and reflection mode, with a mass-to-charge ratio detection range of 140 to 1000, mass spectrometry analysis was performed on the surface of mouse brain tissue slices on ITO slides.

[0085] (12) The mass spectrometry signal (m / z 460.23) of the tanshinone isopropyl ester derivatized product was extracted using Bruker DataAnalysis. The results are as follows: Figure 5 As shown, when the 2,4,6-trimethylpyridinium-3-methylphenylboronic acid synthesis solution is used as the derivatization reagent and CHCA is used as the matrix, the mass spectrometry signal of the tanshinone isopropyl ester derivatization product is 460.23 m / z, and the detection limit is 15 pmol / mm. 2 .

[0086] Example 6: Mass spectrometry imaging analysis of quercetin derived from mouse brain tissue sections

[0087] (1) Frozen mouse brain tissue was taken and coronal sections of brain tissue with a thickness of 14 μm were prepared using a slicer;

[0088] (2) Transfer the brain tissue slices onto indium tin oxide (ITO) slides and dry them in a vacuum desiccator at room temperature for 15 minutes;

[0089] (3) Accurately weigh 15.8 mg of 2,4,6-triphenylpyranium tetrafluoroborate, add 2 mL of methanol-water (50:50, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain 2,4,6-triphenylpyranium tetrafluoroborate (20 mM) solution for later use;

[0090] (4) Accurately weigh 7.44 mg of 3-aminomethylphenyl borate salt, add 4 mL of methanol:water (50:50, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain 3-aminomethylphenyl borate salt (10 mM) solution for later use;

[0091] (5) Take 1 mL each of 2,4,6-triphenylpyranium tetrafluoroborate solution and 4-aminomethylphenylborate solution, add 2 μL of triethylamine, vortex mix well to obtain the derivatization reagent 2,4,6-triphenylpyridinium-3-methylphenylboronic acid solution, for later use;

[0092] (6) Precisely weigh 35 mg CHCA, add 5 mL acetonitrile: water (60:40, v / v) solution containing 0.2% trifluoroacetic acid by volume concentration, vortex mix, and ultrasonic for 5 minutes to obtain CHCA (7 mg / mL) solution, ready for use;

[0093] (7) Precisely weigh quercetin standard, and prepare serial concentration standard solution with 50% methanol solution, with concentrations of 1.08, 0.27, and 0.055 mg / mL, ready for use;

[0094] (8) Drop 0.2 uL of quercetin standard solution on the surface of the same mouse brain tissue section on the ITO glass slide by dry drop method, with each standard solution drop occupying 3 mm 2 , and spaced 1 mm apart, and place in a vacuum desiccator for 15 minutes of drying at room temperature;

[0095] (9) Use the Bruker full-automatic matrix spraying instrument ImagePrep, with a spraying chamber area of about 100 cm 2 , place the ITO glass slide containing the standard drying points on one surface with the standard drying points facing upwards in the middle of the spraying chamber, and spray the 2,4,6-trimethylpyridinium 4-methylphenylboric acid synthesis solution obtained in step (5) into the spraying chamber, with a total of 1 mL of solution, and a total of 12 spraying times, with the final spraying amount of 2,4,6-trimethylpyridinium 4-methylphenylboric acid solution in the synthesis solution being 100 nmol / cm 2 , and naturally dry at room temperature after each layer of spraying, and after the last layer of spraying, place the ITO glass slide in a vacuum desiccator for 15 minutes of drying at room temperature;

[0096] (10) Use the Bruker full-automatic matrix spraying instrument ImagePrep, with a spraying chamber area of about 100 cm 2 , place the ITO glass slide containing the standard drying points on one surface with the standard drying points facing upwards in the middle of the spraying chamber, and spray the CHCA solution prepared in step (6) into the spraying chamber, with a total of 3 mL of solution, and 12 spraying times, with the final spraying amount being 0.21 mg / cm 2 , and naturally dry at room temperature after each layer of spraying, and after the last layer of spraying, place the ITO glass slide containing the standard drying points on one surface with the standard drying points facing upwards in a vacuum desiccator for 15 minutes of drying at room temperature;

[0097] (11) Use the Bruker UltraFlex III MALDI-TOF / TOF mass spectrometer in positive ion, reflection mode, with a mass-to-charge ratio detection range of 140-1000, to perform mass spectrometry analysis on the surface of the mouse brain tissue section on the ITO glass slide;

[0098] (12) Use the Bruker DataAnalysis to extract the mass spectrum signal m / z 522.17 of the quercetin derivative product. The results are as follows:Figure 6 As shown, when the 2,4,6-trimethylpyridinium-3-methylphenylboronic acid synthesis solution is used as the derivatization reagent and CHCA is used as the matrix, the mass spectrum signal m / z 522.17 of the quercetin derivatization product can be obtained, and the detection limit is 1.1 pmol / mm 2 .

[0099] Example 7: Mass spectrometry imaging analysis of surface derivatization of chlorogenic acid in mouse brain tissue sections

[0100] (1) Take the frozen mouse brain tissue and use a microtome to prepare coronal sections of the brain tissue with a thickness of 14 μm;

[0101] (2) Transfer the brain tissue sections to an indium tin oxide (ITO) glass slide and place it in a vacuum desiccator for 15 minutes at room temperature;

[0102] (3) Precisely weigh 15.8 mg of 2,4,6-trimethylpyridinium tetrafluoroborate, add 2 mL of methanol: water (50:50, v / v) solution, vortex well, and ultrasonic for 5 minutes to obtain a 2,4,6-trimethylpyridinium tetrafluoroborate (50 mM) solution, which is ready for use;

[0103] (4) Precisely weigh 7.44 mg of 4-aminomethylphenylboronic acid hydrochloride, add 4 mL of methanol: water (50:50, v / v) solution, vortex well, and ultrasonic for 5 minutes to obtain a 4-aminomethylphenylboronic acid hydrochloride (10 mM) solution, which is ready for use;

[0104] (5) Mix 1 mL of 2,4,6-trimethylpyridinium tetrafluoroborate solution and 1 mL of 4-aminomethylphenylboronic acid hydrochloride solution, add 2 μL of N,N-diisopropylethylamine, vortex well, and obtain a derivatization reagent 2,4,6-trimethylpyridinium-4-methylphenylboronic acid solution, which is ready for use;

[0105] (6) Precisely weigh 35 mg of CHCA, add 5 mL of acetonitrile: water (60:40, v / v) solution containing 0.2% trifluoroacetic acid by volume, vortex well, and ultrasonic for 5 minutes to obtain a CHCA (7 mg / mL) solution, which is ready for use;

[0106] (7) Precisely weigh the chlorogenic acid standard, and prepare a series of concentration standard solution with 50% methanol solution, with concentrations of 1, 0.2, 0.05, 0.01 and 0.005 mg / mL, which is ready for use;

[0107] (8) Using the dry drop method, 0.2 uL of the chlorogenic acid series concentration standard solution is added to the surface of the same mouse brain tissue section on the ITO glass slide, each standard solution point occupies 3 mm 2 , with an interval of 1 mm, and is placed in a vacuum desiccator for 15 minutes at room temperature;

[0108] (9) Using the Brook automatic matrix sprayer ImagePrep, the spraying chamber area is about 100 cm 2 The ITO glass containing the standard dry point of one measured surface was placed in the middle of the spraying chamber with the surface upward, and the 2,4,6-trimethylpyridinium 4-methylphenyl borate synthesis solution obtained in step (5) was sprayed into the spraying chamber, a total of 1 mL of solution was used, and a total of 12 times of spraying was performed, and the final spraying amount of the 2,4,6-trimethylpyridinium 4-methylphenyl borate solution in the synthesis solution system was 100 nmol / cm 2 After each layer was sprayed, it was naturally dried at room temperature, and after the last layer was sprayed, the ITO glass was placed in a vacuum dryer and dried at room temperature for 15 minutes;

[0109] (10) Using the Brook automatic matrix sprayer ImagePrep, the spraying chamber area is about 100 cm 2 The ITO glass containing the standard dry point of one measured surface was placed in the middle of the spraying chamber with the surface upward, and the CHCA solution prepared in step (6) was sprayed into the spraying chamber, a total of 3 mL of solution was used, and a total of 12 times of spraying was performed, and the final spraying amount was 0.21 mg / cm 2 After each layer was sprayed, it was naturally dried at room temperature, and after the last layer was sprayed, the ITO glass containing the standard dry point of one measured surface was placed in a vacuum dryer and dried at room temperature for 15 minutes;

[0110] (11) Using the Brook UltraFlex III MALDI-TOF / TOF mass spectrometer, in positive ion, reflection mode, mass-to-charge ratio detection range 140-1000, mass spectrometric analysis was performed on the surface of the mouse brain tissue section on the ITO glass;

[0111] (12) Using the Brook DataAnalysis, the mass spectrum signal m / z 522.17 of the chlorogenic acid derivative product was extracted. As shown in Figure 7 , when 2,4,6-trimethylpyridinium-3-methylphenyl borate synthesis solution is used as the derivatization reagent and CHCA is used as the matrix, the mass spectrum signal m / z 522.17 of the chlorogenic acid derivative product can be obtained, and the detection limit is 0.95 pmol / mm 2 .

Claims

1. A mass spectrometry imaging method for detecting compounds containing o-dihydroxyl functional groups in tissues, characterized in that, The method includes at least: 1) Synthesize the derivatizing agent pyridinium-methylphenylboronic acid in solution, wherein the derivatizing agent is one or more of formula (I) and / or (II); ； R1 to R5 are one, two, or three of the following: hydrogen or substituents: methyl, phenyl, or benzyl; and the number of substituents is one, two, three, four, or five. 2) The derivatization reagent is sprayed onto the surface of the tissue section to carry out the derivatization reaction. After the reaction is completed, a derivatized product containing an ortho-diphenol hydroxyl compound is obtained. 3) Spray the above-derivatized tissue sections with a matrix to obtain tissue section samples that can be used for mass spectrometry imaging; 4) Mass spectrometry signals of derivatized products on tissue sections were acquired using a laser desorption / sorption ion source. The compounds containing ortho-dihydroxyl functional groups in the tissue were qualitatively identified, and the mass-to-charge ratio information of the derivatized products was extracted to obtain mass spectrometry images. The synthesis process of the derivatization reagent is as follows: a pyranium salt solution and an aminomethylphenylboronic acid salt solution are mixed to obtain a mixed solution, an organic base is added for catalysis, and the mixture is thoroughly mixed to obtain a pyridinium-methylphenylboronic acid solution as the derivatization reagent; The pyranium salt has R1 to R5 as hydrogen or one, two, or three of the substituents methyl, phenyl, or benzyl, and the number of substituents is one, two, three, four, or five, with the anion being BF4; its specific structural formula is (Ⅲ). ； The aminomethylphenylborate salt is one or both of 3-aminomethylphenylborate salt and 4-aminomethylphenylborate salt; the organic base catalyst is one or both of triethylamine or N,N-diisopropylethylamine.

2. The method as described in claim 1, characterized in that, The concentrations of the pyranium salt solution and the aminomethylphenylborate acid salt solution were 10–50 mM and 10–50 mM, respectively; the molar ratio of pyranium salt to aminomethylphenylborate acid salt in the mixed solution synthesis system was 10:1 to 1:

1.

3. The method as described in claim 2, characterized in that, The concentrations of the pyranium salt solution and the aminomethylphenylborate acid salt solution were 20–30 mM and 20–30 mM, respectively; the molar ratio of pyranium salt to aminomethylphenylborate acid salt in the mixed solution synthesis system was 8:1 to 2:

1.

4. The method according to claim 2, wherein the solvent for the pyranium salt and aminomethylphenyl borate salt is an aqueous solution of methanol, wherein the volume concentration of methanol in the aqueous solution is 25-100%.

5. The method as described in claim 2, characterized in that, The volume ratio of the mixture of organic base with pyranium salt solution and aminomethylphenyl borate salt solution is 1:1000 to 4:1000.

6. The method of claim 1, wherein the amount of the derivatization reagent solution sprayed onto the surface of the tissue section to be tested is 100–500 nmol / cm² based on pyridinium-methylphenylboronic acid. 2 .

7. The method as described in claim 1, characterized in that, The matrix used was α-cyano-4-hydroxycinnamic acid, with a mass concentration of 7–10 mg / mL; the solvent was an acetonitrile aqueous solution with a volume concentration of 40–60%, containing 0.1–0.2% trifluoroacetic acid; the spraying amount on one side of the tissue section was 0.07–0.35 mg / cm². 2 .

8. The method as described in claim 1, characterized in that, Animal tissue sections were selected as the tissue sections.

9. The method as described in claim 8, characterized in that, Animal tissue sections include mouse brain tissue sections or rat brain tissue sections.

10. The method according to any one of claims 1 to 9, wherein the compound containing the ortho-dihydroxyl functional group is one or more of endogenous metabolites containing ortho-dihydroxyl groups in tissues and exogenous drugs.

11. The method of claim 10, wherein the compound containing the ortho-dihydroxyl functional group is one or more of catechol neurotransmitters, phenolic acids, and flavonoids.

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