Mass spectrometry imaging method for detecting compounds containing ortho-dihydroxy functional groups in tissues

Abstract

This invention provides a mass spectrometry imaging detection method for compounds containing ovoid dihydroxy functional groups in tissues and its application, belonging to the field of mass spectrometry detection technology. The method includes at least: spraying an oxidizing reagent solution and a derivatizing reagent solution onto the surface of a prepared tissue section, incubating it in a sealed environment containing saturated organic solvent gas, and obtaining the derivatized product of the ovoid dihydroxy functional group compound after the reaction; spraying the derivatized tissue section onto a matrix, and then performing MALDI mass spectrometry imaging analysis. This invention can effectively improve the detection sensitivity of compounds containing ovoid dihydroxy functional groups in tissues, enabling the visual analysis of compounds containing ovoid dihydroxy functional groups in biological tissues, such as endogenous metabolites and exogenous drugs, thus having good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of mass spectrometry detection technology, and relates to a MALDI mass spectrometry imaging method and its application that can detect compounds containing ortho-dihydroxy functional groups in biological tissues with high sensitivity in positive ion mode. Technical Background

[0002] The hydroxyl group is one of the most common functional groups in small drug molecules. Of the 345 organic compounds included in the 21st edition of the National Essential Medicines List (revised in 2019), 165 contain hydroxyl groups, accounting for approximately 50%. Many pharmacodynamic compounds derived from traditional Chinese medicine contain multiple hydroxyl groups. For example, the main component of the traditional Chinese medicine *Ligusticum chuanxiong*, ligustrazine, contains ortho- and tho-dihydroxy groups, exhibiting various pharmacological effects such as anti-oxidative damage, anti-inflammatory analgesia, anticoagulation, platelet aggregation, and vasodilation. Another example is tanshinone isopropyl ester, an effective component screened from numerous metabolites of the compound *Danshen* formula, which has significant anti-hypoxic and anti-ischemic effects on the brain; its structure contains ortho- and tho-dihydroxy groups. Currently, tissue homogenization combined with LC-MS analysis has confirmed the distribution of these pharmacodynamic compounds in cerebrospinal fluid and brain tissue, but information on their localization in brain tissue is still lacking. Currently, most of our knowledge of the tissue localization of these pharmacodynamic compounds comes from whole-body autoradiography or LC-MS analysis after tissue homogenization. However, most pharmacodynamic compounds are unevenly distributed in tissues, and characterizing the tissue distribution of pharmacodynamic compounds is crucial for drug development. Another important class of endogenous small molecules containing ortho- and tho-dihydroxy groups are monoamine neurotransmitters. These are endogenous central neurotransmitters generated within neuronal cell bodies by the hydroxylation of aromatic amino acids, and their molecular structure contains ortho- and tho-dihydroxy groups. They are transported along microtubules or microfilaments within the axon to the nerve ending and stored in vesicles at the nerve ending. When a neuron is excited, the nerve impulse reaches the nerve ending, the vesicles rupture, and the neurotransmitter is released into the synaptic cleft. Most of these diffuse to the postsynaptic membrane, bind to receptors there, and produce the biological effects of intercellular transmission of nerve information. The distribution of these neurotransmitters in the brain is highly uneven.

[0003] 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 based on XY positions on tissue sections. It has been used to measure and visualize the spatial distribution of metabolites and drugs in tissues. However, the low reactivity of hydroxyl groups makes mass spectrometry imaging analysis of these low-abundance and / or poorly ionized small molecule drugs in tissue samples challenging. Tissue surface chemical derivatization techniques introduce specific functional groups into target compounds by reacting them with the active functional groups of the target compound, transforming them into easily analyzable compounds and improving detection sensitivity and selectivity. Kaya et al. reported a pyridinium-containing boric acid molecule that derivatized a monoamine neurotransmitter containing an ortho-dihydroxyl group via borate ester formation (boronic acid-diol reaction). Currently, there is a lack of highly specific and sensitive tissue surface chemical derivatization methods for ortho-dihydroxy compounds for MALDI mass spectrometry imaging analysis. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention aims to provide a mass spectrometry imaging detection method for compounds containing ortho-dihydroxy functional groups in tissues. The present invention is based on the application of a combination of an oxidant and a derivatizing reagent to in situ derivatize compounds containing ortho-dihydroxy functional groups for MALDI mass spectrometry imaging, which significantly improves the detection sensitivity of compounds containing ortho-dihydroxy functional groups in tissues, and realizes the visualization analysis of endogenous metabolites and exogenous drugs containing ortho-dihydroxy functional groups in biological tissues. Therefore, it has good practical application value.

[0005] The technical solution of this invention:

[0006] The first part of this invention provides a mass spectrometry imaging method for detecting compounds containing ortho-dihydroxy functional groups in tissues, the method comprising at least:

[0007] (a) Spray the oxidizing reagent solution onto the surface of the prepared tissue section to carry out the oxidation reaction. After the reaction is completed, the ortho-dihydroxy functional group of the compound containing ortho-dihydroxy functional group in the tissue section is oxidized to an aldehyde group or a ketone group.

[0008] (b) The derivatization reagent solution is sprayed onto the surface of the prepared tissue section to carry out the derivatization reaction. The aldehyde or ketone group of the compound reacts with the acyl trap group of the derivatization reagent to generate a hydrazone. The tissue section is placed in a closed environment containing saturated organic solvent gas for incubation. After incubation, the derivatized product containing the ortho-dihydroxy functional group is obtained.

[0009] (c) The tissue sections after the above derivatization treatment are coated with matrix to obtain tissue section samples that can be used for mass spectrometry imaging;

[0010] (d) Mass spectrometry signals of derivatized products were acquired using a laser desorption / sorption ionization source (MALDI) to qualitatively identify compounds containing ortho-dihydroxy functional groups in tissues and extract mass-to-charge ratio information of derivatized products to obtain mass spectrometry images.

[0011] As a further embodiment of the present invention: the oxidant is sodium periodate, and the concentration of sodium periodate is 5-50 mmol / L, more preferably 15-25 mmol / L, and even more preferably 20 mmol / L; the oxidant is simple to select, the oxidation conditions are mild, and it has good specificity for ortho-dihydroxy groups, making it suitable for in-situ oxidation of compounds containing ortho-dihydroxy groups to compounds containing aldehyde or ketone groups in tissues.

[0012] As a further embodiment of the present invention: the solvent for the oxidant sodium periodate is an aqueous solution of methanol and acetic acid. Preferably, the volume concentration of methanol in the aqueous solution is 25-75%, and the volume concentration of acetic acid is 0.6-10%. More preferably, the volume concentration of methanol is 50%, and the volume concentration of acetic acid is 10%.

[0013] As a further aspect of the present invention: the amount of oxidant sprayed onto one side of the tissue section is 30–1200 nmol / cm². 2 Preferably, it is 180–600 nmol / cm 2 More preferably 240 nmol / cm 2 .

[0014] As a further aspect of the present invention: the concentration of the derivatizing reagent Girard reagent P sprayed is 20-75 mmol / L, more preferably 30-60 mmol / L, and even more preferably 50 mmol / L.

[0015] As a further embodiment of the present invention: a solution of the derivatizing reagent Girard's reagent P, characterized in that the solvent of the derivatizing reagent Girard's reagent P is an aqueous solution of methanol and acetic acid, preferably, the volume ratio of methanol:acetic acid:water is 25-75:10:65-15, and more preferably 50:10:40.

[0016] As a further embodiment of the present invention: the amount of derivatization reagent sprayed onto one side of the tissue section is 240–2700 nmol / cm². 2 Preferably, it is 550–1000 nmol / cm 2 More preferably 850 nmol / cm 2 .

[0017] As a further embodiment of the present invention: the oxidation reaction temperature is 20-40°C, the reaction time is 10-30 minutes, preferably 20 minutes, and the derivatization reaction temperature is room temperature 20-40°C, the time is 10-60 minutes, preferably 30 minutes.

[0018] As a further aspect of the present invention: the saturated organic solvent used for incubation is an aqueous solution of methanol and acetic acid, preferably, the volume ratio of methanol:acetic acid:water is 25-75:10:65-15, more preferably 50:10:40; the incubation temperature is 20-40°C; and the incubation time is 10-60 minutes, preferably 20 minutes.

[0019] As a further embodiment of the present invention: the matrix used is one or more of 2,5-dihydroxybenzoic acid, α-cyano-4-hydroxycinnamic acid (CHCA), or ferulic acid, with a mass concentration of 25 mg / mL for CHCA, 25 mg / mL for 2,5-dihydroxybenzoic acid, and 25 mg / mL for ferulic acid; the solvent is a 60% acetonitrile aqueous solution of CHCA, and an 80% methanol aqueous solution of 2,5-dihydroxybenzoic acid and ferulic acid; all matrix solutions contain 0.2% trifluoroacetic acid, preferably an acetonitrile aqueous solution of CHCA; the spraying amount on one side of the tissue section is 12–36 μL / cm. 2 Preferably 18 μL / cm 2 .

[0020] As a further aspect of the present invention: the tissue sections are selected as animal tissue sections, including mouse brain tissue sections or rat brain tissue sections.

[0021] As a further aspect of the present invention: the compound containing the ortho-dihydroxy functional group is an endogenous metabolite containing ortho-dihydroxy in tissues and an exogenous drug.

[0022] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0023] (1) The oxidant and derivatization reagent used in the above technical solution are simple, the derivatization conditions are mild, and the reaction steps are simple, making it suitable for in-situ derivatization analysis of compounds containing ortho-dihydroxy functional groups in tissues.

[0024] (2) In the above technical method, by derivatizing the compound containing the ortho-dihydroxy functional group, a permanent positive charge is introduced into its structure, thereby improving the MALDI mass spectrometry detection sensitivity of the compound containing the ortho-dihydroxy functional group and realizing in-situ MALDI mass spectrometry imaging analysis of the compound containing the ortho-dihydroxy functional group in the tissue.

[0025] (3) The above technical solution effectively realizes the simultaneous mass spectrometry imaging and visualization analysis of compounds containing ortho-dihydroxy functional groups, such as endogenous metabolites and exogenous drugs, under positive ion conditions, and has good practical application value. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0027] Figure 1 This is the reaction formula of the oxidation of ligustrazine lactone I (a), tanshinone isopropyl ester (b), dopamine and norepinephrine (c) by sodium periodate and derivatization by Girard's reagent P in Example 1 of the present invention;

[0028] Figure 2 These are MALDI mass spectrometry data of ligustrazine lactone I, tanshinone isopropyl ester, dopamine and norepinephrine derivatives on an indium tin oxide slide and mouse brain tissue in Example 1 of this invention.

[0029] Figure 3 These are the MALDI mass spectrometry data of ligustrazine I-derived on an indium tin oxide slide and mouse brain tissue in Example 2 of this invention;

[0030] Figure 4 This is a mass spectrometry image of ligustrazine lactone I in mouse brain (sagittal section) tissue in Example 3 of the present invention;

[0031] Figure 5 This is a mass spectrometry image of ligustrazine lactone I in mouse brain (coronal section) tissue in Example 4 of the present invention;

[0032] Figure 6 This is a mass spectrometry image of ligustrazine lactone I in mouse brain tissue in Example 5 of the present invention;

[0033] Figure 7 This is a mass spectrometry image of ligustrazine lactone I in mouse brain tissue using CHCH and FA as a mixed matrix in Example 6 of the present invention;

[0034] Figure 8 This is a mass spectrometry image of ligustrazine lactone I in the brain tissue of mice that were administered 50% methanol extract of Ligusticum striatum by gavage in Example 7 of this invention; Detailed Implementation

[0035] Example 1: Derivatization and MALDI mass spectrometry analysis of ligustrazine lactone I, dopamine, norepinephrine, and tanshinone isopropyl ester on indium tin oxide slides and mouse brain tissue

[0036] (1) Take mouse brain tissue and prepare coronal sections of brain tissue with a thickness of 14 μm using a slicer;

[0037] (2) Transfer the brain tissue slices onto indium tin oxide (ITO) slides and dry them in a vacuum desiccator for 15 minutes;

[0038] (3) Accurately weigh 107 mg of sodium periodate and place it in a 50 mL volumetric flask. Add methanol:acetic acid:water (50:1:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain sodium periodate (10 mM) solution. Prepare at 0-4℃ in the dark and use immediately.

[0039] (4) Accurately weigh 188 mg of Girard's reagent P and place it in a 50 mL volumetric flask. Add methanol:acetic acid:water (50:10:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain Girard's reagent P (20 mM) solution for later use.

[0040] (5) Accurately weigh 750 mg of CHCA and place it in a 50 mL volumetric flask. Add 60% acetonitrile aqueous solution (0.2% trifluoroacetic acid), vortex to mix, and sonicate for 5 minutes to obtain CHCA (15 mg / mL) solution for later use.

[0041] (6) Accurately weigh 1 mg of ligustrazine lactone I, dopamine, norepinephrine and tanshinone isopropyl ester standards, and prepare standard solutions with 50% methanol for later use.

[0042] (7) Using the dry drop method, 1 μL of the standard solution of ligustrazine lactone I, dopamine, norepinephrine and tanshinone isopropyl ester was dropped onto an ITO glass slide or the surface of a mouse brain tissue slice and then dried in a vacuum desiccator for 5 minutes.

[0043] (8) The above oxidant solution was uniformly sprayed onto one side surface of a mouse brain tissue slice, with a spraying amount of 180 nmol / cm². 2 A total of 1.5 mL of oxidant solution was used to spray 9 layers, with nitrogen flow drying between each layer. The total reaction time was 30 minutes. After the last layer was sprayed, the slices were placed in a vacuum desiccator to dry for 5 minutes.

[0044] (9) The above-mentioned derivatization reagent solution was uniformly sprayed onto one side surface of a mouse brain tissue slice, with a spraying amount of 480 nmol / cm². 2 A total of 2 mL of derivatization reagent solution was used to spray 12 layers, with nitrogen flow drying between each layer. The total reaction time was 40 minutes. After the last layer was sprayed, the slices were placed in a sealed humidified box for incubation.

[0045] (10) The sealed humidified box is a covered glass box with a length, width and height of 12, 12 and 6 cm respectively. Add 50 mL of acetic acid: water (40:60, v / v) solution, place the above brain tissue slices on the upper layer of the humidified box, incubate at room temperature 24℃ for 40 minutes, and then dry in a vacuum desiccator for 5 minutes.

[0046] (11) The above matrix solution was uniformly sprayed onto one side of a mouse brain tissue slice at a spraying volume of 18 μL / cm². 2 .

[0047] A total of 1.5 mL of matrix solution was used to spray 9 layers, with nitrogen flow drying between each layer. The total reaction time was 30 minutes. After the last layer was sprayed, the slices were placed in a vacuum desiccator to dry for 5 minutes.

[0048] (12) Using a Brook UltraFlexⅢ MALDI-TOF / TOF mass spectrometer, in positive ion and reflection mode, with a mass-to-charge ratio detection range of 100-820, mass spectrometry analysis was performed on the standard derivatives on ITO slides or mouse brain tissue slices.

[0049] (13) Based on the reaction formula of ligustrazine lactone I, tanshinone isopropyl ester, dopamine and norepinephrine being oxidized by sodium periodate and derivatized by Girard's reagent P ( Figure 1 The mass spectrometry signals of the derivatized products were extracted using Bruker DataAnalysis. The results are as follows: Figure 2 As shown, when sodium periodate is used as the oxidant, Girard's reagent as the derivatization reagent, and CHCA as the matrix, mass spectrometry signals of the derivatized products of ligustrazine lactone I, tanshinone isopropyl ester, dopamine, and norepinephrine can be obtained.

[0050] Example 2: Derivatization and MALDI mass spectrometry analysis of ligustilide I on indium tin oxide slides and mouse brain tissue

[0051] (1) Take mouse brain tissue and prepare coronal sections of brain tissue with a thickness of 14 μm using a slicer;

[0052] (2) Transfer the brain tissue slices onto ITO slides and dry them in a vacuum desiccator for 15 minutes.

[0053] (3) Accurately weigh 535 mg of sodium periodate and place it in a 50 mL volumetric flask. Add methanol:acetic acid:water (50:10:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain sodium periodate (50 mM) solution. Prepare at 0-4℃ in the dark and use immediately.

[0054] (4) Accurately weigh 470 mg of Girard's reagent P and place it in a 50 mL volumetric flask. Add methanol:acetic acid:water (50:10:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain Girard's reagent P (50 mM) solution for later use.

[0055] (5) Accurately weigh 750 mg of CHCA and place it in a 50 mL volumetric flask. Add 60% acetonitrile aqueous solution (0.2% trifluoroacetic acid), vortex to mix, and sonicate for 5 minutes to obtain CHCA (15 mg / mL) matrix solution for later use.

[0056] (6) Accurately weigh 1.25 g of DHB and place it in a 50 mL volumetric flask. Add 80% methanol aqueous solution (0.2% trifluoroacetic acid), vortex to mix, and sonicate for 5 minutes to obtain a DHB (25 mg / mL) matrix solution for later use.

[0057] (7) Accurately weigh 1.25 g of FA and place it in a 50 mL volumetric flask. Add 80% methanol aqueous solution (0.2% trifluoroacetic acid), vortex to mix, and sonicate for 5 minutes to obtain FA (25 mg / mL) solution for later use.

[0058] (8) Accurately weigh 5 mL of the above CHCA matrix solution and DHB matrix solution, vortex mix, and sonicate for 5 minutes to obtain a mixed matrix solution of CHCA and DHB for later use.

[0059] (9) Accurately weigh 5 mL of the above CHCA matrix solution and FA matrix solution, vortex mix, and sonicate for 5 minutes to obtain a CHCA and FA mixed matrix solution for later use.

[0060] (10) Accurately weigh 1 mg of ligustrazine lactone I standard, and prepare standard solutions with 50% methanol for later use;

[0061] (11) Using the dry drop method, 1 μL of ligustrazine I standard solution was dropped onto an ITO glass slide or the surface of a mouse brain tissue slice and dried in a vacuum desiccator for 5 minutes.

[0062] (12) The above oxidant solution was uniformly sprayed onto one side surface of a mouse brain tissue slice, with a spraying amount of 600 nmol / cm². 2 A total of 1 mL of oxidant solution was used to spray about 6 layers, with nitrogen gas flow drying between each layer. The total reaction time was 20 minutes. After the last layer was sprayed, the slices were placed in a vacuum desiccator to dry for 5 minutes.

[0063] (13) The above-mentioned derivatization reagent solution was uniformly sprayed onto one side surface of a mouse brain tissue slice, with a spraying amount of 900 nmol / cm². 2 A total of 1.5 mL of derivatization reagent solution was used to spray 9 layers, with nitrogen gas flow drying between each layer. The total reaction time was 30 minutes. After the last layer was sprayed, the slices were placed in a sealed humid chamber.

[0064] (14) The sealed humidified box is a covered glass box with a length, width and height of 12, 12 and 6 cm respectively. Add 50 mL of acetic acid: water (40:60, v / v) solution, place the above brain tissue slices on the upper layer of the humidified box, incubate at room temperature 24℃ for 10 minutes, and then dry in a vacuum desiccator for 5 minutes.

[0065] (15) The above matrix solution was uniformly sprayed onto one side surface of a mouse brain tissue section at a spraying volume of 35 μL / cm². 2 A total of 3 mL of matrix solution was used to spray 18 layers, with nitrogen flow drying between each layer. The total reaction time was 60 minutes. After the last layer was sprayed, the slices were placed in a vacuum desiccator to dry for 5 minutes.

[0066] (16) Using a Bruker UltraFlexⅢ MALDI-TOF / TOF mass spectrometer, in positive ion, reflective mode, with a mass-to-charge ratio detection range of 100-820, mass spectrometry analysis was performed on the standard derivatives on ITO slides or mouse brain tissue slices.

[0067] (17) The mass spectrometry signal of the derivatized product of Ligusticum striatum I was extracted using Bruker DataAnalysis, and the results are as follows: Figure 3 As shown, mass spectrometry signals of the derivatives of ligustrazine I can be obtained using CHCA matrix, CHCA and DHB mixed matrix, CHCA and FA mixed matrix, and DHB matrix.

[0068] Example 3: Mass spectrometry imaging analysis of derivatized ligustrazine I on the surface of mouse brain (sagittal section) tissue

[0069] (1) Take mouse brain tissue and prepare coronal sections of brain tissue with a thickness of 14 μm using a slicer;

[0070] (2) Transfer the brain tissue slices onto ITO slides and dry them in a vacuum desiccator for 15 minutes.

[0071] (3) Accurately weigh 214 mg of sodium periodate and place it in a 50 mL volumetric flask. Add methanol:acetic acid:water (50:10:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain sodium periodate (20 mM) solution. Prepare at 0-4℃ in the dark and use immediately.

[0072] (4) Accurately weigh 470 mg of Girard's reagent P and place it in a 50 mL volumetric flask. Add methanol:acetic acid:water (50:10:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain Girard's reagent P (50 mM) solution for later use.

[0073] (5) Accurately weigh 350 mg of CHCA and place it in a 50 mL volumetric flask. Add 60% acetonitrile aqueous solution (0.2% trifluoroacetic acid), vortex to mix, and sonicate for 5 minutes to obtain CHCA (7 mg / mL) solution for later use.

[0074] (6) Accurately weigh 1 mg of ligustrazine lactone I and prepare a series of standard solutions with 50% methanol for later use;

[0075] (7) Using the dry drop method, 0.2 μL of a series of concentrations of ligustrazine lactone I standard solution was added to the surface of mouse brain tissue slices and dried in a vacuum desiccator for 5 minutes;

[0076] (8) Using ImagePrep TM The matrix sprayer sprayed the above-mentioned oxidant solution onto one side of a mouse brain tissue section at a spraying density of 360 nmol / cm².2 A total of 1.5 mL of oxidant solution was used to spray 9 layers, with nitrogen flow drying between each layer. The total reaction time was 30 minutes. After the last layer was sprayed, the slices were placed in a vacuum desiccator to dry for 5 minutes.

[0077] (9) Using ImagePrep TM The matrix sprayer sprayed the above-mentioned derivatization reagent solution onto one side of a mouse brain tissue section at a spraying density of 900 nmol / cm². 2 A total of 1.5 mL of derivatization reagent solution was used to spray 9 layers, with nitrogen flow drying between each layer. The total reaction time was 30 minutes. After the last layer was sprayed, the slices were placed in a sealed humidified box for incubation.

[0078] (10) The sealed humidified box is a covered glass box with a length, width and height of 12, 12 and 6 cm respectively. Add 50 mL of methanol: acetic acid: water (50:10:40, v / v) solution, place the above brain tissue slices on the upper layer of the humidified box, and incubate at 37°C for 20 minutes. After incubation, place the slices in a vacuum desiccator to dry for 5 minutes.

[0079] (11) Using ImagePrep TM The matrix sprayer applied the matrix solution to one side of a mouse brain tissue slice at a spraying volume of 18 μL / cm². 2 A total of 1.5 mL of matrix solution was used to spray 9 layers, with nitrogen gas flow drying between each layer. The total reaction time was 30 minutes. After the last layer was sprayed, the slices were placed in a vacuum desiccator to dry for 5 minutes.

[0080] (12) Using a Brook UltraFlexⅢ MALDI-TOF / TOF mass spectrometer, in positive ion reflection mode, with a spatial resolution of 200×200μm and a mass-to-charge ratio detection range of 100~820, 500 shots were collected per pixel and the position was randomly moved every 100 shots to perform mass spectrometry imaging analysis on mouse brain tissue slices.

[0081] (13) Using Brook's FlexImaging software, targeted mass spectrometry data were extracted from ligustrazine I-derived products in mouse brain tissue sections to obtain their mass spectrometry images. Figure 4 As can be seen, when sodium periodate is used as the oxidant, Girard's reagent as the derivatization reagent, and CHCA as the matrix, mass spectrometry images of the derivatized product of ligustrazine I can be obtained.

[0082] Example 4: Mass spectrometry imaging analysis of derivatized ligustrazine I on the surface of mouse brain (coronal section) tissue

[0083] (1) Take mouse brain tissue and prepare coronal sections of brain tissue with a thickness of 14 μm using a slicer;

[0084] (2) Transfer the brain tissue slices to the same ITO slide and dry them in a vacuum desiccator for 15 minutes.

[0085] (3) Accurately weigh 214 mg of sodium periodate and place it in a 50 mL volumetric flask. Add methanol:acetic acid:water (50:1:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain sodium periodate (20 mM) solution. Prepare at 0-4℃ in the dark and use immediately.

[0086] (4) Accurately weigh 470 mg of Girard's reagent P and place it in a 50 mL volumetric flask. Add methanol:acetic acid:water (50:10:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain Girard's reagent P (50 mM) solution for later use.

[0087] (5) Accurately weigh 350 mg of CHCA and place it in a 50 mL volumetric flask. Add 60% acetonitrile aqueous solution (0.2% trifluoroacetic acid), vortex to mix, and sonicate for 5 minutes to obtain CHCA (7 mg / mL) solution for later use.

[0088] (6) Accurately weigh 1 mg of ligustrazine lactone I and prepare a series of standard solutions with 50% methanol for later use;

[0089] (7) Using the dry drop method, 0.2 μL of a series of concentrations of ligustrazine lactone I standard solution was added to the surface of mouse brain tissue slices and dried in a vacuum desiccator for 5 minutes;

[0090] (8) Using ImagePrep TM The matrix sprayer sprayed the above-mentioned oxidant solution onto one side surface of a mouse brain tissue slice at a spraying density of 360 nmol / cm². 2 A total of 1.5 mL of oxidant solution was used to spray 9 layers, with nitrogen flow drying between each layer. The total reaction time was 30 minutes. After the last layer was sprayed, the slices were placed in a vacuum desiccator to dry for 5 minutes.

[0091] (9) Using ImagePrep TM The matrix sprayer sprayed the above-mentioned derivatization reagent solution onto one side surface of a mouse brain tissue slice at a spraying density of 900 nmol / cm². 2 A total of 1.5 mL of derivatization reagent solution was used to spray 9 layers, with nitrogen flow drying between each layer. The total reaction time was 30 minutes. After the last layer was sprayed, the slices were placed in a sealed humidified box for incubation.

[0092] (10) A sealed humidified box is a covered glass box with a length, width and height of 12, 12 and 6 cm respectively. Add 50 mL of methanol: acetic acid: water (50:10:40, v / v) solution, place the above brain tissue slices on the upper layer of the humidified box, and incubate at room temperature of 24°C for 20 minutes. After incubation, place the slices in a vacuum desiccator to dry for 5 minutes.

[0093] (11) Using ImagePrep TM The matrix sprayer applied the matrix solution to one side of a mouse brain tissue slice at a spraying volume of 35 μL / cm². 2 A total of 3 mL of matrix solution was used to spray 18 layers, with nitrogen flow drying between each layer. The total reaction time was 60 minutes. After the last layer was sprayed, the slices were placed in a vacuum desiccator to dry for 5 minutes.

[0094] (12) Using a Brook UltraFlexⅢ MALDI-TOF / TOF mass spectrometer, in positive ion reflection mode, with a mass-to-charge ratio detection range of 100 to 820 and a spatial resolution of 200×200μm, 500 shots were collected per pixel and the position was randomly moved every 100 shots to perform mass spectrometry imaging analysis on mouse brain tissue slices.

[0095] (13) Using Brook's FlexImaging software, targeted mass spectrometry data were extracted from ligustrazine I-derived products in mouse brain tissue sections to obtain their mass spectrometry images. Figure 5 As can be seen, when sodium periodate is used as the oxidant, Girard's reagent as the derivatization reagent, and CHCA as the matrix, mass spectrometry images of the derivatized product of ligustrazine I can be obtained.

[0096] Example 5: Mass spectrometry imaging analysis of derivatized ligustrazine I on the surface of mouse brain tissue

[0097] (1) Take mouse brain tissue and prepare coronal sections of brain tissue with a thickness of 14 μm using a slicer;

[0098] (2) Transfer the brain tissue slices to the same ITO slide and dry them in a vacuum desiccator for 15 minutes.

[0099] (3) Accurately weigh 107 mg of sodium periodate and place it in a 50 mL volumetric flask. Add methanol:acetic acid:water (50:10:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain sodium periodate (10 mM) solution. Prepare at 0-4℃ in the dark and use immediately.

[0100] (4) Accurately weigh 470 mg of Girard's reagent P and place it in a 50 mL volumetric flask. Add methanol:acetic acid:water (50:10:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain Girard's reagent P (50 mM) solution for later use.

[0101] (5) Accurately weigh 350 mg of CHCA and place it in a 50 mL volumetric flask. Add 60% acetonitrile aqueous solution (0.2% trifluoroacetic acid), vortex to mix, and sonicate for 5 minutes to obtain CHCA (7 mg / mL) solution for later use.

[0102] (6) Accurately weigh 1 mg of ligustrazine lactone I and prepare a series of standard solutions with 50% methanol for later use;

[0103] (7) Using the dry drop method, 0.2 μL of a series of concentrations of ligustrazine lactone I standard solution was added to the surface of mouse brain tissue slices and dried in a vacuum desiccator for 5 minutes;

[0104] (8) Using ImagePrep TM The matrix sprayer sprayed the above-mentioned oxidant solution onto one side surface of a mouse brain tissue slice at a spraying density of 240 nmol / cm². 2 A total of 2 mL of oxidant solution was used to spray 12 layers, with nitrogen flow drying between each layer. The total reaction time was 40 minutes. After the last layer was sprayed, the slices were placed in a vacuum desiccator to dry for 5 minutes.

[0105] (9) Using ImagePrep TM The matrix sprayer sprayed the above-mentioned derivatization reagent solution onto one side surface of a mouse brain tissue slice at a spraying density of 1200 nmol / cm². 2 A total of 2 mL of derivatization reagent solution was used to spray 12 layers, with nitrogen flow drying between each layer. The total reaction time was 40 minutes. After the last layer was sprayed, the slices were placed in a sealed humidified box for incubation.

[0106] (10) A sealed humidified box is a covered glass box with a length, width and height of 12, 12 and 6 cm respectively. Add 50 mL of methanol: acetic acid: water (50:10:40, v / v) solution, place the above brain tissue slices on the upper layer of the humidified box, and incubate at room temperature of 20°C for 20 minutes. After incubation, place the slices in a vacuum desiccator to dry for 5 minutes.

[0107] (11) Using ImagePrep TM The matrix sprayer applied the matrix solution to one side of a mouse brain tissue slice at a spraying volume of 24 μL / cm². 2 A total of 2 mL of matrix solution was used to spray 12 layers, with nitrogen flow drying between each layer. The total reaction time was 40 minutes. After the last layer was sprayed, the slices were placed in a vacuum desiccator to dry for 5 minutes.

[0108] (12) Using a Brook UltraFlexⅢ MALDI-TOF / TOF mass spectrometer, in positive ion reflection mode, with a mass-to-charge ratio detection range of 100 to 820 and a spatial resolution of 200×200μm, 500 shots were collected per pixel and the position was randomly moved every 100 shots to perform mass spectrometry imaging analysis on mouse brain tissue slices.

[0109] (13) Using Brook's FlexImaging software, targeted mass spectrometry data were extracted from ligustrazine I-derived products in mouse brain tissue sections to obtain their mass spectrometry images. Figure 6 As can be seen, when sodium periodate is used as the oxidant, Girard's reagent as the derivatization reagent, and CHCA as the matrix, mass spectrometry images of the derivatized product of ligustrazine I can be obtained.

[0110] Example 6: Mass spectrometry imaging analysis of ligustrazine I derived from mouse brain tissue using CHCA and FA as a mixed matrix

[0111] (1) Take mouse brain tissue and prepare coronal sections of brain tissue with a thickness of 14 μm using a slicer;

[0112] (2) Transfer the brain tissue slices to the same ITO slide and dry them in a vacuum desiccator for 15 minutes.

[0113] (3) Accurately weigh 214 mg of sodium periodate and place it in a 50 mL volumetric flask. Add methanol:acetic acid:water (50:10:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain sodium periodate (20 mM) solution. Prepare at 0-4℃ in the dark and use immediately.

[0114] (4) Accurately weigh 470 mg of Girard's reagent P and place it in a 50 mL volumetric flask. Add methanol:acetic acid:water (50:10:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain Girard's reagent P (50 mM) solution for later use.

[0115] (5) Accurately weigh 750 mg of CHCA and place it in a 50 mL volumetric flask. Add 60% acetonitrile aqueous solution (0.2% trifluoroacetic acid), vortex to mix, and sonicate for 5 minutes to obtain CHCA (15 mg / mL) solution for later use.

[0116] (6) Accurately weigh 1.25 g of FA and place it in a 50 mL volumetric flask. Add 80% methanol aqueous solution (0.2% trifluoroacetic acid), vortex to mix, and sonicate for 5 minutes to obtain FA (25 mg / mL) solution for later use.

[0117] (7) Accurately weigh 5 mL of the above CHCA matrix solution and FA matrix solution, vortex mix, and sonicate for 5 minutes to obtain a mixed CHCA and FA matrix solution for later use.

[0118] (8) Accurately weigh 1 mg of ligustrazine lactone I and prepare a series of standard solutions with 50% methanol for later use;

[0119] (9) Using the dry drop method, 0.2 μL of a series of concentrations of ligustrazine I standard solution was added to the surface of mouse brain tissue slices and dried in a vacuum desiccator for 5 minutes.

[0120] (10) Using ImagePrep TM The matrix sprayer sprayed the above-mentioned oxidant solution onto one side surface of a mouse brain tissue slice at a spraying density of 240 nmol / cm². 2 A total of 1 mL of oxidant solution was used to spray 6 layers. Each layer was allowed to dry naturally between layers and then dried with nitrogen gas between layers. The total reaction time was 20 minutes. After the last layer was sprayed, the slices were placed in a vacuum desiccator and dried for 5 minutes.

[0121] (11) Using ImagePrep TM The matrix sprayer sprayed the above-mentioned derivatization reagent solution onto one side surface of a mouse brain tissue slice at a spraying density of 1200 nmol / cm². 2 A total of 2 mL of derivatization reagent solution was used to spray 12 layers, with nitrogen flow drying between each layer. The total reaction time was 40 minutes. After the last layer was sprayed, the slices were placed in a sealed humidified box for incubation.

[0122] (12) The sealed humidified box is a covered glass box with a length, width and height of 12, 12 and 6 cm respectively. Add 50 mL of methanol: acetic acid: water (50:10:40, v / v) solution, place the above brain tissue slices on the upper layer of the humidified box, and incubate at room temperature of 24°C for 60 minutes. After incubation, place the slices in a vacuum desiccator to dry for 5 minutes.

[0123] (13) Using ImagePrep TM The matrix sprayer applied the above CHCA and FA mixed matrix solution to one side of a mouse brain tissue slice at a spraying volume of 36 μL / cm². 2 A total of 3 mL of matrix solution was used to spray 18 layers, with nitrogen flow drying between each layer. The total reaction time was 60 minutes. After the last layer was sprayed, the slices were placed in a vacuum desiccator to dry for 5 minutes.

[0124] (14) Using a Brook UltraFlexⅢ MALDI-TOF / TOF mass spectrometer, in positive ion reflection mode, with a mass-to-charge ratio detection range of 100 to 820 and a spatial resolution of 200×200μm, 1000 shots were collected per pixel and the position was randomly moved every 100 shots to perform mass spectrometry imaging analysis on mouse brain tissue slices.

[0125] (15) Using Brook's FlexImaging software, targeted mass spectrometry data were extracted from ligustrazine I-derived products in mouse brain tissue sections to obtain their mass spectrometry images. Figure 7 It can be seen that when sodium periodate is used as the oxidant, Girard's reagent as the derivatization reagent, and CHCA and FA as the mixed matrix, mass spectrometry images of the derivatized product of ligustrazine I can be obtained.

[0126] Example 7: Mass spectrometry of ligustilide I in mouse brain tissue after oral administration of 50% methanol extract of Ligusticum striatum.

[0127] (1) 100g of Ligusticum chuanxiong was crushed, 1L of methanol was added, and the mixture was refluxed for 2 hours. The extract was evaporated by rotary evaporation and then freeze-dried to obtain Ligusticum chuanxiong extract.

[0128] (2) C57BL / 6 mice weighing 18-22g were given Ligusticum chuanxiong extract (distilled water suspension) by gavage for 6 consecutive days. The dosage for the first 5 days was 2g / kg (crude drug amount), and the dosage for the 6th day was 100g / kg (crude drug amount). The volume of administration was 0.2mL / 10g body weight. 20 minutes after the last administration, the mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (0.1mL / 10g). The heart was slowly perfused with 20mL of physiological saline. After decapitation, the brain tissue was removed and placed on aluminum foil on dry ice for quick freezing. After freezing solid, it was stored in a -80℃ refrigerator for later use.

[0129] (3) Take mouse brain tissue and prepare coronal sections of brain tissue with a thickness of 14 μm using a slicer;

[0130] (4) Transfer the brain tissue slices to the same ITO slide and dry them in a vacuum desiccator for 15 minutes.

[0131] (5) Accurately weigh 214 mg of sodium periodate and place it in a 50 mL volumetric flask. Add methanol:acetic acid:water (50:10:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain sodium periodate (20 mM) solution. Prepare at 0-4℃ in the dark and use immediately.

[0132] (6) Accurately weigh 470 mg of Girard's reagent P and place it in a 50 mL volumetric flask. Add methanol:acetic acid:water (50:10:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain Girard's reagent P (50 mM) solution for later use.

[0133] (7) Accurately weigh 750 mg of CHCA and place it in a 50 mL volumetric flask. Add 60% acetonitrile aqueous solution (0.2% trifluoroacetic acid), vortex to mix, and sonicate for 5 minutes to obtain CHCA (7 mg / mL) solution for later use.

[0134] (8) Accurately weigh 5 mL of the above CHCA matrix solution and FA matrix solution, vortex mix, and sonicate for 5 minutes to obtain a mixed CHCA and FA matrix solution for later use.

[0135] (9) Using ImagePrep TM The matrix sprayer sprayed the above-mentioned oxidant solution onto one side surface of a mouse brain tissue slice at a spraying density of 240 nmol / cm². 2 A total of 1 mL of oxidant solution was used to spray 6 layers, with nitrogen flow drying between each layer. The total reaction time was 20 minutes. After the last layer was sprayed, the slices were placed in a vacuum desiccator to dry for 5 minutes.

[0136] (10) Using ImagePrep TM The matrix sprayer sprayed the above-mentioned derivatization reagent solution onto one side surface of a mouse brain tissue slice at a spraying density of 1200 nmol / cm². 2 A total of 2 mL of derivatization reagent solution was used to spray 12 layers, with nitrogen flow drying between each layer. The total reaction time was 40 minutes. After the last layer was sprayed, the slices were placed in a sealed humidified box for incubation.

[0137] (11) The sealed humidified box is a covered glass box with a length, width and height of 12, 12 and 6 cm respectively. Add 50 mL of methanol: acetic acid: water (50:10:40, v / v) solution, place the above brain tissue slices on the upper layer of the humidified box, and incubate at room temperature of 24°C for 20 minutes. After incubation, place the slices in a vacuum desiccator to dry for 5 minutes.

[0138] (12) Using ImagePrep TM The CHCA matrix solution was sprayed onto one side of a mouse brain tissue slice using a matrix sprayer at a spraying volume of 18 μL / cm². 2 A total of 1.5 mL of matrix solution was used to spray 9 layers, with nitrogen gas flow drying between each layer. The total reaction time was 30 minutes. After the last layer was sprayed, the slices were placed in a vacuum desiccator to dry for 5 minutes.

[0139] (13) Using a Brook UltraFlexⅢ MALDI-TOF / TOF mass spectrometer, in positive ion reflection mode, with a mass-to-charge ratio detection range of 100 to 820 and a spatial resolution of 100×100μm, 500 shots were collected per pixel and the position was randomly moved every 100 shots to perform mass spectrometry imaging analysis on mouse brain tissue slices.

[0140] (14) Using Brook's FlexImaging software, targeted mass spectrometry data were extracted from ligustrazine I-derived products in mouse brain tissue sections to obtain their mass spectrometry images. Figure 8It can be seen that when sodium periodate is used as the oxidant, Girard's reagent as the derivatization reagent, and CHCA as the matrix, the distribution of the derivatized product of Ligusticum chuanxiong lactone I in the lateral ventricle can be obtained.

Claims

1. A mass spectrometry imaging method for detecting compounds containing ortho-dihydroxy functional groups in tissues, characterized in that, The method includes at least: 1) The oxidizing agent solution was sprayed onto the surface of the tissue section to carry out the oxidation reaction; the oxidizing agent used was sodium periodate (NaIO4). 2) The derivatization reagent solution was sprayed onto the surface of the tissue section to carry out the derivatization reaction. After the derivatization reagent was sprayed, the tissue section was placed in a closed environment containing saturated organic solvent gas for incubation. After incubation, the derivatized product containing the ortho-dihydroxy functional group compound was obtained. The derivatization reagent used was Girard reagent P. 3) The derivatized tissue sections were then coated with a matrix to obtain tissue section samples suitable for mass spectrometry imaging. The matrix used was one or more of 2,5-dihydroxybenzoic acid, α-cyano-4-hydroxycinnamic acid (CHCA), or ferulic acid, with a mass concentration of 25 mg / mL for CHCA, 25 mg / mL for 2,5-dihydroxybenzoic acid, and 25 mg / mL for ferulic acid. The solvent was 60% acetonitrile aqueous solution for CHCA, and 80% methanol aqueous solution for 2,5-dihydroxybenzoic acid and ferulic acid. All matrix solutions contained 0.2% trifluoroacetic acid. The coating amount on one side of the tissue section was 12–36 µL / cm. 2 ; 4) Mass spectrometry signals of derivatized products are acquired using a laser desorption / ionization source to qualitatively identify compounds containing ortho-dihydroxy functional groups in tissues, and mass-to-charge ratio information of derivatized products is extracted to obtain mass spectrometry images.

2. The method as described in claim 1, characterized in that, The concentration of the oxidizing agent sodium periodate was 5–50 mmol / L; the spraying amount on one side of the tissue section was 30–1200 nmol / cm². 2 .

3. The method as described in claim 2, characterized in that, The solvent for the oxidant sodium periodate is an aqueous solution of methanol and acetic acid, wherein the volume concentration of methanol in the aqueous solution is 25-75% and the volume concentration of acetic acid is 0.6-10%.

4. The method as described in claim 1, characterized in that: The concentration of the derivatization reagent Girard's reagent P sprayed was 20–75 mmol / L; the spraying amount on one side of the tissue section was 240–2700 nmol / cm². 2 .

5. The method as described in claim 4, characterized in that, The solvent for the derivatization reagent Girard's reagent P is an aqueous solution of methanol and acetic acid, with a volume ratio of methanol:acetic acid:water of 25–75:10:65–15.

6. The method as described in claim 1, characterized in that, The oxidation reaction temperature is 20–40℃ and the reaction time is 10–30 minutes. The derivatization reaction temperature is room temperature (20–40℃) and the time is 10–60 minutes.

7. The method as described in claim 1, characterized in that, The saturated organic solvent used for incubation is an aqueous solution of methanol and acetic acid, with a volume ratio of methanol:acetic acid:water of 25–75:10:65–15; the incubation temperature is 20–40°C; and the incubation time is 10–60 minutes.

8. The method as described in claim 1, characterized in that, The selected tissue sections were animal tissue sections, including mouse brain tissue sections or rat brain tissue sections.

9. The method according to any one of claims 1 to 8, wherein the compound containing an ortho-dihydroxy functional group is an endogenous metabolite containing an ortho-dihydroxy functional group in tissues or an exogenous drug.

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