Application of pyridinium aldehyde as reactive matrix and MALDI-MS analysis method for detection of sterol metabolites

Abstract

The application provides a MALDI-MS analysis method for detecting solid sterol metabolites by using pyridinium formaldehyde as a reactive matrix, and comprises the following steps: A) attaching or dropping a sample to be analyzed on a substrate, and drying to obtain a sample; B) dispersing a solvent to obtain a reactive matrix solution; the reactive matrix is pyridinium formaldehyde shown in formula (I); C) spraying or dropping the reactive matrix solution in step B) on the sample; and D) performing mass spectrometric analysis on the sample prepared in step C) by using MALDI-MS. The pyridinium formaldehyde can react with alcohol hydroxyl groups at normal temperature and pressure, can be used for in-situ derivatization of various solid sterol metabolites containing hydroxyl groups, has high reaction efficiency, and the product is stable. The spatial distribution information of solid sterol metabolites in a biological sample is obtained by using a derivatization reaction for alcohol hydroxyl groups and using pyridinium formaldehyde compounds as a reactive matrix of target analytes.

Description

Technical Field

[0001] This invention relates to the field of analytical technology, and in particular to the application of pyridinium formaldehyde as a reactive matrix and a MALDI-MS analytical method for detecting sterol metabolites. Background Technology

[0002] Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) is a high-throughput, high-sensitivity in-situ mass spectrometry technique with a spatial resolution of 5-50 μm, comparable to the cellular scale. It can resolve fine brain regions within brain tissue and has therefore been widely used for in-situ metabolomics studies of phospholipids in various tissue samples. MALDI-MS is currently an essential technique for obtaining information on the spatial distribution of metabolites in tissues at the near-cellular scale.

[0003] Cholesterol is an important lipid metabolite, accounting for approximately 2% of the total wet weight of the brain. It is an essential metabolite for maintaining normal brain function, and in-situ characterization of cholesterol metabolism changes at high spatial resolution has attracted widespread attention. However, cholesterol and its metabolites belong to the sterol class of compounds, with a fatty fused ring molecular structure, weak molecular polarity, and most lacking chromophores, making them extremely difficult to ionize in MALDI. Furthermore, the phosphatidylcholine lipids, which are the most abundant in brain tissue, carry a fixed positive charge, causing a masking effect on other analytes. Therefore, direct detection of sterol metabolites in MALDI has low sensitivity, making imaging analysis on tissue sections difficult. Current techniques utilize derivatization to add a fixed positive charge to sterol molecules to improve detection sensitivity; however, the reaction targets the ketone carbonyl group rather than the alcohol hydroxyl group, requiring a three-step spraying process, which is cumbersome.

[0004] Therefore, developing a simple method for tissue-derived steroid metabolites would be a significant technological improvement for detecting steroid metabolites on the MALDI-MS imaging platform. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide an application of pyridinium formaldehyde as a reactive matrix. The pyridinium formaldehyde provided by the present invention improves the desorption / ionization efficiency of the analyte and simplifies the MALDI pretreatment process, thereby improving the detection sensitivity.

[0006] This invention provides, in one aspect, the application of pyridinium formaldehyde as a reactive matrix, as shown in Formula (I), in in-situ and imaging analysis using MALDI-MS;

[0007]

[0008] Wherein, R is a C1 to C5 alkyl group.

[0009] Preferably, R is methyl, ethyl, propyl, butyl, or pentyl.

[0010] A second aspect of the present invention provides a MALDI-MS analytical method for detecting sterol metabolites using pyridinium formaldehyde as a reactive matrix, as shown in formula (I), comprising the following steps:

[0011] A) The sample to be analyzed is attached to or dropped onto the substrate, dried, and the sample is obtained;

[0012] B) The reactive matrix is ​​dispersed in a solvent to obtain a reactive matrix solution; the reactive matrix is ​​pyridinium formaldehyde as shown in formula (I);

[0013] C) Spray or drop the reactive matrix solution described in step B) onto the sample;

[0014] D) Perform mass spectrometry analysis on the sample prepared in step C) using MALDI-MS.

[0015] Preferably, the mass spectrometer is a MALDI-TOF / MS; the laser excitation source is a 355nm and 2kHz solid-state Nd:YAG / 355nm SmartBeam laser.

[0016] Preferably, the mass spectrometry analysis conditions are: a full scan range of m / z 100–1200 Da, and positive ion reflectance mode.

[0017] Preferably, the solvent in step B) is an acetonitrile-water solution; the mass concentration of acetonitrile in the acetonitrile-water solution is 50%–100%; the mass concentration of pyridinium formaldehyde represented by formula (I) in the reactive matrix solution is 0.5–10 mg / mL; and the spraying or dripping thickness of the reactive matrix solution is 0.01–0.4 mg / cm. 2 ;

[0018] Preferably, step C) further includes spraying or dripping an HCCA matrix solution onto the sample after the above reaction is completed;

[0019] The specific method for preparing the HCCA matrix solution is as follows: α-cyano-4-hydroxycinnamic acid is dispersed in a solvent to obtain the HCCA matrix solution.

[0020] Preferably, the spraying thickness of the HCCA matrix solution is 0.001–0.2 mg / cm. 2 ;

[0021] The mass concentration of HCCA in the HCCA matrix solution is 0.05–10 mg / mL.

[0022] Preferably, the substrate comprises a metal target plate or a conductive glass plate;

[0023] More preferably, the conductive glass plate is an indium tin oxide (ITO) glass sheet; the metal target plate is a MALDI metal target plate;

[0024] The samples to be analyzed include solution samples and biological slide samples; the solution samples to be analyzed are cholesterol solutions, ergosterol solutions, vitamin D2 solutions, vitamin D3 solutions, testosterone solutions, or hydrocortisone solutions; the concentration of the solution samples to be analyzed is 5 μg / mL or higher;

[0025] The biological slice samples include mouse brain homogenate slices or mouse brain slices.

[0026] Preferably, the reactive matrix solution is a pyridinium formaldehyde salt solution;

[0027] The pyridinium formaldehyde solution is selected from N-methylpyridinium-2-carboxaldehyde iodide solution, N-methylpyridinium-3-carboxaldehyde iodide solution, or N-methylpyridinium-4-carboxaldehyde iodide solution.

[0028] Compared with existing technologies, this invention provides a MALDI-MS analytical method for detecting steroid metabolites using pyridinium formaldehyde as a reactive matrix, as shown in formula (I). The method comprises the following steps: A) attaching or dropping the sample to be analyzed onto a substrate and drying it to obtain the sample; B) dispersing the reactive matrix in a solvent to obtain a reactive matrix solution; the reactive matrix is ​​pyridinium formaldehyde as shown in formula (I); C) spraying or dropping the reactive matrix solution obtained in step B) onto the sample; D) performing mass spectrometry analysis of the sample prepared in step C) using MALDI-MS. This invention allows pyridinium formaldehyde to undergo a nucleophilic addition reaction with alcohol hydroxyl groups at room temperature and pressure, enabling in-situ derivatization of various hydroxyl-containing steroid metabolites. The reaction is highly efficient, the mechanism is clear, and the product is stable. This invention utilizes a derivatization reaction targeting the hydroxyl group of alcohols, using pyridinium carboxaldehyde compounds as the reactive matrix for the target analyte. This improves the desorption / ionization efficiency of the analyte and simplifies the MALDI pretreatment process, enabling the acquisition of spatial distribution information of sterol metabolites in biological samples at a lower cost and more conveniently. Attached Figure Description

[0029] Figure 1 Equation for the derivatization reaction of N-methylpyridinium-2-carboxaldehyde with cholesterol;

[0030] Figure 2 Elution peaks of cholesterol products with various pyridinium-carboxaldehyde reagents in MALDI mass spectrometry; arrows indicate peaks of cholesterol derivatization products; (a) N-methylpyridinium-2-carboxaldehyde; (b) N-methylpyridinium-3-carboxaldehyde; (c) N-methylpyridinium-4-carboxaldehyde;

[0031] Figure 3 The peaks of various sterols were eluted by a reactive matrix of N-methylpyridinium-2-carboxaldehyde in MALDI mass spectrometry at concentrations of 50 μg / mL and 5 μg / mL. (a) Cholesterol, 50 μg / mL; (b) Ergosterol, 50 μg / mL; (c) Vitamin D3, 50 μg / mL; (d) Cholesterol, 5 μg / mL; (e) Ergosterol, 5 μg / mL; (f) Vitamin D3, 5 μg / mL.

[0032] Figure 4 Peaks in MALDI mass spectrometry for various sterol compounds and their derivatives with N-methylpyridinium-2-carboxaldehyde; (a) Vitamin D2; (b) Vitamin D3; (c) Testosterone; (d) Hydrocortisone;

[0033] Figure 5 Mass spectrometry images obtained in MALDI-MS after adding sterol standards to mouse brain homogenate sections, spraying with pyridinium formaldehyde reagent and HCCA matrix; (a) Vitamin D2; (b) Vitamin D3; (c) Testosterone; (d) Hydrocortisone;

[0034] Figure 6 Spatial distribution of cholesterol was obtained in MALDI-MS by spraying N-methylpyridinium-2-carboxaldehyde solution and HCCA matrix solution onto coronal sections of mouse brain.

[0035] Figure 7 Spatial distribution of cholesterol was obtained by spraying N-methylpyridinium-2-carboxaldehyde solution onto coronal sections of mouse brain and performing MALDI-MS.

[0036] Figure 8 Spectra of various sterol compounds in MALDI mass spectra at a concentration of 50 μg / mL using an HCCA matrix-assisted method. The arrows in the figure indicate [M+H]. + No peak was detected at the corresponding position. (a) Cholesterol; (b) Ergosterol; (c) Vitamin D3. Detailed Implementation

[0037] This invention provides an application of pyridinium formaldehyde as a reactive matrix and a MALDI-MS analytical method for detecting sterol metabolites. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0038] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0039] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0040] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0041] This invention provides a derivatization MALDI-MS analytical method for detecting sterol compounds in biological and chemical samples. It utilizes a derivatization reaction targeting the hydroxyl group of alcohols, uses pyridinium carboxaldehyde compounds as the reactive matrix for the target analytes, and can further assist ionization using traditional MALDI matrices such as HCCA (α-cyano-4-hydroxycinnamic acid). This improves the desorption / ionization efficiency of the analytes and simplifies the MALDI pretreatment process, allowing for the acquisition of spatial distribution information of sterol metabolites in biological samples at a lower cost and more conveniently.

[0042] This invention provides, in one aspect, the application of pyridinium formaldehyde as a reactive matrix, as shown in Formula (I), in in-situ and imaging analysis using MALDI-MS;

[0043]

[0044] Wherein, R is a C1 to C5 alkyl group.

[0045] In this invention, R is methyl, ethyl, propyl, butyl, or pentyl; more preferably methyl, ethyl, or propyl.

[0046] In one embodiment of the present invention, the reactive matrix is ​​N-methylpyridinium-2-carboxaldehyde, N-methylpyridinium-3-carboxaldehyde, N-methylpyridinium-4-carboxaldehyde, N-ethylpyridinium-2-carboxaldehyde, N-ethylpyridinium-3-carboxaldehyde, N-ethylpyridinium-4-carboxaldehyde, N-propylpyridinium-2-carboxaldehyde, N-propylpyridinium-3-carboxaldehyde, or N-propylpyridinium-4-carboxaldehyde.

[0047] In specific embodiments of the present invention, the above-mentioned reactive matrix exists in the form of a salt solution, including but not limited to N-methylpyridinium-2-carboxaldehyde iodide solution, N-methylpyridinium-3-carboxaldehyde iodide solution or N-methylpyridinium-4-carboxaldehyde iodide solution.

[0048] This invention utilizes derivatization to react with the hydroxyl groups of steroid metabolites, effectively enhancing the ionization efficiency of steroid metabolites and obtaining spatial distribution information of steroid metabolites in tissues. This method solves the problems of low sensitivity and complex preprocessing in previous MALDI-MS methods for detecting steroid metabolites, and enables more convenient in-situ imaging analysis of steroid metabolites on biological tissue sections.

[0049] A second aspect of the present invention provides a MALDI-MS analytical method for detecting sterol metabolites using pyridinium formaldehyde as a reactive matrix, as shown in formula (I), comprising the following steps:

[0050] A) The sample to be analyzed is attached to or dropped onto the substrate, dried, and the sample is obtained;

[0051] B) The reactive matrix is ​​dispersed in a solvent to obtain a reactive matrix solution; the reactive matrix is ​​pyridinium formaldehyde as shown in formula (I);

[0052] C) Spray the reactive matrix solution described in step B) onto the sample;

[0053] D) Perform mass spectrometry analysis on the sample prepared in step C) using MALDI-MS.

[0054] The present invention first attaches or drops the sample to be analyzed onto a substrate.

[0055] According to the present invention, the substrate comprises an indium tin oxide (ITO) glass sheet or a MALDI metal target plate;

[0056] The samples to be analyzed include solution samples and biological slide samples; the solution samples to be analyzed include, but are not limited to, cholesterol solutions, ergosterol solutions, vitamin D2 solutions, vitamin D3 solutions, testosterone solutions, or hydrocortisone solutions.

[0057] The concentration of the solution sample to be analyzed is 5 μg / mL or higher; the method of the present invention has high sensitivity and can perform detection within the above concentration range.

[0058] The biological slice samples include, but are not limited to, mouse brain homogenate slices or mouse brain slices.

[0059] In one embodiment, when the sample to be analyzed is a solution, the solution can be dropped onto the surface of an ITO glass slide or a MALDI metal target plate and then dried under vacuum.

[0060] In one embodiment, when the sample to be analyzed is a biological slice, the biological slice to be analyzed is attached to an indium tin oxide (ITO) glass slide and then vacuum dried.

[0061] The present invention does not limit the specific parameters of the above-mentioned vacuum drying; those known to those skilled in the art are acceptable.

[0062] In one embodiment of the present invention, the solvent for the cholesterol and ergosterol solution is a methyl tert-butyl ether-acetonitrile (volume ratio 1:1) solution or acetonitrile.

[0063] In one embodiment of the present invention, the solvent for the vitamin D2 and vitamin D3 solutions is a methanol-acetonitrile (volume ratio 1:1) solution or acetonitrile.

[0064] In one embodiment of the present invention, the solvent for the testosterone and hydrocortisone solution is acetonitrile.

[0065] The reactive matrix is ​​dispersed in a solvent to obtain a reactive matrix solution; the reactive matrix is ​​pyridinium formaldehyde as shown in formula (I).

[0066] The present invention has already clearly described the pyridinium formaldehyde represented by formula (I) above, and will not be repeated here.

[0067] The above-mentioned pyridinium formaldehyde reagent solution was sprayed or dripped onto the sample, and then allowed to undergo in-situ derivatization reaction with sterol metabolites in the sample on the surface.

[0068] The reactive matrix solution is a pyridinium formaldehyde salt solution; more preferably, a pyridinium formaldehyde iodide solution; including but not limited to N-methylpyridinium-2-formaldehyde iodide solution, N-methylpyridinium-3-formaldehyde iodide solution or N-methylpyridinium-4-formaldehyde iodide solution.

[0069] In some preferred embodiments, the solvent in the reactive matrix solution of the present invention is an acetonitrile-water solution; the mass concentration of acetonitrile in the acetonitrile-water solution is 50% to 100%.

[0070] Preferably, the mass concentration of acetonitrile in the acetonitrile aqueous solution is 60% to 90%.

[0071] In some preferred embodiments, the mass concentration of pyridinium formaldehyde of formula (I) in the reactive matrix solution is 0.5–10 mg / mL;

[0072] In some preferred embodiments, the mass concentration of pyridinium formaldehyde of formula (I) in the reactive matrix solution is 1 to 8 mg / mL;

[0073] In some preferred embodiments, the mass concentration of pyridinium formaldehyde of formula (I) in the reactive matrix solution is 2 to 7 mg / mL;

[0074] According to the present invention, the spraying thickness of the reactive matrix solution is 0.01–0.4 mg / cm. 2 More preferably, it is 0.02–0.2 mg / cm³. 2 The optimal value is 0.05–0.1 mg / cm³. 2 .

[0075] Spray the reactive matrix solution described in step B) onto the sample.

[0076] The spraying method described in this invention includes, but is not limited to, spraying using an automatic spraying device; the automatic spraying device spraying includes spraying using an electro-spraying device or a pneumatic spraying device.

[0077] The present invention does not limit the specific parameters of the spraying, as long as the above-mentioned spraying thickness is met.

[0078] In a preferred embodiment of the present invention, step C) further includes spraying or dripping an HCCA matrix solution onto the sample after the reaction is completed.

[0079] The specific method for preparing the HCCA matrix solution is as follows: α-cyano-4-hydroxycinnamic acid is dispersed in a solvent to obtain the HCCA matrix solution.

[0080] This invention uses pyridinium formaldehyde compounds as the reactive matrix for the target analytes and can further assist ionization using traditional MALDI matrices such as HCCA (α-cyano-4-hydroxycinnamic acid), thereby improving the desorption / ionization efficiency of the analytes and simplifying the MALDI pretreatment process. This allows for obtaining spatial distribution information of sterol metabolites in biological samples at a lower cost and more conveniently.

[0081] The spraying thickness of the HCCA matrix solution described in this invention is 0.001–0.2 mg / cm. 2 ;

[0082] The spraying method described in this invention includes, but is not limited to, spraying using an automatic spraying device; the automatic spraying device spraying includes spraying using an electro-spraying device or a pneumatic spraying device.

[0083] The present invention does not limit the specific parameters of the spraying, as long as the above-mentioned spraying thickness is met.

[0084] In some preferred embodiments, the mass concentration of HCCA in the HCCA matrix solution is 0.05–10 mg / mL.

[0085] In some preferred embodiments, the mass concentration of HCCA in the HCCA matrix solution is 1–8 mg / mL.

[0086] In some preferred embodiments, the mass concentration of HCCA in the HCCA matrix solution is 2 to 8 mg / mL.

[0087] The sample prepared in step C) was analyzed by mass spectrometry using MALDI-MS. If imaging analysis was to be performed, the measurement area containing the sample was circled in the mass spectrometry imaging software, and the program was executed. After data acquisition, the mass spectrum of the steroid metabolite derivatization products and the corresponding m / z mass spectra were plotted in the mass spectrometry data analysis software.

[0088] According to the present invention, the mass spectrometer is a MALDI-TOF / MS; the laser excitation source is a 355nm and 2kHz solid-state Nd:YAG / 355nm SmartBeam laser.

[0089] The mass spectrometry analysis conditions were as follows: full scan range of m / z 100–1200 Da, positive ion reflectance mode. Mass spectrometry data were acquired based on non-targeted analysis.

[0090] This invention provides a MALDI-MS analytical method for detecting steroid metabolites using pyridinium formaldehyde as a reactive matrix, as shown in formula (I). The method comprises the following steps: A) attaching or dropping the sample to be analyzed onto a substrate and drying it to obtain the sample; B) dispersing the reactive matrix in a solvent to obtain a reactive matrix solution; wherein the reactive matrix is ​​pyridinium formaldehyde as shown in formula (I); C) spraying the reactive matrix solution obtained in step B) onto the sample; D) performing mass spectrometry analysis of the sample prepared in step C) using MALDI-MS. This invention allows pyridinium formaldehyde to undergo a nucleophilic addition reaction with alcohol hydroxyl groups at room temperature and pressure, enabling in-situ derivatization of various hydroxyl-containing steroid metabolites. The reaction is highly efficient, the mechanism is clear, and the product is stable. This invention utilizes a derivatization reaction targeting the hydroxyl group of alcohols, using pyridinium carboxaldehyde compounds as the reactive matrix for the target analyte. This improves the desorption / ionization efficiency of the analyte and simplifies the MALDI pretreatment process, enabling the acquisition of spatial distribution information of sterol metabolites in biological samples at a lower cost and more conveniently.

[0091] Compared with existing sterol MALDI-MS imaging methods, the method provided by this invention has the following advantages: (1) The derivatization reaction introduces positive ions into sterol molecules, which helps the derivatized products to ionize on MALDI-MS; (2) Pyridinium formaldehyde reagent can be obtained by simple one-step synthesis, the raw materials are cheap, the purification steps are simple, and the operation is relatively easy; (3) This method avoids enzyme spraying and does not require incubation, which reduces the interference of the reactants on the peaks of the target molecules and shortens the pretreatment time; (4) Pyridinium formaldehyde can undergo nucleophilic addition reaction with alcohol hydroxyl groups at room temperature and pressure, which can realize the in-situ derivatization of a variety of hydroxyl-containing sterol metabolites, with high reaction efficiency, clear mechanism, and stable products.

[0092] To further illustrate the present invention, the following examples provide a detailed description of the application of pyridinium formaldehyde as a reactive matrix and the MALDI-MS analytical method for detecting sterol metabolites provided by the present invention.

[0093] Example 1: An example of mass spectrometry analysis performed on a target plate by sequentially adding cholesterol, pyridinium formaldehyde reagent solution, and HCCA matrix solution.

[0094] Step 1) Prepare a cholesterol solution with a concentration of 1 mg / mL, using methyl tert-butyl ether-acetonitrile (1:1 volume ratio) solution. Prepare N-methylpyridinium-2-carboxaldehyde iodide solution, N-methylpyridinium-3-carboxaldehyde iodide solution, and N-methylpyridinium-4-carboxaldehyde iodide solution (collectively referred to as pyridinium-carboxaldehyde reagent solution), each with a concentration of 2 mg / mL, using 70% acetonitrile-water solution as the solvent. Prepare an HCCA matrix solution with a concentration of 5 mg / mL, using 70% acetonitrile-water solution as the solvent.

[0095] 2) Step 2) Add 1 μL of the cholesterol solution prepared in step 1 to 3 positions on the MALDI metal target plate and let it air dry.

[0096] 3) In step 3), add 1 μL of each of the three pyridinium-2-carboxaldehyde reagent solutions prepared in step 1 to the three locations mentioned above, and then vacuum dry. The equations for the derivatization reactions in this step (taking N-methylpyridinium-2-carboxaldehyde as an example) are attached. Figure 1 .

[0097] 4) In step 4), add 1 μL of the matrix HCCA solution prepared in step 1 to each of the above 3 locations and then vacuum dry.

[0098] 5) Step 5) The above points were analyzed using a Bruker UltrafleXtreme MALDI-TOF mass spectrometer. Using the various pyridinium formaldehyde reagents described in this example, the cholesterol derivatization products all showed significant peaks in the mass spectrum (see attached figure). Figure 2 As shown in the figure, the products of the derivatization reactions of cholesterol with N-methylpyridinium-2-carboxaldehyde, N-methylpyridinium-3-carboxaldehyde, and N-methylpyridinium-4-carboxaldehyde all exhibit a peak at m / z 508.4 in the MALDI mass spectrometer, which is consistent with the calculated value of the theoretical products.

[0099] Example 2: An example of adding sterol standard solution and pyridinium formaldehyde reagent solution dropwise onto a target plate and performing mass spectrometry analysis in MALDI-MS.

[0100] Step 1) Prepare cholesterol solution, ergosterol solution, and vitamin D3 solution at a concentration of 50 μg / mL using acetonitrile as the solvent. Prepare N-methylpyridinium-2-carboxaldehyde iodide solution at a concentration of 2 mg / mL using 70% acetonitrile-water solution. Prepare HCCA matrix solution at a concentration of 5 mg / mL using 70% acetonitrile-water solution.

[0101] 2) Step 2) Prepare cholesterol solution, ergosterol solution, and vitamin D3 solution at a concentration of 5 μg / mL. Acetonitrile is used as the solvent for all the above solutions.

[0102] 3) In step 3), select two groups of three positions on the MALDI metal target plate. In the first group, add 1 μL of each of the three sterol solutions prepared in step 1 and dry under vacuum. In the second group, add 1 μL of each of the three sterol solutions prepared in step 2 and dry under vacuum.

[0103] 4) Step 4) Add 1 μL of the pyridinium formaldehyde reagent solution prepared in step 1 to each of the 6 positions in the 2 groups described in step 3, and then vacuum dry.

[0104] 5) Step 5) The above points were analyzed using a Bruker UltrafleXtreme MALDI-TOF mass spectrometer. Using the pyridinium formaldehyde reagent described in this example, various sterol molecules at concentrations of 50 μg / mL and lower were reacted, and the derivatized products all showed significant peaks in the mass spectrum. See attached mass spectrum. Figure 3 As can be seen from the figure, the pyridinium formaldehyde reagent method of this invention has high sensitivity for detecting sterol molecules, and can detect molecules at concentrations above 5 μg / mL.

[0105] Example 3: An example of adding sterol standards to mouse brain homogenate slices, spraying N-methylpyridinium-2-carboxaldehyde solution and matrix HCCA solution using an electrospray device, and performing imaging analysis in MALDI-MS.

[0106] 1) Step 1) Take fresh mouse brain and place it in a 2mL centrifuge tube. Dissolve it in an ultrasonic cell homogenizer in an ice-water bath. Then centrifuge at 13000rpm for 15 minutes at 4℃ to obtain mouse brain homogenate. Transfer it to -80℃ for freezing and shaping.

[0107] 2) Step 2) Take out the homogenized mouse brain from Step 1, prepare 10μm thick homogenate slices at -20℃, and attach them to indium tin oxide (ITO) glass slides. Dry under vacuum.

[0108] 3) Step 3) Prepare a standard solution of steroid metabolites with a concentration of 1 mg / mL. The types and solvents are: vitamin D2, vitamin D3 (solvent is methanol-acetonitrile solution, volume ratio 1:1), testosterone, and hydrocortisone (solvent is acetonitrile).

[0109] 4) Step 4) Add 1 μL of the four sterol standard solutions prepared in step 3 to the homogenized sections and dry them under vacuum.

[0110] 5) Step 5) Prepare N-methylpyridinium-2-carboxaldehyde iodide solution, with 70% acetonitrile-water solution as solvent and concentration of 4 mg / mL; prepare HCCA matrix solution, with 70% acetonitrile-water solution as solvent and concentration of 5 mg / mL.

[0111] 6) Connect the Harvard injection pump to the electrospray device and spray the N-methylpyridinium-2-carboxaldehyde iodide solution prepared in step 5 onto the slices from step 4. The flow rate is 2 μL / min, the voltage is 5 kV, and the average spray thickness is 0.05 mg / cm. 2 .

[0112] 7) In step 7), the HCCA solution prepared in step 5 is sprayed onto the slice from step 6 using an electrospray device at a flow rate of 2 μL / min and a voltage of 5 kV, resulting in an average coating thickness of 0.13 mg / cm. 2 .

[0113] 8) Step 8) The slices prepared in step 7 were analyzed using a Bruker ultrafleXtreme MALDI-TOF mass spectrometer.

[0114] Step 9) Process the imaging data using SCiLSLab software. Mass spectrometry images are plotted for the m / z values ​​of the derivatized products of the four sterol metabolites selected in Step 3. The mass spectra and imaging results of the derivatized products are shown in the appendix. Figure 4 , 5 As can be seen from the figure, the distribution of the theoretical mass spectrometry peaks of the derivatized products of various sterol compounds matches the actual drop positions of the standard solution, indicating that various sterols can be well detected by this method in MALDI mass spectrometry imaging.

[0115] Example 4: An example of using an HTX™ Sprayer pneumatic sprayer to spray N-methylpyridinium-2-carboxaldehyde solution and matrix HCCA solution onto a coronal section of mouse brain, and performing imaging analysis in MALDI-MS.

[0116] 1) Step 1) Take fresh mouse brain, transfer it to -80℃ for freeze-setting, and then prepare 10μm thick coronal sections at -20℃, which are then attached to indium tin oxide (ITO) glass slides. Dry under vacuum.

[0117] 2) Step 2) Prepare N-methylpyridinium-2-carboxaldehyde iodide solution, with 70% acetonitrile-water solution as solvent and concentration of 2 mg / mL; prepare HCCA matrix solution, with 70% acetonitrile-water solution as solvent and concentration of 5 mg / mL, containing 0.2% trifluoroacetic acid.

[0118] 3) Step 3) Use an HTX™ pneumatic sprayer to spray the N-methylpyridinium-2-carboxaldehyde iodide solution prepared in step 2 onto the slice from step 1. The flow rate is 80 μL / min, the air pressure is 6 psi, and the average spray thickness is 0.07 mg / cm. 2 .

[0119] 4) Step 4) Spray the HCCA solution prepared in step 2 onto the slice from step 3 using the HTX™ Sprayer at a flow rate of 120 μL / min and a pressure of 10 psi, achieving an average coating thickness of 0.08 mg / cm. 2 .

[0120] 5) Step 5) The slices prepared in step 4 were analyzed using a Bruker ultrafleXtreme MALDI-TOF mass spectrometer.

[0121] Step 6) Process the imaging data using SCiLSLab software. Plot mass spectra of the m / z values ​​corresponding to the cholesterol derivatization products. See the attached image for the imaging results. Figure 6 As can be seen from the figure, the method described in this invention successfully characterized the concentrated distribution of cholesterol in brain regions such as the corpus callosum and anterior commissure of the rat brain.

[0122] Example 5: An example of using an electrospray device to spray N-methylpyridinium-2-carboxaldehyde solution onto a coronal section of mouse brain and performing imaging analysis in MALDI-MS.

[0123] 1) Step 1) Take fresh mouse brain, transfer it to -80℃ for freeze-setting, and then prepare 10μm thick coronal sections at -20℃, which are then attached to indium tin oxide (ITO) glass slides. Dry under vacuum.

[0124] 2) Step 2) Prepare N-methylpyridinium-2-carboxaldehyde iodide solution, using 70% acetonitrile-water solution as solvent, with a concentration of 4 mg / mL.

[0125] 3) Connect the Harvard injection pump to the electrospray device and spray the N-methylpyridinium-2-carboxaldehyde iodide solution prepared in step 2 onto the slice from step 1. The flow rate is 2 μL / min, the voltage is 5 kV, and the average coating thickness is 0.05 mg / cm. 2 .

[0126] 4) Step 4) The slices prepared in step 3 were analyzed using a Bruker ultrafleXtreme MALDI-TOF mass spectrometer.

[0127] 5) Step 5) Process the imaging data using SCiLSLab software. Create mass spectrometry images corresponding to the m / z values ​​of cholesterol derivatization products. See the attached image for the imaging results. Figure 7 .

[0128] Comparative Example 1

[0129] Step 1) Prepare cholesterol solution, ergosterol solution, and vitamin D3 solution at a concentration of 50 μg / mL using acetonitrile as the solvent. Prepare HCCA matrix solution at a concentration of 5 mg / mL using 70% acetonitrile-water solution as the solvent.

[0130] 2) Step 2) Select three locations on the MALDI metal target plate and add 1 μL of the cholesterol solution, ergosterol solution and vitamin D3 solution prepared in step 1 to each location. Allow them to air dry naturally.

[0131] 3) Step 3) Add 1 μL of the matrix HCCA solution prepared in step 1 to each of the three locations described in step 2, and then vacuum dry.

[0132] 4) Step 4) Analyze the above points using a Bruker UltrafleXtreme MALDI-TOF mass spectrometer. In this comparative example, several sterol molecules at a concentration of 50 μg / mL could not be detected in the MALDI mass spectrometer when only HCCA solution was added. See attached mass spectrum. Figure 8 .

[0133] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of pyridinium formaldehyde as a reactive matrix in MALDI-MS in situ and imaging analysis; Equation (I); in, R is a C1~C5 alkyl group; Pyridium formaldehyde can undergo a nucleophilic addition reaction with alcohol hydroxyl groups at room temperature and pressure, and can be used for in-situ derivatization of sterol metabolites containing alcohol hydroxyl groups.

2. The application according to claim 1, characterized in that, R is methyl, ethyl, propyl, butyl, or pentyl.

3. A MALDI-MS analytical method for detecting sterol metabolites using pyridinium formaldehyde as a reactive matrix, as shown in formula (I), characterized in that, Includes the following steps: A) The sample to be analyzed is attached to or dropped onto the substrate, dried, and the sample is obtained; B) The reactive matrix is ​​dispersed in a solvent to obtain a reactive matrix solution; the reactive matrix is ​​pyridinium formaldehyde as shown in formula (I); C) Spray or drop the reactive matrix solution described in step B) onto the sample; D) Perform mass spectrometry analysis on the sample prepared in step C) using MALDI-MS; Equation (I); Wherein, R is a C1~C5 alkyl group; Pyridium formaldehyde can undergo a nucleophilic addition reaction with alcohol hydroxyl groups at room temperature and pressure, and can be used for in-situ derivatization of sterol metabolites containing alcohol hydroxyl groups; The mass concentration of pyridinium formaldehyde (I) in the reactive matrix solution is 0.5–10 mg / mL; the spraying or dripping thickness of the reactive matrix solution is 0.01–0.4 mg / cm. 2 .

4. The method according to claim 3, characterized in that, The mass spectrometer was a MALDI-TOF / MS; the laser excitation source was a 355 nm and 2 kHz solid-state Nd:YAG / 355 nm SmartBeam laser.

5. The method according to claim 3, characterized in that, The mass spectrometry analysis conditions were as follows: full scan range of m / z 100~1200 Da, positive ion reflectance mode.

6. The method according to claim 3, characterized in that, Step B) The solvent is an acetonitrile-water solution; the mass concentration of acetonitrile in the acetonitrile-water solution is 50%~100%.

7. The method according to claim 3, characterized in that, Step C) also includes spraying or dripping HCCA matrix solution onto the sample after the above reaction is completed; The specific method for preparing the HCCA matrix solution is as follows: α-cyano-4-hydroxycinnamic acid is dispersed in a solvent to obtain the HCCA matrix solution.

8. The method according to claim 7, characterized in that, The spraying thickness of the HCCA matrix solution is 0.001~0.2 mg / cm. 2 ; The HCCA concentration in the HCCA matrix solution is 0.05~10 mg / mL.

9. The method according to claim 3, characterized in that, The substrate includes a metal target plate or a conductive glass plate; The samples to be analyzed include solution samples and biological slide samples; the solution samples to be analyzed are cholesterol solution, ergosterol solution, vitamin D2 solution, vitamin D3 solution, testosterone solution, or hydrocortisone solution; The biological slice samples include mouse brain homogenate slices or mouse brain slices.

10. The method according to claim 3, characterized in that, The reactive matrix solution is a pyridinium formaldehyde salt solution; The pyridinium formaldehyde solution is selected from N-methylpyridinium-2-carboxaldehyde iodide solution, N-methylpyridinium-3-carboxaldehyde iodide solution, or N-methylpyridinium-4-carboxaldehyde iodide solution.

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