A nucleotide analysis method based on paired derivatization technology

Abstract

The present invention relates to the field of analytical detection technology. More specifically, the present invention relates to a method for analyzing nucleotides based on paired derivatization technology. 4-Ethylbenzylamine (4-EBA) and 4-Methylbenzylamine (4-MBA) are used as derivatization reagents in the detection of nucleotides. This method improves the accuracy and precision of nucleotide quantitative determination. Although the analytical strategy of the present invention does not use an isotopic internal standard, it can achieve similar results as using an isotopic internal standard.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and more particularly to a method for analyzing nucleotides based on paired derivatization technology. Background Art

[0002] Nucleotides are important biological molecules composed of nitrogen bases, pentose sugars and phosphate groups. They are distributed in the nuclei and cytoplasm of various organs, tissues and cells of the body, and participate in the basic life activities of the body such as heredity, growth and development as nucleic acid components. In addition, there are a considerable number of free nucleotides in organisms, which are involved in many key cellular processes: energy metabolism and signal transduction, etc. Analyzing the level of nucleotides can provide information for understanding the relationship between nucleotides and diseases, which is of great significance for the elucidation of the pathogenesis of related diseases and clinical diagnosis and treatment. At present, many detection methods have been developed and used for the analysis of nucleotides. Such as nuclear magnetic resonance (NMR) [1] , capillary electrophoresis (CE) [2] , high performance liquid chromatography (HPLC) [3] , capillary electrophoresis mass spectrometry (CE-MS) [4] and liquid chromatography-mass spectrometry (LC-MS) [5] Among them, LC-MS has the characteristics of high specificity, good separation, and high sensitivity, and is currently the most powerful platform for analyzing nucleotides.

[0003] However, the following problems need to be solved in LC-MS detection of nucleotides: (1) The phosphate groups of nucleotides will bind to liquid phase steel and mass spectrometer needles, resulting in tailing of chromatographic peaks and reduced sensitivity. The effects may be complexation, oxidation, or epoxidation. [6] (2) Due to the high polarity of nucleotides, their retention on reversed-phase chromatography columns is weak, which is not conducive to the chromatographic separation of nucleotides. In addition, their ionization efficiency in the electrospray ionization source is poor, resulting in poor mass spectrometry detection sensitivity. (3) In addition, in order to accurately quantify the target analyte and minimize the matrix effect of the target analyte in the detection, LC-MS usually uses stable isotope internal standards for quantitative analysis. However, isotope internal standards are difficult to obtain or very expensive, and the development of corresponding methods to solve this problem is urgent.

[0004] In order to solve the above problems, derivatization technology is generally used; derivatization technology is to quantitatively convert the target compound that is difficult to analyze and detect in the sample into another compound that is easy to analyze and detect through a chemical reaction, and the target compound can be qualitatively or quantitatively analyzed through the analysis and detection of the latter. At present, the derivatization reagents for mass spectrometry analysis of nucleotides include: 3-aminomethylpyridine (AMPy), trimethylsilylated diazomethane (TMSD), N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA), 2-(diazomethyl)-N-methyl-N-phenyl-benzamide (2-DMBA), etc. Among them, in the analytical method for LC-MS / MS determination of nucleotides based on chemical derivatization, 3-aminomethylpyridine (AMPy) [7] was used to derivatize nucleotides, but the reaction products were still not retained on reversed-phase chromatography columns. Li et al. [8] The trimethylsilylated diazomethane (TMSD) derivatization strategy was used to determine the nucleotides in A549 and HCT116 cells. [9] The development of N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization to measure nucleotides in cells. After these derivatization reagents react with nucleotides, the products can be retained on reversed-phase chromatography columns, but none of these methods can provide a one-to-one internal standard. Liu et al.

[10] They synthesized a pair of light and heavy stable isotope labeling reagents, 2-(diazomethyl)-N-methyl-N-phenyl-benzamide (2-DMBA) and d5-2-(diazomethyl)-N-methyl-N-phenyl-benzamide (d5-2-DMBA) to provide a one-to-one internal standard. They used this method to perform sensitive and accurate RNA detection on 8 cells. On this basis, they

[11] Another pair of light and heavy stable isotope labeling reagents, 2-(diazomethyl)phenyl)(9-methyl-1,3,4,9-tetrahydro-2H-pyridin[3,4-b]indol-2-yl)methane (DMPI) and d3-(2-(diazomethyl)phenyl)(9-methyl-1,3,4,9-tetrahydro-2H-pyridin[3,4-b]indol-2-yl)methane (d3-DMPI), were synthesized. Using this method, they determined the nucleotide content of single mammalian cells. However, the complex and tedious synthesis of these reagents limited their application.

[0005] In this study, we optimized 4-methylbenzylamine (4-MBA) as an efficient pre-column derivatization reagent and used 4-ethylbenzylamine (4-EBA) as a one-to-one internal standard for derivatized nucleotides. We developed a 4-MBA / 4-EBA-based paired derivatization method that is accurate and sensitive, allowing for the simultaneous quantification of 12 nucleotides by liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis. Summary of the Invention

[0006] Purpose of the Invention

[0007] The present invention provides a 4-MBA / 4-EBA paired derivatization strategy, which avoids the use of isotopes and achieves a nucleotide LC-MS / MS analysis method with high sensitivity and high selectivity.

[0008] Technical Solution

[0009] The present invention solves the above-mentioned problems by adopting the following technical solutions: Figure 1 As shown:

[0010] 4-Ethylbenzylamine (4-EBA) and 4-methylbenzylamine (4-MBA) are used as derivatization reagents to detect the free nucleotide content in cells or tissues.

[0011] The product of the reaction between 4-ethylbenzylamine (4-EBA) and 12 nucleotide standards is used as an internal standard. Protein precipitation is performed on biological samples, and free nucleotides in the supernatant are chemically labeled with 4-methylbenzylamine (4-MBA). After mixing, the free nucleotides are detected and analyzed using LC-MS / MS. This method includes the following steps:

[0012] (1) Prepare nucleotide standard solution using acetonitrile and water in a volume ratio of 1:1;

[0013] (2) Using acetonitrile and water in a volume ratio of 1:1 as solvent, EDC, 4-EBA, and NMM were added in sequence and mixed well to obtain a 4-EBA derivatization solution;

[0014] (3) preparing a nucleotide mixed standard solution from the nucleotide mother solution obtained in step (1), adding the 4-EBA derivatization solution and mixing, and reacting to obtain a derivatized internal standard solution;

[0015] (4) Using acetonitrile and water in a volume ratio of 1:1 as solvent, EDC, 4-MBA, and NMM were added in sequence and mixed evenly to obtain a 4-MBA derivatization solution;

[0016] (5) drawing a standard curve: selecting a nucleotide standard and diluting it with a solvent to form a series of standard solutions, adding a 4-MBA derivatization solution to carry out a derivatization reaction, adding the internal standard solution obtained in step (3), and diluting it to obtain the test solutions for each standard curve;

[0017] (6) The LC-MS / MS method was used to detect the test solutions of each standard curve and establish the standard curve of each nucleotide; wherein, the liquid chromatography conditions were as follows: chromatographic column: Shimadzu Shim-pack GIST C18 column (2.1×100 mm, 2.0 μm); flow rate: 0.3 mL / min; column temperature: 35°C; injection volume: 4 μL; mobile phase: A is pure water containing 5 mM NH4HCO3 and adjusted to pH 7.8, and B is pure acetonitrile; elution gradient: 0-3 min, 5% B; 3-7 min, 5%-20% B; 7-10 min, 20%-25% B; 10-15 min, 25%-30% B; 15-20 min, 30%-60% B; 20-22 min, 60%-80% B; 22-24 min, 80% B; 24-26 min, 80%-5% B; and then equilibrate for 4 min.

[0018] Mass spectrometry conditions were as follows: positive ion mode, nebulizer gas flow rate of 3 L / min, drying gas and heater gas flow rates of 10 L / min, interface temperature of 250°C, DL tube temperature of 250°C, and heating block temperature of 400°C. Multiple reaction monitoring (MRM) mode was used to detect derivatized nucleotides.

[0019] (7) After the biological sample is extracted and protein precipitated, the obtained supernatant is dried with nitrogen gas to obtain sample A;

[0020] (8) Sample A obtained in step (7) is dissolved in acetonitrile and water in a volume ratio of 1:1, 4-MBA derivatization solution is added, and the mixture is mixed until the nucleotide reaction is complete. The derivatized internal standard solution obtained in step (3) is then added, and the mixture is diluted with water, mixed, and centrifuged to obtain supernatant sample B.

[0021] (9) The supernatant sample B obtained in step (8) is subjected to reverse phase liquid chromatography-electrospray ionization mass spectrometry analysis to perform quantitative analysis of nucleotides.

[0022] In step (4), the derivatization temperature, derivatization time, and the molar ratio of 4-MBA, EDC, and NMM to nucleotides were optimized to obtain a good labeling efficiency (e.g. Figure 2First, the reaction temperature (20–90°C) was optimized. In our results, the peak area of ​​the derivative reached a maximum at 60°C. The reaction time (0.5–7 h) was then optimized. From 0.5 to 4 h, the peak area of ​​the derivative increased over time and gradually stabilized after 4 h. Therefore, 4 h was selected as the optimal reaction time. The molar ratio of substrate to 4-MBA was then optimized from 1:1000 to 1:50,000. The peak area reached a maximum at 1:5000. Therefore, a molar ratio of 1:5000 was selected. The optimal EDC concentration was then investigated. The molar ratio of substrate to EDC was optimized within a range of 1:1000 to 1:50,000, and a molar ratio of 1:5000 was also selected. Finally, the molar ratio of substrate to NMM was optimized within a range of 1:1000 to 1:50,000. The peak area of ​​the derivative showed little change at different molar ratios. Therefore, a molar ratio of substrate to NMM of 1:1000 was selected. In general, the reaction was carried out with 4-MBA at 60°C for 4 h, and the molar ratios of 4-MBA, EDC and NMM to nucleotides were set to 5000, 5000 and 1000 respectively. Under these conditions, the derivatization reaction was relatively complete (see attached). Figure 3 In step (2), the reaction of 4-EBA is also carried out under the optimized reaction conditions.

[0023] The biological sample is tissue or cell.

[0024] Method for removing protein from the tissue in step (7): Add the tissue to a 2 mL centrifuge tube, add a pre-cooled mixed solution of methanol and water in a volume ratio of 4:1, place it in a grinder and grind it for 60 seconds each time, with a 10-second interval in between, for a total of 10 times. After grinding, centrifuge the homogenate at 13000 rpm and 4°C for 10 minutes, and remove the supernatant.

[0025] Method for removing protein from cells in step (7): After washing the cells with PBS buffer, place them in a 1.5 mL centrifuge tube and centrifuge at 3000 rpm for 5 min to precipitate the cells. Discard the supernatant, add a pre-cooled mixed solution of methanol and water with a volume ratio of 4:1, and incubate at 4°C for 30 min. After vortex mixing, centrifuge at 13000 rpm and 4°C for 10 min, and remove the supernatant.

[0026] In step (8), in the nucleotide LC-MS / MS analysis method based on paired derivatization technology, the volume ratio of biological sample A, internal standard reaction solution and water is 1:1:1.

[0027] The principle of this reaction (as shown below):

[0028] Although there are many reports on the use of derivatization strategies combined with liquid chromatography-mass spectrometry to quantify nucleotides, the limited availability of isotope internal standards remains a challenge in analyzing nucleotides. Compounds with similar structures to the target compounds can be used as substitutes for isotope internal standards. There are reports on the use of synthetic paired derivatization reagents for quantitative analysis, but the synthesis of these reagents is complex and tedious, which to some extent limits their application.

[0029] In this study, we propose for the first time the use of readily available 4-MBA or 4-EBA as paired derivatization reagents for the quantitative analysis of nucleotides. The structurally similar homologues 4-MBA and 4-EBA act as paired derivatization reagents. In the presence of the condensing agent EDC and a base, along with the catalyst NMM, they react with nucleotides. 4-MBA or 4-EBA and the nucleotides shed a molecule of H2O, forming a phosphoramide-like structure. Because the derivatization reagents contain a benzene ring, they increase the hydrophobicity of the nucleotides, significantly improving their retention on reversed-phase chromatography columns and reducing interference. Furthermore, the presence of amino groups in the 4-MBA or 4-EBA structure increases the ionization efficiency of the modified nucleotides in the ESI source, significantly enhancing the sensitivity of their mass spectrometric detection. A mixed standard solution of nucleotides derivatized with 4-EBA is used as a derivatized internal standard solution; free nucleotides in the sample are derivatized with 4-MBA (biological samples such as cells or tissues are processed to obtain a supernatant, and 4-MBA is added for derivatization). The free nucleotide content in the sample supernatant is analyzed by LC-MS / MS (the peak area of ​​4-MBA-modified nucleotides is calculated / the peak area of ​​4-EBA-modified nucleotides is divided to obtain a relative result. The above calculation can reduce the matrix effect of the quantitative analysis of nucleotides in the sample. The changes in the content of nucleotides secreted by cells or tissues are evaluated, and then the condition is judged in combination with other data to obtain objective results.

[0030] Calculation formula: relative value = 4-MBA modified nucleotide peak area / 4-EBA modified nucleotide peak area.

[0031]

[0032] Reaction formula for nucleotide derivatization with 4-MBA and 4-EBA

[0033] Beneficial effects

[0034] (1) 4-MBA or 4-EBA as derivatization reagents has not been reported in the literature and was discovered by the inventors for the first time. The labeling efficiency of the derivatization reaction involved in the present invention is 81.5% to 100%, while Sheng et al. [7] The labeling efficiency of 3-aminomethylpyridine (AMPy) is 74.4% to 89.3%. The derivatization reaction of the present invention has a high labeling efficiency, which is conducive to quantitative analysis.

[0035] (2) The derivatization reaction of the present invention can significantly improve the retention of nucleotides on reverse phase chromatography columns and increase mass spectrometry response. Figure 4 ), that is, before the reaction, the limit of quantification of nucleotides was 0.0333~1.6640pmol, and after 4-MBA labeling, the limit of quantification of nucleotides was 0.0002~0.0166pmol, and the sensitivity was improved by 10.0~166.4 times after the reaction. Because biological samples are difficult to obtain, this method uses 30mg of colon tissue and 800μL of extraction solvent to extract and take 400μL of supernatant to quantify nucleotides. There are literature reports

[12] Without derivatization, 500 μL of 50 mg of tissue must be added and 400 μL of supernatant must be taken to quantify nucleotides. Therefore, this method requires less sample volume.

[0036] (3) The paired derivatization strategy of the present invention is that the derivatization reagents 4-MBA and 4-EBA are a pair of homologues, differing only by one "-CH2" group. After the nucleotide standard reacts with 4-EBA, it is added as an internal standard to the sample to be tested after derivatization with 4-MBA, so that each analyte has a one-to-one corresponding homologue as an internal standard, thereby achieving the purpose of reducing the matrix effect (Appendix Figure 5 ), which can effectively improve the accuracy and precision of nucleotide quantification. Li et al. [8] The trimethylsilylation diazomethane (TMSD) derivatization strategy was used with ATP- 13 C 10 , 15 N5 and AMP- 13 C 10 , 15 N5 is an internal standard for nucleotide quantification. Its intra-day precision and inter-day precision are 2.1% to 14.7% and 3.9% to 14.7%, respectively; its intra-day accuracy and inter-day accuracy are -12.0% to 13.5%; and its matrix effect is 91.9% to 110.9%. Zhang et al. [9] The strategy of derivatizing nucleotides with N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) was also used. 13 C 10 , 15 N5 and AMP- 13 C 10 , 15N5 was used as the internal standard for nucleotide quantification. Its intra-day precision and inter-day precision were 1.3%-14.4% and 3.4%-14.8%, respectively. Its intra-day accuracy and inter-day accuracy were -10.8%-9.4% and -10.4%-11.2%, respectively. The matrix effect was 91.1%-113.9%. In the present invention, a paired derivatization strategy was used to analyze nucleotides in the colon and cells. In the quantitative methodology for colon tissue, the intra-day precision and inter-day precision were 2.3% to 13.7% and 0.9% to 13.8%, respectively, the intra-day accuracy and inter-day accuracy were -7.3% to 10.3% and -9.4% to 6.9%, respectively, and the matrix effect was 87.2% to 114.6%. In the quantitative methodology for cells, the intra-day precision and inter-day precision were 1.4% to 14.0% and 2.6% to 11.9%, respectively, the intra-day accuracy and inter-day accuracy were -11.0% to 10.7% and -11.2% to 12.2%, respectively, and the matrix effect was 86.3% to 115.4%. It can be seen that the analytical strategy of the present invention, although not using an isotopic internal standard, can achieve similar results as using an isotopic internal standard. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is the technical roadmap of the present invention;

[0038] Figure 2 Comparison of peak areas of nucleotide derivatization products under different conditions in the present invention (A is the derivatization temperature, B is the derivatization time, C is the molar ratio of 4-MBA to nucleotide, D is the molar ratio of EDC to nucleotide, and E is the molar ratio of NMM to nucleotide 4-MBA);

[0039] Figure 3 Comparative chromatograms of the original nucleotides before and after the derivatization reaction of each nucleotide in the present invention (A is AMP, B is ADP, C is ATP, D is GMP, E is GDP, F is GTP, G is CMP, H is CDP, I is CTP, J is UMP, K is UDP, and L is UTP);

[0040] Figure 4 : A comparison of the LC-MS / MS detection effects before and after derivatization in the present invention (A is a diagram of the LC-MS / MS detection effect before chemical labeling, and B is a diagram of the LC-MS / MS detection effect after chemical labeling);

[0041] Figure 5The chromatograms are shown after derivatization of nucleotides with 4-MBA and 4-EBA in the present invention (A is a chromatogram of 4-EBA-ATP, 4-MBA-ATP, 4-EBA-ADP, 4-MBA-ADP, 4-EBA-AMP, and 4-MBA-AMP; B is a chromatogram of 4-EBA-GTP, 4-MBA-GTP, 4-EBA-GDP, 4-MBA-GDP, 4-EBA-GMP, and 4-MBA-GMP; C is a chromatogram of 4-EBA-CTP, 4-MBA-CTP, 4-EBA-CDP, 4-MBA-CDP, 4-EBA-CMP, and 4-MBA-CMP; and D is a chromatogram of 4-EBA-UTP, 4-MBA-UTP, 4-EBA-UDP, 4-MBA-UDP, 4-EBA-UMP, and 4-MBA-UMP);

[0042] Figure 6 Comparison of nucleotide content between the DSS-induced acute colitis model of the present invention and the normal control group (A is AMP, B is GMP, C is CMP, D is UMP, E is ADP, F is GDP, G is CDP, H is UDP, I is ATP, J is GTP, K is CTP, and L is UTP) *p < 0.05, **p < 0.01, ***p < 0.001;

[0043] Figure 7 This is a comparison of the nucleotide content in LPS-stimulated RAW264.7 cells of the present invention and the normal control group (A is AMP, B is GMP, C is CMP, D is UMP, E is ADP, F is GDP, G is CDP, H is UDP, I is ATP, J is GTP, K is CTP, and L is UTP) **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION

[0044] The 4-methylbenzylamine in the present invention can be abbreviated as 4-MBA, the 4-ethylbenzylamine can be abbreviated as 4-EBA, and the 1-(3-

[0045] (2-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride can be abbreviated as EDC, N-methylmorpholine can be abbreviated as NMM, electrospray ionization source can be abbreviated as ESI, multiple reaction monitoring can be abbreviated as MRM, and liquid chromatography tandem mass spectrometry can be abbreviated as LC-MS / MS.

[0046] Example 1

[0047] 1. Prepare a 10 mM stock solution of each nucleotide standard using acetonitrile and water in a 1:1 volume ratio.

[0048] 2. Prepare the working solution: (1) Dissolve EDC, 4-MBA, and NMM in a 1:1 volume ratio mixture of acetonitrile and water to prepare a 4-MBA derivatization solution, where the concentrations of the 4-MBA solution are 62.5 mM, the EDC solution is 62.5 mM, and the NMM solution are 12.5 mM. (2) Dissolve EDC, 4-EBA, and NMM in a 1:1 volume ratio mixture of acetonitrile and water to prepare a 4-EBA derivatization solution, where the concentrations of the 4-EBA solution are 62.5 mM, the EDC solution is 62.5 mM, and the NMM solution are 12.5 mM.

[0049] 3. Prepare a nucleotide mixed standard solution using the nucleotide mother solution in step (1). Take 20 μL of the nucleotide mixed standard, add 80 μL of 4-EBA derivatization solution and mix well. React at 60°C for 4 h with an oscillation rate of 300 rpm. After the reaction is completed, place on ice for 2 min to terminate the reaction to obtain a derivatized internal standard solution.

[0050] 4. Use LC-MS / MS to detect each standard curve test solution, and use the nucleotide concentration as the horizontal axis and the peak area ratio of the 4-MBA labeled nucleotide in the standard curve test solution and the corresponding 4-EBA labeled nucleotide in the derivatized internal standard solution as the vertical axis to establish a standard curve for various nucleotides.

[0051] 5. Biological samples were precipitated by adding pre-cooled methanol-water solution (methanol: water = 4:1, v / v), centrifuged at 13000 rpm, 4°C for 10 min, and the supernatant was removed and dried under nitrogen at room temperature.

[0052] 6. The biological sample treated in step 5 was redissolved in 20 μL of a solution of acetonitrile and water in a volume ratio of 1:1, 4-MBA derivatization reagent was added and mixed, and the reaction was carried out at 60°C for 4 h with an oscillation rate of 300 rpm. After the reaction was completed, it was placed on ice for 2 min to terminate the reaction. 45 μL of the biological sample reaction solution was taken into a 1.5 mL centrifuge tube, 45 μL of the derivatized internal standard solution was added, and then 45 μL of ultrapure water was added. The mixture was vortexed for 30 seconds to mix evenly, and centrifuged at 13000 rpm and 4°C for 10 min. The supernatant was taken for LC-MS / MS analysis.

[0053] The mass spectrometer used in the LC-MS / MS method was a Shimadzu 8045 mass spectrometer (Tokyo, Japan) equipped with an electrospray ionization source (ESI). The liquid chromatography unit was equipped with an LC-40B XR pump, a CTO-40S column oven, and a SIL-40C autosampler. A reverse-phase chromatography column Shim-pack GIST C18 column (2.1×100mm, 2.0μm) was used; flow rate: 0.3mL / min; column temperature: 35℃; injection volume: 4μL; mobile phase: A is pure water with 5mM NH4HCO3, and the pH is adjusted to 7.8, and B is pure acetonitrile; elution gradient: 0-3min, 5% B; 3-7min, 5%-20% B; 7-10min, 20%-25% B; 10-15min, 25-30% B; 15-20min, 30%-60% B; 20-22min, 60%-80% B; 22-24min, 80% B; 24-26min, 80%-5% B; then balance for 4min.

[0054] Mass spectrometry conditions were as follows: positive ion mode, nebulizer gas flow rate of 3 L / min, drying gas and heater gas flow rates of 10 L / min, interface temperature of 250°C, DL tube temperature of 250°C, and heating block temperature of 400°C. Multiple reaction monitoring (MRM) mode was used to detect derivatized nucleotides.

[0055] Example 2: Analysis of nucleotides in colonic tissue in DSS-induced acute colitis

[0056] Six- to eight-week-old C57BL / 6J male mice (18-20 g) were acclimated for one week and then divided into two groups: a control group and a UC model group. UC was induced in the model group by adding 3% (w / v) dextran sodium sulfate (DSS) to drinking water for 7 days, while the control group drank pure water during this period. Mice were sacrificed on day 8, and colon tissue was obtained. Collected samples should be processed promptly to prevent degradation of the test substance.

[0057] Take 30 mg of colon tissue in a 2 mL centrifuge tube, add 800 μL of pre-cooled methanol and water mixed solution with a volume ratio of 4:1, add magnetic beads, and place it in a grinder for grinding. Grind for 60 seconds each time with a 10-second interval in between, and grind a total of 10 times. After grinding, centrifuge the homogenate at 13000 rpm and 4°C for 10 minutes, remove the supernatant, and blow dry with nitrogen at room temperature.

[0058] The dried sample was reconstituted with 20 μL of acetonitrile aqueous solution (acetonitrile: water = 1:1, v / v), and 80 μL of 4-MBA derivatization solution was added. After the reaction, 45 μL was taken into a 1.5 mL EP tube, 45 μL of derivatization internal standard solution was added, and then 45 μL of pure water was added. The mixture was vortexed to mix evenly, and the mixture was centrifuged at 13000 rpm and 4°C for 10 min. The supernatant was taken for LC-MS / MS analysis.

[0059] The standard curve ranges for each nucleotide are 5-500 μM for AMP, ADP, and ATP, 0.5-50 μM for CDP and CTP, and 1-100 μM for the remaining nucleotides. Figure 6 shown.

[0060] Example 3: Analysis of Nucleotides in LPS-Stimulated RAW264.7 Cells

[0061] The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and in a cell culture incubator at 37°C and 5% CO2. They were divided into a control group and a lipopolysaccharide (LPS) model group. The model group was stimulated with 200 ng / mL LPS for 12 hours, and then the cells were collected.

[0062] A certain number of cultured cells were washed with PBS buffer, placed in a 1.5 mL centrifuge tube, and centrifuged at 3000 rpm, 4°C for 5 minutes. The supernatant was discarded, and 1 mL of a pre-chilled methanol:water mixture (4:1 by volume) was added. The tube was incubated at 4°C for 30 minutes, vortexed, and centrifuged at 13000 rpm, 4°C for 10 minutes. The supernatant was dried under nitrogen. The collected protein was detected using a bicinchoninic acid (BCA) protein assay kit according to the manufacturer's recommended protocol for quantification of cell protein.

[0063] The dried sample was reconstituted with 20 μL of acetonitrile aqueous solution (acetonitrile: water = 1:1, v / v), and 80 μL of 4-MBA derivatization solution was added. After the reaction, 45 μL was taken into a 1.5 mL EP tube, 45 μL of derivatization internal standard solution was added, and then 45 μL of pure water was added. The mixture was vortexed to mix evenly, and the mixture was centrifuged at 13000 rpm and 4°C for 10 min. The supernatant was taken for LC-MS / MS analysis.

[0064] The ranges of the standard curves for each nucleotide are 0.2-20 μM for AMP, ADP, and UDP, 2-200 μM for ATP and GTP, 0.005-0.5 μM for GMP, 0.02-2 μM for GDP, 0.05-5 μM for CMP and CDP, 0.5-50 μM for CTP, 0.1-10 μM for UMP, 0.2-20 μM for UDP, and 5-500 μM for UTP. The measured nucleotide contents are compared as follows: Figure 7 shown.

[0065] The results showed that in vitro, LPS stimulation significantly reduced nucleotide levels in RAW264.7 cells, suggesting that under inflammatory conditions, cells release nucleotides from the intracellular compartment to the extracellular compartment for signal transduction. In vivo, GDP showed the most significant change (p < 0.001), while CMP, UMP, CDP, UDP, and GTP remained unchanged. This suggests that ulcerative colitis is most closely linked to GDP metabolism and the inflammatory signaling response it mediates, potentially providing new clues and targets for the prevention and treatment of ulcerative colitis.

[0066] In summary, inflammatory bowel diseases (IBDs), represented by Crohn's disease (CD) and ulcerative colitis (UC), are chronic and relapsing diseases that cause gastrointestinal inflammation. The prevalence is high in developed Western countries, with up to 2 million people suffering from these diseases in Europe.

[13] The incidence of IBD has been increasing in developing countries in South America, Asia, Africa, and Eastern Europe. [13,14] . Studies have shown that abnormal nucleotide metabolism is significantly correlated with the occurrence and development of IBD. Under inflammatory stress conditions, nucleotides are released into the extracellular space through ubiquitin-binding proteins and gap junction hemichannels (such as connexin 43), bind to specific purinergic receptors, and induce the occurrence of inflammatory responses. Different nucleotides activate different purinergic receptor subtypes. P2X and P2Y11 receptors are mainly activated by extracellular ATP, P2Y2 receptors are mainly activated by ATP and UTP, P2Y1, P2Y12, and P2Y13 receptors are mainly activated by ADP, and P2Y12 and P2Y13 receptors are mainly activated by ADP. P2Y4 is activated by UTP; P2Y6 and P2Y14 are activated by UDP

[15] Therefore, analyzing nucleotides in tissues and identifying abnormal nucleotides in UC will help to elucidate the pathological mechanism of UC and is also an urgent need for discovering new targets for treating UC.

[0067] In the early stages of the inflammatory response, macrophages, as effector cells, first enter the infected area, and then activate and recruit neutrophils, forming the body's first line of defense against infection. Therefore, in Examples 2 and 3, we applied established methods to quantitatively analyze nucleotide levels in in vitro and in vivo inflammatory models, aiming to explore the changes in nucleotides after inflammation in the body. Example 2 is an in vivo sample analysis. We used DSS to model C57BL / 6J mice. The clinical manifestations and pathological characteristics of this animal colitis are similar to those of human UC, and it is currently a classic method for studying the pathogenesis and drug treatment of UC. After modeling, colon tissues of the blank control group and the DSS model group were taken for analysis. Example 3 is an in vitro cell level analysis. In the study, we used LPS to stimulate RAW264.7 cells for modeling. LPS is the main component of the cell wall of Gram-negative bacteria, which can stimulate cells to produce NO and TNF-α, forming an inflammatory response. Nucleotide quantitative analysis was performed on the cell lysates of the blank control group and the LPS-stimulated model group. It can be demonstrated through Examples 2 and 3 that the method provided by this patent can be used to determine the content of nucleotides in biological samples in vivo and in vitro.

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Claims

1. The use of 4-ethylbenzylamine and 4-methylbenzylamine as derivatization reagents in detecting nucleotides in biological samples, characterized in that: The nucleotide is one or more of AMP, ADP, ATP, GMP, GDP, GTP, CMP, CDP, CTP, UMP, UDP and UTP; the biological sample is a cell or tissue; the application adopts the LC-MS / MS method, wherein the liquid chromatography conditions are: C18 column, mobile phase A is pure water containing NH4HCO3, and mobile phase B is pure acetonitrile.

2. The use according to claim 1, characterized in that Free nucleotides in the sample were derivatized with 4-methylbenzylamine, and standard nucleotides were derivatized with 4-ethylbenzylamine.

3. The use according to claim 1, characterized in that Using acetonitrile and water in a volume ratio of 1:1 as solvent, EDC, 4-methylbenzylamine and N-methylmorpholine are mixed evenly to obtain a 4-methylbenzylamine derivatization solution; using acetonitrile and water in a volume ratio of 1:1 as solvent, EDC, 4-ethylbenzylamine and N-methylmorpholine are mixed evenly to obtain a 4-ethylbenzylamine derivatization solution.

4. A method for detecting nucleotides, characterized in that: Follow these steps to achieve this: (1) Prepare a nucleotide standard solution using acetonitrile and water in a 1:1 volume ratio as solvent; (2) Using acetonitrile and water in a volume ratio of 1:1 as solvent, EDC, 4-ethylbenzylamine, and N-methylmorpholine were added in sequence and mixed evenly to obtain a 4-ethylbenzylamine derivatization solution; (3) preparing a nucleotide mixed standard solution from the nucleotide mother solution obtained in step (1), adding 4-ethylbenzylamine derivatization solution and mixing, and reacting to obtain a derivatized internal standard solution; (4) Using acetonitrile and water in a volume ratio of 1:1 as solvent, EDC, 4-methylbenzylamine, and N-methylmorpholine were added in sequence and mixed evenly to obtain a 4-methylbenzylamine derivatization solution; (5) Drawing a standard curve: Select a nucleotide standard and dilute it with a solvent to form a series of standard solutions, add 4-methylbenzylamine derivatization solution to carry out derivatization reaction, add the internal standard solution obtained in step (3), and dilute it to obtain the test solutions for each standard curve; (6) The standard curve test solutions were detected by LC-MS / MS method to establish the standard curve of each nucleotide; the liquid chromatography conditions were as follows: chromatographic column: Shimadzu Shim-pack GIST C18 column, 2.1×100 mm, 2.0 μm; flow rate: 0.3 mL / min; column temperature: 35°C; injection volume: 4 μL; mobile phase: A is pure water containing 5 mM NH4HCO3 and adjusted to pH 7.8, B is pure acetonitrile; elution gradient: 0-3 min, 5% B; 3-7 min, 5%-20% B; 7-10 min, 20%-25% B; 10-15 min, 25%-30% B; 15-20 min, 30%-60% B; 20-22 min, 60%-80% B; 22-24 min, 80% B; 24-26 min, 80%-5% B; then equilibrate for 4 min; Mass spectrometry conditions were as follows: positive ion mode, nebulizer gas flow rate of 3 L / min; drying and heating gas flow rate of 10 L / min, interface temperature of 250°C, DL tube temperature of 250°C, and heating block temperature of 400°C; multiple reaction monitoring (MRM) mode was used to detect derivatized nucleotides; (7) After extracting the cell or tissue sample and precipitating the protein, the obtained supernatant is dried with nitrogen gas to obtain sample A; (8) Dissolve the sample A obtained in step (7) in acetonitrile and water in a volume ratio of 1:1, add the 4-methylbenzylamine derivatization solution, mix and react until the nucleotide reaction is complete, then add the derivatization internal standard solution obtained in step (3), dilute with water, mix, and centrifuge to obtain the supernatant sample B; (9) The supernatant sample B obtained in step (8) was subjected to reverse phase liquid chromatography-electrospray ionization mass spectrometry analysis for quantitative analysis of nucleotides; The nucleotide is one or more of AMP, ADP, ATP, GMP, GDP, GTP, CMP, CDP, CTP, UMP, UDP and UTP.

5. The method according to claim 4, characterized in that Wherein, in step (4), the derivatization temperature is 60°C, the derivatization time is 4 h, the molar ratio of nucleotide to 4-methylbenzylamine is 1:5000; the molar ratio of nucleotide to EDC is 1:5000; and the molar ratio of nucleotide to N-methylmorpholine is 1:1000; The method for removing protein from tissue samples is as follows: the tissue is placed in a 2 mL centrifuge tube, and a pre-cooled mixed solution of methanol and water with a volume ratio of 4:1 is added. The tissue is placed in a grinder and ground for 60 seconds each time, with a 10-second interval in between, for a total of 10 times. After grinding, the homogenate is centrifuged at 13,000 rpm and 4°C for 10 minutes, and the supernatant is removed. Method for removing protein from cell samples: After washing the cells with PBS buffer, place them in a 1.5 mL centrifuge tube and centrifuge at 3000 rpm for 5 minutes to precipitate the cells. Discard the supernatant and add a pre-cooled mixed solution of methanol and water with a volume ratio of 4:

1. Incubate at 4°C for 30 minutes. Vortex mix and centrifuge at 13000 rpm at 4°C for 10 minutes. Remove the supernatant.

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