Quantitative analysis method for glycometabolism pathway energy metabolite in mouse kidney tissue based on LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) detection method

Abstract

The invention provides a method for quantitative analysis of glycometabolism pathway energy metabolites in mouse kidney tissues based on an LC-MS / MS detection method, and belongs to the technical field of physiology and biochemistry. The method comprises the following steps: (1) extracting metabolites in mouse kidney tissues; (2) carrying out ultra-high performance liquid chromatography separation by taking the metabolite obtained in the step (1) as a sample injection sample; (3) putting the sample subjected to high performance liquid chromatography separation in the step (2) into a mass spectrometer, and performing mass spectrometry in a multi-reaction monitoring mode and a negative ion mode; and (4) collecting data obtained in the step (3), and carrying out quantitative analysis on the energy metabolites of the 22 glycometabolism pathways in the mouse kidney tissue. The method has good precision and accuracy, and has a wide linear range. The method can be applied to research on aristolochic acid I-induced mouse renal toxicity, and provides support for further research on biological functions of central carbon metabolism.

Description

Technical Field

[0001] The invention relates to the field of physiological and biochemical technology, and in particular to a method for quantitatively analyzing energy metabolites in a sugar metabolism pathway in mouse kidney tissue based on an LC-MS / MS detection method. Background Art

[0002] Central carbon metabolism (CCM), also known as energy metabolism, is one of the most basic cellular pathways in all organisms. It includes glycolysis, the pentose phosphate pathway (PPP), and the tricarboxylic acid (TCA) cycle. Among them, glycolysis is the core pathway of energy metabolism, which breaks down glucose into pyruvate, accompanied by the generation of ATP and NADH in the process, which are used for the basic energy needs of the cell. The pentose phosphate pathway can ultimately convert glucose into 5-phosphoribose and NADPH. 5-phosphoribose then further reacts to produce ribose, which is used to form DNA and RNA. Its structure can be mainly divided into sugar phosphates (such as glucose 6-phosphate), phosphate carboxylic acids (such as phosphoenolpyruvate), and nucleotide energy metabolites (such as ATP, NAD and NADP).

[0003] Since the metabolites in glycolysis and the pentose phosphate pathway are highly polar, and most metabolites contain phosphorylated groups and a large number of isomers, it is difficult to quantify them, and there are limitations on their biological function research. Therefore, it is urgent to establish an accurate and reliable detection method to explore its pathophysiological effects in vivo. At present, many mass spectrometry-based methods can be used to quantify central carbon metabolites, such as gas chromatography-mass spectrometry (GC-MS), liquid chromatography (LC-MS) and ion pair chromatography (IPC-MS). Among them, LC-MS has higher stability and sensitivity than GC-MS. At the same time, although IPC-MS can cover more central carbon metabolites, this method of adding ion pair reagents to the mobile phase will affect the sensitivity of the mass spectrometer. In addition, although the derivatization method can improve the detection sensitivity, the pretreatment process of this method is relatively complicated and cannot adapt to the characteristics of the structural diversity of central carbon metabolites.

[0004] Aristolochic acids (AAs) are produced by plants of the Aristolochaceae family and can cause aristolochic acid nephropathy (AAN), which is characterized by elevated serum creatinine (SCR) and tubular proteinuria. Aristolochic acid I (AAI) monomer is the most abundant and toxic component of AAs, which can cause necrosis and apoptosis of renal tubular epithelial cells, lymphocyte infiltration, and significant interstitial fibrosis. Many studies based on proteomics, transcriptomics, and non-targeted metabolomics have shown that AAI can disrupt metabolic biosynthesis and impair mitochondrial function; AAI can affect fatty acid β-oxidation and central carbon metabolism. However, there are no studies using targeted metabolomics to explore the effects of aristolochic acid nephropathy on central carbon metabolism and to elucidate its nephrotoxic mechanism.

[0005] Therefore, it is urgent to establish a method that can accurately quantify energy metabolites of glycolysis and pentose phosphate pathway to reveal the molecular mechanism of the effects of kidney-injury compounds (such as AAI) on central carbon metabolism (CCM) metabolites in mouse kidney tissue and provide an experimental basis for the preparation of drugs to prevent or treat kidney injury. Summary of the invention

[0006] The purpose of the present invention is to provide a method for quantitatively analyzing energy metabolites in the sugar metabolism pathway in mouse kidney tissue based on LC-MS / MS detection method.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for quantitatively analyzing energy metabolites in the sugar metabolism pathway in mouse kidney tissue based on LC-MS / MS detection method, comprising the following steps:

[0009] (1) Extract metabolites from mouse kidney tissue;

[0010] (2) using the metabolites obtained in step (1) as injection samples for ultra-high performance liquid chromatography separation, wherein the ultra-high performance liquid chromatography separation conditions are: using an Atlantis PremierBEH Z-HILIC column, mobile phase A is an aqueous solution of ammonium bicarbonate, and mobile phase B is an aqueous solution of ammonium bicarbonate-acetonitrile; the mobile phase elution gradient conditions are: 0-1 min, phase A is maintained at 10%, and phase B is maintained at 90%; 1-3 min, phase A linearly changes to 20%, and phase B linearly changes to 80%; 3-5 min, phase A is maintained at 20%, and phase B is maintained at 80%; 5-6 min, phase A linearly changes to 40%, and phase B linearly changes to 60%; 6-8 min, phase A is maintained at 40%, and phase B is maintained at 60%; 8-8.5 min, phase A linearly changes to 10%, and phase B linearly changes to 90%; 8.5-12 min, phase A is maintained at 10%, and phase B is maintained at 90%;

[0011] (3) placing the sample separated by high performance liquid chromatography obtained in step (2) in a mass spectrometer, and performing mass spectrometry analysis in multiple reaction monitoring mode and negative ion mode; the ion source used in the mass spectrometry analysis is an electrospray ion source, the spray voltage is 3500-3700V, the sheath gas flow rate is 25-35arb, the auxiliary gas flow rate is 8-12arb, and the ion transfer tube temperature and the nebulizer temperature are both 320-380°C;

[0012] (4) Collecting the data obtained in step (3), quantitatively analyzing 22 energy metabolites of the sugar metabolism pathway in mouse kidney tissue.

[0013] Preferably, the method for extracting metabolites from mouse kidney tissue in step (1) is: crushing the mouse kidney tissue and mixing it with an extraction solvent on ice for reaction, and taking the liquid part after solid-liquid separation to obtain metabolites;

[0014] The on-ice extraction solvent is obtained by mixing acetonitrile, methanol and water in a volume ratio of 1.6-2.4:1.6-2.4:0.8-1.2.

[0015] Preferably, the mass volume ratio of the mouse kidney tissue minced material and the extraction solvent on ice when mixed is 8-12 mg:40-60 μL.

[0016] Preferably, the mixing reaction is carried out by vortexing, the vortexing speed is 2500-3500 rpm, and the time is 30-90 s;

[0017] The solid-liquid separation method is centrifugation, the temperature during the centrifugation is 3-5°C, the speed is 10000-15000rpm, the time is 8-12min, and the centrifugation is performed twice.

[0018] Preferably, in step (2), the specification of the chromatographic column Atlantis PremierBEH Z-HILIC is 2.5 μm, 2.1×100 mm, the column temperature is 35-45° C., and the injection volume is 1-3 μL.

[0019] Preferably, the concentrations of the ammonium bicarbonate aqueous solution and the ammonium bicarbonate-acetonitrile aqueous solution in step (2) are both 4-6 mmol / L; the ammonium bicarbonate-acetonitrile aqueous solution is prepared by dissolving ammonium bicarbonate in an acetonitrile aqueous solution, and the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 85-95:8-12.

[0020] Preferably, the flow rate of the mobile phase during elution in step (2) is 0.2 to 0.4 mL / min.

[0021] Preferably, the software used for data collection in step (4) is ThermoFisher Xcalibur software, and the software used for quantitative analysis is ThermoFisher QuanBrowser (Version 4.5.445.18) software.

[0022] Preferably, the 22 sugar metabolism pathway energy metabolites in step (4) are glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, 2-phosphoglyceric acid, 3-phosphoglyceric acid, phosphoenolpyruvate, 6-phosphogluconate, ribulose-5-phosphate, xylulose-5-phosphate, ribose-5-phosphate, sedoheptulose-7-phosphate, erythrose-4-phosphate, acetyl-CoA, succinyl-CoA, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and reduced nicotinamide adenine dinucleotide phosphate.

[0023] The present invention also provides an application of the method in studying the molecular mechanism of the effects of kidney injury compounds on metabolites in kidney tissue and in preparing drugs for preventing or treating kidney injury, wherein the kidney injury compounds include aristolochic acid.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention is based on the LC-MS / MS system, and establishes a method for quantitatively detecting 22 energy metabolites involved in glycolysis and pentose phosphate pathway in mouse kidney tissue at the same time, and the method has good precision and accuracy, and has a wide linear range. The method can be applied to the study of mouse nephrotoxicity induced by aristolochic acid I, and provides support for further exploring the biological function of central carbon metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0027] Figure 1 is the chromatogram of each metabolite standard in pure solvent in Example 1;

[0028] Figure 2 is the chromatogram of each metabolite in the mouse kidney tissue in Example 1;

[0029] Figure 3 The concentration changes of metabolites Acetyl-CoA (Ace-CoA), Succinyl-CoA (Suc-CoA), G6P and F6P in the kidney tissue samples of mice in the control group and the low-concentration and high-concentration AAI treatment groups in Example 2;

[0030] Figure 4 The concentration changes of metabolites GAP, 2-PG, PP and 6-PG in the kidney tissue samples of mice in the control group and the low-concentration and high-concentration AAI treatment groups in Example 2;

[0031] Figure 5 The concentration changes of metabolites RU5P / X5P, R5P, E4P and S7P in the kidney tissue samples of mice in the control group and the low-concentration and high-concentration AAI treatment groups in Example 2;

[0032] Figure 6 The concentration changes of metabolites ATP, ADP, AMP and NAD in the kidney tissue samples of mice in the control group and the low-concentration and high-concentration AAI treatment groups in Example 2;

[0033] Figure 7 The concentration changes of metabolites NADH, NADP and NADPH in kidney tissue samples of mice in the control group and low-concentration and high-concentration AAI treatment groups in Example 2 are shown. DETAILED DESCRIPTION

[0034] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] Example 1

[0036] 1. Preparation of Standard Stock and Working Solutions

[0037] Preparation of stock solutions: Acetyl-CoA and Succinyl-CoA were dissolved in methanol to a concentration of 1 mg / mL. GAP, E4P, 2-PG, 3-PG, RU5P, X5P, and R5P were dissolved in water to a concentration of 1 mg / mL. F6P, G6P, FBP, PP, S7P, and 6-PG were dissolved in water to a concentration of 10 mg / mL. ATP, ADP, and AMP standards were prepared freshly when used.

[0038] Preparation of working solution: Use precipitant (acetonitrile: methanol: water = 2:2:1) to dilute the stock solution in proportion to prepare the working solution. Internal standard (G6P- 13 C6) working standard solution was prepared at a concentration of 200 ng / mL. All solutions were stored at -20°C.

[0039] 2. Extraction of Metabolites from Mouse Kidney Tissue

[0040] First, the frozen kidney tissue was ground into powder using a cryogenic grinder (Shanghai Jingxin Industrial Development Co., Ltd., China). Next, 50 μL of ice extraction solvent (acetonitrile: methanol: water = 2:2:1) was added to each 10 mg of ground tissue. The mixture was vortexed at 3000 rpm for 1 min and then centrifuged twice at 4 ° C, 13000 rpm, each for 10 minutes. Finally, the second supernatant was collected and transferred to an MS vial for analysis.

[0041] 3. Chromatographic parameters

[0042] Chromatographic column: Atlantis PremierBEH Z-HILIC (2.5 μm, 2.1×100 mm); column temperature was 40°C, and injection volume was 2 μL.

[0043] Mobile phase: Phase A: 5 mmol / L ammonium bicarbonate aqueous solution, Phase B: 5 mmol / L ammonium bicarbonate, dissolved in acetonitrile / water (90:10, v / v).

[0044] Gradient elution was used, and the elution gradient of the chromatographic mobile phase is shown in Table 1. The chromatograms of each metabolite standard in pure solvent are shown in Figure 1 The chromatograms of metabolites in mouse kidney tissue are shown in Figure 2 shown.

[0045] Table 1 Ultra-high performance liquid chromatography mobile phase elution gradient

[0046]

[0047]

[0048] 4. Mass spectrometry parameters

[0049] The mass spectrometry in ultra-high performance liquid chromatography tandem mass spectrometry was analyzed in multiple reaction monitoring (MRM) and negative ion mode. The ion source was an electrospray ion source (ESI), the spray voltage was 3600 V, the sheath gas flow rate was 30 arb, the auxiliary gas flow rate was 10 arb, and the ion transfer tube temperature and nebulizer temperature were both 350 °C. The mass spectrometry parameters are shown in Table 2.

[0050] Table 2 Mass spectrometry parameters

[0051]

[0052]

[0053] 5. Methodological Validation

[0054] At present, there is no unified standard for method validation of endogenous substances. Commonly used methods include: (a) surrogate matrix method; (b) using isotope-labeled analytes as surrogates; (c) adding real analytes to real matrices. The surrogate matrix method usually uses pure solvents, buffers or 5% BSA, but the matrix effects of these surrogate matrices are different from those of the real matrix. The method of using isotope-labeled analytes as surrogates has high accuracy, but this method is usually expensive and difficult to implement. In addition, there is a method in which the real matrix is ​​treated to remove the analyte, such as activated carbon adsorption, but this method is not ideal and cannot completely remove the endogenous analyte to be measured. Therefore, the present invention uses the method of adding a standard to the real matrix for method validation. ThermoFisherXcalibur software was used for data acquisition, and ThermoFisher QuanBrowser (Version4.5.445.18) software was used for quantitative analysis. Ttest statistical analysis was performed using the software Graphpad Prism (Version 9.5.0.730).

[0055] (1) Linear

[0056] The ratio of the peak area of ​​the analyte to the internal standard was used as the ordinate, and the concentration of each metabolite was used as the abscissa. At least six concentration points of each metabolite were plotted to construct a linear regression equation with a weighting coefficient of 1 / X. Linear range (ng / mL), linear equation, linear correlation coefficient (R 2 ), detection limit (ng / mL) and quantification limit (ng / mL) are shown in Table 3.

[0057] Table 3 Linear equations, linear correlation coefficients, detection limits and quantification limits of each metabolite

[0058]

[0059]

[0060]

[0061] It can be seen from Table 3 that the correlation coefficients R 2 >0.99, and the accuracy of each concentration point on the standard curve was within ±15%, showing a good linear relationship. Due to the lack of blank matrix without endogenous substances, the instrument's lower limit of detection (LLOD) and lower limit of quantification (LLOQ) data were obtained in pure solvents, and the signal-to-noise ratio (S / N) was calculated to be 3:1 and 10:1, respectively.

[0062] (2) Precision and accuracy

[0063] Precision and accuracy were evaluated by analyzing low, medium, and high concentration quality control samples (LQCs, MQCs, and HQCs). Six samples were analyzed in parallel for each concentration on a single day to evaluate the intra-day precision and accuracy of the method; the test was repeated on three consecutive days to evaluate the inter-day precision and accuracy. Accuracy was calculated by relative error (RE), with the formula: RE = [(measured concentration - theoretical concentration) / theoretical concentration] × 100%. Precision is expressed as relative standard deviation (RSD, %). The deviation of the precision and accuracy of the method should be less than ±15%. The detection precision and accuracy of each metabolite are shown in Table 4.

[0064] Table 4 Detection precision and accuracy of each metabolite

[0065]

[0066]

[0067]

[0068]

[0069] It can be seen from Table 4 that the detection precision and accuracy deviations of each metabolite are all less than ±15%, indicating that the method of the present invention has good detection precision and accuracy.

[0070] (3) Matrix effect and extraction recovery

[0071] To evaluate the matrix effect and extraction recovery of endogenous metabolites, six samples of unspiked samples and standard spiked samples (LQCs, MQCs, HQCs) were analyzed in parallel. The value of the matrix effect was calculated by the following formula: MF = [real sample with standard spiked / (real sample without standard spiked + pure solvent standard sample)]. The extraction recovery was evaluated by comparing the standard concentration spiked directly into the QC sample with the standard concentration spiked into the QC sample after extraction, and six samples were repeated for each concentration. The results are shown in Table 5.

[0072] Table 5 Matrix effects and extraction recoveries of each metabolite

[0073]

[0074]

[0075]

[0076]

[0077] It can be seen from Table 5 that the matrix effect and extraction recovery rate of most metabolites are within the range of 80% to 120%, indicating that the method of the present invention has good matrix effect and extraction recovery rate.

[0078] (4) Stability

[0079] The stability study mainly evaluates the stability of the autosampler and the stability of the stock solution. The stability of the autosampler was evaluated by analyzing low, medium and high concentration quality control samples after storage at 4°C for 48 hours, with six samples in parallel for each concentration. The stability of the stock solution was evaluated by storing the stock solution at -20°C for 7 days. The ratio of the compound concentration after storage to the concentration before storage was calculated, and it was considered stable within the range of 85% to 115%. The test results are shown in Table 6.

[0080] Table 6 Stability of each metabolite

[0081]

[0082]

[0083]

[0084]

[0085] As can be seen from Table 6, the stability of each metabolite is in the range of 85% to 115%, indicating that both the autosampler and the stock solution have good stability.

[0086] Example 2

[0087] 1. Animal experiments and sample collection

[0088] C57BL / 6J male mice (20 ± 5 g, 7 weeks old) were obtained from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. (Jiangsu, China). Mice were fed with standard feed and autoclaved water in an SPF animal room with an ambient temperature of 22 ± 3 °C, a relative humidity of 60% ± 10%, and 12 h of light per day. All animal experiments were performed in accordance with the ethical guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai University (approval number: ECSHU 2024-108).

[0089] Aristolochic acid I (AAI) was suspended in 0.5% sodium carboxymethylcellulose (CMC-Na) solution for later use. Before the formal experiment, mice were allowed to adapt to the environment for one week. 18 mice were randomly divided into 3 groups (n=6 in each group). Two groups of mice were given 0.2 mg kg -1 ·d -1 and 20 mg kg -1 ·d -1 The control group was orally administered with an equal volume of 0.5% CMC-Na solution every day. The body weights of 12 mice were weighed every day before each administration. The medication was continued for 7 days.

[0090] 24 hours after the last administration, mice were anesthetized with chloral hydrate and kidney tissues were collected. Kidney tissues from mice were collected, immediately frozen in liquid nitrogen, and stored at -80°C for metabolomics analysis.

[0091] 2. Analysis results

[0092] According to the method established in Example 1, it was applied to mouse kidney tissue samples to explore the changes in glycolysis, pentose phosphate pathway, and related energy metabolites in mouse kidney tissue under low and high concentrations of aristolochic acid (AAI) compared with the control group. Finally, 19 metabolites were detected in the kidney tissue samples, and FBP and 3-PG were not detected. The changes in the concentrations of each metabolite in the kidney tissue of mice in the control group and the low and high concentration AAI treatment groups are as follows Figure 3 to Figure 7 shown.

[0093] Depend on Figure 3 to Figure 7 It can be seen that compared with the control group, R5P, S7P, AMP and NADH in the low-dose administration group were significantly upregulated; GAP, 2-PG, PP and E4P in the high-dose group were significantly upregulated, while Acetyl-CoA (Ace-CoA), Succinyl-CoA (Suc-CoA), R5P, ATP, ADP, AMP, NAD, NADH and NADP were significantly downregulated.

[0094] Aristolochic acid preferentially targets proximal tubular epithelial cells, which in turn causes nephrotoxicity. Renal tubular epithelial cells mainly rely on fatty acid β-oxidation (FAO) for energy, and impaired FAO is a key mechanism for renal injury. Acetyl-CoA plays a crucial role between fatty acid β-oxidation and the tricarboxylic acid cycle. Afshinnia et al. showed that renal acetyl-CoA carboxylase levels were increased in patients with renal disease. This finding is consistent with our results that decreased acetyl-CoA levels in AAI-induced mouse nephropathy may lead to lipid metabolism disorders. Succinyl-CoA showed a downward trend, indicating that the metabolic process of the tricarboxylic acid cycle was impaired, resulting in reduced energy generation. At the same time, the reduction in NAD and NADH also indicated that fatty acid β-oxidation and TCA cycle activities were decreased, which further affected glycolysis and energy metabolism. Glyceraldehyde-3-phosphate (GAP), 2-phosphoglycerate (2-PG), (PP), and erythrose-4-phosphate (E4P) were significantly upregulated, reflecting the accumulation of downstream metabolites of glycolysis and pentose phosphate pathways. This may be due to the inhibition of pyruvate kinase (PK) activity, which leads to the obstruction of downstream metabolism, resulting in accumulation in the intermediate links. The reduction of R5P and NADP indicates that the PPP pathway is significantly inhibited, which weakens the antioxidant capacity of cells and aggravates the damage of cells to oxidative stress. At the same time, ATP, ADP and AMP in the high-dose group were significantly reduced, which directly indicates that energy metabolism is impaired.

[0095] The quantitative method provided by the present invention was successfully applied to the study of aristolochic acid-induced nephrotoxicity in mice. The results showed that aristolochic acid significantly affected the central carbon metabolism of mouse kidney tissue, leading to the accumulation of glycolysis intermediate metabolites and the inhibition of PPP pathway activity, and directly affecting the generation of energy such as ATP. This provides a basis for further revealing the molecular mechanism of aristolochic acid nephrotoxicity and potential intervention targets in the future.

[0096] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for quantitative analysis of energy metabolites in the sugar metabolism pathway in mouse kidney tissue based on LC-MS / MS detection method, characterized in that: The steps include: (1) Extract metabolites from mouse kidney tissue; (2) using the metabolites obtained in step (1) as injection samples for ultra-high performance liquid chromatography separation, wherein the ultra-high performance liquid chromatography separation conditions are: using an Atlantis PremierBEH Z-HILIC column, mobile phase A is an aqueous solution of ammonium bicarbonate, and mobile phase B is an aqueous solution of ammonium bicarbonate-acetonitrile; the mobile phase elution gradient conditions are: 0-1 min, phase A is maintained at 10%, and phase B is maintained at 90%; 1-3 min, phase A linearly changes to 20%, and phase B linearly changes to 80%; 3-5 min, phase A is maintained at 20%, and phase B is maintained at 80%; 5-6 minutes, phase A changes linearly to 40%, and phase B changes linearly to 60%; 6-8 minutes, phase A is maintained at 40%, and phase B is maintained at 60%; 8-8.5min, phase A changes linearly to 10%, and phase B changes linearly to 90%; 8.5-12min, phase A is maintained at 10%, and phase B is maintained at 90%; (3) placing the sample separated by high performance liquid chromatography obtained in step (2) in a mass spectrometer, and performing mass spectrometry analysis in multiple reaction monitoring mode and negative ion mode; the ion source used in the mass spectrometry analysis is an electrospray ion source, the spray voltage is 3500-3700V, the sheath gas flow rate is 25-35arb, the auxiliary gas flow rate is 8-12arb, and the ion transfer tube temperature and the nebulizer temperature are both 320-380°C; (4) Collecting the data obtained in step (3), quantitatively analyzing 22 energy metabolites of the sugar metabolism pathway in mouse kidney tissue.

2. The method according to claim 1, characterized in that The method for extracting metabolites from mouse kidney tissue in step (1) is: crushing the mouse kidney tissue and mixing it with an extraction solvent on ice for reaction, and taking the liquid part after solid-liquid separation to obtain metabolites; The on-ice extraction solvent is obtained by mixing acetonitrile, methanol and water in a volume ratio of 1.6-2.4:1.6-2.4:0.8-1.

2.

3. The method according to claim 2, characterized in that The mass-to-volume ratio of the mouse kidney tissue minced material and the extraction solvent on ice was 8-12 mg: 40-60 μL.

4. The method according to claim 2, characterized in that The mixing reaction is carried out by vortexing, the vortexing speed is 2500-3500 rpm, and the time is 30-90 s; The solid-liquid separation method is centrifugation, the temperature during the centrifugation is 3-5°C, the speed is 10000-15000rpm, the time is 8-12min, and the centrifugation is performed twice.

5. The method according to claim 1, characterized in that In step (2), the specification of the chromatographic column Atlantis Premier BEH Z-HILIC is 2.5 μm, 2.1×100 mm, the column temperature is 35-45° C., and the injection volume is 1-3 μL.

6. The method according to claim 1, characterized in that The concentrations of the ammonium bicarbonate aqueous solution and the ammonium bicarbonate-acetonitrile aqueous solution in step (2) are both 4-6 mmol / L; the ammonium bicarbonate-acetonitrile aqueous solution is prepared by dissolving ammonium bicarbonate in an acetonitrile aqueous solution, and the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 85-95:8-12.

7. The method according to claim 1, characterized in that The flow rate of the mobile phase during elution in step (2) is 0.2 to 0.4 mL / min.

8. The method according to claim 1, characterized in that The software used for data acquisition in step (4) was ThermoFisher Xcalibur software, and the software used for quantitative analysis was ThermoFisher QuanBrowser (Version 4.5.445.18) software.

9. The method according to claim 1, characterized in that The 22 sugar metabolism pathway energy metabolites described in step (4) are glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, 2-phosphoglyceric acid, 3-phosphoglyceric acid, phosphoenolpyruvate, 6-phosphogluconate, ribulose-5-phosphate, xylulose-5-phosphate, ribose-5-phosphate, sedoheptulose-7-phosphate, erythrose-4-phosphate, acetyl-CoA, succinyl-CoA, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and reduced nicotinamide adenine dinucleotide phosphate.

10. Use of the method according to any one of claims 1 to 9 in studying the molecular mechanism of the effects of kidney-damaging compounds on metabolites in kidney tissue and in preparing drugs for preventing or treating kidney damage, characterized in that: The kidney damaging compounds include aristolochic acid.

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