Relative quantitative quasi-targeted metabonomics analysis method for liver arginine anabolites

Abstract

The invention relates to a quasi-targeting metabonomics analysis method based on liquid chromatography-mass spectrometry (LC-MS). The quasi-targeting metabonomics analysis method is used for simultaneously determining the relative content of 21 arginine anabolites in the liver. The method comprises the following steps: preparing a metabolite and an internal standard solution, optimizing multiple reaction monitoring (MRM) parameters, carrying out homogenization and acetonitrile precipitation pretreatment on a liver tissue sample, preparing a quality control (QC) sample, and establishing a standard curve through series dilution. The method meets analysis requirements in the aspects of linearity, precision, stability and the like, is suitable for relative quantitative research of arginine metabolites in liver tissues, and provides reliable technical support for related metabolic pathway analysis.

Description

Technical Field

[0001] This invention relates to the field of pseudo-targeted metabolomics technology, specifically a method for relative quantitative pseudo-targeted metabolomics analysis of liver arginine biosynthetic metabolites. Background Technology

[0002] Arginine, a semi-essential amino acid, is a core component of the urea cycle, nitric oxide (NO) synthesis, and polyamine metabolism. Its metabolites, such as citrulline, ornithine, putrescine, and spermidine, are widely involved in key physiological and pathological processes including cell proliferation, immune regulation, vascular homeostasis, and tumorigenesis. Recent studies have shown that abnormal arginine metabolism is closely related to the development of various diseases, including cardiovascular disease, immune dysfunction, and cancer. Therefore, establishing efficient and accurate analytical methods for arginine biosynthetic metabolites is crucial for in-depth exploration of disease mechanisms, discovery of novel biomarkers, and monitoring of the efficacy of targeted therapies.

[0003] Currently, the detection of arginine biosynthetic metabolites mainly relies on techniques such as high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), gas chromatography-mass spectrometry (GC-MS), and enzyme-linked immunosorbent assay (ELISA), which have been widely applied to the analysis of biological samples such as plasma and urine. However, these methods all have certain limitations: ELISA is susceptible to interference from antibody cross-reactions, affecting detection specificity; GC-MS and HPLC usually require complex sample pretreatment, such as derivatization steps, resulting in lengthy analytical procedures and high costs. Liquid chromatography-mass spectrometry (LC-MS) combines the high separation efficiency of liquid chromatography with the high sensitivity and selectivity of mass spectrometry, significantly improving the specificity and sensitivity of the method. In addition, arginine and its metabolites are mostly highly polar compounds, and the separation effect on the traditional reversed-phase HPLC stationary phase is poor. Hydrophilic interaction chromatography (HILIC), using a silica gel stationary phase, has excellent retention and separation capabilities for polar small molecules. Therefore, this study selected a HILIC column to achieve efficient separation of arginine metabolites. Existing literature has established non-targeted metabolomics methods for arginine-related metabolites in plasma. While non-targeted metabolomics, as a profiling technique, can detect many components, its accuracy in determining the relative abundance of each component is not high. The pseudo-targeted metabolomics technique, in its method establishment, uses high-resolution mass spectrometry and standards to obtain ion-pair information of metabolites, and employs multiple reaction detection (MRM) to measure metabolite abundance during sample analysis. Compared to non-targeted techniques, it has advantages such as good linearity and repeatability, and higher accuracy.

[0004] Based on this, this study uses LC-MS technology to establish an efficient, rapid, and accurate method for relative quantitative analysis of arginine synthesis metabolism in the liver, aiming to provide reliable technical support for in-depth research on arginine synthesis metabolism. Summary of the Invention

[0005] The purpose of this invention is to provide a pseudo-targeted metabolomics analysis method based on HPLC-MS for the relative quantification of 21 arginine biosynthetic metabolites in the liver. This method is stable, reliable, reproducible, and precise, and provides guidance for establishing methods to determine the content of arginine biosynthetic metabolites in the liver.

[0006] To achieve the above objectives, the technical solution adopted is: a method for relative quantitative targeted metabolomics analysis of liver arginine synthesis metabolites, comprising the following steps: The arginine biosynthetic metabolites are: ornithine, L-citrulline, N-acetyl-L-ornithine, proline, glutamine, symmetrical N,N-dimethylarginine, creatinine, N,N-dimethylglycine, S-adenosyl homocysteine, urea, betaine, cysteine, S-adenosylmethionine, N-acetyl-L-glutamic acid, asymmetrical dimethylarginine, aspartic acid, L-glutamic acid, homocysteine, arginine, α-ketoglutarate, and fumaric acid. The two internal standards are L-glutamic acid D3 and 2,5-dihydroxybenzoic acid. (1) Preparation of reference solution and internal standard solution: Accurately weigh 21 kinds of arginine biosynthetic metabolites and 2 kinds of internal standard substances and place them in volumetric flasks. Add 50% methanol water to dissolve and dilute to obtain the standard solution. (2) Use of internal standard solution: L-glutamic acid D3 is selected as internal standard in positive ion mode, and 2,5-dihydroxybenzoic acid is selected as internal standard in negative ion mode; (3) Optimization of MRM parameters: The precursor ion MS1 ​​of 21 arginine biosynthetic metabolites was determined using a 4000QTRAP instrument, and the fragment ion with higher response intensity was selected as the daughter ion MS2; together with the precursor ion, candidate ion pairs were constructed, and the optimal DP and CE were selected for each ion pair based on the response intensity. (4) Pretreatment of tissue samples: Accurately weigh liver tissue into a centrifuge tube, add distilled water to homogenize, take the homogenate, add internal standard and acetonitrile solution, vortex to mix, centrifuge to take the supernatant, blow dry with nitrogen, add 50% methanol water to reconstitute, and the sample is obtained. (5) Preparation of QC samples: Take an appropriate amount of liver tissue homogenate from the blank group and the model group, vortex mix, and process according to the method in step (4); (6) Establishment of standard curve: The QC samples from step (5) are arranged according to 2 7 The internal standard was diluted using a series of dilutions to ensure that the concentration of the internal standard was the same. The series of diluted solutions were injected into LC-MS, and the data were analyzed using Analyst 1.6.3. A standard curve was plotted with the ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard as the x-axis and the number of dilutions as the y-axis. (7) Relative content determination: Using the same method and under the same detection conditions, the liver tissue sample was injected into LC-MS after tissue sample pretreatment in step (1), and the mass spectrometry data was exported. Analyst 1.6.3 was used for data analysis. The relative content of 21 arginine biosynthetic metabolites in the liver tissue sample could be directly calculated using the method in step (6).

[0007] Further, in step (1), 2 mg of each of the 21 kinds of arginine biosynthetic metabolites and 2 kinds of internal standard references are accurately weighed and placed in a 2 mL volumetric flask. 2 mL of 50% methanol water is added to dissolve them, shake well and make up to volume to prepare a 1 mg / mL reference stock solution. An appropriate amount of each reference stock solution is taken and diluted with 50% methanol water to 1 μg / mL. The 1 μg / mL solution of each reference is injected into LC-MS for qualitative analysis.

[0008] Furthermore, in step (2), the concentration of the positive ion mode and the internal standard concentration of the negative ion are 0.5 μg / mL.

[0009] Further, in step (4), 60 mg of liver tissue is weighed into a centrifuge tube, 300 μL of water is added, and the tissue is homogenized using a cryogenic high-throughput tissue homogenizer. 200 μL of homogenate is taken and 20 μL of internal standard solution is added. The mixture is vortexed for 3 min, centrifuged at 4 ℃ and 12000 rpm for 10 min. 100 μL of supernatant is taken, dried with N2, and then reconstituted with 100 μL of 50% methanol solution. The mixture is vortexed for 3 min, centrifuged at 4 ℃ and 12000 rpm for 10 min, and then 60 μL of supernatant is taken for LC-MS analysis.

[0010] Furthermore, in steps (5) and (6), the detection conditions include: Mass spectrometry conditions: electrospray ionization source, positive and negative ion multiple reaction monitoring (MRM) modes; ionization voltage +5500V, -4500V; ion source temperature 550 ℃; spray gas 50 psi; auxiliary heating gas 50 psi; Chromatographic conditions: Phenomenex Hilic column (4.6 × 100 mm, 2.6 μm); mobile phase A: 0.1% formic acid acetonitrile, mobile phase B: 0.1% formic acid aqueous solution; flow rate: 0.8 mL / min; gradient elution: 0–7 min, 75%–50% A, 7–8 min, 50%–75% A; equilibration: 1 min; column temperature: 30 °C; injection volume: 1 μL.

[0011] Furthermore, in steps (3) and (4), the parameters of Analyst 1.6.3 are set as follows: the mass spectrometry parameters of 21 arginine biosynthetic metabolites and 2 internal standards, namely Molecule List Name, Precursor Name, Precursor m / z, Precursor RT, and Product m / z, are imported into the mass spectrometer's built-in analysis software Analyst 1.6.3 to establish a pseudo-targeted qualitative analysis method for LC-MS / MS. The peak areas of each analyte and internal standard are imported, and a relative quantitative analysis method is established through a standard curve.

[0012] Furthermore, the ion pair information for each analyte is as follows: ornithine 133.3→72.1, L-citrulline 176.1→113.2, N-acetyl-L-ornithine 175.1→114.7, proline 116.1→70.2, glutamine 147.1→130.2, symmetrical N,N-dimethylarginine 104.0→58.2, creatinine 114.4→44.4, N,N-dimethylglycine 239.0→57.1, S-adenosyl homocysteine ​​385.1→136.2, urea 61.0→44.1, betaine 118.1→59.3, cysteine ​​122.0→76.0, and S-adenosylmethionine 399. 1→250.2, N-acetyl-L-glutamic acid 190.3→129.8, asymmetric dimethylarginine 203.2→70.3, aspartic acid 134.0→74.1, L-glutamic acid 148.1→84.3, homocysteine ​​136.1→90.2, arginine 175.1→70.1, α-ketoglutarate 145.5→145.5, fumaric acid 115.0→70.8, L-glutamic acid D3 150.9→87, 2,5-dihydroxybenzoic acid 152.6→107.9.

[0013] Furthermore, the mass spectrometry reaction parameters of the 21 arginine biosynthetic metabolites are shown in Tables 1 and 2: Table 1. Parameters for multiple reaction detection of 21 arginine biosynthetic metabolites using positive ion mode with internal standards

[0014] Table 2. Multiple reaction monitoring parameters of two arginine biosynthetic metabolites under negative ion mode.

[0016] This invention establishes a pseudo-targeted metabolomics analysis method for the relative quantification of 21 arginine biosynthetic metabolites based on liquid chromatography-mass spectrometry (LC-MS), and validates the method. Characteristic peaks, retention times, and mass spectrometry parameters of each compound are determined using standards. The relative content of the 21 arginine biosynthetic metabolites in the liver is calculated based on the peak area ratio of the analyte to the internal standard.

[0017] This invention establishes a method for determining the relative content of 21 arginine biosynthetic metabolites in the liver based on LC-MS technology and a pseudo-targeted metabolomics approach. This method features simple tissue sample pretreatment, easy operation, high sensitivity, and good stability, and can provide a reference for the determination of the relative content of arginine biosynthetic metabolites in biological samples. Attached Figure Description

[0018] Figure 1 Chromatograms of arginine biosynthetic metabolites in positive ion mode multiple reaction detection; Figure 2 Chromatograms of arginine biosynthetic metabolites in negative ion mode multiple reaction detection channel; Figure 3 .QC sample PCA one-dimensional score plot; Figure 4 OPLS-DA plot of the sample determined by the established pseudo-targeted metabolomics analysis method. Detailed Implementation

[0019] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. The described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0020] Example 1 The relative quantitative pseudo-targeted metabolomics analysis method for acid synthesis metabolites of the present invention includes the following steps: the arginine synthesis metabolites are ornithine, L-citrulline, N-acetyl-L-ornithine, proline, glutamine, symmetrical N,N-dimethylarginine, creatinine, N,N-dimethylglycine, S-adenosyl homocysteine, urea, betaine, cysteine, S-adenosylmethionine, N-acetyl-L-glutamic acid, asymmetrical dimethylarginine, aspartic acid, L-glutamic acid, homocysteine, arginine, α-ketoglutarate, and fumaric acid, and the two internal standards are L-glutamic acid D3 and 2,5-dihydroxybenzoic acid, respectively. (1) Preparation of reference solution and internal standard solution: Accurately weigh 21 kinds of arginine biosynthetic metabolites and 2 kinds of internal standard substances and place them in volumetric flasks. Add 50% methanol water to dissolve and dilute to obtain the standard solution. (2) Use of internal standard solution: L-glutamic acid D3 is selected as internal standard in positive ion mode, and 2,5-dihydroxybenzoic acid is selected as internal standard in negative ion mode; (3) Optimization of MRM parameters: The precursor ion MS1 ​​of 21 arginine biosynthetic metabolites was determined using a 4000QTRAP instrument, and the fragment ion with higher response intensity was selected as the daughter ion MS2; together with the precursor ion, candidate ion pairs were constructed, and the optimal DP and CE were selected for each ion pair based on the response intensity. (4) Pretreatment of tissue samples: Accurately weigh liver tissue into a centrifuge tube, add distilled water to homogenize, take the homogenate, add internal standard and acetonitrile solution, vortex to mix, centrifuge to take the supernatant, blow dry with nitrogen, add 50% methanol water to reconstitute, and the sample is obtained. (5) Preparation of QC samples: Take an appropriate amount of liver tissue homogenate from the blank group and the model group, vortex mix, and process according to the method in step (4); (6) Establishment of standard curve: The QC samples from step (5) are arranged according to 2 7 The internal standard was diluted using a series of dilutions to ensure that the concentration of the internal standard was the same. The series of diluted solutions were injected into LC-MS, and the data were analyzed using Analyst 1.6.3. A standard curve was plotted with the ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard as the x-axis and the number of dilutions as the y-axis. (7) Relative content determination: Using the same method and under the same detection conditions, the liver tissue sample was injected into LC-MS after tissue sample pretreatment in step (1), and the mass spectrometry data was exported. Analyst 1.6.3 was used for data analysis. The relative content of 21 arginine biosynthetic metabolites in the liver tissue sample could be directly calculated using the method in step (6).

[0021] In step (1), 2 mg of each of the 21 arginine biosynthetic metabolites and 2 internal standard references were accurately weighed and placed in a 2 mL volumetric flask. 2 mL of 50% methanol water was added to dissolve them, and the solution was shaken and diluted to a final volume to prepare a 1 mg / mL reference stock solution. An appropriate amount of each reference stock solution was taken and diluted with 50% methanol water to 1 μg / mL. The 1 μg / mL solution of each reference was injected into LC-MS for qualitative analysis.

[0022] In step (2), the positive ion mode and the concentration of the negative ion internal standard are 0.5 μg / mL.

[0023] In step (4), 60 mg of liver tissue was weighed into a centrifuge tube, 300 μL of water was added, and the tissue was homogenized using a cryogenic high-throughput tissue homogenizer. 200 μL of homogenate was taken and 20 μL of internal standard solution was added. The mixture was vortexed for 3 min, centrifuged at 4 ℃ and 12000 rpm for 10 min. 100 μL of supernatant was taken, dried with N2, and then reconstituted with 100 μL of 50% methanol solution. The mixture was vortexed for 3 min, centrifuged at 4 ℃ and 12000 rpm for 10 min, and then 60 μL of supernatant was taken for LC-MS analysis.

[0024] In steps (5) and (6), the detection conditions include: mass spectrometry conditions: electrospray ionization source, positive and negative ion multiple reaction monitoring (MRM) mode; ionization voltage +5500V, -4500V; ion source temperature 550 ℃; spray gas 50 psi; auxiliary heating gas 50 psi; chromatographic conditions: Phenomenex Hilic column (4.6×100 mm, 2.6 μm); mobile phase A: 0.1% formic acid acetonitrile, mobile phase B: 0.1% formic acid aqueous solution; flow rate 0.8 mL / min; gradient elution: 0-7 min, 75%-50% A, 7-8 min, 50%-75% A; equilibration 1 min; column temperature 30℃; injection volume 1 μL.

[0025] In steps (3) and (4), the parameters of Analyst 1.6.3 are set as follows: the mass spectrometry parameters of 21 arginine biosynthetic metabolites and 2 internal standards, namely Molecule List Name, Precursor Name, Precursor m / z, PrecursorRT, and Product m / z, are imported into the mass spectrometer's built-in analysis software Analyst 1.6.3 to establish a pseudo-targeted qualitative analysis method for LC-MS / MS. The peak areas of each analyte and internal standard are imported, and a relative quantitative analysis method is established through the standard curve.

[0026] The ion pair information for each analyte is as follows: ornithine 133.3→72.1, L-citrulline 176.1→113.2, N-acetyl-L-ornithine 175.1→114.7, proline 116.1→70.2, glutamine 147.1→130.2, symmetrical N,N-dimethylarginine 104.0→58.2, creatinine 114.4→44.4, N,N-dimethylglycine 239.0→57.1, S-adenosyl homocysteine ​​385.1→136.2, urea 61.0→44.1, betaine 118.1→59.3, cysteine ​​122.0→76.0, and S-adenosylmethionine 399. 1→250.2, N-acetyl-L-glutamic acid 190.3→129.8, asymmetric dimethylarginine 203.2→70.3, aspartic acid 134.0→74.1, L-glutamic acid 148.1→84.3, homocysteine ​​136.1→90.2, arginine 175.1→70.1, α-ketoglutarate 145.5→145.5, fumaric acid 115.0→70.8, L-glutamic acid D3 150.9→87, 2,5-dihydroxybenzoic acid 152.6→107.9.

[0027] The mass spectrometry reaction parameters of the 21 arginine biosynthetic metabolites are shown in Tables 1 and 2. Table 1. Parameters for multiple reaction detection of 21 arginine biosynthetic metabolites using positive ion mode with internal standards

[0028] Table 2. Multiple reaction monitoring parameters of two arginine biosynthetic metabolites under negative ion mode.

[0030] Example 2: Establishment of the pseudo-targeting method 1. Materials and Methods 1.1 Instruments AB Qtrap 4000 mass spectrometer (AB SCIEX, USA); LC-40B×3 chromatograph (Shimadzu Corporation, Japan); 1-16R benchtop high-speed refrigerated centrifuge (Hunan Kecheng Instrument Equipment Co., Ltd.); LPD2500 multi-tube vortex mixer (Lept Scientific Instruments (Beijing) Co., Ltd.); FB2085 0.0001 g electronic balance (Shanghai Sunny Hengping Scientific Instruments Co., Ltd.); SCIENTZ-48L refrigerated high-throughput tissue homogenizer (Ningbo Xinzhi Biotechnology Co., Ltd.); QPN-5L nitrogen evaporator (Shanghai Quanpu Scientific Instruments Co., Ltd.).

[0031] 1.2 Test Drugs Ornithine (purity ≥98%, batch number: MR028349), L-citrulline (purity ≥98%, batch number: AB1007), N-acetyl-L-ornithine (purity ≥98%, batch number: MR004026), N-acetyl-L-glutamic acid (purity ≥98%, batch number: MR003231), glutamine (purity ≥98%, batch number: AB2459), L-proline (purity ≥98%, batch number: AB2453), α-ketoglutarate (purity ≥98%, batch number: ABH0317), symmetrical N,N-dimethylarginine (purity ≥98%, batch number: MR003352), creatinine (purity ≥98%, batch number: AB2384), N,N-dimethylglycine (purity ≥98%) The following reference standards were obtained from Chengdu Abok Biotechnology Co., Ltd.: S-adenosyl homocysteine ​​(purity ≥98%, batch number: MR024623), asymmetric dimethylarginine (purity ≥98%, batch number: MR0131172), and spermidine (purity ≥98%, batch number: MR014869). Fumaric acid (purity ≥98%, batch number: PS010304), urea (purity ≥98%, batch number: PSD240812-174), betaine (purity ≥98%, batch number: PS012048), cysteine ​​(purity ≥98%, batch number: PSD240812-175), S-adenosylmethionine (purity ≥98%, batch number: PSS240812-178), aspartic acid (purity ≥98%, batch number: PSS240812-173), homocysteine ​​(purity ≥98%, batch number: PSD240814-177), arginine (purity ≥98%, batch number: PS020741), 2,5-dihydroxybenzoic acid (purity ≥98%) The reference standards (batch number: PSD240930-581) and L-D3 glutamic acid (purity ≥98%, batch number: PSD240930-581) are from Chengdu Pusi Biotechnology Co., Ltd.

[0032] 1.3 Reagents Acetonitrile (mass spectrometry grade, Beijing Bailingwei Technology Co., Ltd.), formic acid (mass spectrometry grade, Thermo Fisher Scientific (China) Co., Ltd.), methanol (mass spectrometry grade, Beijing Bailingwei Technology Co., Ltd.).

[0033] 1.4 Methods 1.4.1 Chromatographic conditions Phenomenex Hilic column (4.6 × 100 mm, 2.6 μm); mobile phase A: 0.1% formic acid acetonitrile, mobile phase B: 0.1% formic acid aqueous solution; flow rate: 0.8 mL / min; gradient elution: 0–7 min, 75%–50% A, 7–8 min, 50%–75% A; equilibration: 1 min; column temperature: 30 °C; injection volume: 1 μL.

[0034] 1.4.2 Mass Spectrometry Conditions Multiple reaction detection (MRM) mode for positive ions: curtain gas 40 psi; ionization voltage 5500 V; ion source temperature 500 °C; spray gas 50 psi; auxiliary heating gas 50 psi. Mass spectrometry reaction parameters for 21 arginine biosynthetic metabolites and 2 internal standards are shown in Tables 2 and 3.

[0035] 1.4.3 Preparation of 21 Arginine Synthetic Metabolites and Internal Standard Solutions Twenty-one arginine synthesis reference standards were accurately weighed, dissolved in 50% methanol-water, and shaken well to obtain the following concentrations: ornithine 1.09 mg / mL, L-citrulline 1.07 mg / mL, N-acetyl-L-ornithine 1.13 mg / mL, proline 1.07 mg / mL, glutamine 1.02 mg / mL, symmetric N,N-dimethylarginine 1.05 mg / mL, creatinine 1.06 mg / mL, N,N-dimethylglycine 1.02 mg / mL, S-adenosyl homocysteine ​​1.01 mg / mL, urea 1.04 mg / mL, betaine 1.02 mg / mL, cysteine ​​1.07 mg / mL, and S-adenosylmethionine 1.05 mg / mL. Single reference stock solution containing: N-acetyl-L-glutamic acid 1.00 mg / mL, asymmetric dimethylarginine 1.01 mg / mL, aspartic acid 1.03 mg / mL, L-glutamic acid 1.03 mg / mL, homocysteine ​​1.00 mg / mL, arginine 1.01 mg / mL, α-ketoglutarate 1.02 mg / mL, fumaric acid 1.03 mg / mL, L-glutamic acid D3 1.03 mg / mL, and 2,5-dihydroxybenzoic acid 1.04 mg / mL.

[0036] 1.4.4 Optimization of mass spectrometry parameters for the analyte Using a 4000QTRAP syringe pump, 1 μg / mL of each analyte and internal standard solution was injected. First, Q1 was optimized to determine the precursor ion, then Q3 was optimized to determine the daughter ion, and finally, the ion pair parameters CE and DP were optimized to obtain the MRM parameters for each analyte and internal standard. The ID, MRM ion pair, retention time, optimal CE, and optimal DP of each compound and internal standard were imported into a pre-defined MRM monitoring list, establishing a pseudo-targeted analysis method based on LC-QTRAP-MS / MS. Nineteen compounds were analyzed in positive ion mode, and two compounds in negative ion mode. The MRM monitoring lists are shown in Tables 1 and 2.

[0037] 1.4.5 Tissue Sample Processing Accurately weigh 60 mg of liver tissue into a centrifuge tube, add 300 μL of distilled water at a weight-to-volume ratio (mg:μL, 1:5), and homogenize at 2500 rpm for 3 min at 4℃. Take 200 μL of the homogenate, add 20 μL of 5 μg / mL internal standard solution, add 300 mL of acetonitrile to precipitate the protein, and vortex at 2500 rpm for 3 min at 4℃. Centrifuge at 12000 rpm for 10 min at 4℃, and dry the supernatant with N2. Redissolve the tissue in 100 μL of 50% methanol aqueous solution, vortex at 2500 rpm for 3 min at 4℃, centrifuge at 12000 rpm for 10 min at 4℃, and take 60 μL of the supernatant for LC-MS analysis.

[0038] 1.4.5 Preparation of QC Samples 10 μL of each liver tissue homogenate sample was mixed to form a total of 20 biological samples. The blank group (n=10) and the model group (n=10) constituted the QC samples, which were processed according to the tissue sample pretreatment method.

[0039] Example 3: Methodological Investigation of Relative Quantification of 21 Arginine Synthetic Metabolites in the Liver Using Pseudo-Targeted Metabolomics (1) Linear Take QC samples and process them according to biological sample pretreatment methods, adding 50% methanol and water according to 2 7 Dilution levels (1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128) were used to ensure the same internal standard concentration. Linear regression was performed with the analyte-to-internal standard peak area ratio as the x-axis and the dilution level as the y-axis. The linear coefficients for all 21 arginine biosynthetic metabolites were >0.95, indicating good linearity of the method.

[0040] (2) Precision test QC samples were taken and processed according to the biological sample pretreatment method. After redissolving in 50% methanol-water, 60 μL of the supernatant was centrifuged and injected for analysis. The injection was repeated 6 times. The relative standard deviation (RSD) of the peak area ratio of 21 analytes to the internal standard was calculated to evaluate the intra-day precision of the method. The RSD values ​​were all <15%. After 3 consecutive days of injection, the relative standard deviation (RSD) of the peak area ratio of 21 analytes to the internal standard was calculated to evaluate the inter-day precision of the method. The RSD values ​​were all <15%, indicating that the method has good precision.

[0041] (3) Stability test Stability was assessed by calculating changes in QC samples during the execution of metabolomics analysis sequences, with QC samples injected every three times. The PCA one-dimensional score plots of the QC samples were examined; all QC samples were within ±2 SD (std.dev), indicating the stability of the established targeted metabolomics method.

[0042] Systematic methodological studies have shown that the linearity of all 21 arginine biosynthetic metabolites is greater than 0.95, indicating good linearity. The precision and stability of this method meet the requirements of the target-related methodology, indicating that this method can be used for the relative quantitative determination of 21 arginine biosynthetic metabolites in liver samples.

[0043] Example 4: Application of a pseudo-targeted metabolomics method for the relative quantification of 21 arginine biosynthetic metabolites in the liver. The validated method was applied to the determination of liver contents in the blank and model groups of mice with cantharidin-induced liver injury. The relative contents of each analyte were calculated using the corresponding regression standard curves. The results are shown in Tables 5 and 6. OPLS-DA analysis results are as follows. Figure 4 As shown, further analysis revealed that, compared with the blank control group, the levels of L-citrulline, betaine, urea, aspartic acid, L-glutamic acid, fumaric acid, and creatinine in the model group were significantly increased (P < 0.05), while the levels of S-adenosylmethionine, N-acetyl-L-glutamic acid, cysteine, and S-adenosylhomocysteine ​​were significantly decreased (P < 0.05).

[0044] Table 3.21 Linear Equations and Correlation Coefficients of Arginine Synthetic Metabolites Serial Number Analytes Dilution factor Linear equations linear coefficients 1 ornithine 2~128 y = 0.216x - 2.23 0.9716 2 L-citrulline 2~128 y=0.0117-0.105 0.9774 3 N-acetyl-L-ornithine 2~128 y = 8.81e-x + 0.000498 0.9578 4 proline 2~128 y = 0.187x - 1.62 0.9892 5 glutamine 2~128 y = 0.22x - 1.64 0.9883 6 α-Ketoglutarate 2~128 y = 0.106x - 0.525 0.9594 7 Symmetric N,N-dimethylarginine 2~128 y = 0.00579x - 0.0533 0.9757 8 Creatinine 2~128 y = 0.000878x - 0.00514 0.9720 9 N,N-Dimethylglycine 2~128 y = 0.0626x - 0.601 0.9844 10 S-adenosylhomocysteine 2~128 y = 0.00423x - 0.0445 0.9555 11 fumaric acid 2~128 y = 0.0501x - 0.495 0.9658 12 urea 2~128 y = 0.104x - 0.965 0.9755 13 betaine 2~128 y = 0.216x - 2.04 0.9832 14 Cysteine 2~128 y = 0.00121x - 0.0104 0.9845 15 S-adenosylmethionine 2~128 y = 0.00203x - 0.0257 0.9506 16 N-acetyl-L-glutamic acid 2~128 y=0.00635-0.0611 0.9748 17 asymmetric dimethylarginine 2~128 y = 0.0106x - 0.118 0.9536 18 Aspartic acid 2~128 y = 0.707x - 0.654 0.9838 19 L-glutamic acid 2~128 y = 0.435x - 3.46 0.9829 20 homocysteine 2~128 y = 0.00126x - 0.00995 0.9733 21 Arginine 2~128 y=0.000741-0.00629 0.9717 Table 4. Intra-day and inter-day precision of QC samples of 21 arginine biosynthetic metabolites (n=6) Serial Number Analytes Intraday Precision RSD% Daytime precision RSD% 1 ornithine 12.60 12.61 2 L-citrulline 12.91 13.18 3 N-acetyl-L-ornithine 10.06 11.70 4 proline 13.75 11.61 5 glutamine 14.47 10.58 6 α-Ketoglutarate 14.27 5.95 7 Symmetric N,N-dimethylarginine 9.79 14.13 8 Creatinine 12.19 10.28 9 N,N-Dimethylglycine 13.05 10.26 10 S-adenosylhomocysteine 13.97 11.70 11 fumaric acid 12.77 10.19 12 urea 13.80 10.84 13 betaine 14.10 11.11 14 Cysteine 14.19 10.31 15 S-adenosylmethionine 9.53 12.80 16 N-acetyl-L-glutamic acid 9.92 14.84 17 asymmetric dimethylarginine 10.27 12.78 18 Aspartic acid 14.44 9.19 19 L-glutamic acid 14.06 9.80 20 homocysteine 11.99 13.67 21 Arginine 13.36 13.79 Table 5. Relative contents of 21 arginine biosynthetic metabolites in the liver of the control group

[0045] Continued from Table 5.

[0046] Table 6. Relative contents of 21 arginine biosynthetic metabolites in the liver of the model group

[0048] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0049] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for relative quantitative analysis of liver arginine synthesis metabolites using pseudo-targeted metabolomics, characterized by: Includes the following steps: The arginine biosynthetic metabolites are: ornithine, L-citrulline, N-acetyl-L-ornithine, proline, glutamine, symmetrical N,N-dimethylarginine, creatinine, N,N-dimethylglycine, S-adenosyl homocysteine, urea, betaine, cysteine, S-adenosylmethionine, N-acetyl-L-glutamic acid, asymmetrical dimethylarginine, aspartic acid, L-glutamic acid, homocysteine, arginine, α-ketoglutarate, and fumaric acid. The two internal standards are L-glutamic acid D3 and 2,5-dihydroxybenzoic acid. (1) Preparation of reference solution and internal standard solution: Accurately weigh 21 kinds of arginine biosynthetic metabolites and 2 kinds of internal standard substances and place them in volumetric flasks. Add 50% methanol water to dissolve and dilute to obtain the standard solution. (2) Use of internal standard solution: L-glutamic acid D3 is selected as internal standard in positive ion mode, and 2,5-dihydroxybenzoic acid is selected as internal standard in negative ion mode; (3) Optimization of MRM parameters: The precursor ion MS1 ​​of 21 arginine biosynthetic metabolites was determined using a 4000QTRAP instrument, and the fragment ion with higher response intensity was selected as the daughter ion MS2; together with the precursor ion, candidate ion pairs were constructed, and the optimal DP and CE were selected for each ion pair based on the response intensity. (4) Pretreatment of tissue samples: Accurately weigh liver tissue into a centrifuge tube, add distilled water to homogenize, take the homogenate, add internal standard and acetonitrile solution, vortex to mix, centrifuge to take the supernatant, blow dry with nitrogen, add 50% methanol water to reconstitute, and the sample is obtained. (5) Preparation of QC samples: Take an appropriate amount of liver tissue homogenate from the blank group and the model group, vortex mix, and process according to the method in step (4); (6) Establishment of standard curve: The QC samples from step (5) are arranged according to 2 7 The internal standard was diluted using a series of dilutions to ensure that the concentration of the internal standard was the same. The series of diluted solutions were injected into LC-MS, and the data were analyzed using Analyst 1.6.

3. A standard curve was plotted with the ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard as the x-axis and the number of dilutions as the y-axis. (7) Relative content determination: Using the same method and under the same detection conditions, the liver tissue sample was injected into LC-MS after tissue sample pretreatment in step (1), and the mass spectrometry data was exported. Analyst 1.6.3 was used for data analysis. The relative content of 21 arginine biosynthetic metabolites in the liver tissue sample could be directly calculated using the method in step (6).

2. The method for relative quantitative targeted metabolomics analysis of hepatic arginine biosynthetic metabolites according to claim 1, characterized in that: In step (1), 2 mg of each of the 21 arginine biosynthetic metabolites and 2 internal standard references were accurately weighed and placed in a 2 mL volumetric flask. 2 mL of 50% methanol water was added to dissolve them, and the solution was shaken and diluted to a final volume to prepare a 1 mg / mL reference stock solution. An appropriate amount of each reference stock solution was taken and diluted with 50% methanol water to 1 μg / mL. The 1 μg / mL solution of each reference was injected into LC-MS for qualitative analysis.

3. The method for relative quantitative pseudo-targeted metabolomics analysis of hepatic arginine biosynthesis metabolites according to claim 1, characterized in that: In step (2), the positive ion mode and the internal standard concentration of negative ions are 0.5 μg / mL.

4. The method for relative quantitative targeted metabolomics analysis of hepatic arginine biosynthesis metabolites according to claim 1, characterized in that: In step (4), 60 mg of liver tissue was weighed into a centrifuge tube, 300 μL of water was added, and the tissue was homogenized using a cryogenic high-throughput tissue homogenizer. 200 μL of homogenate was taken and 20 μL of internal standard solution was added. The mixture was vortexed for 3 min, centrifuged at 4 ℃ and 12000 rpm for 10 min. 100 μL of supernatant was taken, dried with N2, and then reconstituted with 100 μL of 50% methanol solution. The mixture was vortexed for 3 min, centrifuged at 4 ℃ and 12000 rpm for 10 min, and then 60 μL of supernatant was taken for LC-MS analysis.

5. The method for relative quantitative targeted metabolomics analysis of hepatic arginine biosynthetic metabolites according to claim 1, characterized in that: In steps (5) and (6), the detection conditions include: Mass spectrometry conditions: electrospray ionization source, positive and negative ion multiple reaction monitoring (MRM) modes; ionization voltage +5500V, -4500V; ion source temperature 550 ℃; spray gas 50 psi; auxiliary heating gas 50 psi; Chromatographic conditions: Phenomenex Hilic column (4.6 × 100 mm, 2.6 μm); mobile phase A: 0.1% formic acid acetonitrile, mobile phase B: 0.1% formic acid aqueous solution; flow rate: 0.8 mL / min; gradient elution: 0–7 min, 75%–50% A, 7–8 min, 50%–75% A; equilibration: 1 min; column temperature: 30 °C; injection volume: 1 μL.

6. The method for relative quantitative pseudo-targeted metabolomics analysis of hepatic arginine biosynthetic metabolites according to claim 1, characterized in that: In steps (3) and (4), the parameters of Analyst 1.6.3 are set as follows: the mass spectrometry parameters of 21 arginine biosynthetic metabolites and 2 internal standards, namely Molecule List Name, Precursor Name, Precursor m / z, Precursor RT, and Product m / z, are imported into the mass spectrometer's built-in analysis software Analyst 1.6.3 to establish a pseudo-targeted qualitative analysis method for LC-MS / MS. The peak areas of each analyte and internal standard are imported, and a relative quantitative analysis method is established through a standard curve.

7. The method for relative quantitative pseudo-targeted metabolomics analysis of hepatic arginine biosynthesis metabolites according to claim 1, characterized in that: The ion pair information for each analyte is as follows: ornithine 133.3→72.1, L-citrulline 176.1→113.2, N-acetyl-L-ornithine 175.1→114.7, proline 116.1→70.2, glutamine 147.1→130.2, symmetrical N,N-dimethylarginine 104.0→58.2, creatinine 114.4→44.4, N,N-dimethylglycine 239.0→57.1, S-adenosyl homocysteine ​​385.1→136.2, urea 61.0→44.1, betaine 118.1→59.3, cysteine ​​122.0→76.0, and S-adenosylmethionine 399. 1→250.2, N-acetyl-L-glutamic acid 190.3→129.8, asymmetric dimethylarginine 203.2→70.3, aspartic acid 134.0→74.1, L-glutamic acid 148.1→84.3, homocysteine ​​136.1→90.2, arginine 175.1→70.1, α-ketoglutarate 145.5→145.5, fumaric acid 115.0→70.8, L-glutamic acid D3 150.9→87, 2,5-dihydroxybenzoic acid 152.6→107.

9.

8. The method for relative quantitative pseudo-targeted metabolomics analysis of hepatic arginine biosynthesis metabolites according to claim 1, characterized in that: The mass spectrometry reaction parameters of the 21 arginine biosynthetic metabolites are shown in Tables 1 and 2. Table 1. Parameters for multiple reaction detection of 21 arginine biosynthetic metabolites using positive ion mode with internal standards Table 2. Multiple reaction monitoring parameters of two arginine biosynthetic metabolites under negative ion mode. 。

Images