Method for simultaneously detecting five biological sample metabolites based on mass spectrum

Abstract

The invention discloses a method for simultaneously detecting five biological sample metabolites based on mass spectrometry, and belongs to the technical field of biological sample metabolite detection. On the basis of an advanced UPLC-MS / MS (Ultra Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) combined technology, absolute quantification can be carried out on five standard substances (N-Lactoyl-Phenylalanine, N-Acetyl-L-Lucine, L-Anserine, L-Carnosine and L-Phenylalanine) in a sample, and the linearity, the lower limit of quantification, the accuracy, the precision and the stability are verified one by one from the perspective of methodology.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological sample metabolite detection, and in particular relates to a method for simultaneously detecting five biological sample metabolites based on mass spectrometry. Background Art

[0002] L-Anserine: Anserine is a type of histidine dipeptide naturally present in vertebrates. It is water-soluble and has significant antioxidant and anti-aging functions. Anserine can activate immune cells, thereby improving the body's immunity. In addition, anserine can promote the growth and differentiation of bone cells, increase bone density, and thus improve bone health.

[0003] L-Carnosine: It is a dipeptide composed of beta-alanine and histidine, which can inhibit many biochemical changes caused by aging. Carnosine has the functions of protecting cell membranes, maintaining endocrine balance, anti-oxidation, regulating immunity, increasing skin elasticity, anti-aging, and nourishing the body.

[0004] L-Phenylalanine: One of the essential amino acids for the human body, it can promote protein synthesis and regulate the functions of the nerve and immune systems. Because it can stimulate the central nervous system, improve brain blood circulation, and increase blood supply to the brain, it helps improve memory.

[0005] N-Acetyl-L-leucine (N-acetyl-L-leucine): It is an acetylated derivative of leucine, which has multiple functions and effects such as regulating metabolism, promoting protein synthesis, stabilizing hemodynamics, improving central nervous system function and enhancing immunity.

[0006] N-Lactoyl-Phenylalanine: It is a peptide conjugate of lactic acid and phenylalanine. It is one of the metabolites during acute exercise. It suppresses appetite by entering the brain, thereby producing an "anti-hunger" effect. It can be used to study obesity and metabolic-related diseases.

[0007] In summary, these substances have the effects of anti-aging, improving immunity, and promoting brain vitality. The establishment of absolute quantitative methods for these substances can evaluate the content of these active substances in medicinal plants, evaluate the efficacy, and study the metabolic mechanism of drugs in animals. At present, there is no absolute quantitative method based on mass spectrometry for these five substances. Summary of the invention

[0008] In view of the above situation, in order to overcome the defects of the prior art, the present invention has established a method for simultaneously detecting five metabolites: L-Anserine (L-Anserine), L-Carnosine (L-Carnosine), L-Phenylalanine (L-Phenylalanine), N-Acetyl-L-leucine (N-Acetyl-L-Leucine) and N-Lactoyl-Phenylalanine (N-Lactoylphenylalanine), and performing absolute quantitative mass spectrometry analysis.

[0009] In order to achieve the above object, the following technical solution is adopted: The present invention provides a method for simultaneously detecting five metabolites of biological samples based on mass spectrometry, comprising the following steps:

[0010] S1. Preparation of linear solution: Use a pipette to take 10 μL of each of the 5 10 mg / mL standard stock solutions, add 950 μL of 80% methanol, mix well, and prepare a linear stock solution 1 with a concentration of 100,000 ng / mL. The diluent is 80% methanol, and then dilute with the linear stock solution 1 to obtain the remaining linear point stock solutions. The standard stock solutions are L-angesone, L-carnosine, L-phenylalanine, N-acetyl-L-leucine and N-lactylphenylalanine;

[0011] S2. Preparation of internal standard working solution: First, prepare the three isotopic internal standards L-Arginine, L-Tryptophan, and L-ProLine into 1 mg / mL internal standard stock solutions, dilute the three stock solutions to obtain internal standard stock solutions, and then dilute them to obtain working solutions of different concentrations for subsequent experiments;

[0012] S3. Plasma / tissue sample pretreatment, including:

[0013] S301. Take out the sample from the refrigerator;

[0014] S302. Place the grinding beads in a 2 mL grinding tube, pre-cool the grinding tube, take it out and put it on a thousandth balance to peel it, weigh 100 mL of plasma sample / 100 mg of tissue sample and put it into the grinding tube;

[0015] S303. The packaged samples are placed on a grinder and ground at a frequency of 70 Hz and a time of 2 min;

[0016] S304. Use a pipette to draw 400 μL or 500 μL of mass spectrometry water and add it to the ground powder, vortex mix, prepare a homogenate, place it in an ice bath for 15 minutes, and further dilute it according to the type of sample to be tested;

[0017] S305. Centrifuge at 12000g for 10 min at 4°C, transfer 100 μL of the supernatant to a 1.5 mL centrifuge tube, add 400 μL of the extract containing the isotope internal standard, vortex mix for 1 min, and transfer to a sample bottle; take out the chromatographic sample tray, place the samples on the tray in order, and perform the above steps simultaneously with the preparation of linearity and quality control products. The volume ratio of water to methanol in the extract is 1 / 4;

[0018] S4. Preparation of quality control and blank samples, including:

[0019] S401. Use a pipette to add 100 μL of low and high quality control solutions, add 400 μL of the extract mixed with the isotope internal standard, vortex to mix, name them QCL and QCH respectively, centrifuge at 12000g for 10 min at 4°C, take the supernatant to the injection bottle for loading on the machine, the concentrations of the low and high quality control solutions are 250 ng / mL and 10000 ng / mL respectively, and the volume ratio of water to methanol in the extract is 1 / 4;

[0020] S402. Take an appropriate amount of the extract mixed with the isotope internal standard into a sample injection bottle for loading onto the machine, wherein the volume ratio of water to methanol in the extract is 1 / 4;

[0021] S5. Perform chromatography and mass spectrometry analysis.

[0022] Furthermore, the chromatographic conditions are:

[0023] Column: ACQUITY UPLC HSS T3, 100 × 2.1 mm, 1.8 μm;

[0024] Mobile phase: Phase A: 0.1% formic acid in water,

[0025] Phase B: acetonitrile;

[0026] Column temperature: 40°C;

[0027] Injection volume: 2 μL.

[0028] Furthermore, the gradient elution procedure of the chromatographic column is:

[0029] 0-0.5min, flow rate 0.3mL / min, mobile phase A 99%, mobile phase B 1%;

[0030] 0.5-5.0min, flow rate 0.3mL / min, mobile phase A reduced from 99% to 65%;

[0031] 5.0-5.5 min, flow rate 0.3 mL / min, mobile phase A reduced from 65% to 0%;

[0032] 5.5-6.5 min, flow rate 0.3 mL / min, mobile phase A 0%, mobile phase B 100%;

[0033] 6.5-6.6 min, flow rate 0.3 mL / min, flowability A increased from 0% to 99%;

[0034] 6.6-8.0min, flow rate 0.3mL / min, mobile phase A 99%, mobile phase B 1%.

[0035] Furthermore, the mass spectrometry conditions are: electrospray ionization source, positive and negative ion ionization mode, ion source temperature 300° C., ion source voltage 4000 V, negative mode -3500 V, nebulizer gas 35 psi, and scanning using multiple reaction monitoring.

[0036] Furthermore, diluting the linear master liquor 1 to obtain the remaining linear point master liquors specifically includes the following steps: diluting the linear master liquor 1 into a linear master liquor 2 with a concentration of 50,000 ng / mL, diluting the linear master liquor 2 into a linear master liquor 3 with a concentration of 20,000 ng / mL and a linear master liquor 4 with a concentration of 5,000 ng / mL, respectively, diluting the linear master liquor 3 into a linear master liquor 5 with a concentration of 2,000 ng / mL, diluting the linear master liquor 4 into a linear master liquor 6 with a concentration of 500 ng / mL, diluting the linear master liquor 5 into a linear master liquor 7 with a concentration of 200 ng / mL, and diluting the linear master liquor 6 into a linear master liquor 8 with a concentration of 50 ng / mL.

[0037] Furthermore, the step S2 specifically includes: preparing three isotope internal standards L-Arginine, L-Tryptophan, and L-ProLine into 1 mg / mL internal standard mother solutions, diluting the three mother solutions to obtain 5 ug / mL internal standard stock solutions, and then diluting the stock solutions again to 20 ng / mL, 10 ng / mL, and 1 ng / mL working solutions, respectively, for subsequent experiments.

[0038] The beneficial effects of the present invention are as follows: based on the advanced UPLC-MS / MS coupling technology, the present invention can perform absolute quantification of five standard substances (N-Lactoyl-Phenylalanine, N-Acetyl-L-leucine, L-Anserine, L-Carnosine, L-Phenylalanine) in a sample, and verify the linearity, lower limit of quantification, accuracy, precision and stability one by one from a methodological perspective. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the recorded regression equation and correlation coefficient of the sample N-Acetyl-L-leucine of the present invention;

[0040] Figure 2 is the recorded regression equation and correlation coefficient of the sample L-Anserine of the present invention;

[0041] Figure 3 is the recorded regression equation and correlation coefficient of the sample L-Carnosine of the present invention;

[0042] Figure 4 is the recorded regression equation and correlation coefficient of the sample L-Phenylalanine of the present invention;

[0043] Figure 5 The recorded regression equation and correlation coefficient of the sample N-Acetyl-L-leucine of the present invention are shown below.

[0044] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0046] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0047] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0048] Example 1

[0049] Sample preparation

[0050] The following steps are involved:

[0051] 1.1. Preparation of linear solution: Use a pipette to take 10 μL of each of the 5 10 mg / mL standard stock solutions, add 950 μL of 80% methanol, mix well, and prepare a linear stock solution 1 with a concentration of 100,000 ng / mL. The diluent is 80% methanol, and then dilute with the linear stock solution 1 to obtain the remaining linear point stock solutions, specifically comprising the following steps: dilute the linear stock solution 1 to a linear stock solution 2 with a concentration of 50,000 ng / mL, dilute the linear stock solution 2 to a linear stock solution 3 with a concentration of 20,000 ng / mL and a linear stock solution 4 with a concentration of 5,000 ng / mL, dilute the linear stock solution 3 to a linear stock solution 5 with a concentration of 2,000 ng / mL, dilute the linear stock solution 4 to a linear stock solution 6 with a concentration of 500 ng / mL, dilute the linear stock solution 5 to a linear stock solution 7 with a concentration of 200 ng / mL, and dilute the linear stock solution 6 to a linear stock solution 8 with a concentration of 50 ng / mL;

[0052] The standard substance mother solution is L-angesine, L-carnosine, L-phenylalanine, N-acetyl-L-leucine and N-lactylphenylalanine;

[0053] 1.2. Preparation of internal standard working solution: First, prepare the three isotopic internal standards L-Arginine, L-Tryptophan, and L-ProLine into 1 mg / mL internal standard stock solutions, dilute the three stock solutions to obtain 5 ug / mL internal standard stock solutions, and then dilute the stock solutions again to 20 ng / mL, 10 ng / mL, and 1 ng / mL working solutions for subsequent experiments;

[0054] 1.3. Plasma / tissue sample pretreatment, including:

[0055] 1.3.1. Take the sample out of the refrigerator;

[0056] 1.3.2. Place the grinding beads in a 2 mL grinding tube, pre-cool the grinding tube, take it out and put it on a thousandth balance to peel it, weigh 100 mL of plasma sample / 100 mg of tissue sample and put it into the grinding tube;

[0057] 1.3.3. Put the packaged samples on the grinder and grind them at a frequency of 70 Hz and a time of 2 min;

[0058] 1.3.4. Use a pipette to draw 400μL or 500μL of mass spectrometry water and add it to the ground powder. Vortex to mix well and prepare a homogenate. Place it in an ice bath for 15 minutes. Further dilution can be performed according to the type of sample to be tested.

[0059] 1.3.5. Centrifuge at 12000g for 10 min at 4°C, transfer 100 μL of the supernatant to a 1.5 mL centrifuge tube, add 400 μL of the extract containing the isotope internal standard, vortex mix for 1 min, and transfer to a sample bottle; take out the chromatographic sample tray and place the samples on the tray in order. The above steps are carried out simultaneously with the preparation of linearity and quality control products. The volume ratio of water to methanol in the extract is 1 / 4;

[0060] 1.4. Preparation of quality control and blank samples, including:

[0061] 1.4.1. Use a pipette to add 100 μL of low and high quality control solutions, add 400 μL of extract mixed with isotope internal standard, vortex mix, name them QCL and QCH respectively, centrifuge at 12000g for 10 min at 4°C, take the supernatant to the injection bottle for loading on the machine, the concentrations of the low and high quality control solutions are 250 ng / mL and 10000 ng / mL respectively, and the volume ratio of water to methanol in the extract is 1 / 4;

[0062] 1.4.2. Take an appropriate amount of the extract mixed with the isotope internal standard into the injection bottle for loading onto the machine. The volume ratio of water to methanol in the extract is 1 / 4;

[0063] Example 2

[0064] Chromatographic analysis

[0065] 2.1. Chromatographic conditions:

[0066] Column: ACQUITY UPLC HSS T3, 100 × 2.1 mm, 1.8 μm;

[0067] Mobile phase: Phase A: 0.1% formic acid in water,

[0068] Phase B: acetonitrile;

[0069] Column temperature: 40°C;

[0070] Injection volume: 2 μL.

[0071] 2.2. The gradient elution procedure of the chromatographic column is:

[0072] 0-0.5min, flow rate 0.3mL / min, mobile phase A 99%, mobile phase B 1%;

[0073] 0.5-5.0min, flow rate 0.3mL / min, mobile phase A reduced from 99% to 65%;

[0074] 5.0-5.5 min, flow rate 0.3 mL / min, mobile phase A reduced from 65% to 0%;

[0075] 5.5-6.5 min, flow rate 0.3 mL / min, mobile phase A 0%, mobile phase B 100%;

[0076] 6.5-6.6 min, flow rate 0.3 mL / min, flowability A increased from 0% to 99%;

[0077] 6.6-8.0min, flow rate 0.3mL / min, mobile phase A 99%, mobile phase B 1%.

[0078] 2.3. Under the above chromatographic conditions, the retention times of the five standards are shown in the following table.

[0079] Table 1 Retention time of 5 kinds of standards

[0080] Serial number substance Internal Standard RT 1 N-Lactoyl-Phenylalanine L-Tryptophan 6.469 2 L-Anserine L-Tryptophan 0.711 3 L-Carnosine L-Arginine 0.707 4 L-Phenylalanine L-Proline 3.778 5 N-Acetyl-L-leucine L-Arginine 5.949

[0081] Example 3

[0082] Mass spectrometry quantitative analysis

[0083] The mass spectrometry conditions were as follows: electrospray ionization source, positive and negative ion ionization modes, ion source temperature of 300°C, ion source voltage of 4000V, negative mode of -3500V, nebulizer gas of 35psi, and scanning with multiple reaction monitoring.

[0084] Example 4

[0085] 4.1. Limit of quantification and linearity

[0086] 4.1.1.Lower limit of quantification and acceptance criteria

[0087] The lower limit of quantitation is the lowest concentration of the analyte in a sample that can be reliably quantified with acceptable accuracy and precision. The lower limit of quantitation is generally the lowest point of the standard curve and should be appropriate for the expected concentration and test purpose. Linearity refers to the degree to which the measurement result is proportional to the concentration of the target in the sample within a given measurement range.

[0088] Acceptance criteria:

[0089] The signal-to-noise ratio is greater than or equal to 10 (S / N ≥ 10);

[0090] The quantitative lower limit detection result CV≤20%.

[0091] 4.1.2. Linearity and acceptance criteria

[0092] Linearity refers to the degree to which the measurement result is proportional to the concentration of the target substance in the sample within a given measurement range. Select several concentration levels within the expected measurement range to evaluate linearity, and the selected concentration levels should cover the entire expected measurement range.

[0093] Acceptance criteria: r ≥ 0.9900.

[0094] 4.1.3. The results of the lower limit of quantification, linearity and linear range are shown in the table below.

[0095]

[0096] 4.2. Accuracy

[0097] 4.2.1. Accuracy refers to the closeness of the result measured by the detection method to the true value or reference value. Recovery test is often used for evaluation, adding pure target substance to blank matrix for sample processing and testing, and calculating the ratio of the result to the theoretical value.

[0098] 4.2.2. Acceptance criteria:

[0099] R%=(QC 回收样 –B 空白 ) / S 加标量 ×100%;

[0100] R% is the recovery rate, QC 回收样 For the matrix plus the recovery point sample, B 空白 For blank matrix samples, S 加标量 Add concentrations for theory.

[0101] The recovery rate R% of each target was in the range of 85% to 115%;

[0102] The CV of the signal intensity detection results of the three recovery points of each target object was ≤15%.

[0103] 4.2.3. The accuracy verification results are shown in the table below.

[0104] Table 2 Accuracy verification results

[0105]

[0106] Note: QCL is the low value quality control, QCH is the high value quality control.

[0107] Results Analysis

[0108] The linear correlation coefficients of the five standards were all greater than 0.9900, meeting the acceptance criteria. The QCL and QCH results of the injector stability of the 33 bile acid samples after treatment were all within the range of CV ≤ 15%, meeting the acceptance criteria. The qualitative and quantitative test results of the five standards showed that they could meet the industry requirements in terms of linearity, accuracy and stability. The method for these five standards can achieve accurate qualitative and quantitative detection.

[0109] The ratio of the peak area of ​​each test substance to the internal standard was used as the ordinate (y), and the corresponding theoretical concentration was used as the abscissa (x). The linearity was evaluated by multivariate regression analysis, and the regression equation and correlation coefficient (r) were recorded. The recorded regression equations and correlation coefficients (r) of the five standards are shown in Figure 1-5 .

[0110] The results of SmapleA, SampleB and SampleC and two standard points C3, C6 and QC are shown in the table below.

[0111] Table 3 Results of SmapleA, SampleB and SampleC and two standard points C3, C6 and QC

[0112]

[0113] Three isotopic internal standards, L-Arginine, L-Tryptophan, and L-Proline, were used in two experiments. The response differences between L-Arginine and L-Proline in the standard and sample were too large, which may be due to instability and other reasons. They are not suitable as internal standards to calculate the concentration of the substance in the sample. The following table shows the response peak area values ​​of the same internal standard in the standard and sample (the table is randomly selected data). Therefore, L-Tryptophan was finally used as the internal standard to calculate the sample concentration.

[0114] The results of the first experiment are shown in the table below.

[0115] Table 4 Results of the first experiment

[0116]

[0117]

[0118] The results of the second experiment are shown in the table below.

[0119] Table 5 Results of the first experiment

[0120]

[0121] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0122] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for simultaneously detecting five metabolites of biological samples based on mass spectrometry, characterized in that: The following steps are involved: S1. Preparation of linear solution: Use a pipette to take 10 μL of each of the 5 10 mg / mL standard stock solutions, add 950 μL of 80% methanol, mix well, and prepare a linear stock solution 1 with a concentration of 100,000 ng / mL. The diluent is 80% methanol, and then dilute with the linear stock solution 1 to obtain the remaining linear point stock solutions. The standard stock solutions are L-angesone, L-carnosine, L-phenylalanine, N-acetyl-L-leucine and N-lactylphenylalanine; S2. Preparation of internal standard working solution: First, prepare the three isotopic internal standards L-Arginine, L-Tryptophan, and L-ProLine into 1 mg / mL internal standard stock solutions, dilute the three stock solutions to obtain internal standard stock solutions, and then dilute them to obtain working solutions of different concentrations for subsequent experiments; S3. Plasma / tissue sample pretreatment, including: S301. Take out the sample from the refrigerator; S302. Place the grinding beads in a 2 mL grinding tube, pre-cool the grinding tube, take it out and put it on a thousandth balance to peel it, weigh 100 mL of plasma sample / 100 mg of tissue sample and put it into the grinding tube; S303. The packaged samples are placed on a grinder and ground at a frequency of 70 Hz and a time of 2 min; S304. Use a pipette to draw 400 μL or 500 μL of mass spectrometry water and add it to the ground powder, vortex mix, prepare a homogenate, place it in an ice bath for 15 minutes, and further dilute it according to the type of sample to be tested; S305. Centrifuge at 12000g for 10 min at 4°C, transfer 100 μL of the supernatant to a 1.5 mL centrifuge tube, add 400 μL of the extract containing the isotope internal standard, vortex mix for 1 min, and transfer to a sample bottle; take out the chromatographic sample tray, place the samples on the tray in order, and perform the above steps simultaneously with the preparation of linearity and quality control products. The volume ratio of water to methanol in the extract is 1 / 4; S4. Preparation of quality control and blank samples, including: S401. Use a pipette to add 100 μL of low and high quality control solutions, add 400 μL of the extract mixed with the isotope internal standard, vortex to mix, name them QCL and QCH respectively, centrifuge at 12000g for 10 min at 4°C, take the supernatant to the injection bottle for loading on the machine, the concentrations of the low and high quality control solutions are 250 ng / mL and 10000 ng / mL respectively, and the volume ratio of water to methanol in the extract is 1 / 4; S402. Take an appropriate amount of the extract mixed with the isotope internal standard into a sample injection bottle for loading onto the machine, wherein the volume ratio of water to methanol in the extract is 1 / 4; S5. Perform chromatography and mass spectrometry quantitative analysis.

2. The method for simultaneously detecting five biological sample metabolites based on mass spectrometry according to claim 1, characterized in that: The chromatographic conditions are: Column: ACQUITY UPLC HSS T3, 100 × 2.1 mm, 1.8 μm; Mobile phase: Phase A: 0.1% formic acid in water, Phase B: acetonitrile; Column temperature: 40°C; Injection volume: 2 μL.

3. The method for simultaneously detecting five biological sample metabolites based on mass spectrometry according to claim 2, characterized in that: The gradient elution program of the chromatographic column is: 0-0.5min, flow rate 0.3mL / min, mobile phase A 99%, mobile phase B 1%; 0.5-5.0min, flow rate 0.3mL / min, mobile phase A reduced from 99% to 65%; 5.0-5.5 min, flow rate 0.3 mL / min, mobile phase A reduced from 65% to 0%; 5.5-6.5 min, flow rate 0.3 mL / min, mobile phase A 0%, mobile phase B 100%; 6.5-6.6 min, flow rate 0.3 mL / min, flowability A increased from 0% to 99%; 6.6-8.0min, flow rate 0.3mL / min, mobile phase A 99%, mobile phase B 1%.

4. The method for simultaneously detecting five biological sample metabolites based on mass spectrometry according to claim 3, characterized in that: The mass spectrometry conditions are: electrospray ionization source, positive and negative ion ionization modes, ion source temperature of 300° C., ion source voltage of 4000 V, negative mode of -3500 V, nebulizer gas of 35 psi, and scanning using multiple reaction monitoring.

5. The method for simultaneously detecting five biological sample metabolites based on mass spectrometry according to claim 4, characterized in that: Dilution with linear master liquor 1 to obtain the remaining linear point master liquors specifically includes the following steps: diluting linear master liquor 1 into linear master liquor 2 with a concentration of 50,000 ng / mL, diluting linear master liquor 2 into linear master liquor 3 with a concentration of 20,000 ng / mL and linear master liquor 4 with a concentration of 5,000 ng / mL, respectively, diluting linear master liquor 3 into linear master liquor 5 with a concentration of 2,000 ng / mL, diluting linear master liquor 4 into linear master liquor 6 with a concentration of 500 ng / mL, diluting linear master liquor 5 into linear master liquor 7 with a concentration of 200 ng / mL, and diluting linear master liquor 6 into linear master liquor 8 with a concentration of 50 ng / mL.

6. The method for simultaneously detecting five biological sample metabolites based on mass spectrometry according to claim 5, characterized in that: The step S2 specifically includes: preparing three isotope internal standards L-Arginine, L-Tryptophan, and L-ProLine into 1 mg / mL internal standard mother solutions, diluting the three mother solutions to obtain 5 ug / mL internal standard stock solutions, and then diluting the stock solutions again to 20 ng / mL, 10 ng / mL, and 1 ng / mL working solutions, respectively, for subsequent experiments.

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