Method for detecting 19 amino acids in brain tissue and bone marrow of mouse

Abstract

The invention discloses a method for detecting 19 amino acids in brain tissue and bone marrow of a mouse. The method comprises the following steps: step 1, preparing a standard substance; step 2, sampling brain tissues and bone marrow of the mouse; step 3, removing proteins in brain tissues and bone marrow samples; step 4, preparing a derivatization reagent; step 5, separating and determining 19 amino acids in the brain tissue and the bone marrow sample by HPLC-FLD; and 6, calculating a result. The method for detecting 19 amino acids in brain tissues and bone marrow of mice has the advantages of simple operation steps, high accuracy and good sensitivity, and can realize accurate detection of 19 amino acids in brain tissues and bone marrow samples.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical chemical detection, and more specifically, to a method for detecting 19 amino acids in mouse brain tissue and bone marrow. Background Technology

[0002] Mouse brain tissue and bone marrow, as vital functional units, play a crucial role in maintaining the dynamic balance of amino acid metabolism, which is essential for maintaining normal nervous system function and immune regulation. Amino acids in brain tissue not only participate in neurotransmitter synthesis and energy supply but also form a close connection with systemic metabolism through the blood-brain barrier. Meanwhile, bone marrow, as the primary site of hematopoiesis and immune cell production, has an amino acid microenvironment whose levels directly affect cell proliferation, differentiation, and stress responses. Existing research suggests that the brain-bone marrow axis may have a synergistic regulatory mechanism under various pathological conditions. Therefore, establishing a detection method capable of simultaneously analyzing the content of multiple amino acids in both tissues is of great significance for revealing the molecular mechanisms of related diseases. Current analyses of amino acids in biological samples mostly focus on single tissue types or are limited to the detection of a few high-abundance amino acids. While liquid chromatography-mass spectrometry (LC-MS) offers high sensitivity and resolution, its high operating cost and complex pretreatment process limit its application in batch sample screening. High-performance liquid chromatography-fluorescence detection, though less expensive, often faces challenges such as insufficient separation and uneven derivatization efficiency when simultaneously detecting multiple amino acids with significantly different polarities. Optimizing chromatographic conditions, pretreatment conditions, and derivatization methods is necessary to improve its sensitivity and accuracy. This is especially true when simultaneously processing samples with significantly different matrices, such as brain tissue (high lipid content) and bone marrow (complex composition). Establishing a unified sample pretreatment procedure to achieve efficient impurity removal and accurate determination becomes a key challenge in developing this method.

[0003] Therefore, in order to solve the problems of poor tissue compatibility and low detection throughput in existing technologies and to provide a reliable analytical tool for cross-disciplinary research on neuroimmunology, it is essential to develop a high-performance liquid chromatography-fluorescence analysis method applicable to both mouse brain tissue and bone marrow, capable of simultaneously detecting 19 amino acids. This experiment aims to achieve this goal, using homoserine as an internal standard and OPA as a derivatizing reagent, and employing a pre-column derivatization method to establish a highly sensitive, efficient, and easy-to-operate method for determining amino acids in mouse brain tissue and bone marrow. Summary of the Invention

[0004] One object of the present invention is to provide a method for detecting the amino acid content in mouse brain tissue and bone marrow.

[0005] To achieve these objectives according to the present invention, a method for determining the amino acid content in mouse brain tissue and bone marrow is provided, comprising the following steps:

[0006] Step 1: Preparation of standard products;

[0007] Step 2: Sampling of mouse brain tissue and bone marrow;

[0008] Step 3: Remove proteins from brain tissue and bone marrow samples;

[0009] Step 4: Preparation of derivatizing reagents;

[0010] Step 5: HPLC-FLD separation and determination of 19 amino acids in brain tissue and bone marrow samples;

[0011] Step 6: Calculate the results.

[0012] Preferably, the specific method for preparing the standard in step one is as follows:

[0013] Aspartic acid, glutamic acid, serine, histidine, glutamyl gum, arginine, citrulline, glycine, threonine, tyrosine, γ-aminobutyric acid, tryptophan, methionine, valine, phenylalanine, isoleucine, leucine, lysine, and ornithine were used as standards, and stock solutions of 1.0 mg / mL were prepared with ultrapure water for later use.

[0014] Preferably, the specific method for sampling mouse brain tissue and bone marrow in step two is as follows:

[0015] Mice were euthanized by decapitation. The scalp was immediately cut open, and the dura mater and skull were removed. The cerebral cortex and hippocampus were separated on an ice plate. Residual blood was washed away with physiological saline, the water was blotted dry, and the mice were weighed. The cortex was added to ultrapure water at a ratio of 1:16 (mg:μL), and the hippocampus was added to ultrapure water at a ratio of 1:8 (mg:μL). The mixtures were then manually homogenized using a glass homogenizer. The hind limb muscles were carefully dissected, and the tibia was removed. 1 mL of physiological saline was drawn into a syringe and gently inserted into the medullary cavity. The needle was aligned with a centrifuge tube, and the bone marrow fluid was flushed out. The mixture was centrifuged, and the supernatant was collected. The homogenate and supernatant were stored at -80°C for later use.

[0016] Preferably, the specific method for removing proteins from the epidermis in step three is as follows:

[0017] Take the brain tissue homogenate and bone marrow supernatant from step two. Use a pipette to take an appropriate amount of homogenate and add 15% 2000 PEG at a ratio of 1:1 (v / v). Add homoserine (1 mg / mL, IS), vortex for 30 min to precipitate the protein, centrifuge at 15000×g at 4℃ for 20 min, take the supernatant, derivatize and determine by HPLC-FLD.

[0018] Preferably, the specific method for preparing the derivatizing reagent in step four is as follows:

[0019] Accurately weigh 25.75 mg of o-phthalaldehyde (OPA) into a 10 mL EP tube, dissolve it in 3.94 mL of methanol, then add 60 μL of β-mercaptoethanol, vortex to mix, and obtain a concentration of 40 mmol·L⁻¹. -1 The OPA solution should be stored at 4°C in the dark for later use.

[0020] Preferably, in step five, HPLC-FLD is used to separate and determine 19 amino acids in brain tissue and bone marrow samples, under the following conditions:

[0021] Detector: Waters 2475 fluorescence detector

[0022] Chromatographic column: COSMOSIL 5C18-MS-II analytical column (150mm × 4.6mm, 5μm)

[0023] Mobile phase A: Methanol;

[0024] Mobile phase B: Sodium acetate aqueous solution, pH = 6.4

[0025] Gradient elution was performed with the following elution program: 0-10 min: 10%-30% A; 10-16 min: 30%-35% A; 16-22 min: 35%-50% A; 22-28 min: 50%-60% A; 28-38 min: 60%-70% A; 38-40 min: 70% A; 40-41 min: 10% A; 41-46 min: 10% A.

[0026] Column temperature: 35℃;

[0027] Flow rate: 0.9 mL / min -1 ;

[0028] Detection wavelength: Excitation wavelength: 340nm; Emission wavelength: 450nm

[0029] Injection volume: 20 μL

[0030] Preferably, the result calculation in step five is as follows:

[0031] Calculate the content of aspartic acid according to formula (1); calculate the content of glutamic acid according to formula (2); calculate the content of serine according to formula (3); calculate the content of histidine according to formula (4); calculate the content of glutamine according to formula (5); calculate the content of arginine according to formula (6); calculate the content of citrulline according to formula (7); calculate the content of glycine according to formula (8); calculate the content of threonine according to formula (9); calculate the content of tyrosine according to formula (10); calculate the content of γ-aminobutyric acid according to formula (11); calculate the content of tryptophan according to formula (12); calculate the content of methionine according to formula (13); calculate the content of valine according to formula (14); calculate the content of phenylalanine according to formula (15); calculate the content of isoleucine according to formula (16); calculate the content of leucine according to formula (17); calculate the content of ornithine according to formula (18); calculate the content of lysine according to formula (19).

[0032] Y = 0.1343X + 0.0175 (1)

[0033] Y = 0.1169X + 0.0139 (2)

[0034] Y = 0.1793X + 0.0571 (3)

[0035] y = 0.0451x + 0.0025 (4)

[0036] y = 0.1554x + 0.0127 (5)

[0037] y = 0.1535x + 0.0014 (6)

[0038] y = 0.1606x + 0.0067 (7)

[0039] y = 0.1481x + 0.0618 (8)

[0040] y = 0.0584x + 0.0031 (9)

[0041] y = 0.1059x + 0.0301 (10)

[0042] y = 0.3215x + 0.0262 (11)

[0043] y = 0.0666x + 0.00005(12)

[0044] Y = 0.1293x + 0.0069 (13)

[0045] Y = 0.2355x + 0.0082 (14)

[0046] Y = 0.0936x + 0.1026 (15)

[0047] Y = 0.1727x + 0.2069 (16)

[0048] Y = 0.1472x + 0.017 (17)

[0049] Y = 0.0384x + 0.0108 (18)

[0050] Y = 0.0523x + 0.0086 (19)

[0051] In the formula:

[0052] Y: The ratio of the peak area of ​​the analyte to that of the internal standard;

[0053] X: The concentration of the analyte, in micrograms per milliliter.

[0054] The present invention has at least the following beneficial effects:

[0055] 1. The Waters-2475 fluorescence detector provided by this invention has high sensitivity and good stability.

[0056] 2. The determination of amino acids in mouse brain tissue and bone marrow using the method of the present invention is simple and convenient, and can achieve rapid determination of 19 amino acids in biological samples.

[0057] Other advantages, objectives and features of the present invention will be apparent from the following description. Attached Figure Description

[0058] Figure 1 This is a high-performance liquid chromatogram of 19 amino acid standards and internal standards in the embodiments of the present invention. Peak 1 is aspartic acid, peak 2 is glutamic acid, peak 3 is serine, peak 4 is histidine, peak 5 is glutamine, peak 6 is internal standard homoserine, peak 7 is arginine, peak 8 is citrulline, peak 9 is glycine, peak 10 is threonine, peak 11 is tyrosine, peak 12 is γ-aminobutyric acid, peak 13 is tryptophan, peak 14 is methionine, peak 15 is valine, peak 16 is phenylalanine, peak 17 is isoleucine, peak 18 is leucine, peak 19 is ornithine, and peak 20 is lysine.

[0059] Figure 2This is a high-performance liquid chromatogram (HPLC) of 19 amino acids in the mouse cerebral cortex as described in the embodiments of the present invention. Peak 1 is aspartic acid, peak 2 is glutamic acid, peak 3 is serine, peak 4 is histidine, peak 5 is glutamine, peak 7 is arginine, peak 8 is citrulline, peak 9 is glycine, peak 10 is threonine, peak 11 is tyrosine, peak 12 is γ-aminobutyric acid (GABA), peak 13 is tryptophan, peak 14 is methionine, peak 15 is valine, peak 16 is phenylalanine, peak 17 isoleucine, peak 18 is leucine, peak 19 is ornithine, and peak 20 is lysine.

[0060] Figure 3 This is a high-performance liquid chromatogram (HPLC) of 19 amino acids in the mouse hippocampus as described in the embodiments of the present invention. Peak 1 is aspartic acid, peak 2 is glutamic acid, peak 3 is serine, peak 4 is histidine, peak 5 is glutamine, peak 7 is arginine, peak 8 is citrulline, peak 9 is glycine, peak 10 is threonine, peak 11 is tyrosine, peak 12 is γ-aminobutyric acid (GABA), peak 13 is tryptophan, peak 14 is methionine, peak 15 is valine, peak 16 is phenylalanine, peak 17 isoleucine, peak 18 is leucine, peak 19 is ornithine, and peak 20 is lysine.

[0061] Figure 4 This is a high-performance liquid chromatogram (HPLC) of 19 amino acids in mouse bone marrow as described in the embodiments of the present invention. Peak 1 is aspartic acid, peak 2 is glutamic acid, peak 3 is serine, peak 4 is histidine, peak 5 is glutamine, peak 7 is arginine, peak 8 is citrulline, peak 9 is glycine, peak 10 is threonine, peak 11 is tyrosine, peak 13 is tryptophan, peak 14 is methionine, peak 15 is valine, peak 16 is phenylalanine, peak 17 is isoleucine, peak 18 is leucine, peak 19 is ornithine, and peak 20 is lysine. Detailed Implementation

[0062] The specific embodiments of the present invention will be further described below with reference to examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0063] <Example>

[0064] 1 Instrument

[0065] High Performance Liquid Chromatography (Waters)

[0066] 2 reagents

[0067] Water: Ultrapure water (filtered through a 0.45μm aqueous microporous membrane).

[0068] Methanol: Chromatographically pure methanol from Tedia, USA (filtered through a 0.22 μm organic microporous membrane).

[0069] Sodium acetate solution: The pH of ultrapure water was adjusted to 6.4 with acetic acid (filtered through a 0.22μm aqueous microporous membrane).

[0070] Standards: Aspartic acid, glutamic acid, serine, histidine, glutamyl gum, arginine, citrulline, glycine, threonine, tyrosine, γ-aminobutyric acid, tryptophan, methionine, valine, phenylalanine, isoleucine, leucine, lysine, ornithine (all purchased from Shanghai Aladdin Reagent Co., Ltd.)

[0071] 3. Detection methods

[0072] (1) Preparation of standard and test sample solutions:

[0073] a) Preparation of standard solutions:

[0074] Aspartic acid, glutamic acid, serine, histidine, glutamyl gum, arginine, citrulline, glycine, threonine, tyrosine, γ-aminobutyric acid, tryptophan, methionine, valine, phenylalanine, isoleucine, leucine, lysine, and ornithine were used as standards. Each stock solution was prepared with ultrapure water to a concentration of 1.0 mg / mL and stored separately at -20°C for later use. When needed, the solutions were diluted with methanol solution to the required concentration.

[0075] b) Preparation of the test sample solution:

[0076] Mouse cerebral cortex, hippocampal homogenates, and bone marrow supernatants were aliquoted into 1.5 mL centrifuge tubes and stored at -80°C. The supernatant samples were taken, Hse internal standard solution (1 mg / mL) was added, and the mixture was vortexed for 30 s to precipitate proteins. The mixture was then centrifuged at 15000 × g for 20 min at 4°C. The supernatant was collected as the test sample. 50 μL of the test sample was added to 200 μL of sodium borate buffer (adjusted to pH 10.5 with NaOH), and 100 μL of OPA derivatizing reagent was added. The mixture was vortexed for 1 min and then analyzed by HPLC-FLD.

[0077] (2) High performance liquid chromatography detection

[0078] Chromatographic conditions: Detector: Waters 2475 fluorescence detector; Column: COSMOSIL 5C18-MS-II analytical column (150 mm × 4.6 mm, 5 μm); Column temperature: 35℃; Flow rate: 0.9 mL / min -1 Detection wavelength: Excitation wavelength: 340 nm; Emission wavelength: 450 nm; Injection volume: 20 μL; Mobile phase: Methanol:sodium acetate solution (pH = 6.4)

[0079] Prepare the standard solution at a concentration of 0.01-50 μg / mL. -1 Six series of standard solutions with different concentrations were prepared within the range. After each solution was injected, linear regression was performed using the ratio of the peak area of ​​the analyte to the internal standard as Y and the concentration of the analyte as X. The correlation coefficient γ ≥ 0.9991.

[0080] The standard and sample solutions were injected separately, and the chromatographic peaks of each component in the sample were qualitatively determined based on the retention time of each standard. The content of amino acids in the sample was calculated using the internal standard method.

[0081] 4 Calculation of Results

[0082] Calculate the content of aspartic acid according to formula (1); calculate the content of glutamic acid according to formula (2); calculate the content of serine according to formula (3); calculate the content of histidine according to formula (4); calculate the content of glutamine according to formula (5); calculate the content of arginine according to formula (6); calculate the content of citrulline according to formula (7); calculate the content of glycine according to formula (8); calculate the content of threonine according to formula (9); calculate the content of tyrosine according to formula (10); calculate the content of γ-aminobutyric acid according to formula (11); calculate the content of tryptophan according to formula (12); calculate the content of methionine according to formula (13); calculate the content of valine according to formula (14); calculate the content of phenylalanine according to formula (15); calculate the content of isoleucine according to formula (16); calculate the content of leucine according to formula (17); calculate the content of ornithine according to formula (18); calculate the content of lysine according to formula (19).

[0083] Y = 0.1343X + 0.0175 (1)

[0084] Y = 0.1169X + 0.0139 (2)

[0085] Y = 0.1793X + 0.0571 (3)

[0086] y = 0.0451x + 0.0025 (4)

[0087] y = 0.1554x + 0.0127 (5)

[0088] y = 0.1535x + 0.0014 (6)

[0089] y = 0.1606x + 0.0067 (7)

[0090] y = 0.1481x + 0.0618 (8)

[0091] y = 0.0584x + 0.0031 (9)

[0092] y = 0.1059x + 0.0301 (10)

[0093] y = 0.3215x + 0.0262 (11)

[0094] y = 0.0666x + 0.00005(12)

[0095] Y = 0.1293x + 0.0069 (13)

[0096] Y = 0.2355x + 0.0082 (14)

[0097] Y = 0.0936x + 0.1026 (15)

[0098] Y = 0.1727x + 0.2069 (16)

[0099] Y = 0.1472x + 0.017 (17)

[0100] Y = 0.0384x + 0.0108 (18)

[0101] Y = 0.0523x + 0.0086 (19)

[0102] In the formula:

[0103] Y: The ratio of the peak area of ​​the analyte to that of the internal standard;

[0104] X: The concentration of the analyte, in micrograms per milliliter.

[0105] 5 Exclusivity

[0106] Under the patented conditions for the determination of various amino acids in the experiment, no peak was observed at the retention time of the analyte in the chromatogram of the blank solvent, indicating that this method has good specificity.

[0107] 6. Linearity and Sensitivity

[0108] The linearity of aspartic acid, glutamic acid, serine, histidine, glutamyl glutamate, arginine, citrulline, glycine, threonine, tyrosine, γ-aminobutyric acid, tryptophan, methionine, valine, phenylalanine, isoleucine, leucine, lysine, and ornithine under the patented conditions was demonstrated by the standard curve over a wide range of concentrations (0.01, 0.1, 1.0, 10, 25, 50 μg / mL). -1 The concentration and the concentration showed a linear relationship (γ≥0.9991). The retention time and linear range of each amino acid, and the regression equations are shown in Table 1 (signal-to-noise ratio of detection limit ≥3 and signal-to-noise ratio of quantitation limit ≥10).

[0109] Table 1. Retention times and linear regression equations for 119 amino acids

[0110]

[0111] 7 Recovery rate

[0112] Recovery was determined by adding known amounts of standards to the sample matrix. Standards at concentrations of 0.5 μg / mL, 2.0 μg / mL, and 5.0 μg / mL were added, with five replicates for each concentration, and measurements were taken within one day to calculate the recovery rate.

[0113] 8 Precision

[0114] Precision was determined by adding known amounts of standards to the sample matrix. Standards at concentrations of 0.5 μg / mL, 2.0 μg / mL, and 5.0 μg / mL were added, with five replicates for each concentration, and measurements were performed continuously for three days to calculate inter-day precision. Precision was expressed as relative standard deviation. The recoveries and precision of the three concentrations (0.5 μg / mL, 2.0 μg / mL, and 5.0 μg / mL) in mouse cortex are shown in Table 2; the recoveries and precision of the three concentrations (0.5 μg / mL, 2.0 μg / mL, and 5.0 μg / mL) in mouse hippocampus are shown in Table 3; and the recoveries and precision of the three concentrations (0.5 μg / mL, 2.0 μg / mL, and 5.0 μg / mL) in mouse bone marrow are shown in Table 4.

[0115] Table 2. Recovery and precision of spiked mouse cortical samples (mean ± standard deviation)

[0116]

[0117]

[0118] Table 3. Recovery and precision of spiked mouse hippocampal samples (mean ± standard deviation)

[0119]

[0120]

[0121]

[0122] Table 4. Recovery and precision of spiked mouse bone marrow samples (mean ± standard deviation)

[0123]

[0124]

[0125] 9. Stability

[0126] Stability was determined by adding known amounts of standards to the sample matrix. Three concentrations of standards (0.5 μg / mL, 2.0 μg / mL, and 5.0 μg / mL) were added, with three parallel samples for each concentration. The samples were incubated at 4℃ for 0 h, 24 h, and 48 h, respectively, and the stability was measured. A sample was considered stable if the decrease in concentration was less than 10%. The stability of mouse cortical samples with concentrations of 0.5 μg / mL, 2.0 μg / mL, and 5.0 μg / mL at 4℃ is shown in Table 5; the stability of mouse hippocampal samples with concentrations of 0.5 μg / mL, 2.0 μg / mL, and 5.0 μg / mL at 4℃ is shown in Table 6; and the stability of mouse bone marrow samples with concentrations of 0.5 μg / mL, 2.0 μg / mL, and 5.0 μg / mL at 4℃ is shown in Table 7. It is best to complete the analysis of actual samples within two days.

[0127] Table 5 Stability of spiked dermal samples

[0128]

[0129]

[0130]

[0131] Table 6. Stability of Spiked Hippocampal Samples

[0132]

[0133]

[0134]

[0135] Table 7 Stability of Bone Marrow Sample Spikes

[0136]

[0137]

[0138] As can be seen from the above examples, the embodiments of the present invention have high sensitivity, accuracy and precision in detecting aspartic acid, glutamic acid, serine, histidine, glutamyl gum, arginine, citrulline, glycine, threonine, tyrosine, γ-aminobutyric acid, tryptophan, methionine, valine, phenylalanine, isoleucine, leucine, lysine and ornithine in mouse brain tissue and bone marrow.

Claims

1. A method for detecting the content of 19 amino acids in mouse brain tissue and bone marrow, characterized in that, Includes the following steps: Step 1: Preparation of standard products; Step 2: Sampling of mouse brain tissue and bone marrow; Step 3: Remove proteins from brain tissue and bone marrow samples; Step 4: Preparation of derivatizing reagents; Step 5: HPLC-FLD separation and determination of 19 amino acids in brain tissue and bone marrow samples; Step 6: Calculate the results.

2. The method for detecting amino acid content in mouse brain tissue and bone marrow as described in claim 1, characterized in that, The specific method for preparing the standard in step one is as follows: Aspartic acid, glutamic acid, serine, histidine, glutamyl gum, arginine, citrulline, glycine, threonine, tyrosine, γ-aminobutyric acid, tryptophan, methionine, valine, phenylalanine, isoleucine, leucine, lysine, and ornithine were used as standards, and stock solutions of 1.0 mg / mL were prepared with ultrapure water for later use.

3. The method for detecting amino acid content in mouse brain tissue and bone marrow as described in claim 1, characterized in that, The specific method for sampling mouse brain tissue and bone marrow in step two is as follows: Mice were euthanized by decapitation. The scalp was immediately cut open, and the dura mater and skull were removed. The cerebral cortex and hippocampus were separated on an ice plate. Residual blood was washed away with physiological saline, the water was blotted dry, and the mice were weighed. The cortex was added to ultrapure water at a ratio of 1:16 (mg:μL), and the hippocampus was added to ultrapure water at a ratio of 1:8 (mg:μL). The mixtures were then manually homogenized using a glass homogenizer. The hind limb muscles were carefully dissected, and the tibia was removed. 1 mL of physiological saline was drawn into a syringe and gently inserted into the medullary cavity. The needle was aligned with a centrifuge tube, and the bone marrow fluid was flushed out. The mixture was centrifuged, and the supernatant was collected. The homogenate and supernatant were stored at -80°C for later use.

4. The method for detecting amino acid content in mouse brain tissue and bone marrow as described in claim 1, characterized in that, The specific method for removing proteins from brain tissue and bone marrow in step three is as follows: Take the brain tissue homogenate and bone marrow supernatant from step two. Use a pipette to take an appropriate amount of homogenate and add 15% 2000 PEG at a ratio of 1:1 (v / v). Add homoserine (1 mg / mL, IS), vortex for 30 min to precipitate the protein, centrifuge at 15000×g for 20 min at 4℃, take the supernatant, derivatize and determine by HPLC-FLD.

5. The method for detecting amino acid content in mouse brain tissue and bone marrow as described in claim 1, characterized in that, The specific method for preparing the derivatizing reagent OPA in step four is as follows: Accurately weigh 25.75 mg of o-phthalaldehyde (OPA) into a 10 mL EP tube, add 3.94 mL of methanol to dissolve, then add 60 μL of β-mercaptoethanol, vortex to mix, to obtain an OPA solution with a concentration of 40 mmol·L -1 -1 at 4 ℃ in the dark for later use.

6. The method for detecting amino acid content in mouse brain tissue and bone marrow as described in claim 1, characterized in that, In step five, HPLC-FLD was used to separate and determine 19 amino acids in brain tissue and bone marrow samples. The determination conditions were as follows: Detector: Waters 2475 fluorescence detector Chromatographic column: COSMOSIL 5C18-MS-II analytical column (150mm × 4.6mm, 5μm) Mobile phase A: Methanol; Mobile phase B: Sodium acetate aqueous solution, pH = 6.4 Gradient elution, elution program as follows: 0-10 min: 10%-30% A; 10-16 min: 30%-35% A; 16-22 min: 35%-50% A; 22-28 min: 50%-60% A; 28-38 min: 60%-70% A; 38-40 min: 70% A; 40-41min: 10%A; 41-46min: 10%A; Column temperature: 35℃; Flow rate: 0.9 mL / min -1 ; Detection wavelength: Excitation wavelength: 340nm; Emission wavelength: 450nm Injection volume: 20 μL.

7. The method for detecting amino acid content in mouse brain tissue and bone marrow as described in claim 1, characterized in that, The specific calculation of the results in step six is ​​as follows: Calculate the content of aspartic acid according to formula (1); calculate the content of glutamic acid according to formula (2); calculate the content of serine according to formula (3); calculate the content of histidine according to formula (4); calculate the content of glutamine according to formula (5); calculate the content of arginine according to formula (6); calculate the content of citrulline according to formula (7); calculate the content of glycine according to formula (8); calculate the content of threonine according to formula (9); calculate the content of tyrosine according to formula (10); calculate the content of γ-aminobutyric acid according to formula (11); calculate the content of tryptophan according to formula (12); calculate the content of methionine according to formula (13); calculate the content of valine according to formula (14); calculate the content of phenylalanine according to formula (15); calculate the content of isoleucine according to formula (16); calculate the content of leucine according to formula (17); calculate the content of ornithine according to formula (18); calculate the content of lysine according to formula (19). Y = 0.1343X + 0.0175 (1) Y = 0.1169X + 0.0139 (2) Y = 0.1793X + 0.0571 (3) y = 0.0451x + 0.0025 (4) y = 0.1554x + 0.0127 (5) y = 0.1535x + 0.0014 (6) y = 0.1606x + 0.0067 (7) y = 0.1481x + 0.0618 (8) y = 0.0584x + 0.0031 (9) y = 0.1059x + 0.0301 (10) y = 0.3215x + 0.0262 (11) y = 0.0666x + 0.00005(12) Y = 0.1293x + 0.0069 (13) Y = 0.2355x + 0.0082 (14) Y = 0.0936x + 0.1026 (15) Y = 0.1727x + 0.2069 (16) Y = 0.1472x + 0.017 (17) Y = 0.0384x + 0.0108 (18) Y = 0.0523x + 0.0086 (19) In the formula: Y: The ratio of the peak area of ​​the analyte to that of the internal standard; X: The concentration of the analyte, in micrograms per milliliter.

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