A method of liquid chromatography tandem mass spectrometry for rapid analysis of multiple amino acids in plasma

Abstract

The present invention discloses a method for liquid chromatography tandem mass spectrometry for rapid analysis of multiple amino acids in plasma. The method comprises the following steps: S1. Preparation of an amino acid standard curve working solution, including accurately weighing powders of 26 amino acid standards respectively, adding them to pure water, and fully mixing to prepare single amino acid stock solutions of 10 mg / mL. The present invention relates to the technical field of liquid chromatography tandem mass spectrometry for simultaneous detection of multiple amino acids. For the method of liquid chromatography tandem mass spectrometry for rapid analysis of multiple amino acids in plasma, after performing a protein precipitation reaction on the plasma, the treated sample can be used for liquid chromatography tandem mass spectrometry analysis. Combining with the pretreatment mode of 96-well plates, the pretreatment process of 96 plasma samples only takes 40 minutes. Additionally, by using an optimized combination of mobile phase and chromatographic column, the sample injection analysis time is only 10 minutes. The processing scheme of this device is simple and efficient, and no derivatization reaction is required.

Description

Technical Field

[0001] The present invention relates to the technical field of liquid chromatography tandem mass spectrometry for analyzing multiple amino acids in plasma, and specifically to a method for rapidly analyzing multiple amino acids in plasma by liquid chromatography tandem mass spectrometry. Background Art

[0002] Amino acids are one of the important substances in the body's metabolism, with a wide range of biological functions. They are the basic building blocks of biological protein molecules and an important material basis for maintaining homeostasis. Their physiological functions involve all aspects of human growth and development, muscle and bone growth, hormone secretion, etc. For example, leucine can promote the repair of bone, skin and muscle tissues; glutamic acid can maintain and promote brain cell function; isoleucine can stabilize and regulate blood sugar, etc. Amino acids participate in many metabolic pathways in the body. For example, glutamic acid, aspartic acid, arginine, citrulline, ornithine, and γ-aminobutyric acid can be used to evaluate the urea cycle metabolism; serine, glutamic acid, aspartic acid, tyrosine, tryptophan, taurine, and γ-aminobutyric acid can be used to evaluate the metabolism of neurotransmitters; taurine can be used to evaluate sulfur metabolism. When the amino acid level in the human body is abnormal, it will affect the normal progress of the body's metabolism and ultimately lead to the occurrence of diseases. Thus, it is very necessary to monitor the content of amino acids in the blood, which is of great significance in the prediction, diagnosis and mechanism research of diseases.

[0003] High performance liquid chromatography is a commonly used method for detecting amino acids in clinical practice. Especially after the combination of high performance liquid chromatography and tandem mass spectrometry, it can achieve high-sensitivity and high-specificity detection of compounds. In recent years, this detection system has developed rapidly in clinical laboratories in our country.

[0004] At present, there are mainly two technical solutions for detecting amino acids in plasma by liquid chromatography-tandem mass spectrometry. One is to first derivatize the amino acids in plasma using derivatizing reagents such as phenyl isothiocyanate and dansyl chloride, and then process the derivatized plasma. After the processed plasma is separated from various amino acids on a C18 reverse chromatographic column, it enters the mass spectrometer for detection. A main purpose of derivatization is to enhance the interaction between amino acids and the stationary phase of the chromatographic column. Since most amino acids have large polarities, they are difficult to retain on a common C18 reverse chromatographic column and cannot achieve the separation of various amino acids on the chromatographic column. By reacting with amino acids using a derivatizing reagent, derivatives with relatively smaller polarities can be generated, thereby achieving retention and separation on the reverse chromatographic column. Another technical solution is to add an ion-pairing reagent of perfluorocarboxylic acid type to the mobile phase. The ion-pairing reagent pairs with amino acids through ionic interactions, thereby reducing the polarity of amino acids and achieving retention and separation on a C18 reverse chromatographic column. The above two technical solutions have their respective disadvantages. The pretreatment process of the derivatization method is relatively complex, there are many interfering reactions, and some derivatizing reagents are highly toxic. The use of ion-pairing reagents will accelerate the contamination of the ion source of the mass spectrometer, increase the maintenance cost of the mass spectrometer, and thus increase the detection cost. In addition, the instrument time for detecting one sample by the above two methods is generally as long as dozens of minutes, and the detection efficiency is low. In addition, there are amino acid isomers with the same molecular weight and amino acids with very close molecular weights in plasma, which also requires good separation of such amino acids on the chromatograph to prevent interference due to the same or close mass-to-charge ratios during mass spectrometry detection and affect the detection results. Summary of the Invention

[0005] (1) Technical problems to be solved

[0006] In view of the deficiencies of the prior art, the present invention provides a method for liquid chromatography-tandem mass spectrometry for rapid analysis of multiple amino acids in plasma, and solves the problems mentioned in the background art.

[0007] (2) Technical solutions

[0008] To achieve the above object, the present invention is realized through the following technical solutions: A method for liquid chromatography-tandem mass spectrometry for rapid analysis of multiple amino acids in plasma, the method comprising the following steps:

[0009] S1. Preparation of the working solution of the amino acid standard curve, including:

[0010] Accurately weigh the powders of 26 amino acid standards respectively, add them to pure water, and mix well to prepare a single amino acid stock solution of 10 mg / mL;

[0011] Calculated by molar concentration, use a pipette to add a certain volume of individual amino acid stock solutions to centrifuge tubes, a total of 26 amino acid stock solutions, and then make up the corresponding volume of pure water, mix well to prepare the highest concentration mixed solution of the standard curve working solution at 1000 μmol / L;

[0012] Then use pure water to dilute the 1000 μmol / L standard solution to 700 μmol / L and 400 μmol / L respectively;

[0013] Gradually dilute the 1000 (STD13), 700 (STD12), 400 (STD11) μmol / L standard mixed solution with pure water to 100, 70, 40, 10, 7, 4, 1, 0.7, 0.4 μmol / L (STD10 - STD1); thus completing the preparation of the standard curve working solution (STD13 - STD1);

[0014] S2. Sample pretreatment for quantitative detection of 26 amino acids in plasma, including:

[0015] Add 100 μL of the plasma to be measured or the amino acid standard curve working solution, 100 μL of the amino acid internal standard mixed solution (the amino acid internal standard used is the stable isotope compound of the amino acid, and the concentration of each amino acid internal standard is 50 μmol / L), and 800 μL of acetonitrile into a 96 - well plate. First, mix preliminarily with a pipette, then place it on a vortex mixer to mix well. After vortexing, place the 96 - well plate in a 4 - degree refrigerator and let it stand for 30 minutes. Take it out and centrifuge for 10 minutes, then take the supernatant and transfer it to the sample tray for liquid chromatography - tandem mass spectrometry analysis;

[0016] S3. Experiments of liquid chromatography and tandem mass spectrometry: Use a high - performance liquid chromatography - tandem mass spectrometer to detect the processed standard curve working solution and the samples;

[0017] S4. Calculation of the amino acid concentration in the sample, including:

[0018] In the process of amino acid quantification by liquid chromatography - tandem mass spectrometry, a stable isotope of the amino acid is used as the internal standard. Taking the ratio of the peak area of the amino acid standard to the peak area of its corresponding internal standard as the Y - axis and the concentration of the amino acid standard curve working solution as the X - axis, establish a standard curve. When establishing the standard curve, select 1 / X 2 As the weight of linear fitting, the concentration of each amino acid in the sample is calculated through the fitting equation of the standard curve.

[0019] Preferably, in step S2, the volume ratio of plasma: amino acid internal standard mixed solution: acetonitrile is 1:1:8.

[0020] Preferably, when using acetonitrile as the precipitant, compared with using methanol as the precipitant, there is no need to add two sample pretreatment steps of nitrogen blowing and solvent re-dissolution, and the supernatant after centrifugation can directly enter liquid chromatography-tandem mass spectrometry for analysis.

[0021] Preferably, in step S3, the liquid chromatography uses Shimadzu LC-20ADX, the chromatographic column is a hydrophilic column that can be used to separate highly polar compounds, the column temperature is 35 °C, the injection volume is 10 μL. The mobile phase consists of phase A and phase B. Phase A is an aqueous solution containing 0.2% formic acid and 10 mM ammonium formate, and phase B is an acetonitrile solution containing 0.2% formic acid and 10 mM ammonium formate. The mobile phase passes through the chromatographic column at a flow rate of 0.6 mL / min in a gradient manner.

[0022] Preferably, in step S3, the mass spectrometer is an ABSciex API4000 mass spectrometer, using electrospray ionization mode, and scanning the positively charged amino acid ions in multiple reaction monitoring mode.

[0023] Preferably, in step S3, the mass spectrometry conditions are as follows: curtain gas: 35 psi; collision gas: 6 psi; auxiliary heating gas: 50 psi; nebulizing gas: 60 psi; ionization voltage: 5500 V; ion source temperature: 500 °C.

[0024] Beneficial effects

[0025] The present invention provides a liquid chromatography-tandem mass spectrometry method for rapidly analyzing multiple amino acids in plasma. It has the following beneficial effects:

[0026] For the liquid chromatography-tandem mass spectrometry method for rapidly analyzing multiple amino acids in plasma, although 100 μL of plasma is used when processing samples, since only 10 μL of the supernatant after centrifugation is required for liquid chromatography-tandem mass spectrometry detection, when ensuring that the volume ratio of plasma sample: amino acid internal standard mixed solution: acetonitrile is 1:1:8, when performing sample pretreatment in the implementation of the present invention, the plasma volume can be reduced as needed, and combinations such as 50 μL plasma + 50 μL amino acid internal standard mixed solution + 400 μL acetonitrile; 10 μL plasma + 10 μL amino acid internal standard mixed solution + 80 μL acetonitrile can be used, which has great flexibility in clinical applications.

[0027] In addition, the two commonly used plasma precipitants are acetonitrile and methanol. The present invention preferably uses acetonitrile as the plasma precipitant. There are two reasons for this. One is that at the same dilution ratio of plasma, acetonitrile has a better precipitation effect on the proteins in plasma. Additionally, when methanol is used as the precipitant, the supernatant after centrifugation cannot directly enter liquid chromatography-tandem mass spectrometry for analysis, and two sample pretreatment steps of nitrogen blowing to dryness and solvent reconstitution are required. Compared with using acetonitrile as the plasma precipitant, the pretreatment time becomes longer. Therefore, acetonitrile is more preferably used as the pretreatment reagent.

[0028] This method has good resolution for isomers among 26 amino acids, such as leucine and isoleucine (both with a molecular weight of 131.2), as well as amino acids with very close molecular weights, such as glutamic acid (molecular weight of 147.1) and glutamine (molecular weight of 146.1), and aspartic acid (molecular weight of 133.1) and asparagine (molecular weight of 132.1).

[0029] By performing a protein precipitation reaction on plasma, the treated sample can be used for liquid chromatography-tandem mass spectrometry analysis. Combining with the pretreatment mode of 96-well plates, the pretreatment process for 96 plasma samples only takes 40 minutes. Additionally, by using an optimized combination of mobile phase and chromatographic column, the analysis time of the sample is only 10 minutes, making the processing scheme of this device simple and efficient, without the need for derivatization reactions. Brief Description of the Drawings

[0030] Figure 1 It is a table of 26 amino acids and their corresponding internal standard names involved in the present invention;

[0031] Figure 2 It is a table of 26 amino acids and their corresponding internal standard names involved in the present invention;

[0032] Figure 3 It is the mobile phase gradient table of the present invention;

[0033] Figure 4 It is a table of linear equations and correlation coefficients of 26 amino acids of the present invention;

[0034] Figure 5 It is a table of within-batch and between-batch imprecision of 26 amino acids of the present invention;

[0035] Figure 6 It is a table of within-batch and between-batch imprecision of 26 amino acids of the present invention;

[0036] Figure 7 It is a table of spiked recovery rates of 26 amino acids of the present invention;

[0037] Figure 8 It is a table of spiked recovery rates of 26 amino acids of the present invention;

[0038] Figure 9 This is the chromatogram of 26 amino acids of the present invention;

[0039] Figure 10 This is the chromatogram of leucine and isoleucine of the present invention;

[0040] Figure 11 This is the chromatogram of glutamic acid, glutamine, aspartic acid and asparagine of the present invention. Detailed implementation manners

[0041] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention.

[0042] Please refer to Figures 1-11 , the present invention provides a technical solution:

[0043] Embodiment 1:

[0044] A liquid chromatography tandem mass spectrometry method for rapidly analyzing multiple amino acids in plasma, the method comprising the following steps:

[0045] S1. Preparation of the amino acid standard curve working solution, including:

[0046] Weigh the powders of 26 amino acid standards accurately respectively, add them to pure water, mix well fully, and prepare single amino acid stock solutions with a concentration of 10 mg / mL;

[0047] Calculated according to the molar concentration, use a pipette to add a certain volume of the single amino acid stock solution to centrifuge tubes respectively, a total of 26 amino acid stock solutions, and then make up the corresponding volume of pure water, mix well fully, and prepare the highest concentration mixture of the standard curve working solution with a concentration of 1000 μmol / L;

[0048] Then dilute the 1000 μmol / L standard solution to 700 μmol / L and 400 μmol / L respectively with pure water;

[0049] Gradually dilute the standard mixture solutions of 1000 (STD13), 700 (STD12), 400 (STD11) μmol / L to 100, 70, 40, 10, 7, 4, 1, 0.7, 0.4 μmol / L (STD10 - STD1) with pure water; thus, the preparation of the standard curve working solution (STD13 - STD1) can be completed;

[0050] S2. Sample pretreatment for quantitatively detecting 26 amino acids in plasma, including:

[0051] Mix 100 μL of the plasma to be tested or the working solution of the amino acid standard curve with 100 μL of the amino acid internal standard mixture (the amino acid internal standard used is the stable isotope compound of the amino acid, and the concentration of each amino acid internal standard is 50 μmol / L) and 800 μL of acetonitrile, add them to a 96-well plate, first mix them preliminarily with a pipette, then place them on a vortex mixer to mix, after vortexing, place the 96-well plate in a refrigerator at 4 °C for 30 minutes, take it out and centrifuge for 10 minutes, then take the supernatant and transfer it to a sample tray for liquid chromatography-tandem mass spectrometry analysis;

[0052] S3. Experiments on liquid chromatography and tandem mass spectrometry: Use a high-performance liquid chromatography-tandem mass spectrometer to detect the processed standard curve working solution and the samples;

[0053] S4. Calculation of the amino acid concentration in the sample, including:

[0054] In the process of amino acid quantification by liquid chromatography-tandem mass spectrometry, the stable isotope of the amino acid is used as the internal standard. Taking the ratio of the peak area of the amino acid standard to the peak area of its corresponding internal standard as the Y-axis and the concentration of the amino acid standard curve working solution as the X-axis, a standard curve is established. When establishing the standard curve, 1 / X 2 is selected as the weight for linear fitting, and the concentration of each amino acid in the sample is calculated through the fitting equation of the standard curve.

[0055] Among them, the methodological verification steps for 26 amino acids are as follows:

[0056] 1). Linearity of 26 amino acids, including: The linear evaluation of this method is carried out by pretreating the standard curve working solution (STD1-STD13) in the way of parallelly processing 3 replicated samples for each concentration level of the working solution, and then entering the liquid chromatography-tandem mass spectrometry system for analysis. Taking the concentration of the standard substance as the X-axis and the ratio of the peak area of the standard substance to its internal standard as the Y-axis, a standard curve is established. The linearity of the standard curves of 26 amino acids is good, and the correlation coefficient R is greater than 0.99;

[0057] 2). Between-batch and within-batch imprecision of 26 amino acids, including: The evaluation of the imprecision of this method is carried out by detecting the quality control products at 3 concentration levels of low, medium, and high, dividing them into 3 batches for detection, and parallelly processing 5 samples for each concentration of each batch for determination, so as to verify the within-batch and between-batch imprecision of the method. The results show that the CV% of the between-batch and within-batch imprecision of 26 amino acids are all within 15%, and the imprecision of the method is good;

[0058] 3) Accuracy between and within batches of 26 amino acids, including: The accuracy of this method was evaluated by adding low- and high-concentration amino acid standard mixtures to plasma samples with known concentrations to prepare spiked samples at two concentrations. Each sample at each concentration was divided into 5 parts and repeated for 3 batches. The spiked recovery rates at the two concentrations were calculated respectively (spiked recovery rate calculation formula: ((average detected concentration of spiked sample - background concentration) / theoretical spiked concentration) * 100%). The experimental results showed that the spiked recovery rates within and between batches of these 26 amino acids in the two concentration ranges were both between 85% and 115%, and the accuracy of the method was good.

[0059] Example 2: The difference between this example and Example 1 is that,

[0060] In step S2, the volume ratio of plasma: amino acid internal standard mixed solution: acetonitrile is 1:1:8;

[0061] When using acetonitrile as the precipitant, compared with using methanol as the precipitant, there is no need to add two sample pretreatment steps of nitrogen blowing dry and solvent re-dissolution, and the supernatant after centrifugation can directly enter liquid chromatography-tandem mass spectrometry for analysis;

[0062] In step S3, the liquid chromatography uses Shimadzu LC-20ADX, the chromatographic column is a hydrophilic column that can be used to separate highly polar compounds, the column temperature is 35 degrees, and the injection volume is 10 uL. The mobile phase consists of phase A and phase B. Phase A is an aqueous solution containing 0.2% formic acid and 10 mM ammonium formate, and phase B is an acetonitrile solution containing 0.2% formic acid and 10 mM ammonium formate. The mobile phase passes through the chromatographic column at a flow rate of 0.6 mL / min in a gradient manner;

[0063] In step S3, the mass spectrometer is ABSciex API4000 mass spectrometer, using electrospray ionization mode, and scanning positively charged amino acid ions in multiple reaction monitoring mode;

[0064] In step S3, the mass spectrometry conditions are: curtain gas: 35 psi; collision gas: 6 psi; auxiliary heating gas: 50 psi; spray gas: 60 psi; ionization voltage: 5500 V; ion source temperature: 500 °C.

[0065] The working steps are as follows:

[0066] During operation, the first step: accurately weigh the powders of 26 standard amino acid samples respectively, add them to pure water, mix well thoroughly to prepare single amino acid stock solutions with a concentration of 10 mg / mL. Calculated according to molar volume, use a pipette to add a certain volume of the single amino acid stock solution into a centrifuge tube, make up the corresponding volume of pure water, mix well thoroughly to prepare the highest concentration mixture of the standard curve working solution with a concentration of 1000 μmol / L, and then dilute this standard solution to 700 μmol / L and 400 μmol / L respectively with water. Then gradient dilute the standard mixture solutions of 1000 (STD13), 700 (STD12), and 400 (STD11) μmol / L to 100, 70, 40, 10, 7, 4, 1, 0.7, 0.4 μmol / L with pure water. Complete the preparation of the standard curve working solution (STD13 - STD1).

[0067] The second step: Add the mixed solution of the plasma or amino acid standard sample to be measured, the standard mixture solution of amino acid isotopes (the concentration of each amino acid isotope standard is 50 μmol / L), and acetonitrile to a 96-well plate according to a volume ratio of 1:1:8. First, mix preliminarily with a pipette gun, then place it on a vortex mixer to mix well. After vortexing, place the 96-well plate in a 4°C refrigerator and let it stand for 30 minutes. Take it out and centrifuge for 10 minutes, then take the supernatant and transfer it to a sample tray for liquid chromatography-tandem mass spectrometry analysis;

[0068] The liquid chromatography uses Shimadzu LC-20ADX, the chromatographic column is a hydrophilic column that can be used to separate highly polar compounds, the column temperature is 35°C, and the injection volume is 10 μL. The mobile phase consists of phase A and phase B. Phase A is an aqueous solution containing 0.2% formic acid and 10 mM ammonium formate, and phase B is an acetonitrile solution containing 0.2% formic acid and 10 mM ammonium formate. The mobile phase passes through the chromatographic column at a flow rate of 0.6 mL / min in a gradient manner. The gradient change mode is as Figure 3 ; The mass spectrometer is ABSciex API4000 mass spectrometer, using electrospray ionization mode, and scanning the positively charged amino acid ions in multiple reaction monitoring mode;

[0069] The third step: In the process of amino acid quantification by liquid chromatography-mass spectrometry method, amino acid isotope standards are used as internal standards to calibrate and eliminate the influence on the analysis results caused by the fluctuations of the instrument response during the pretreatment process, and improve the accuracy of the analysis results. Therefore, taking the ratio of the peak area of the amino acid standard to the peak area of its corresponding internal standard as the Y-axis and the concentration of the amino acid standard curve working solution as the X-axis, establish a standard curve. When establishing the standard curve, select 1 / X 2 as the weight for linear fitting. The concentration of each amino acid in the sample is calculated through the fitting equation of the standard curve;

[0070] Total: Although 100 uL of plasma was used when processing the sample, since only 10 uL of the centrifuged supernatant is required for liquid chromatography-tandem mass spectrometry detection, when ensuring that the volume ratio of plasma sample: amino acid internal standard mixed solution: acetonitrile is 1:1:8, the plasma volume can be reduced as needed during the sample pretreatment in the implementation of the present invention. Combinations such as 50 uL plasma + 50 uL amino acid internal standard mixed solution + 400 uL acetonitrile; 10 uL plasma + 10 uL amino acid internal standard mixed solution + 80 uL acetonitrile can be adopted, which has great flexibility in clinical applications.

[0071] In addition, the two commonly used plasma precipitants are acetonitrile and methanol. The present invention preferably uses acetonitrile as the plasma precipitant for two reasons. One is that at the same dilution ratio of plasma, acetonitrile has a better precipitation effect on the proteins in plasma. In addition, when methanol is used as the precipitant, the centrifuged supernatant cannot be directly analyzed by liquid chromatography-tandem mass spectrometry, and two sample pretreatment steps of nitrogen blowing and solvent reconstitution are required. Compared with using acetonitrile as the plasma precipitant, the pretreatment time becomes longer. Therefore, acetonitrile is more preferably used as the pretreatment reagent.

[0072] This method has good resolution for isomers among 26 amino acids, such as leucine and isoleucine (both with a molecular weight of 131.2), as well as amino acids with very close molecular weights, such as glutamic acid (molecular weight of 147.1) and glutamine (molecular weight of 146.1), and aspartic acid (molecular weight of 133.1) and asparagine (molecular weight of 132.1);

[0073] The advantages of this device are that the pretreatment scheme is simple, efficient, and no derivatization reaction is required. Only the protein precipitation reaction treatment of plasma is needed, and the processed sample can be used for liquid chromatography-tandem mass spectrometry analysis. Combining with the pretreatment mode of 96-well plates, the pretreatment process of 96 plasma samples only takes 40 minutes. In addition, by using an optimized combination of mobile phase and chromatographic column, the analysis time of the sample is only 10 minutes.

[0074] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations.

Claims

1. A method for liquid chromatography tandem mass spectrometry for rapid analysis of multiple amino acids in plasma, characterized in that: The method includes the following steps: S1. Preparation of the amino acid standard curve working solution, including: Accurately weigh the powders of 26 amino acid standards respectively, add them to pure water, and mix well to prepare a single amino acid stock solution with a concentration of 10 mg / mL; Calculated according to the molar concentration, use a pipette to add a certain volume of each single amino acid stock solution to centrifuge tubes, a total of 26 amino acid stock solutions, and then make up the corresponding volume of pure water, mix well to prepare the highest concentration mixture of the standard curve working solution with a concentration of 1000 μmol / L; Then dilute the 1000 μmol / L standard solution to 700 μmol / L and 400 μmol / L respectively with pure water; Gradually dilute the standard mixtures of 1000 μmol / L, 700 μmol / L, and 400 μmol / L to 100 μmol / L, 70 μmol / L, 40 μmol / L, 10 μmol / L, 7 μmol / L, 4 μmol / L, 1 μmol / L, 0.7 μmol / L, and 0.4 μmol / L with pure water; thus, the preparation of the standard curve working solution is completed; S2. Sample pretreatment for quantitative detection of 26 amino acids in plasma, including: Add 100 μL of the plasma to be tested or the amino acid standard curve working solution, 100 μL of the amino acid internal standard mixture, and 800 μL of acetonitrile to a 96-well plate. First, mix them preliminarily with a pipette, then place them on a vortex mixer to mix well. After vortexing, place the 96-well plate in a 4°C refrigerator for 30 minutes, take it out and centrifuge for 10 minutes, then take the supernatant and transfer it to a sample tray for liquid chromatography-tandem mass spectrometry analysis; S3. Experiments of liquid chromatography and tandem mass spectrometry: Use a high-performance liquid chromatography-tandem mass spectrometer to detect the standard curve working solution and samples processed in step S2; S4. Calculation of the amino acid concentration in the sample, including: In the process of quantifying amino acids by liquid chromatography-tandem mass spectrometry, stable isotopes of amino acids are used as internal standards. The ratio of the peak area of the amino acid standard to the peak area of its corresponding internal standard is used as the Y-axis, and the concentration of the amino acid standard curve working solution is used as the X-axis to establish a standard curve. When establishing the standard curve, 1 / X 2 is selected as the weight for linear fitting, and the concentration of each amino acid in the sample is calculated through the fitting equation of the standard curve.

2. The method of liquid chromatography tandem mass spectrometry for rapid analysis of multiple amino acids in plasma according to claim 1, characterized in that: In step S2, the volume ratio of plasma: amino acid internal standard mixed solution: acetonitrile is 1:1:

8.

3. A method for liquid chromatography-tandem mass spectrometry for rapid analysis of multiple amino acids in plasma according to claim 2, characterized in that: When acetonitrile is used as the precipitant, compared with using methanol as the precipitant, there is no need to add two sample pretreatment steps of nitrogen blowing to dryness and solvent re-dissolution, which can directly make the supernatant after centrifugation enter the liquid chromatography-tandem mass spectrometry for analysis.

4. A method for liquid chromatography tandem mass spectrometry for rapid analysis of multiple amino acids in plasma according to claim 1, characterized in that: In step S3, the liquid chromatography uses Shimadzu LC-20ADX, the chromatographic column is a hydrophilic column that can be used to separate highly polar compounds, the column temperature is 35°C, and the injection volume is 10 μL. The mobile phase consists of phase A and phase B. Phase A is an aqueous solution containing 0.2% formic acid and 10 mM ammonium formate, and phase B is an acetonitrile solution containing 0.2% formic acid and 10 mM ammonium formate. The mobile phase passes through the chromatographic column at a flow rate of 0.6 mL / min in a gradient manner.

5. A method for liquid chromatography tandem mass spectrometry for rapid analysis of multiple amino acids in plasma according to claim 1, characterized in that: In step S3, the mass spectrometer is an ABSciex API4000 mass spectrometer, using electrospray ionization mode, and scanning the positively charged amino acid ions in multiple reaction monitoring mode.

6. A method for liquid chromatography-tandem mass spectrometry for rapid analysis of multiple amino acids in plasma according to claim 5, characterized in that: In step S3, the mass spectrometry conditions are: curtain gas: 35 psi; Collision gas: 6 psi; auxiliary heating gas: 50 psi; Spray gas: 60 psi; Ionization voltage: 5500 V; Ion source temperature: 500 °C.

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