CN117630263A

CN117630263A - Method for detecting various free amino acids in plasma based on liquid chromatography-tandem mass spectrometry

Info

Publication number: CN117630263A
Application number: CN202311867385.7A
Authority: CN
Inventors: 戴辉; 高平; 翟立斐; 谢晓磊
Original assignee: Shanghai Ruizhi Technology Co ltd
Current assignee: Shanghai Ruizhi Technology Co ltd
Priority date: 2023-12-29
Filing date: 2023-12-29
Publication date: 2024-03-01

Abstract

The invention discloses a method for detecting various free amino acids in plasma based on liquid chromatography-tandem mass spectrometry. Specifically, the invention discloses a method for detecting lysine, ornithine, histidine, arginine, glycine, serine, alanine, asparagine, aspartic acid, threonine, glutamine, glutamic acid, citrulline, proline, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine and tryptophan in blood plasma based on liquid chromatography-tandem mass spectrometry. The principle of the method of the invention is as follows: the pH value of a plasma sample is regulated to enable amino acid in the sample to be free, macromolecules such as proteins and pigments in the plasma are intercepted by adopting an ultrafiltration mode, so that the amino acid is extracted and purified, various amino acids are separated by a chromatographic column, and the quantitative detection is carried out on a liquid chromatography-tandem mass spectrometry system by adopting a multi-reaction monitoring (MRM) mode.

Description

Method for detecting various free amino acids in plasma based on liquid chromatography-tandem mass spectrometry

Technical Field

The invention relates to the technical field of analysis and detection, in particular to a method for detecting various free amino acids in plasma based on liquid chromatography-tandem mass spectrometry.

Background

Amino acids are the basic units constituting proteins, and up to now, 300 or more natural amino acids have been found. Different amino acids play different functions in the metabolic process of human body, and they regulate vital activities and maintain health. Amino acids can play the following roles in the human body by metabolism: (1) synthesizing tissue proteins; (2) amino-containing substances such as acids, hormones, antibodies, and creatine; (3) converted into carbohydrates and fats; (4) oxidized into carbon dioxide and water and urea to produce energy. Therefore, the existence of amino acids in human body not only provides an important raw material for synthesizing protein, but also provides a material basis for promoting growth, normal metabolism and life maintenance. If one of the human bodies lacks or decreases, the normal life metabolism of the human body may be impaired, and even cause the occurrence of various diseases or termination of life activities.

Amino acids are the basic units constituting proteins, which are the main contributors to vital activities, so amino acid nutrition monitoring is closely related to human health.

1) Assessing the nutritional status of the human body, avoiding sub-health status, and avoiding occurrence of related diseases or complications of the diseases;

2) Prediction of risk for chronic diseases (e.g., liver cirrhosis, diabetes, etc.);

3) Diagnosis of amino acid metabolic diseases (heredity) and other disease etiology, diagnosis as early as possible, intervention as early as possible;

4) The amino acid formulation supplements the nutritional monitoring of the patient for treatment, assisting in the adjustment of the clinical treatment supplementation regimen.

By accurately detecting 21 amino acids in a human body, the balance state of amino acids in the human body can be comprehensively and accurately measured, and the detailed amino acid metabolism condition in the human body is revealed. Intuitively judging the amino acid level in the body, providing the assessment results of nutrition, metabolism and health condition of the individual, and providing corresponding nutrition improvement and intervention suggestions.

Amino acid analysis methods include pre-column derivatization-ion exchange chromatography, gas chromatography, liquid chromatography, capillary capacitance, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the like. Traditional amino acid analysis is mainly accomplished by pre-column derivatization-ion exchange chromatography and pre-column derivatization-liquid chromatography separation-photometric detection of phthalic aldehyde (OPA) and fluorenylmethyl chloroformate (FMOC). The pre-column derivatization-ion exchange chromatography has high repeatability, but the analysis time is long and the cost is high. The liquid chromatography method is one of the most widely used methods in amino acid analysis, wherein the pre-column derivatization reagent used is phthalaldehyde, dimethylaminonaphthalene sulfonyl chloride, fluorescein isothiocyanate and the like. The pre-column derivatization liquid chromatography can be repeated and sensitive, but has weak specificity for analytes. Moreover, both methods cannot distinguish between 14N and 15N-labeled amino acids and cannot perform isotopic labeling quantification.

With the development of modern amino acid analysis technology, mass spectrometry plays an important role in amino acid analysis as a detection tool with high selectivity and high sensitivity. LC-MS/MS can accurately quantify the mass-to-charge ratio (m/z) of amino acid, and can obtain more amino acid detection information in one analysis process. Compared with the common capillary electrophoresis method, the pre-column derivative liquid chromatography method and other methods, the method has higher detection limit, precision and detection accuracy, and provides a technical basis for faster and more accurate amino acid quantitative detection.

At present, the liquid chromatography tandem mass spectrometry technology is mainly used for detecting amino acids in blood plasma, one is that derivatization of amino acids in blood plasma is carried out by using derivatization reagents such as phenyl isothiocyanate, dansyl chloride and the like, then the derivatized blood plasma is treated, and the treated blood plasma is subjected to separation of various amino acids on a C18 reverse chromatographic column and then enters a mass spectrum for detection. One of the main purposes of derivatization is to enhance the interaction of amino acids with the stationary phase of a chromatographic column, and most of the amino acids are difficult to retain on a common C18 reverse chromatographic column because of the relatively high polarity, so that the separation of various amino acids on the chromatographic column cannot be realized, and the derivatization reagent reacts with the amino acids to generate derivatives with relatively low polarity, so that the retention and separation on the reverse chromatographic column are realized. In another technical scheme, the ionic pairing reagent of perfluorocarboxylic acid is added into the mobile phase, and the ionic pairing reagent and amino acid are paired through ionic interaction, so that the polarity of the amino acid is reduced, and the retention and separation on a C18 reverse chromatographic column are realized. The two technical schemes have the respective defects that the pretreatment flow of the derivatization method is complex, the interference reaction is more, and the toxicity of part of derivatization reagents is stronger. The use of ion pairing reagents can accelerate contamination of the ion source of the mass spectrometer, increase the maintenance cost of the mass spectrometer, and further increase the detection cost. In addition, the two schemes generally have the detection time of one sample of tens of minutes, and the detection efficiency is low. In addition, the presence of amino acid isomers of the same molecular weight in plasma also requires good chromatographic separation of such amino acids to prevent interference due to the same mass-to-charge ratio during mass spectrometry detection, affecting the detection results.

The patent CN110018266B is a method for rapidly and quantitatively analyzing 48 amino acids, and mainly uses n-propanol and 3-methylpyridine as derivatization agents, and performs liquid chromatography-tandem mass spectrometry detection on the sample after derivatization of the amino acids. However, the pretreatment is complex and takes a long time, the sample injection analysis time is more than 20 minutes, and the detection efficiency is low. Patent CN112730723a, a method for detecting 22 free amino acids in plasma by ultra-high performance liquid chromatography-tandem mass spectrometry: the sample is extracted by methanoic acid and ethyl acetate, and is redissolved for detection after nitrogen is blown dry, and the sample analysis and detection time is 15 minutes. The pretreatment requires long nitrogen blowing time, and the chromatographic mobile phase is added with the heptafluorobutyric acid ion pair reagent, so that the mass spectrometer is polluted, and the maintenance cost of the mass spectrometer is increased. Patent CN116087371a, a method for rapidly analyzing liquid chromatography tandem mass spectrometry of multiple amino acids in plasma, by subjecting plasma to protein precipitation treatment, and then loading the sample to liquid chromatography tandem mass spectrometry detection. The method uses protein precipitation to extract amino acid, the sample is not clean, the matrix effect is very large, and the pollution to chromatographic columns and mass spectrometers is easy to generate. The used hydrophilic chromatographic column is easy to cause the retention time drift of the target object in the subsequent detection of a large number of samples, and the chromatography is unstable. The Hilic mode is not suitable for mass sample detection due to poor chromatographic stability. In the patent CN114624338A, the method for quantitatively analyzing free amino acid in a biological sample by utilizing liquid chromatography-tandem mass spectrometry is characterized in that methanol is adopted to precipitate plasma, supernatant is centrifugally taken, nitrogen is dried, and the sample is loaded into the liquid chromatography-tandem mass spectrometry for detection after redissolution. After protein precipitation, nitrogen blowing and re-dissolution are adopted for sample loading, so that the sample purifying effect is poor, and a chromatographic column and a mass spectrometer are easy to be polluted; the inability to separate the isomeric leucine and isoleucine from the spectra presented, as measured by the analysis of the waters T3 column, resulted in inaccurate quantification of both isomers.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the prior art, and provides a method for detecting various free amino acids in plasma based on liquid chromatography tandem mass spectrometry, which comprises the following steps: the method comprises the steps of preprocessing a plasma sample, adding a proper amount of isotope internal standard into the plasma sample, adding a releasing agent, carrying out vortex oscillation for a set time 1 to enable the samples to be mixed uniformly, transferring all the samples into an ultrafiltration tube, carrying out high-speed centrifugation for a set time 2, taking out the ultrafiltration tube, transferring extracting solution centrifuged out from the lower layer into a sample plate, and carrying out detection by a sample loading liquid chromatograph-tandem mass spectrometer.

The principle of the method of the invention is as follows: the pH value of a plasma sample is regulated to enable amino acid in the sample to be free, macromolecules such as proteins and pigments in the plasma are intercepted by adopting an ultrafiltration mode, so that the amino acid is extracted and purified, various amino acids are separated by a chromatographic column, and the quantitative detection is carried out on a liquid chromatography-tandem mass spectrometry system by adopting a multi-reaction monitoring (MRM) mode.

The aim of the invention can be achieved by the following scheme:

in a first aspect, the present invention provides a method for detecting a plurality of free amino acids in plasma based on liquid chromatography tandem mass spectrometry, the method comprising the steps of:

s1, mixing a plasma sample and an internal standard solution, and adding a release agent to adjust the pH to be less than 5; and performing ultrafiltration;

s2, performing liquid chromatography-tandem mass spectrometry detection on the lower layer extract obtained after ultrafiltration in the step S1.

As an embodiment of the present invention, in step S1, the volume ratio of the plasma sample, the internal standard solution and the release agent is 80 to 120:10-30:40-60.

As one embodiment of the present invention, in the step S1, the internal standard solution IS an aqueous hydrochloric acid solution containing alanine-IS, arginine-IS, aspartic acid-IS, citrulline-IS, glutamic acid-IS, glycine-IS, leucine-IS, methionine-IS, ornithine-IS, phenylalanine-IS, tyrosine-IS, valine-IS, threonine-IS, serine-IS, asparagine-IS, proline-IS, isoleucine-IS, histidine-IS, tryptophan-IS, lysine-IS, glutamine-IS at a concentration of 2 μg/mL-20 μg/mL.

As one embodiment of the present invention, in the step S1, the releasing agent is hydrochloric acid solution with the concentration of 0.02M-0.2M.

As an embodiment of the present invention, in step S1, the pH is adjusted to less than 5; a pH value of the solution is larger, tryptophan, tyrosine and valine are combined with protein, and cannot be ultrafiltered.

As an embodiment of the present invention, in step S1, the mixing is performed by vortex oscillation; the vortex oscillation time is 2-20min.

As an embodiment of the present invention, in step S1, the ultrafiltration device includes any one of an ultrafiltration tube and an ultrafiltration plate; the specification of the ultrafiltration tube is 1-10KD,1.0-5.0ml; the specification of the super filter plate is 1-10KD,200-2000 mu L.

As one embodiment of the invention, in the step S1, when an ultrafiltration tube is adopted, the speed of ultrafiltration is 12000-15000r/min, and the time is 15-30min; when an ultrafiltration plate is adopted, the pressure of ultrafiltration is 0.1-0.4MPa, and the time is 15-30 minutes.

Preferably, the means for ultrafiltration is a ultrafiltration plate. In some embodiments, the sample after the step S1 is transferred to a 3kd,350 μl ultrafiltration plate, a 96-well receiving plate is placed under the ultrafiltration plate, holes are located in one-to-one correspondence, the assembled ultrafiltration plate and receiving plate are placed on a positive pressure device, press-filtered at 0.2MPa for 20 minutes, the 96-well receiving plate is removed, and directly detected by a liquid chromatograph-tandem mass spectrometer.

The method extracts the amino acid from the plasma sample by ultrafiltration without derivatization, is simple and quick, and can basically finish the pretreatment of the amino acid within 30 minutes. After the isotope internal standard is added for dilution, the matrix effect and interference are reduced, and the accuracy and stability of quantification are ensured.

In step S2, the sample plate is a 96-well sample plate.

In the invention, the liquid chromatography-tandem mass spectrometer used in the liquid chromatography-tandem mass spectrometry is RZ-500 series of Shanghai Ruikang biotechnology Co.

As an embodiment of the present invention, in step S2, parameters of the liquid chromatography-tandem mass spectrometry include:

chromatographic conditions:

mobile phase a: an aqueous solution containing 0.01% -0.5% formic acid;

mobile phase B: acetonitrile solution containing 0.01% -0.3% formic acid;

chromatographic column model: c18 30x100mm 3 μm;

column temperature: 40 ℃;

sample injection amount: 2. Mu.L;

the gradient elution mode is adopted, and the flow rate and the gradient are as follows:

mass spectrometry conditions:

electrospray ion source (ESI), positive ion MRM scan analysis, ion source parameters: spraying voltage is 3000V, the temperature of a transmission pipe is 280 ℃, the temperature of an ion source is 350 ℃, the sheath gas is 40Arb, and the auxiliary gas is 25Arb; the Q1/Q3 ion channels are respectively selected as follows:

the invention uses the reversed phase chromatographic system, and can ensure that 21 amino acids have chromatographic retention on the premise of not adding an ion pair reagent in a mobile phase by selecting the water-resistant reversed phase C18 chromatographic column as the chromatographic column of the amino acids. Acetonitrile added with 0.1% formic acid and water added with 0.2% formic acid are used as mobile phases, and the chromatographic separation of 21 amino acids is completed within 6 minutes by adopting a gradient elution mode, so that each amino acid has a good signal, and isomers can reach a baseline separation. The short chromatographic separation time can greatly improve the detection flux of amino acid and the detection efficiency. Can be used for rapid extraction of a large number of samples.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention can rapidly realize the extraction of amino acid by ultrafiltration, and can finish the extraction and purification within 30 min.

2. Compared with the traditional protein precipitation method, the method has cleaner and thorough sample purification, can greatly reduce the loss of chromatographic columns and mass spectrometers during sample injection analysis and detection, and improves the detection sensitivity.

3. The pretreatment of the invention does not generate dilution to the sample basically, and can detect the plasma sample with lower concentration of amino acid content.

4. The invention adopts an ultra-high performance liquid chromatography-tandem mass spectrometry method to detect 21 amino acids, and the method simultaneously detects the peak time and the ion pair of a target object. The specificity is high, and cross reaction interference can be greatly avoided, so that the accuracy is improved.

5. The invention adopts the isotope internal standard method for quantification, can greatly eliminate the matrix effect and can achieve accurate quantification.

6. The method provided by the invention has the advantages that the high throughput is realized, the pretreatment is performed once, 21 amino acids can be detected simultaneously by one needle of sample injection, and the types of the detected amino acids are very comprehensive.

7. The invention has short analysis time, can complete detection and analysis within 6 minutes, and has important significance for detecting a large number of samples.

8. The method of the invention can realize the baseline separation of isomers without derivatization.

9. The method has the advantages of good detection repeatability, good specificity and high sensitivity.

10. The method has the advantages of simple sample treatment and high automation degree.

Drawings

Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:

FIG. 1 is a chromatogram of the lower limit of Lys quantification (1. Mu.g/mL);

FIG. 2 is a chromatogram of the lower limit of Orn quantitation (1. Mu.g/mL);

FIG. 3 is a chromatogram of the lower limit of His quantitation (1. Mu.g/mL);

FIG. 4 is a chromatogram of Arg lower limit of quantitation (1. Mu.g/mL);

FIG. 5 is a chromatogram of the Gly lower limit of quantitation (2. Mu.g/mL);

FIG. 6 is a chromatogram of the lower limit of Ser quantitation (1. Mu.g/mL);

FIG. 7 is a chromatogram of the lower limit of Ala quantification (2. Mu.g/mL);

FIG. 8 is a chromatogram of the Asn lower limit of quantitation (1. Mu.g/mL);

FIG. 9 is a chromatogram of the Asp lower limit of quantitation (0.2. Mu.g/mL);

FIG. 10 is a chromatogram of the Thr lower limit of quantitation (1. Mu.g/mL);

FIG. 11 is a graph of Gln lower limit of quantitation (4. Mu.g/mL);

FIG. 12 is a chromatogram of the lower limit of Glu ration (1. Mu.g/mL);

FIG. 13 is a plot of the lower limit of Cit quantification (1. Mu.g/mL);

FIG. 14 is a chromatogram of Pro lower limit of quantitation (2. Mu.g/mL);

FIG. 15 is a chromatogram of the Val lower limit of quantitation (2. Mu.g/mL);

FIG. 16 is a chromatogram of the lower limit of Met quantitation (0.5. Mu.g/mL);

FIG. 17 is a chromatogram of the lower limit of Ile quantification (2. Mu.g/mL);

FIG. 18 is a chromatogram of the Leu lower limit of quantitation (1. Mu.g/mL);

FIG. 19 is a chromatogram of the Tyr lower limit of quantitation (1. Mu.g/mL);

FIG. 20 is a chromatogram of the lower limit of Phe quantitation (2. Mu.g/mL);

FIG. 21 depicts a chromatogram of the Trp lower limit of quantitation (1. Mu.g/mL);

FIG. 22 is a total ion flow diagram of 21 amino acids.

Detailed Description

The invention will now be described in detail with reference to the drawings and specific examples. The following examples, which are presented to provide those of ordinary skill in the art with a detailed description of the invention and to provide a further understanding of the invention, are presented in terms of implementation and operation. It should be noted that the protection scope of the present invention is not limited to the following embodiments, and several adjustments and improvements made on the premise of the inventive concept are all within the protection scope of the present invention.

Example 1

The embodiment provides a method for detecting various free amino acids in plasma based on liquid chromatography tandem mass spectrometry, which comprises the following steps: and (3) preprocessing the plasma sample, adding a proper amount of internal standard solution into the plasma sample, adding a releasing agent, carrying out vortex oscillation to uniformly mix the samples, transferring all the samples into an ultrafiltration tube, carrying out high-speed centrifugation, taking out the ultrafiltration tube, transferring the extracting solution centrifuged out of the lower layer into a sample plate, and carrying out detection by a sample liquid chromatograph-tandem mass spectrometer.

Preparation of the reagent:

releasing agent: an aqueous solution containing 0.2M hydrochloric acid;

internal standard solution: an aqueous hydrochloric acid solution containing alanine-IS, arginine-IS, aspartic acid-IS, citrulline-IS, glutamic acid-IS, glycine-IS, leucine-IS, methionine-IS, ornithine-IS, phenylalanine-IS, tyrosine-IS, valine-IS, threonine-IS, serine-IS, asparagine-IS, proline-IS, isoleucine-IS, histidine-IS, tryptophan-IS, lysine-IS, glutamine-IS at a concentration of 20 μg/mL.

Standard stock solution: respectively accurately weighing 10mg of 21 amino acid (lysine, ornithine, histidine, arginine, glycine, serine, alanine, asparagine, aspartic acid, threonine, glutamine, glutamic acid, citrulline, proline, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine and tryptophan) standard substances in 21 brown reagent bottles, respectively sucking 1mL of hydrochloric acid aqueous solution into each brown reagent bottle by using a pipette, carrying out ultrasonic treatment for 5min, and carrying out vortex mixing to obtain 21 amino acid stock solutions, wherein the concentration of the 21 amino acid stock solutions is 10mg/mL.

Pretreatment of the sample: adding 100 mu L of sample into a reaction test tube, adding 20 mu L of internal standard solution, adding 50 mu L of release agent, and uniformly mixing for 3min by vortex oscillation; all the liquid in the reaction tube is transferred into a 3KD ultrafiltration tube, the ultrafiltration tube is covered with a cover, and the reaction tube is put into a centrifugal machine to be centrifuged for 20min at 14000 r/min. Transferring the lower layer solution in the centrifuged ultrafiltration tube to a 96-well sample plate, and detecting by a liquid chromatograph-tandem mass spectrometer.

The liquid chromatograph tandem mass spectrometer is RZ-500 series of Shanghai Ruikang biotechnology Co.

Chromatographic conditions:

mobile phase a: an aqueous solution containing formic acid;

mobile phase B: acetonitrile solution containing formic acid;

chromatographic column model: c18 30x100mm 3 μm;

column temperature: 40 ℃;

sample injection amount: 2. Mu.L;

the flow rate and gradient are shown in Table 1 by gradient elution:

TABLE 1 high performance liquid chromatography separation flow rate and gradient

Mass spectrometry conditions:

TABLE 2 parameter Table of parent and child ions

The total ion flow diagram of the 21 amino acids is shown in figure 22.

Linear range and sensitivity

In the test, a blank plasma (self-made) sample without amino acid is used, each amino acid with different concentration is respectively added into the blank plasma, and 6 amino acid standard sample series with different concentration is prepared, wherein the concentration of each amino acid is respectively as follows:

alanine: 2. Mu.g/mL, 4. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, 100. Mu.g/mL;

arginine: 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 50 μg/mL;

asparagine: 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 50 μg/mL;

aspartic acid: 0.2. Mu.g/mL, 0.4. Mu.g/mL, 1. Mu.g/mL, 2. Mu.g/mL, 4. Mu.g/mL, 10. Mu.g/mL;

citrulline: 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 50 μg/mL;

glutamic acid: 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 50 μg/mL;

glutamine: 4. Mu.g/mL, 8. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, 80. Mu.g/mL, 200. Mu.g/mL;

glycine: 2. Mu.g/mL, 4. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, 100. Mu.g/mL;

histidine: 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 50 μg/mL;

leucine: 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 50 μg/mL;

isoleucine: 2. Mu.g/mL, 4. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, 100. Mu.g/mL;

lysine: 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 50 μg/mL;

methionine: 0.5 μg/mL, 1 μg/mL, 2.5 μg/mL, 5 μg/mL, 10 μg/mL, 25 μg/mL;

ornithine: 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 50 μg/mL;

phenylalanine: 2. Mu.g/mL, 4. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, 100. Mu.g/mL;

proline: 2. Mu.g/mL, 4. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, 100. Mu.g/mL;

serine: 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 50 μg/mL;

threonine: 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 50 μg/mL;

tryptophan: 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 50 μg/mL;

tyrosine: 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 50 μg/mL;

valine: 2. Mu.g/mL, 4. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, 100. Mu.g/mL.

And (3) carrying out experimental treatment on the prepared sample according to the sample pretreatment method, detecting the sample by using a mass spectrometer, and carrying out linear fitting on detection data by using mass spectrometer software to obtain a linear range, a standard curve equation, a linear correlation coefficient and a detection limit, wherein the linear range, the standard curve equation, the linear correlation coefficient and the detection limit are respectively shown in Table 3. Typical profiles for the lower limit of quantitation are shown in FIGS. 1-21.

TABLE 3 linear ranges and equations for amino acids

As can be seen from Table 3, the amino acid curves have good correlation (R > 0.995).

Precision and accuracy detection

In the test, a blank plasma (self-made) sample without amino acid is used, and amino acid standard solutions with different volumes are respectively added into the blank plasma to obtain amino acid standard samples with low concentration, medium concentration and high concentration (the theoretical addition amount shown in Table 4 for details), and six samples are respectively prepared for each concentration. The sample pretreatment step is carried out, and the sample pretreatment is detected by a mass spectrometer, so that the data are shown in Table 4.

TABLE 4 recovery and precision of blank serum addition

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As can be seen from Table 4, the precision and accuracy of the sample detection were good (CV <15%, DIFF < 15%).

Detection of clinical samples

10 plasma samples of normal people are selected, and the plasma samples are subjected to experimental treatment according to the pretreatment step of the samples and then detected by a mass spectrometer, so that data are shown in tables 5 and 6.

TABLE 5 clinical sample test results

TABLE 6 clinical sample test results

As can be seen from tables 5 and 6, the results of each amino acid in the plasma of normal human tested by this method were within the reference range.

The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.

Claims

1. A method for detecting a plurality of free amino acids in plasma based on liquid chromatography tandem mass spectrometry, the method comprising the steps of:

2. The method according to claim 1, wherein in step S1, the volume ratio of the plasma sample, the internal standard solution and the release agent is 80-120:10-30:40-60.

3. The method according to claim 1, wherein in step S1, the internal standard solution IS an aqueous hydrochloric acid solution containing alanine-IS, arginine-IS, aspartic acid-IS, citrulline-IS, glutamic acid-IS, glycine-IS, leucine-IS, methionine-IS, ornithine-IS, phenylalanine-IS, tyrosine-IS, valine-IS, threonine-IS, serine-IS, asparagine-IS, proline-IS, isoleucine-IS, histidine-IS, tryptophan-IS, lysine-IS, glutamine-IS at a concentration of 2 μg/mL to 20 μg/mL.

4. The method of claim 1, wherein in step S1, the releasing agent is a hydrochloric acid solution with a concentration of 0.02M to 0.2M.

5. The method according to claim 1, wherein in step S1, the mixing is performed by vortexing; the vortex oscillation time is 2-20min.

6. The method according to claim 1, wherein in step S1, the ultrafiltration device comprises any one of an ultrafiltration tube and an ultrafiltration plate; the specification of the ultrafiltration tube is 1-10KD,1.0-5.0ml; the specification of the super filter plate is 1-10KD,200-2000 mu L.

7. The method according to claim 6, wherein in step S1, when a ultrafiltration tube is used, the ultrafiltration speed is 12000-15000r/min, and the ultrafiltration time is 15-30min; when an ultrafiltration plate is adopted, the pressure of ultrafiltration is 0.1-0.4MPa, and the time is 15-30 minutes.

8. The method of claim 1, wherein the plurality of free amino acids comprises lysine, ornithine, histidine, arginine, glycine, serine, alanine, asparagine, aspartic acid, threonine, glutamine, glutamic acid, citrulline, proline, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan.

9. The method according to claim 1, wherein in step S2, the chromatographic conditions of the liquid chromatography-tandem mass spectrometry:

mobile phase a: an aqueous solution containing 0.01% -0.5% formic acid;

mobile phase B: acetonitrile solution containing 0.01% -0.3% formic acid;

chromatographic column model: c18 30x100mm 3 μm;

column temperature: 40 ℃;

sample injection amount: 2. Mu.L;

time (minutes) Flow rate (mL/min) A％ B％ 0 0.5 100 0 2.0 0.5 100 0 3.0 0.5 70 30 4.0 0.5 70 30 4.1 0.5 100 0 6.0 0.5 100 0

。

10. The method according to claim 1, wherein in step S2, the mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry:

analyte(s) Q1 parent separation (m/z) Q3 fragment separation (m/z) RF voltage (V) Collision energy CE (V) Alanine ALA 90.2 44.125 30 10.98 Alanine internal standard ALA-IS 94.2 48.125 30 10.69 Arginine ARG 175.2 116.125 30 13.89 Arginine internal standard ARG-IS 182.1 123.125 40 13.59 Asparagine ASN 133.2 74.113 30 15.57 Asparagine internal standard ASN-IS 139.088 92.125 30 10.64 Asparagus ASP 134.1 88.05 30 10.01 Aspartic acid internal standard ASP-IS 137.1 91.125 30 10.3 Citrulline CIT 176.3 113.125 30 15.19 Citrulline internal standard CIT-IS 183.4 120.125 35 16.25 Glutamine GLN 147.088 84.054 70 17.51 Glutamine internal standard GLN-IS 152.088 88.125 30 17.43 Glutamic acid GLU 148.2 84.125 70 15.53 Glutamic acid internal standard GLU-IS 153.1 107.1 70 16.63 Glycine GLY 76.1 30.2 30 10.39 Glycine internal standard GLY-IS 78.1 32.2 30 10.9 Histidine HIS 156.3 110.125 120 12.54 Histidine internal standard HIS-IS 159.1 113.125 120 12.71 Isoleucine ILE 132.2 86.196 100 10.14 Isoleucine internal standard ILE-IS 142.2 96.2 100 10.7 Leucine LEU 132.2 86.196 100 10.14 Leucine internal standard LEU-IS 135.2 89.196 120 10 Lysine LYS 147.1 84.125 50 15.45 Lysine internal standard LYS-IS 155.1 90.196 70 16.33 Methionine MET 150.3 104.125 70 10.1 Methionine internal standard MET-IS 153 107.1 80 10.5 Ornithine ORN 133.3 70.125 40 16.67 Ornithine internal standard ORN-IS 140.3 77.125 60 17.64 Phenylalanine PHE 166.1 103.125 110 25.47 Phenylalanine internal standard PHE-IS 171.1 106.125 90 27.03 Proline PRO 116.3 70.125 90 14.94 Proline internal standard PRO-IS 123.3 77.125 90 14.73 Serine SER 106.2 60.125 30 10.86 Serine internal standard SER-IS 109.1 62.0 30 11.7 Threonine THR 120.2 74.125 40 10.48 Threonine internal standard THR-IS 125.088 78.125 30 11.4 Tryptophan TRP 205.1 146.125 80 16.96 Tryptophan internal standard TRP-IS 210.3 150.125 110 18.4 Tyrosine TYR 182.2 136.125 90 12.88 Tyrosine internal standard TYR-IS 186.2 140.125 100 13.3 Valine VAL 118.3 72.196 80 10.52 Valine internal standard VAL-IS 126.3 80.137 150 10.9

。