CN114624338A - Method for quantitatively analyzing free amino acids in biological sample by using liquid chromatography-tandem mass spectrometry - Google Patents
Method for quantitatively analyzing free amino acids in biological sample by using liquid chromatography-tandem mass spectrometry Download PDFInfo
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Abstract
The invention belongs to the field of analysis and detection, and discloses a method for quantitatively analyzing free amino acids in a biological sample by using liquid chromatography-tandem mass spectrometry. The method comprises the following steps: step 1), pretreating a biological sample to obtain a treated biological sample; step 2), placing the biological sample treated in the step 1) into a refrigerated centrifuge for centrifugation, collecting supernatant, and concentrating the supernatant to be nearly dry under a nitrogen blowing instrument; step 3), uniformly mixing the sample obtained in the step 2) with a solvent, filtering, and storing at 4 ℃ to be detected; and 4) carrying out ultra performance liquid chromatography-tandem mass spectrometry combined analysis on the biological sample obtained in the step 3) to obtain the type and content of free amino acid in the biological sample. The invention can analyze the content of amino acid in different types of biological samples such as serum, plasma, tissue and the like, the analyte of the biological sample to be detected does not need to be derived, and the pretreatment method of the biological sample is simple.
Description
Technical Field
The invention belongs to the field of analysis and detection, and particularly relates to a method for quantitatively analyzing free amino acids in a biological sample by using liquid chromatography-tandem mass spectrometry.
Background
Amino acids are one of the important metabolites of the body and have a wide range of biological functions. Not only are the basic components of biomacromolecule active substances such as various proteins and enzymes in the living body and important precursors of nitrogen-containing biomacromolecule substances such as polypeptides, neurotransmitters and polyamines, but also they are involved in the synthesis of carbohydrate and lipid metabolism, purine and pyrimidine synthesis, and the like. Imbalance in amino acid metabolism can cause physiological dysfunction, affect normal progress of body metabolism and further cause diseases, and become one of the causes or manifestations of various diseases. Therefore, amino acid is used as an important target in the metabolic process, and monitoring the content of amino acid in a biological sample has important significance for assisting clinical diagnosis and clarifying the influence on the physiological function of an organism.
The existing amino acid analysis method mainly comprises a direct analysis method and a derivatization indirect analysis method. There are numerous methods for analyzing amino acids, including amino acid analyzers, liquid chromatography, gas chromatography, capillary electrophoresis, and mass spectrometry. The determination method of the amino acid analyzer has good reproducibility and reliable result, but the determination method is complex in hardware configuration, high in maintenance cost, poor in flexibility, low in resolution and long in analysis period. Derivatization is needed in both liquid chromatography and gas chromatography, but the method causes complicated sample processing steps, long analysis time, difficulty in realizing high-throughput detection and distortion of results due to low content of amino acid. The mass spectrometry technology has the advantages of high accuracy, high sensitivity, good stability and good repeatability, and the liquid chromatography with high separation capacity becomes the most common method for amino acid analysis at present. Liquid chromatography-mass spectrometry mainly improves the retention behavior of amino acids on a chromatographic column by derivatizing the amino acids with phenyl isothiocyanate, o-phthalaldehyde, 6-aminoquinoline-N-hydroxysuccinimidyl carbamate and the like or analyzes the amino acids by adding volatile ions to reagents such as alkyl sulfonate solutions of sodium pentane sulfonate, sodium N-hexyl sulfonate and the like, but both methods can pollute the system.
In recent years, researchers have tried to separate amino acids by using normal phase chromatographic packing such as silica gel, amino group, cyano group and the like or a hydrophilic interaction (HILIC) high performance liquid chromatography column with a polar group on the surface, and although the liquid chromatography-mass spectrometry based on the HILIC chromatography column can realize the analysis of the amino acids without derivatization, the retention capacity of the HILIC chromatography column on the substance to be analyzed is comprehensively influenced by various factors such as the content of an organic modifier, the flow rate, the pH value of a mobile phase buffer system, the type and the concentration of a buffer salt and the like. Therefore, in practical application, the chromatographic parameters need to be strictly controlled, and the application conditions are more strict. Furthermore, in the analysis of the amino acid content using a HILIC column, it is necessary to add an ammonium formate buffered salt solution to the mobile phase. The buffer salt is easy to separate out and remain, causes pollution and blockage to a chromatographic system and a chromatographic column and influences the retention capacity of the chromatogram. However, the indirect analysis method of derivatizing amino acids increases the number of experimental steps in the whole analysis and detection process, and the results are easily affected by the derivatization reagent, the derivatization step, the generated derivative and other factors, so that it is difficult to directly and rapidly analyze and detect the content of amino acids in a sample.
The amino acid plays an important role in the fields of biotechnology development, protein research and food and medicine industries, so the development of an amino acid analysis and detection method which is simple and convenient to operate, high in sensitivity, good in selectivity, high in detection flux and suitable for various biological samples is particularly important.
Disclosure of Invention
The invention aims to solve the technical problems in the background technology and provides a liquid chromatography-mass spectrometry analysis method suitable for the content of amino acid in various biological samples such as serum, brain tissues and the like. Compared with the existing method, the method has the advantages of simple sample pretreatment method, no need of derivatization, no addition of buffer salt reagent in a mobile phase, high sensitivity and high selectivity, is suitable for various biological samples, provides a new reference method for analysis of amino acid content in biological samples such as serum and brain tissues, and provides important technical support for evaluation of amino acid content in common clinical and biological samples and related biological and medical researches.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for quantitative analysis of free amino acids in a biological sample using liquid chromatography-tandem mass spectrometry, the method comprising the steps of:
step 1), pretreating a biological sample to obtain a treated biological sample;
step 2), placing the biological sample treated in the step 1) into a refrigerated centrifuge for centrifugation, collecting supernatant, and concentrating the supernatant to be nearly dry under a nitrogen blowing instrument;
step 3), uniformly mixing the sample obtained in the step 2) with a solvent, filtering, and storing at 4 ℃ to be detected;
and 4) carrying out ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis on the biological sample obtained in the step 3) to obtain the type and content of free amino acid in the biological sample.
In the technical scheme of the invention, the biological sample comprises a serum sample, a plasma sample, a brain tissue sample and a cell sample.
In the technical solution of the present invention, in step 1), when the biological sample is a serum sample or a plasma sample, the step of pretreating the serum sample or the plasma sample comprises: and (2) centrifuging the serum sample or the plasma sample for 10-20min under the conditions of 12000-15000rpm at 4 ℃, taking the upper layer of serum or plasma for standby, adding a methanol solution with the volume being 3-5 times that of the sample into the upper layer of serum or plasma sample, shaking and uniformly mixing, and standing for 10-15 min to precipitate the protein in the sample.
In the technical scheme of the invention, in the step 1), when the biological sample is a brain tissue sample, the step of pretreating the brain tissue sample comprises the following steps: adding 500 μ L of methanol/water mixed solvent into 50mg brain tissue sample, grinding the brain tissue sample in tissue grinder at 4 deg.C and 120Hz for 10min, wherein the volume ratio of methanol to water in the methanol/water mixed solvent is 1: 1.
In the technical scheme of the invention, in the step 2), the condition of freeze centrifugation is that centrifugation is carried out at 12000-15000rpm for 10-20min at 4 ℃.
In the technical scheme of the invention, in the step 3), when the biological sample is a serum sample, adding 150-250 μ L of solvent to each 50 μ L of serum sample, and uniformly mixing; when the biological sample is a brain tissue sample, adding 500 mu L of solvent into every 50mg of brain tissue sample and uniformly mixing;
preferably, in the step 3), a 0.22 μm cellulose acetate filter membrane is adopted for filtration; the solvent is a methanol/water mixed solvent or an acetonitrile/water mixed solvent, the volume ratio of methanol to water in the methanol/water mixed solvent is 1:1, and the volume ratio of acetonitrile to water in the acetonitrile/water mixed solvent is 1: 1.
In the technical scheme of the invention, an analytical column used for the combined analysis of the ultra-high performance liquid chromatography and the tandem mass spectrometry is ACQUITY UPLC HSS T3, and the specification is as follows: 2.1X 100mm, 1.8. mu.m.
In the technical scheme of the invention, in the step 4), the mobile phase in the ultra performance liquid chromatography-tandem mass spectrometry combined analysis is as follows: phase A is 0.1% formic acid-water, phase B is 0.1% formic acid-acetonitrile; the gradient elution procedure used was: the initial ratio is 100% for phase A and 0% for phase B, and the time is 0.5 min; reducing the phase A to 40% and increasing the phase B to 60% in 0.5-5 min; recovering to the initial gradient equilibrium system at 5.1min to 7.0 min; the flow rate of the mobile phase in the whole elution process is always 0.2 mL/min; the temperature of the column oven is 40 ℃; the sample injection volume is 5-10 mu L; the cleaning liquid of the sample injection needle is a methanol/water mixed solvent, and the volume ratio of methanol to water in the methanol/water mixed solvent is 1: 1.
In the technical scheme of the invention, in the step 4), amino acids existing in a qualitatively screened biological sample are quantified by using retention time locking and characteristic ion pair locking modes and the content of the amino acids is quantified by an external standard method, and the ionization mode of the tandem mass spectrum is electrospray ionization in which a positive ion mode and a negative ion mode are mixed; the detection mode is multiplex reaction detection (MRM); the ion source temperature is 200 ℃, the desolventizing temperature is 400 ℃, the desolventizing air flow rate is 10L/min, the capillary voltage is 4.0kV, the taper hole voltage is 30kV, the collision gas is argon, the pressure is 270kPa, and the collision energy is 20V.
The conditions of the liquid chromatography-tandem mass spectrometry are as follows:
compared with the prior art, the invention has the following beneficial effects:
1. the biological sample analyte to be detected does not need derivatization, and the pretreatment method of the biological sample is simple.
2. The invention uses the ACQUITY UPLC HSS T3 chromatographic column of a special stationary phase, the chromatographic column has better retention and separation effects on small molecular organic compounds with water solubility and large polarity, and different types of amino acids can be retained and separated on the chromatographic column without adding a buffer salt solution in a mobile phase.
3. The invention can analyze the content of amino acid in different types of biological samples such as serum, plasma, tissue and the like.
4. The invention develops an analysis method which does not need derivatization and addition of ion pair reagents, has high sensitivity and high selectivity, and aims to realize high-throughput determination of the content of common free amino acids in biological samples such as serum, plasma, body fluid, tissues and the like.
Drawings
FIG. 1(A) is a total ion flow chromatogram of an HSS T3-based column separation of 18 free amino acid standards; the total ion flow chromatogram for the separation of 18 free amino acids in the serum sample of fig. 1(B) is based on an HSS T3 column, wherein the retention times of the amino acids in fig. 1(B) and fig. 1(a) are the same and the chromatographic peaks corresponding to the retention times are the same amino acid.
FIG. 2 is a chromatogram of a detected ion of three free amino acid standards, wherein (a) the chromatogram is an extracted ion chromatogram of a separation of histidine on a HSS T3 chromatographic column; (b) the figure is an extracted ion chromatogram of alanine based HSS T3 chromatographic column separation; (c) the figure is an extracted ion chromatogram of an asparagine based HSS T3 column separation.
FIG. 3 is a chromatogram of a detected ion of three free amino acid standards, wherein (a) is a chromatogram of an extracted ion of aspartic acid based on HSS T3 column separation; (b) the figure is an extracted ion chromatogram of an isoleucine based HSS T3 chromatographic column separation; (c) the figure is an extracted ion chromatogram of a separation of leucine on a HSS T3 chromatography column.
FIG. 4 is a chromatogram of a detected ion of three free amino acid standards, wherein (a) the chromatogram is an extracted ion chromatogram of a separation of lysine on a HSS T3 chromatographic column; (b) the figure is an extracted ion chromatogram of a methionine based HSS T3 chromatography column separation; (c) the figure is an extracted ion chromatogram of a serine based HSS T3 chromatography column separation.
FIG. 5 is a chromatogram of detected ions of three standard free amino acids, wherein (a) the chromatogram is an extracted ion chromatogram of a separation of threonine on a HSS T3 column; (b) the figure is an extracted ion chromatogram of valine based on HSS T3 chromatographic column separation; (c) the figure is an extracted ion chromatogram of a proline based on an HSS T3 chromatography column separation.
FIG. 6 shows a chromatogram of a detection ion for three free amino acid standards, glutamic acid, glutamine, and tryptophan.
FIG. 7 is a chromatogram of the detected ions of three free amino acid standards, glycine, phenylalanine, and tyrosine.
FIG. 8 is a standard curve of six free amino acids, wherein A: (ii) histidine; b: alanine; c: asparagine; d: aspartic acid; e: isoleucine; f: (ii) leucine.
FIG. 9 is a standard curve of six free amino acids, wherein G: lysine; h: methionine; i: serine; j: threonine; k: valine; l: proline.
FIG. 10 is a standard curve of six free amino acids, wherein M: glutamic acid; n: (ii) glutamine; o: tryptophan; p: glycine; q: phenylalanine; r: tyrosine.
Detailed Description
The present invention will be described in further detail with reference to examples and comparative examples, but the embodiments of the present invention are not limited thereto.
Example 1
The standard substance is histidine, alanine, asparagine, aspartic acid, isoleucine, leucine, lysine, methionine, serine, threonine, valine, proline, glutamic acid, glutamine, tryptophan, glycine, phenylalanine and tyrosine.
And (3) standard product configuration:
weighing 100mg of each of the 18 amino acid standards, dissolving the 18 amino acid standards in a methanol-water mixed solution (v/v,1:1), diluting step by step and mixing each standard to obtain 18 amino acid mixed standard samples with the final concentration of 1ppm, and diluting step by step to the concentrations of 500, 200, 100, 50, 10 and 5pbb for establishing a standard sample curve.
The standard substance is subjected to amino acid determination, wherein the UPLC-MS/MS adopted by the instrumental analysis of the amino acid content is UPLC-MS 8060 of Shimadzu, and the used analytical column is ACQUITY UPLC HSS T3(2.1 × 100mm,1.8 μm); the mobile phase used consisted of 0.1% formic acid-water phase a and 0.1% formic acid-acetonitrile phase B. Gradient elution procedure: the initial ratio is 100% for phase A and 0% for phase B, and the time is 0.5 min; 0.5-5 min, reducing the phase A to 40%, and increasing the phase B to 60%; the system was returned to the initial gradient equilibrium at 5.1min to 8.0 min. The flow rate of the mobile phase in the whole elution process is always 0.2 mL/min; the temperature of the column oven is 40 ℃; the sample injection volume is 5 mu L; the cleaning solution of the sample injection needle is methanol/water (v/v,1: 1). The ionization mode of the tandem mass spectrum is electrospray ionization in a positive ion mode; the detection mode is multiplex reaction detection (MRM); the ion source temperature is 200 ℃, the desolvation temperature is 400 ℃, the flow rate of desolvation gas is 10L/min, the capillary voltage is 4.0kV, the taper hole voltage is 30kV, the collision gas is argon, and the pressure is 270 kPa.
The parameters of the monitored ion pairs and the corresponding collision energy in the analysis and detection method for the content of the 18 free amino acid standard amino acids are detailed in the following table 1.
The total ion flow chromatogram of the 18 free amino acid standards obtained by the above analysis method was based on HSS T3 column separation, as shown in fig. 1 (a). Fig. 2-7 are extracted ion chromatograms of 18 free amino acid standards based on HSS T3 chromatographic column separations.
The method for quantitatively analyzing the free amino acids in the serum sample by using the serum sample as a biological sample through liquid chromatography-tandem mass spectrometry comprises the following steps:
step 1), obtaining a serum sample: centrifuging the blood sample obtained from the mouse body at 4 deg.C under 10,000 Xg for 10min, and collecting the upper serum; transferring 50 mu L of serum sample into a 1.5mL centrifuge tube, adding a methanol solution with the volume equivalent to 3 times of the sample volume, shaking and uniformly mixing, and standing for 10-15 min to precipitate protein in the sample;
step 2), placing the sample obtained in the step 1) at 4 ℃ and centrifuging at 12000rpm for 10min, collecting supernatant sample clear liquid and concentrating to be nearly dry under a nitrogen blowing instrument; using a methanol/water mixed solvent (v/v,1:1) to determine the volume of the sample to be 0.5-1.0 mL, carrying out vortex oscillation and uniform mixing, then using a 0.22 mu m cellulose acetate filter membrane to filter the sample, and storing at 4 ℃ to be detected;
step 3), performing instrumental analysis on the content of the amino acids in the sample obtained in the step 2), wherein the UPLC-MS/MS is the UPLC-MS 8060 of Shimadzu, and the analytical column is ACQUITY UPLC HSS T3(2.1 × 100mm,1.8 μm); the mobile phase used consisted of a phase a of 0.1% formic acid-water and a phase B of 0.1% formic acid-acetonitrile. Gradient elution procedure: the initial proportion is 100 percent of phase A and 0 percent of phase B, and the time lasts for 0.5 min; reducing the phase A to 40% and increasing the phase B to 60% in 0.5-5 min; the system was returned to the initial gradient equilibrium at 5.1min to 8.0 min. The flow rate of the mobile phase in the whole elution process is always 0.2 mL/min; the temperature of the column oven is 40 ℃; the sample injection volume is 5 mu L; the cleaning solution of the sample injection needle is methanol/water (v/v,1: 1). The ionization mode of the tandem mass spectrum is electrospray ionization in a positive ion mode; the detection mode is multiplex reaction detection (MRM); the ion source temperature is 200 ℃, the desolvation temperature is 400 ℃, the flow rate of desolvation gas is 10L/min, the capillary voltage is 4.0kV, the taper hole voltage is 30kV, the collision gas is argon, and the pressure is 270 kPa.
The total ion current chromatogram of the separation of 18 free amino acids in the serum sample based on the HSS T3 chromatographic column, obtained by the above analysis method, is shown in FIG. 1 (B).
The monitored ion pairs and corresponding collision energy parameters in the analytical detection method for the amino acid content in a sample are detailed in table 1 below.
TABLE 1 amino acid monitoring ion-pair and Collision energy parameters
Detecting the content of amino acid in the sample according to the UPLC-MS/MS conditions in the table 1, taking mixed standard substance solutions with different mass concentrations to carry out UPLCMS/MS determination, drawing a standard curve by taking the concentration of each amino acid as an abscissa and the peak area of a quantitative ion mass spectrum as an ordinate, and analyzing the content of the amino acid in the sample: and obtaining the standard curves of the two, and solving a corresponding linear regression equation and a correlation coefficient.
FIGS. 8-10 are standard curves for 18 free amino acids, as follows:
histidine standard curve: according to the quantitative ion pair (as shown in table 1), the standard curve (as shown in fig. 8A) is retrieved by taking the histidine concentration as the abscissa and the quantitative ion peak area as the ordinate, and the function relationship of the standard curve is obtained as follows: y ═ (30077.4) X + (0), correlation coefficient R2＝0.9981244。
Alanine standard curve: according to the quantitative ion pair (shown in table 1), taking the alanine concentration as the abscissa and the quantitative ion peak area as the ordinate, the standard curve (shown in fig. 8B) is retrieved, and the function relationship of the standard curve is obtained as: y ═ (11003.6) X + (0), correlation coefficient R2＝0.9957335。
Standard curve for asparagine: based on the quantitative ion pair (shown in Table 1), the standard curve (shown in FIG. 8C) is retrieved with asparagine concentration as abscissa and the quantitative ion peak area as ordinate to obtain the function relationship of the standard curveThe method comprises the following steps: y ═ (4647.43) X + (0), correlation coefficient R2＝0.9970109。
Standard curve for aspartic acid: according to the quantitative ion pair (as shown in table 1), the standard curve (as shown in fig. 8D) is retrieved by taking the aspartic acid concentration as the abscissa and the quantitative ion peak area as the ordinate, and the function relationship of the standard curve is obtained as: y ═ (10943.0) X + (0), correlation coefficient R2＝0.9960386。
Isoleucine standard curve: according to the quantitative ion pair (as shown in table 1), the standard curve (as shown in fig. 8E) is retrieved by taking the isoleucine concentration as the abscissa and the quantitative ion peak area as the ordinate, and the function relationship of the standard curve is obtained as: y ═ (8714.00) X + (0), correlation coefficient R2＝0.9998885。
Leucine standard curve: according to the quantitative ion pair (as shown in table 1), the standard curve (as shown in fig. 8F) is retrieved by taking the leucine concentration as the abscissa and the quantitative ion peak area as the ordinate, and the function relationship of the standard curve is obtained as follows: y ═ (43756.6) X + (0), correlation coefficient R2＝0.9999542。
Lysine standard curve: according to the quantitative ion pair (as shown in table 1), the standard curve (as shown in fig. 9G) is retrieved by taking the lysine concentration as the abscissa and the quantitative ion peak area as the ordinate, and the function relationship of the standard curve is obtained as: y ═ (30098.6) X + (0), correlation coefficient R2＝0.9984927。
Methionine standard curve: based on the quantitative ion pair (as shown in table 1), the standard curve (as shown in fig. 9H) is retrieved with the methionine concentration as the abscissa and the quantitative ion peak area as the ordinate, and the function relationship of the standard curve is obtained as: y ═ (18482.5) X + (0), correlation coefficient R2＝0.9995012。
Serine standard curve: according to the quantitative ion pair (shown in table 1), the standard curve (shown in fig. 9I) is retrieved by taking the serine concentration as the abscissa and the quantitative ion peak area as the ordinate, and the function relationship of the standard curve is obtained as follows: y ═ (9933.69) X + (0), correlation coefficient R2＝0.9975547。
Threonine standard curve: according to the quantitative ion pair (shown in Table 1), the ion peak area was quantified using the threonine concentration as the abscissaFor the ordinate receipt standard curve (as shown in fig. 9J), the standard curve function relationship is obtained as: y ═ (16270.7) X + (0), correlation coefficient R2＝0.9993402。
Valine standard curve: according to the quantitative ion pair (shown in table 1), the standard curve (shown in fig. 9K) is retrieved by taking the valine concentration as the abscissa and the quantitative ion peak area as the ordinate, and the function relationship of the standard curve is obtained as follows: y ═ (64463.1) X + (0), correlation coefficient R2＝0.9982837。
Proline standard curve: according to the quantitative ion pair (as shown in table 1), the standard curve (as shown in fig. 9L) is retrieved by taking the proline concentration as the abscissa and the quantitative ion peak area as the ordinate, and the function relationship of the standard curve is obtained as follows: y ═ (71507.9) X + (0), correlation coefficient R2＝0.9973854。
Standard curve for glutamic acid: according to the quantitative ion pair (as shown in table 1), the standard curve (as shown in fig. 10M) is retrieved by taking the glutamic acid concentration as the abscissa and the quantitative ion peak area as the ordinate, and the function relationship of the standard curve is obtained as: y ═ (8460.51) X + (0), correlation coefficient R2＝0.9999456。
Standard curve of glutamine: according to the quantitative ion pair (shown in table 1), the standard curve (shown in fig. 10N) is retrieved by taking the glutamine concentration as the abscissa and the quantitative ion peak area as the ordinate, and the function relationship of the standard curve is obtained as follows: y ═ (11079.7) X + (0), correlation coefficient R2＝0.9999321。
Tryptophan standard curve: according to the quantitative ion pair (as shown in table 1), the standard curve (as shown in fig. 10O) is retrieved by taking the tryptophan concentration as the abscissa and the quantitative ion peak area as the ordinate, and the function relationship of the standard curve is obtained as: y ═ (15734.1) X + (0), correlation coefficient R2＝0.9999879。
Glycine standard curve: according to the quantitative ion pair (as shown in table 1), the standard curve (as shown in fig. 10P) is retrieved by taking the glycine concentration as the abscissa and the quantitative ion peak area as the ordinate, and the function relationship of the standard curve is obtained as: y ═ (950.571) X + (0), correlation coefficient R2＝0.9985857。
Phenylalanine standard curve: according to quantitative ion pairs (As shown in table 1), the phenylalanine concentration is used as the abscissa, the quantitative ion peak area is used as the ordinate, and the standard curve (as shown in fig. 10Q) is retrieved, so as to obtain the standard curve function relationship as: y ═ (26210.0) X + (0), correlation coefficient R2＝0.9999878。
Tyrosine standard curve: based on the quantitative ion pair (as shown in table 1), the standard curve (as shown in fig. 10R) is retrieved with the tyrosine concentration as the abscissa and the quantitative ion peak area as the ordinate, and the function relationship of the standard curve is obtained as: y ═ (8763.37) X + (0), correlation coefficient R2＝0.9999597。
The results show that: the 18 amino acids have good linear relation in a wide mass concentration range, and the correlation coefficient R2All above 0.995.
The content of amino acids in the serum samples was calculated from a standard curve of 18 free amino acids, and the detection limit of the standard solution at a signal-to-noise ratio S/N-3 and the quantification limit at S/N-10:
the method comprises the following steps of measuring free amino acids in a serum sample by using a parameter method in Table 1, quantifying the amino acids in a qualitatively screened biological sample by using retention time locking and characteristic ion pair locking modes and the content of the amino acids by using an external standard method, wherein UPLC-MS (ultra Performance liquid chromatography-Mass Spectrometry) is used for analyzing the amino acid content range in the serum sample and the detection limit and the quantitative limit of an analysis method are shown in the following table 2:
TABLE 2 UPLC-MS analysis of amino acid content range in serum samples and analysis method detection limit, quantitative limit
Wherein, the content of the amino acid in the serum sample is calculated by substituting the response value of the serum sample on the instrument into a standard curve constructed by a standard sample. From table 2, the amino acid content in the actual serum sample can be seen, as well as the detection limit of the instrument for 18 amino acids and the linear range of the curve for the 18 amino acid standard samples.
Example 2
This example is the same as the test method of example 1, except that a brain tissue sample is used as the biological sample to be tested:
the brain tissue sample is obtained by placing mouse brain tissue sample into a clean 1.5mL centrifuge tube, adding 500. mu.L of methanol-water (v/v,1:1) solution, and grinding in a tissue grinder at 4 deg.C and 120Hz for 10 min. The other detection procedures were the same as in example 1.
In order that the invention may be more clearly understood by those skilled in the art, the present invention will now be described in detail with reference to the specific embodiments and the accompanying drawings. The following examples are intended to illustrate the actual operation of the process and are not intended to be limiting in the practice. Apart from the operating examples described below, the details may be optimized or modified, and not all embodiments need be or are not exhaustive. Any modifications made on the basis of the examples of the present invention, which are common knowledge, are within the scope of the present invention.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include such modifications and variations.
Claims (10)
1. A method for quantitative analysis of free amino acids in a biological sample using liquid chromatography-tandem mass spectrometry, the method comprising the steps of:
step 1), pretreating a biological sample to obtain a treated biological sample;
step 2), placing the biological sample treated in the step 1) into a refrigerated centrifuge for centrifugation, collecting supernatant, and concentrating the supernatant to be nearly dry under a nitrogen blowing instrument;
step 3), uniformly mixing the sample obtained in the step 2) with a solvent, filtering, and storing at 4 ℃ to be detected;
and 4) carrying out ultra performance liquid chromatography-tandem mass spectrometry combined analysis on the biological sample obtained in the step 3) to obtain the type and content of free amino acid in the biological sample.
2. The method of claim 1, wherein the biological sample comprises serum sample, plasma sample, brain tissue sample, cell sample.
3. The method for quantitative analysis of free amino acids in biological samples by liquid chromatography-tandem mass spectrometry as claimed in claim 2, wherein in step 1), when the biological sample is a serum sample or a plasma sample, the step of pretreating the serum sample or the plasma sample comprises: and (2) centrifuging the serum sample or the plasma sample for 10-20min under the conditions of 12000-15000rpm at 4 ℃, taking the upper layer of serum or plasma for standby, adding a methanol solution with the volume being 3-5 times that of the sample into the upper layer of serum or plasma sample, then oscillating and uniformly mixing, and standing for 10-15 min to precipitate the protein in the sample.
4. The method for quantitative analysis of free amino acids in biological samples by liquid chromatography-tandem mass spectrometry as claimed in claim 2, wherein in step 1), when the biological sample is a brain tissue sample, the step of pretreating the brain tissue sample comprises: adding 500 μ L of methanol/water mixed solvent into 50mg brain tissue sample, grinding the brain tissue sample in a tissue grinder at 4 deg.C and 120Hz for 10min, wherein the volume ratio of methanol to water in the methanol/water mixed solvent is 1: 1.
5. The method for quantitative analysis of free amino acids in biological samples by liquid chromatography-tandem mass spectrometry as claimed in claim 1, wherein the conditions of the refrigerated centrifugation in step 2) are 12000-15000rpm centrifugation at 4 ℃ for 10-20 min.
6. The method as claimed in claim 1, wherein in step 3), when the biological sample is a serum sample, 150-250 μ L of solvent is added to 50 μ L of serum sample; when the biological sample is a brain tissue sample, adding 500 mu L of solvent into every 50mg of brain tissue sample and uniformly mixing;
preferably, in the step 3), a 0.22 μm cellulose acetate filter membrane is adopted for filtration; the solvent is a methanol/water mixed solvent or an acetonitrile/water mixed solvent, the volume ratio of methanol to water in the methanol/water mixed solvent is 1:1, and the volume ratio of acetonitrile to water in the acetonitrile/water mixed solvent is 1: 1.
7. The method of claim 1, wherein the analytical column used in the hplc-tandem mass spectrometry analysis is acquired UPLC HSS T3, specification: 2.1X 100mm,1.8 μm.
8. The method for quantitative analysis of free amino acids in biological samples by liquid chromatography-tandem mass spectrometry as claimed in claim 1, wherein in step 4), the mobile phase in the ultra performance liquid chromatography-tandem mass spectrometry is as follows: phase A is 0.1% formic acid-water, phase B is 0.1% formic acid-acetonitrile; the gradient elution procedure used was: the initial ratio is 100% for phase A and 0% for phase B, and the time is 0.5 min; reducing the phase A to 40% and increasing the phase B to 60% in 0.5-5 min; recovering to the initial gradient equilibrium system at 5.1min to 7.0 min; the flow rate of the mobile phase in the whole elution process is always 0.2 mL/min; the temperature of the column oven is 40 ℃; the sample injection volume is 5-10 mu L; the sample injection needle cleaning solution is a methanol/water mixed solvent, and the volume ratio of methanol to water in the methanol/water mixed solvent is 1: 1.
9. The method for quantitative analysis of free amino acids in biological samples by liquid chromatography-tandem mass spectrometry as claimed in claim 1, wherein in step 4), the ionization mode of tandem mass spectrometry is electrospray ionization in which positive and negative ion modes are mixed; the detection mode is multiplex reaction detection (MRM); the ion source temperature is 200 ℃, the desolventizing temperature is 400 ℃, the desolventizing air flow rate is 10L/min, the capillary voltage is 4.0kV, the taper hole voltage is 30kV, the collision gas is argon, the pressure is 270kPa, and the collision energy is 20V.
10. The method of claim 1, wherein the conditions of the liquid chromatography-tandem mass spectrometry are as follows:
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