CN113009030A - Amino acid high-throughput target detection method and application thereof - Google Patents
Amino acid high-throughput target detection method and application thereof Download PDFInfo
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Abstract
The invention provides an amino acid high-flux target detection method and application thereof. The method specifically comprises the following steps: s1: extracting metabolites; s2: preparing a standard solution; s3: and (5) performing detection and analysis on the machine. The amino acid high-flux target detection method disclosed by the invention has high sensitivity, and can accurately determine the component content of various different amino acids while accurately identifying the amino acids.
Description
Technical Field
The invention relates to the technical field of amino acid detection, in particular to an amino acid high-flux target detection method and application thereof.
Background
Humans must ingest food in order to survive and maintain normal metabolic activity of the body. The protein is a main carrier of biological functions and is also a nutrient substance required by a human body. Amino acids are building block molecules of proteins, and thus are involved in many metabolic pathways of the organism and play a key role. The metabolic condition of the amino acid is judged by analyzing the content of the amino acid, so that the method can help prevent and diagnose a plurality of diseases, not only can protect the health of a human body, but also has important significance for regulating the order of the production and metabolism of various organisms.
However, because of the variety and content of amino acids, there are many difficulties in analyzing the metabolites of amino acids, and how to efficiently and accurately measure the metabolites of amino acids while increasing the flux is one of the problems to be solved at present.
Disclosure of Invention
In order to solve the above technical problems, a first aspect of the present invention provides a method for detecting an amino acid high-throughput target, comprising the following steps: s1: extracting metabolites; s2: preparing a standard solution; s3: and (5) performing detection and analysis on the machine.
As a preferred technical solution, the step S1:
1) placing a sample to be detected in an EP tube, adding the extracting solution, and uniformly mixing by vortex;
2) homogenizing and performing ice-water bath ultrasound, and repeating the steps of homogenizing and ultrasound to obtain a sample solution;
3) standing the sample liquid;
4) centrifuging the sample liquid after standing;
5) and taking the supernatant to an LC sample injection bottle for UHPLC-MS/MS analysis.
As a preferred technical scheme, the extracting solution is prepared from acetonitrile: methanol: water ═ 1-3: (1-3): 1 configuration.
As a further preferable technical scheme, the extracting solution is prepared by mixing acetonitrile: methanol: water 2: 2: 1 configuration.
As a preferable technical scheme, the extracting solution needs to be pre-cooled at the temperature of minus 20 ℃.
As a preferred technical scheme, the extracting solution contains isotope internal standard mixture.
As a preferred technical solution, the number of times of repeating the homogenization sonication step is 2 to 5 times.
As a preferred technical scheme, the vortex mixing time is 20-40 s.
As a preferable technical proposal, the homogenizing frequency is 30-50Hz, and the homogenizing time is 200-280 s; the ultrasonic time of ice water bath is 3-8 min.
As a preferable technical scheme, the standing temperature is-50 to-30 ℃, and the standing time is 0.5 to 1.5 hours.
As a preferred technical scheme, the centrifugation temperature is 3-5 ℃, the centrifugation speed is 10000-15000rpm, and the centrifugation time is 10-20 min.
As a preferred technical solution, the step S2: respectively preparing standard substance stock solutions by placing the standard substances in volumetric flasks; taking a standard substance stock solution in a volumetric flask to prepare a mixed standard solution; the standard solutions were diluted sequentially to give a series of calibration solutions.
As a preferable technical scheme, the concentration of the standard substance stock solution is 10 mmol/L.
As a preferred technical solution, the mobile phase conditions in step S3 are:
liquid chromatography column: ACQUITY UPLC BEH Amide, Specification: 100X 2.1mm, particle size: 1.7 μm;
liquid chromatography phase A: 0.5-1.5% volume fraction aqueous formic acid solution, phase B: 0.5-1.5% volume fraction formic acid/acetonitrile liquid;
the temperature of the column incubator is 30-40 ℃; setting a sample tray at 3-5 ℃; the injection volume is 0.5-1.5. mu.L.
As a preferred technical solution, the mobile phase conditions in step S3 are:
liquid chromatography phase A: 1% volume fraction aqueous formic acid, phase B: 1% volume fraction formic acid/acetonitrile;
the temperature of the column incubator is 35 ℃; the sample tray is set to 4 ℃; the injection volume was 1. mu.L.
As a further preferable technical solution, in the step S3, the mass spectrum condition is:
an ion source: AJS-ESI; detection mode: a multiple reaction monitoring mode;
ion source parameters:
in positive ion mode (+), Capillaryvoltage +4000V, nzzle voltage +500V, gas (N2) temperature 300 ℃, gas (N2) flow 5L/min, sheath gas (N2) temperature 250 ℃, sheath gas flow 11L/min, and nebulizer 45 psi.
In negative ion mode (+), Capillaryvoltage-3500V, nzule voltage-500V, gas (N2) temperature-300 ℃, gas (N2) flow-5L/min, sheath gas (N2) temperature-250 ℃, sheath gas flow-11L/min, and nebulizer-45 psi.
The second aspect of the invention provides an application of the amino acid high-throughput target detection method in biochemical, pharmaceutical and clinical research, and can be used for detecting the amino acid high-throughput targets of organisms such as animals, plants, bacteria, fungi and the like.
Has the advantages that:
1) the amino acid high-flux target detection method has the advantages of safe and nontoxic extraction solvent, short treatment period and high separation efficiency.
2) The high-throughput target detection method for amino acid has high mass spectrum sensitivity and short analysis time, and can accurately determine the component content of the amino acid while accurately identifying the contained amino acid.
3) The amino acid high-flux target detection method can simultaneously carry out quantitative analysis on various different amino acids, and has the advantages of simple operation, good repeatability and accurate and reliable detection result.
Description of the drawings:
FIGS. 1 to 5 are ion chromatograms of 25 amino acid standard solution extractions;
FIG. 1 shows ion chromatograms of Glycine standard solution extraction from top to bottom, RT 1.859; extracting an ion chromatogram map of the L-Alanine standard solution, and carrying out RT 1.543; extracting an ion chromatogram map of a beta-Alanine standard solution, RT 1.395; 4-Aminobiutic acid standard solution extraction ion chromatogram, RT 1.271; extracting an ion chromatogram map by using an L-Serine standard solution, and performing RT 2.600; an IS-5 standard solution extraction ion chromatogram, RT 2.599; an IS-3 standard solution extraction ion chromatogram, RT 1.272;
FIG. 2 shows an L-Proline standard solution extraction ion chromatogram, RT1.382, from top to bottom; an L-Valine standard solution extraction ion chromatogram, RT 1.184; extracting an ion chromatogram map of the L-Threonine standard solution, RT 2.801; 4-Hydroxyproline standard solution extraction ion chromatogram, RT 2.079; an L-Ornithine standard solution extraction ion chromatogram, RT 6.114; an ion chromatogram extracted from the L-Asparagine standard solution, RT 2.912; an ion chromatogram map of the L-Aspartic acid standard solution is extracted, and the RT 2.472;
FIG. 3 shows an IS-6 standard solution extraction ion chromatogram, RT2.468, from top to bottom; extracting an ion chromatogram of the L-Lysine standard solution, RT 5.672; extracting an ion chromatogram of the L-Glutamine standard solution, RT 2.528; extracting an ion chromatogram map of the L-Glutamic acid standard solution, RT 2.064; an ion chromatogram extracted from the L-Methionine standard solution is RT 1.201; IS-8 standard solution extraction ion chromatogram, RT 2.665; an IS-2 standard solution extraction ion chromatogram, RT 1.203;
FIG. 4 shows an IS-4 standard solution extraction ion chromatogram, RT2.063, in sequence from top to bottom; extracting an ion chromatogram from the L-Histidine standard solution, RT 5.857; 5-Hydroxylysine standard solution extraction ion chromatogram, RT 6.536; an ion chromatogram extracted from an L-phenylalkane standard solution is RT 1.049; 3-methyl-L-histidine standard solution extraction ion chromatogram, RT 5.509; 1-methyl-L-histidine standard solution extraction ion chromatogram, RT 4.903; extracting an ion chromatogram of the L-Arginine standard solution, RT 5.415;
FIG. 5 shows an ion chromatogram for extraction of L-Citruline standard solution, RT2.904, from top to bottom; an IS-7 standard solution extraction ion chromatogram, RT 5.414; extracting an ion chromatogram of the L-Tyrosine standard solution, RT 1.330; extracting an ion chromatogram map from the L-Tryptophan standard solution, and performing RT 1.039; an IS-1 standard solution extraction ion chromatogram, RT 1.010;
FIGS. 6-10 are sample solution extracted ion chromatograms of example 1;
FIG. 6 shows the ion chromatogram for Glycine standard solution extraction from top to bottom, RT 1.854; an L-Alanine standard solution extraction ion chromatogram, RT 1.536; extracting an ion chromatogram map of a beta-Alanine standard solution, RT 1.393; 4-Aminobutric acid standard solution extraction ion chromatogram, RT 1.265; extracting an ion chromatogram of the L-Serine standard solution, RT 2.594; an IS-5 standard solution extraction ion chromatogram, RT 2.591; an IS-3 standard solution extraction ion chromatogram, RT 1.265;
FIG. 7 is a sequence of L-Proline standard solution extraction ion chromatograms from top to bottom, RT 1.379; an L-Valine standard solution extraction ion chromatogram, RT 1.179; extracting an ion chromatogram map of the L-Threonine standard solution, RT 2.073; 4-Hydroxyproline standard solution extraction ion chromatogram, RT 2.067; an L-Ornithine standard solution extraction ion chromatogram, RT 6.115; an ion chromatogram extracted from the L-Asparagine standard solution, RT 2.904; extracting an ion chromatogram map of the L-Aspartic acid standard solution, RT 2.469;
FIG. 8 shows an IS-6 standard solution extraction ion chromatogram, RT2.467, from top to bottom; extracting an ion chromatogram of the L-Lysine standard solution, RT 5.665; extracting an ion chromatogram of the L-Glutamine standard solution, RT 2.519; extracting an ion chromatogram map of the L-Glutamic acid standard solution, RT 2.061; an ion chromatogram extracted from the L-Methionine standard solution is RT 1.198; IS-8 standard solution extraction ion chromatogram, RT 5.657; an IS-2 standard solution extraction ion chromatogram, RT 1.198;
FIG. 9 shows an IS-4 standard solution extraction ion chromatogram, RT2.060, from top to bottom; extracting an ion chromatogram from the L-Histidine standard solution, RT 5.854; 5-Hydroxylysine standard solution extraction ion chromatogram, RT 6.54; an ion chromatogram extracted from an L-phenylalkane standard solution is RT 1.047; 3-methyl-L-histidine standard solution extraction ion chromatogram, RT 5.503; 1-methyl-L-histidine standard solution extraction ion chromatogram, RT 4.911; extracting an ion chromatogram of the L-Arginine standard solution, RT 5.405;
FIG. 10 shows an ion chromatogram for extraction of L-Citruline standard solution, RT2.896, from top to bottom; IS-7 standard solution extraction ion chromatogram, RT 5.403; extracting an ion chromatogram of the L-Tyrosine standard solution, RT 1.330; extracting an ion chromatogram map from the L-Tryptophan standard solution, and performing RT 1.038; IS-1 standard solution extraction ion chromatogram, RT 1.039.
Detailed Description
In order to solve the above problems, the present invention provides a method for detecting an amino acid high-throughput target, comprising the following steps: s1: extracting metabolites; s2: preparing a standard solution; s3: and (5) performing detection and analysis on the machine.
As a preferred technical solution, the step S1:
1) placing a sample to be detected in an EP tube, adding the extracting solution, and uniformly mixing by vortex;
2) homogenizing and performing ice-water bath ultrasound, and repeating the steps of homogenizing and ultrasound to obtain a sample solution;
3) standing the sample liquid;
4) centrifuging the sample liquid after standing;
5) and taking the supernatant to an LC sample injection bottle for UHPLC-MS/MS analysis.
As a preferred technical scheme, the extracting solution is prepared from acetonitrile: methanol: water ═ 1-3: (1-3): 1 configuration.
As a further preferable technical scheme, the extracting solution is prepared by mixing acetonitrile: methanol: water 2: 2: 1 configuration.
As a preferable technical scheme, the extracting solution needs to be pre-cooled at the temperature of minus 20 ℃.
As a preferred technical scheme, the extracting solution contains isotope internal standard mixture.
As a preferred technical solution, the number of times of repeating the homogenization sonication step is 2 to 5 times.
As a preferred technical scheme, the vortex mixing time is 20-40 s.
As a preferable technical proposal, the homogenizing frequency is 30-50Hz, and the homogenizing time is 200-280 s; the ultrasonic time of ice water bath is 3-8 min.
As a preferable technical scheme, the standing temperature is-50 to-30 ℃, and the standing time is 0.5 to 1.5 hours.
As a preferred technical scheme, the centrifugation temperature is 3-5 ℃, the centrifugation speed is 10000-15000rpm, and the centrifugation time is 10-20 min.
As a preferred technical solution, the step S2: respectively preparing standard substance stock solutions by placing the standard substances in volumetric flasks; taking a standard substance stock solution in a volumetric flask to prepare a mixed standard solution; the standard solutions were diluted sequentially to give a series of calibration solutions.
As a preferable technical scheme, the concentration of the standard substance stock solution is 10 mmol/L.
As a preferred technical solution, the mobile phase conditions in step S3 are:
liquid chromatography column: ACQUITY UPLC BEH Amide, Specification: 100X 2.1mm, particle size: 1.7 μm;
liquid chromatography phase A: 0.5-1.5% volume fraction aqueous formic acid solution, phase B: 0.5-1.5% volume fraction formic acid/acetonitrile;
the temperature of the column incubator is 30-40 ℃; setting a sample tray at 3-5 ℃; the injection volume is 0.5-1.5. mu.L.
As a further preferable technical solution, in the step S3, the mobile phase conditions are:
liquid chromatography phase A: 1% volume fraction aqueous formic acid, phase B: 1% volume fraction formic acid/acetonitrile;
the temperature of the column incubator is 35 ℃; the sample tray is set to 4 ℃; the injection volume was 1. mu.L.
As a preferred technical solution, the gradient elution conditions in step S3 are:
0-3.0 min: mobile phase A: 15% -20%, mobile phase B: 80% -85%;
3.0-5.0 min: mobile phase A: 20% -50%, mobile phase B: 20% -50%;
5.0-8.8 min: mobile phase A: 50% -50%, mobile phase B: 50% -50%;
8.7-13.00 min: mobile phase A: 15% -20%, mobile phase B: 80% -85%;
the gradient elution flow rate was 0.400 mL/min.
As a preferred technical solution, the mass spectrum condition in step S3 is:
an ion source: AJS-ESI; detection mode: a multiple reaction monitoring mode;
ion source parameters:
in positive ion mode (+), Capillaryvoltage +4000V, nzzle voltage +500V, gas (N2) temperature 300 ℃, gas (N2) flow 5L/min, sheath gas (N2) temperature 250 ℃, sheath gas flow 11L/min, and nebulizer 45 psi.
In negative ion mode (+), Capillaryvoltage-3500V, nzule voltage-500V, gas (N2) temperature-300 ℃, gas (N2) flow-5L/min, sheath gas (N2) temperature-250 ℃, sheath gas flow-11L/min, and nebulizer-45 psi.
The standard solution was introduced into the mass spectrum before UHPLC-MS/MS analysis was performed. For each amino acid, selecting a plurality of parent ion-daughter ion pairs (transitions) with the highest signal intensity, optimizing the MRM parameters of the parent ion-daughter ion pairs, and selecting the ion pair with the best response for quantitative analysis and other ion pairs for qualitative analysis of the amino acid.
The second aspect of the invention provides an application of the amino acid high-throughput target detection method in biochemical, pharmaceutical and clinical research, and can be used for detecting the amino acid high-throughput targets of organisms such as animals, plants, bacteria, fungi and the like.
The present invention will be specifically described below by way of examples. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and that the insubstantial modifications and adaptations of the present invention by those skilled in the art based on the above disclosure are still within the scope of the present invention.
In addition, the starting materials used are all commercially available, unless otherwise specified.
Examples
Example 1
A method for detecting an amino acid high-throughput target comprises the following steps: s1: extracting metabolites; s2: preparing a standard solution; s3: and (5) performing detection and analysis on the machine.
The step S1:
1) placing 21.6mg of sample to be tested in an EP tube, adding 1000ul of the extracting solution, and mixing uniformly by vortex;
2) homogenizing and performing ice-water bath ultrasound, and repeating the steps of homogenizing and ultrasound to obtain a sample solution;
3) standing the sample liquid;
4) centrifuging the sample liquid after standing;
5) and taking the supernatant to an LC sample injection bottle for UHPLC-MS/MS analysis.
The sample to be detected is rice flour.
The extracting solution is acetonitrile according to the volume ratio: methanol: water 2: 2: 1 configuration.
The extract is pre-cooled at-20 deg.C.
The extract contains a mixture of isotopic internal standards.
The number of times of repeating the homogenization sonication step was 3 times.
The vortex mixing time was 30 s.
The homogenizing frequency is 40Hz, and the homogenizing time is 240 s; the ultrasonic time of ice-water bath is 5 min.
The standing temperature is-40 ℃, and the standing time is 1 h.
The centrifugation temperature is 4 ℃, the centrifugation speed is 12000rpm, and the centrifugation time is 15 min.
The step S2: respectively preparing standard substance stock solutions by placing the standard substances in volumetric flasks; taking a standard substance stock solution in a volumetric flask to prepare a mixed standard solution; the standard solutions were diluted sequentially to give a series of calibration solutions.
The concentration of the standard substance stock solution is 10 mmol/L.
Amino acid series calibration solution concentrations (nmol/L) as described in Table 1.
| Cal-01.d | Cal-02.d | Cal-03.d | Cal-04.d | Cal-05.d | Cal-06.d | Cal-07.d | Cal-08.d | |
| Conc. | 40000.00 | 20000.00 | 10000.00 | 5000.00 | 2500.00 | 1250.00 | 625.00 | 312.50 |
| Cal-09.d | Cal-10.d | Cal-11.d | Cal-12.d | Cal-13.d | Cal-14.d | Cal-15.d | Cal-16.d | |
| Conc. | 156.25 | 78.13 | 39.06 | 19.53 | 9.77 | 4.88 | 2.44 | 1.22 |
Mobile phase conditions in said step S3:
liquid chromatography column: ACQUITY UPLC BEH Amide, Specification: 100X 2.1mm, particle size: 1.7 μm;
liquid chromatography phase A: 1% volume fraction aqueous formic acid, phase B: 1% volume fraction formic acid/acetonitrile solution;
the temperature of the column incubator is 35 ℃; the sample tray is set to 4 ℃; the injection volume was 1. mu.L.
The gradient elution conditions in step S3 are as follows:
0-3.0 min: mobile phase A: 15% -20%, mobile phase B: 80% -85%;
3.0-5.0 min: mobile phase A: 20% -50%, mobile phase B: 20% -50%;
5.0-8.8 min: mobile phase A: 50% -50%, mobile phase B: 50% -50%;
8.7-13.00 min: mobile phase A: 15% -20%, mobile phase B: 80% -85%;
the gradient elution rate was 0.400 mL/min.
Mass spectrum condition in the step S3:
an ion source: AJS-ESI; detection mode: a multiple reaction monitoring mode;
ion source parameters:
in positive ion mode (+), Capillaryvoltage +4000V, nzzle voltage +500V, gas (N2) temperature 300 ℃, gas (N2) flow 5L/min, sheath gas (N2) temperature 250 ℃, sheath gas flow 11L/min, and nebulizer 45 psi.
In negative ion mode (+), Capillaryvoltage-3500V, nzule voltage-500V, gas (N2) temperature-300 ℃, gas (N2) flow-5L/min, sheath gas (N2) temperature-250 ℃, sheath gas flow-11L/min, and nebulizer-45 psi.
Example 2
A method for detecting an amino acid high-throughput target, which is implemented in the same manner as in example 1, except that the homogenization frequency is 45 Hz.
Example 3
The specific implementation mode of the method for detecting the amino acid high-flux target is the same as that of the example 1, except that the temperature of the column oven is 38 ℃.
Comparative example 1
The specific implementation mode of the method for detecting the high-flux target of the amino acid is the same as that in example 1, except that the extracting solution comprises the following components in percentage by volume: methanol: water-4: 2: 1 configuration.
Comparative example 2
The specific implementation mode of the method for detecting the high-flux target of the amino acid is the same as that in example 1, except that the extracting solution comprises the following components in percentage by volume: methanol: water 1: 4: 1 configuration.
Comparative example 3
The amino acid high-throughput target detection method is implemented in the same way as in example 1, except that the liquid chromatography A phase: 3% volume fraction aqueous formic acid, phase B: 1% volume fraction formic acid/acetonitrile.
Comparative example 4
The amino acid high-throughput target detection method is implemented in the same way as in example 1, except that the liquid chromatography A phase: 1% volume fraction aqueous formic acid, phase B: 3% volume fraction formic acid/acetonitrile.
Performance testing
The standard solution was introduced into the mass spectrum before UHPLC-MS/MS analysis was performed. For each amino acid, selecting a plurality of parent ion-daughter ion pairs (transitions) with the highest signal intensity, optimizing the MRM parameters of the parent ion-daughter ion pairs, and selecting the ion pair with the best response for quantitative analysis and other ion pairs for qualitative analysis of the amino acid.
TABLE 2 ion pair parameters for amino acid analysis
| Compound Name | Prec Ion | Prod Ion | Polarity | Quantifier/Qualifier |
| 1-Methyhistidine | 170.1 | 124.0 | Positive | Quantifier |
| 1-Methyhistidine | 170.1 | 83.0 | Positive | Qualifier |
| 3-Methyhistidine | 170.1 | 96.0 | Positive | Quantifier |
| 3-Methyhistidine | 170.1 | 126.0 | Positive | Qualifier |
| 4-Aminobutyric acid | 104.1 | 87.1 | Positive | Quantifier |
| 4-Aminobutyric acid | 104.1 | 69.1 | Positive | Qualifier |
| Hydroxyproline | 132.1 | 86.0 | Positive | Quantifier |
| Hydroxyproline | 132.1 | 68.1 | Positive | Qualifier |
| 5-Hydroxylysine | 163.1 | 128.0 | Positive | Quantifier |
| 5-Hydroxylysine | 163.1 | 82.0 | Positive | Qualifier |
| beta-Alanine | 90.1 | 72.1 | Positive | Quantifier |
| beta-Alanine | 90.1 | 30.1 | Positive | Qualifier |
| Glycine | 76.1 | 30.2 | Positive | Quantifier/Qualifier |
| Alanine | 90.1 | 44.1 | Positive | Quantifier/Qualifier |
| Arginine | 175.1 | 70.0 | Positive | Quantifier |
| Arginine | 175.1 | 116.0 | Positive | Qualifier |
| Asparagine | 133.1 | 87.0 | Positive | Quantifier |
| Asparagine | 133.1 | 116.0 | Positive | Qualifier |
| Aspartic acid | 134.1 | 88.1 | Positive | Quantifier |
| Aspartic acid | 134.1 | 116.0 | Positive | Qualifier |
| Citrulline | 176.1 | 159.0 | Positive | Quantifier |
| Citrulline | 176.1 | 113.0 | Positive | Qualifier |
| Glutamic acid | 148.1 | 84.1 | Positive | Quantifier |
| Glutamic acid | 148.1 | 130.0 | Positive | Qualifier |
| Glutamine | 147.1 | 130.0 | Positive | Quantifier |
| Glutamine | 147.1 | 84.1 | Positive | Qualifier |
| Histidine | 156.1 | 110.0 | Positive | Quantifier |
| Histidine | 156.1 | 93.0 | Positive | Qualifier |
| Lysine | 147.1 | 84.1 | Positive | Quantifier |
| Lysine | 147.1 | 130.0 | Positive | Qualifier |
| Methionine | 150.1 | 104.0 | Positive | Quantifier |
| Methionine | 150.1 | 56.1 | Positive | Qualifier |
| Ornithine | 133.1 | 70.0 | Positive | Quantifier |
| Ornithine | 133.1 | 116.0 | Positive | Qualifier |
| Phenylalanine | 166.1 | 120.0 | Positive | Quantifier |
| Phenylalanine | 166.1 | 77.1 | Positive | Qualifier |
| Proline | 116.1 | 70.2 | Positive | Quantifier/Qualifier |
| Serine | 106.1 | 60.1 | Positive | Quantifier |
| Serine | 106.1 | 88.1 | Positive | Qualifier |
| Threonine | 120.1 | 74.1 | Positive | Quantifier |
| Threonine | 120.1 | 56.1 | Positive | Qualifier |
| Tryptophan | 205.1 | 188.0 | Positive | Quantifier |
| Tryptophan | 205.1 | 146.0 | Positive | Qualifier |
| Tyrosine | 182.1 | 136.0 | Positive | Quantifier |
| Tyrosine | 182.1 | 91.0 | Positive | Qualifier |
| Valine | 118.1 | 72.1 | Positive | Quantifier |
| Valine | 118.1 | 55.1 | Positive | Qualifier |
In the performance test, all mass spectrum data acquisition and amino acid quantitative analysis Work are completed by Agilent Mass Hunter Work Station Software (B.08.00, Agilent Technologies) by selecting IS 1-8 as an internal standard.
TABLE 3 internal standards
TABLE 4 internal standards corresponding to amino acids
| L-Tryptophan | IS 1 | L-Aspartic acid | IS 6 |
| L-Phenylalanine | IS 1 | L-Glutamine | IS 4 |
| L-Valine | IS 2 | L-Serine | IS 5 |
| L-Methionine | IS 2 | L-Citrulline | IS 5 |
| 4-Aminobutyric acid | IS 3 | L-Asparagine | IS 5 |
| L-Tyrosine | IS 3 | 1-Methyl-L-histidine | IS 7 |
| L-Proline | IS 3 | L-Arginine | IS 7 |
| Beta-Alanine | IS 3 | 3-Methyl-L-histidine | IS 7 |
| L-Alanine | IS 4 | L-Lysine | IS 8 |
| Glycine | IS 4 | L-Histidine | IS 7 |
| L-Glutamic acid | IS 4 | L-Ornithine | IS 7 |
| 4-Hydroxyproline | IS 4 | 5-Hydroxylysine | IS 7 |
| L-Threonine | IS 4 |
1. Chromatographic separation
The sample liquid and the standard solution in example 1 were chromatographed.
2. Calibration curve
UPLC-MRM-MS/MS analysis was performed on the standard solution. y represents the ratio of the peak areas of the amino acid and the corresponding internal standard substance, and x represents the concentration of the amino acid (nmol/L). The regression analysis was performed by the least squares method, and the standard solution recovery (accuracy) and the correlation coefficient (R2) were the best when the weight was set to 1/x. If the recovery rate of a standard concentration exceeds the range of 80-120%, the concentration calibration point is excluded.
3. Method detection limit and quantification limit
And sequentially diluting the standard solution by 2 times, performing UHPLC-MRM-MS analysis, and calculating the detection limit and the quantitative limit of the method according to the signal-to-noise ratio of the standard solution. The lowest detection limit (LLOD) is defined as the concentration of amino acid corresponding to a signal-to-noise ratio of 3, and the lowest limit of quantitation (LLOQ) is defined as the concentration of amino acid corresponding to a signal-to-noise ratio of 10 (US FDA gulideline for biological assay).
4. Method precision and accuracy
QC samples were taken for UHPLC-MRM-MS analysis and precision was assessed by the standard relative deviation (RSD) of repeat injection. Accuracy was assessed by Recovery on spiking, and the percentage value of measured concentration to spiked concentration was the spiked Recovery. The number of times of repeated sample injection of the QC sample is 5.
TABLE 5QC sample conditions
| Compound Name | 3-Methyl-L-histidine | L-Serine | L-Phenylalanine | L-Tryptophan | L-Threonine |
| Concentration(nmol/L) | 1500.00 | 2000.00 | 2000.00 | 2000.00 | 2000.00 |
| Compound Name | L-Citrulline | L-Aspartic acid | L-Asparagine | L-Arginine | L-Lysine |
| Concentration(nmol/L) | 2000.00 | 2000.00 | 2000.00 | 2000.00 | 2000.00 |
| Compound Name | L-Valine | L-Methionine | 5-Hydroxylysine | L-Glutamic acid | 4-Aminobutyric acid |
| Concentration(nmol/L) | 2000.00 | 2000.00 | 2000.00 | 2000.00 | 2000.00 |
| Compound Name | 1-Methyl-L-histidine | Beta-Alanine | L-Glutamine | L-Ornithine | Glycine |
| Concentration(nmol/L) | 2000.00 | 2000.00 | 2000.00 | 2000.00 | 2000.00 |
| Compound Name | L-Histidine | L-Proline | L-Alanine | L-Tyrosine | 4-Hydroxyproline |
| Concentration(nmol/L) | 2000.00 | 2000.00 | 2000.00 | 2000.00 | 2000.00 |
Test results and analysis
1. The chromatographic separation is shown in figure 1 and figure 2 of the attached drawings of the specification
2. Table 6 example 1 performance tests 2-3 specific results
| Compound Name | LLOD(nmol/L) | LLOQ(nmol/L) | ULOQ(nmol/L) | R^2 |
| L-Tryptophan | 4.88 | 9.77 | 20000.00 | 0.9995 |
| L-Phenylalanine | 19.53 | 39.06 | 40000.00 | 0.9998 |
| L-Valine | 2.44 | 9.77 | 40000.00 | 0.9993 |
| L-Methionine | 4.88 | 9.77 | 40000.00 | 0.9997 |
| 4-Aminobutyric acid | 4.88 | 19.53 | 20000.00 | 0.9996 |
| L-Proline | 39.06 | 78.13 | 20000.00 | 0.9993 |
| L-Tyrosine | 2.44 | 156.25 | 40000.00 | 0.9983 |
| Beta-Alanine | 19.53 | 39.06 | 5000.00 | 0.9994 |
| L-Alanine | 19.53 | 39.06 | 20000.00 | 0.9987 |
| Glycine | 1.22 | 156.25 | 20000.00 | 0.9995 |
| L-Threonine | 9.77 | 156.25 | 40000.00 | 0.9992 |
| L-Glutamic acid | 4.88 | 39.06 | 40000.00 | 0.9992 |
| 4-Hydroxyproline | 19.53 | 156.25 | 20000.00 | 0.9993 |
| L-Glutamine | 4.88 | 156.25 | 40000.00 | 0.9998 |
| L-Aspartic acid | 4.88 | 39.06 | 40000.00 | 0.9997 |
| L-Serine | 1.22 | 39.06 | 40000.00 | 0.9997 |
| L-Asparagine | 19.53 | 39.06 | 10000.00 | 0.9975 |
| L-Citrulline | 1.22 | 39.06 | 20000.00 | 0.9996 |
| 1-Methyl-L-histidine | 39.06 | 78.13 | 20000.00 | 0.9998 |
| L-Arginine | 1.22 | 78.13 | 40000.00 | 0.9996 |
| 3-Methyl-L-histidine | 7.32 | 14.65 | 15000.00 | 0.9997 |
| L-Lysine | 9.77 | 39.06 | 40000.00 | 0.9999 |
| L-Histidine | 4.88 | 39.06 | 40000.00 | 0.9997 |
| L-Ornithine | 1.22 | 39.06 | 40000.00 | 0.9998 |
| 5-Hydroxylysine | 4.88 | 9.77 | 20000.00 | 0.9997 |
3. TABLE 7 precision and accuracy of the method of example 1
| Compound Name | Recovery | RSD | Compound Name | Recovery | RSD |
| 3-Methyl-L-histidine | 98.8 | 1.6％ | L-Glutamic acid | 102.4 | 3.9％ |
| L-Serine | 96.5 | 3.1％ | 4-Aminobutyric acid | 93.3 | 3.0％ |
| L-Phenylalanine | 94.6 | 1.3％ | 1-Methyl-L-histidine | 102.0 | 1.8％ |
| L-Tryptophan | 95.1 | 1.6％ | Beta-Alanine | 109.8 | 2.9％ |
| L-Threonine | 89.2 | 3.2％ | L-Glutamine | 87.8 | 3.7％ |
| L-Citrulline | 90.7 | 1.5％ | L-Ornithine | 103.7 | 2.2％ |
| L-Aspartic acid | 97.3 | 3.9％ | Glycine | 91.7 | 3.1％ |
| L-Asparagine | 101.0 | 2.8％ | L-Histidine | 97.4 | 2.5％ |
| L-Arginine | 93.0 | 2.3％ | L-Proline | 107.1 | 2.7％ |
| L-Lysine | 94.3 | 0.9％ | L-Alanine | 93.2 | 3.7％ |
| L-Valine | 103.0 | 2.2％ | L-Tyrosine | 106.2 | 2.7％ |
| L-Methionine | 102.2 | 1.8％ | 4-Hydroxyproline | 87.9 | 2.2％ |
| 5-Hydroxylysine | 101.6 | 2.5％ |
4. Table 8 example 1 actual quantitative test results of sample liquid
5. TABLE 9 test results of examples 2-3 and comparative examples 1-4 (as represented by the partial test results of 3-Methyl-L-histidine, due to the large amount of data).
| LLOQ(nmol/L) | Recovery | RSD | |
| Comparative example 1 | 18.22 | 92.0 | 2.3％ |
| Comparative example 2 | 18.00 | 93.5 | 2.3％ |
| Comparative example 3 | 23.25 | 88.8 | 3.2％ |
| Comparative example 4 | 24.40 | 86.7 | 2.9％ |
As can be seen by comparing FIG. 1 and FIG. 2, all amino acids exhibited symmetrical chromatographic peaks; the chromatographic separation of each amino acid is well realized, and the chromatographic peak shapes in the biological sample and the standard solution have no obvious difference.
Table 6 was analyzed to determine the minimum detection limits (LLODs) of 1.22-39.06 nmol/L and the minimum quantification limits (LLOQs) of 9.77-156.25 nmol/L for the above amino acids; the correlation coefficient (R2) of all amino acids is larger than 0.9975, which shows that the peak area of the chromatographic peak and the concentration of the compound have good quantitative relation and can meet the requirement of targeted metabonomics analysis.
The analysis of Table 7 shows that the average recovery rate of all amino acids is between 87.8% and 109.8%, and the standard relative deviation is less than 3.9%, which indicates that the amino acid high-throughput target detection method of the invention has high recovery rate and accurate detection result.
Table 8 shows that the retention time of each amino acid in the biological sample is moderate.
Comparing example 1 with comparative example 1 and comparative example 2, it can be seen that the component ratio of the extracting solution in the comparative example is changed, so that amino acid cannot be well recovered, and the accuracy of detection is affected; comparing example 1 with comparative example 3 and comparative example 4, it can be seen that the liquid chromatogram A phase and B phase selected by the invention can well detect and separate various amino acids, improve the recovery rate and ensure the result to be accurate and reliable.
The analysis of the data shows that the method can accurately and reliably detect the content of the target amino acid in the sample within the concentration range shown above.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in other forms, and any person skilled in the art may modify or change the technical content of the above disclosure into equivalent embodiments with equivalent changes, but all those simple modifications, equivalent changes and modifications made to the above embodiments according to the technical spirit of the present invention still belong to the protection scope of the present invention.
Claims (10)
1. The method for detecting the amino acid high-throughput target is characterized by comprising the following steps of: s1: extracting metabolites; s2: preparing a standard solution; s3: and (5) performing detection and analysis on the machine.
2. The method for detecting the high-throughput target of claim 1, wherein the step S1:
1) placing a sample to be detected in an EP tube, adding the extracting solution, and uniformly mixing by vortex;
2) homogenizing and performing ice-water bath ultrasound, and repeating the steps of homogenizing and ultrasound to obtain a sample solution;
3) standing the sample liquid;
4) centrifuging the sample liquid after standing;
5) and taking the supernatant to an LC sample injection bottle for UHPLC-MS/MS analysis.
3. The method for detecting the high-throughput target of the amino acid according to claim 2, wherein the volume ratio of the extracting solution to acetonitrile: methanol: water ═ 1-3: (1-3): 1 configuration.
4. The method for detecting the high-throughput target of amino acid as claimed in claim 2, wherein the homogenization frequency is 30-50Hz, and the homogenization time is 200-280 s; the ultrasonic time of ice water bath is 3-8 min.
5. The method for detecting the high-throughput target of the amino acid according to claim 2, wherein the standing temperature is-50 ℃ to-30 ℃, and the standing time is 0.5 h to 1.5 h.
6. The method for detecting the high-throughput target of claim 1, wherein the step S2: respectively preparing standard substance stock solutions by placing the standard substances in volumetric flasks; taking a standard substance stock solution in a volumetric flask to prepare a mixed standard solution; the standard solutions were diluted sequentially to give a series of calibration solutions.
7. The method for detecting the high-throughput target of claim 1, wherein the mobile phase conditions in step S3 are as follows:
liquid chromatography column: ACQUITY UPLC BEH Amide, Specification: 100X 2.1mm, particle size: 1.7 μm;
liquid chromatography phase A: 0.5-1.5% volume fraction aqueous formic acid solution, phase B: 0.5-1.5% volume fraction formic acid/acetonitrile;
the temperature of the column incubator is 30-40 ℃; setting a sample tray at 3-5 ℃; the injection volume is 0.5-1.5. mu.L.
8. The method for detecting the high-throughput target of claim 7, wherein the mobile phase conditions in step S3 are as follows:
liquid chromatography phase A: 1% volume fraction aqueous formic acid, phase B: 1% volume fraction formic acid/acetonitrile;
the temperature of the column incubator is 35 ℃; the sample tray is set to 4 ℃; the injection volume was 1. mu.L.
9. The method for detecting the high-throughput target of claim 1, wherein the mass spectrum conditions in the step S3 are as follows:
an ion source: AJS-ESI; detection mode: a multiple reaction monitoring mode;
ion source parameters:
in positive ion mode (+), Capillaryvoltage +4000V, nzzle voltage +500V, gas (N2) temperature 300 ℃, gas (N2) flow 5L/min, sheath gas (N2) temperature 250 ℃, sheath gas flow 11L/min, nebulizer 45 psi;
in negative ion mode (+), Capillaryvoltage-3500V, nzule voltage-500V, gas (N2) temperature-300 ℃, gas (N2) flow-5L/min, sheath gas (N2) temperature-250 ℃, sheath gas flow-11L/min, and nebulizer-45 psi.
10. Use of a method for high-throughput target detection of amino acids according to any one of claims 1-9 in biochemical, pharmaceutical and clinical research.
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