CN114236025A - Liquid phase mass spectrum method for simultaneously determining 43 amino acids without using ion pair reagent and non-derivatization - Google Patents
Liquid phase mass spectrum method for simultaneously determining 43 amino acids without using ion pair reagent and non-derivatization Download PDFInfo
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Abstract
The invention discloses a method for simultaneously determining 43 amino acids without using an ion pair reagent and non-derivatization, which comprises the steps of firstly adding 41 isotope labeled internal standards, pretreating a biological sample by a protein precipitation/extraction method, then performing chromatographic separation and mass spectrometry detection, respectively taking the relative retention time and a qualitative ion pair of each amino acid as qualitative bases, and making a standard curve for quantification by using a standard product. The method realizes the purpose of simultaneously analyzing and detecting 43 amino acids in one sample by applying HPLC-MS/MS technology without using ion pair reagent and non-derivatization treatment, has the advantages of no ion pair pollution to instruments, simple and quick operation, high specificity, high flux and low cost, effectively monitors the level of amino acids in a human body in real time, provides sufficient basis for clinical disease diagnosis, and is easy to popularize and popularize clinically.
Description
Technical Field
The invention belongs to the field of amino acid detection, and relates to a liquid mass spectrometry method for simultaneously determining 43 amino acids without using an ion pair reagent and non-derivatization.
Background
Amino acids are the basic constituent substances of proteins, which are the material basis of life. Changes in body amino acid concentrations provide information for the diagnosis of related diseases, and serve as specific biomarkers to identify diseases. For example: phenylalanine blood disease (PKU) is a congenital disorder of amino acid metabolism abnormality, and can be measured by blood phenylalanine due to a deficiency in tyrosine synthase leading to accumulation of phenylalanine and lack of tyrosine.
The detection technology of amino acid comprises high performance liquid chromatography, an amino acid automatic analyzer, capillary electrophoresis, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry combined method and the like. High performance liquid chromatography generally uses an optical detector to perform derivatization treatment on a sample, and has the disadvantages of complicated pretreatment steps, high cost of derivatization reagents, long analysis time and the like. The amino acid automatic analyzer is derived by ninhydrin, and can be used for visible light photometric detection, with high cost, long analysis time and large sample consumption. Under the action of a high-voltage electric field, a capillary electrophoresis method takes a capillary as a separation channel, is a high-efficiency micro-separation platform for analyzing small volume, and is not good enough when a complex sample is quantified. The gas chromatography-mass spectrometry analysis of amino acid needs to derive the amino acid into easily gasified derivatives, has complex derivation steps and much interference in derivation reaction, is not suitable for thermolabile amino acid, and cannot realize the determination of all amino acids by using 1 chromatographic column. The liquid chromatography-mass spectrometry combined method utilizes the advantages of the high separation capability of ultra-high performance liquid chromatography and the structure identification of triple quadrupole mass spectrometry to make up for the defects of high performance liquid chromatography separation and the defects of sensitivity and specificity of an optical/electrochemical detector, and is considered to be the method with the greatest prospect for biological sample analysis by virtue of the advantages of excellent separation capability, high specificity and sensitivity, short analysis time and the like.
At present, the liquid chromatography-mass spectrometry combined method is used for analyzing amino acids in biological samples, and the pretreatment method is divided into a derivation method and a non-derivation method. The derivative reagent comprises 6-aminoquinoline-N-hydroxysuccinimide carbamate (AQC), N-butanol hydrochloride, Phenyl Isothiocyanate (PITC), o-phthalaldehyde (OPA) + 9-Fluorenylmethylchloroformate (FMOC), N- (4-nitrophenoxycarbonyl) -L-phenylalanine 2-methoxyethyl ((S) -NIFE), iTRAQ reagent in an iITARTM kit, and the like, and the derivative after the amino acid derivative is indirectly detected, so that the amino acid content in the organism is judged. The derivatization operation can be helpful for liquid phase separation of amino acid isomers and improvement of detection sensitivity, but derivatization pretreatment is complex in derivatization step, difficult in product production, poor in stability of derivatization reagent and high in cost, and development of a cheap non-derivatization method which can effectively separate multiple groups of amino acid isomers, is simple and convenient in pretreatment method and has high sensitivity and stability is an important direction in amino acid research.
According to the method for quantifying the amino acid in the non-derivatization pretreatment sample, an ion pair reagent is mostly used for detection, the ion pair reagent can improve the separation degree of amino acid isomers, but in mass spectrometry detection, the ion pair reagent can cause pollution of the whole LC-MS, so that instrument signals are reduced and noise is enhanced, and the ion pair reagent is not used in the mass spectrometry detection. At present, no effective method for directly detecting more than 40 endogenous amino acids and 41D/13C/15N labeled isotope amino acids in a body exists for detecting non-derivatized amino acids under the condition of not adding an ion pair reagent.
Disclosure of Invention
In view of the technical problems in the prior art, the present invention aims to provide a liquid mass spectrometry method for simultaneously measuring 43 amino acids and 41D/13C/15N labeled isotope amino acids without adding an ion pair reagent and without derivatization. The method has the advantages of no ion pair pollution to instruments, simple pretreatment, strong specificity, high detection sensitivity, short liquid phase sample running time and high flux.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention firstly provides a method for simultaneously determining 43 amino acids without using an ion pair reagent and non-derivatization, which comprises the following steps:
1) pretreating a sample, and adding an internal standard mixed solution in the pretreatment process to obtain a sample to be detected;
2) carrying out qualitative and internal standard quantitative detection on 43 amino acids in a sample by using a UPLC-MS/MS method, wherein the internal standard quantitative detection comprises the following steps: preparing a standard curve by using the mixed standard solutions with eight concentrations, wherein the concentration ratio of the standard solution to the internal standard is taken as an X axis, and the peak area ratio of the standard solution to the internal standard is taken as a Y axis; carrying out linear regression analysis to obtain a regression equation, substituting the peak area of the corresponding amino acid into the regression equation, and calculating the concentration of the amino acid in the sample;
wherein the liquid phase chromatographic conditions are as follows: a chromatographic column: c18 column, mobile phase a: water containing 0.1% formic acid +10mM ammonium formate + 20% acetonitrile, mobile phase B: containing 0.1% formic acid +10mM ammonium formate + 90% acetonitrile in water; gradient elution conditions: 0-3.25min, 95% B; changing from 95% B to 55% B in 3.25-6.25 min; gradually changing from 55% B to 50% B in 6.25-6.5 min; changing from 50% B to 0% B gradually for 6.5-6.8 min; 6.8-7.3min, 0% B; 7.31-7.8min, 20% B; changing from 20% B to 95% B in 7.8-8 min; 8-10min, flow rate 0.3mL/min, 95% B.
The mass spectrum adopts a multi-ion reaction monitoring mode of positive ion electrospray ionization;
the 43 amino acids are: lysine (Lys), histidine (His), arginine (Arg), alanine (Ala), glutamic acid (Glu), isoleucine (Ile), phenylalanine (Phe), serine (Ser), valine (Val), threonine (Thr), tyrosine (Tyr), asparagine (Asn), tryptophan (Trp), glutamine (Gln), methionine (Met), aspartic acid (Asp), glycine (Gly), leucine (Leu), proline (Pro), cysteine (Cys), phosphoethanolamine (PEtN), gamma-aminobutyric acid (GABA), taurine (Tau), beta-aminoisobutyric acid (bAib), ethanolamine (EtN), ornithine (Orn), anserine (Ans), carnosine (Car), citrulline (Cit), hydroxyproline (Hyp), 1-methylhistidine (1MHis), 3-methylhistidine (3MHis), Hydroxylysine (Hylys), homocitrulline (Hcit), homocysteine (Hcy), alpha-aminoadipic acid (Aad), alpha-aminobutyric acid (Abu), sarcosine (Sar), phosphoserine (P-Ser), beta alanine (beta-Ala), kynurenine (Kyn), argininosuccinic acid (Asa), cystathionine (Cth);
the internal standard mixed solution comprises 41 kinds of amino acid internal standards which are respectively as follows: D8-L-lysine hydrochloride, 13C 6-L-histidine hydrochloride, D7-arginine, D3-alanine, D5-glutamic acid, D10-L-isoleucine, D5-phenylalanine, D3-L-serine, D8-valine, D2-L-threonine, D6-tyrosine, D3-L-asparagine, D3-L-tryptophan, D5-glutamine, D3-methionine, D3-aspartic acid, D5-glycine, D3-leucine, D3-L-proline, 13C 3-L-cysteine, D4-phosphorylethanolamine, D6-gamma-aminobutyric acid, D4-taurine, D3-3-aminoisobutyric acid hydrochloride, D4623-L-proline, 13C 2-ethanolamine, D7-ornithine, D4-L-anserine, D4-L-carnosine, D2-L-citrulline, D4-L-hydroxyproline, D3-1-methyl-L-histidine, D3-L-homocitrulline, D4-homocysteine, D3-alpha-aminoadipic acid, D6-L-2-aminobutyric acid, D3-sarcosine, 13C3, 15N-L-phosphoserine, D4-beta-alanine, D4-L-kynurenine, 13C6,15N 4-arginosuccinic acid, and D4-cystathionine.
The separation/determination of 4-group isomer of 43 amino acids and the separation/determination of 2-group isomer of 41 isotope-labeled amino acids; effective separation of isomers and selection of characteristic ion pairs can eliminate conventional analytical interference.
The 43 amino acid group 4 isomers include: sarcosine, beta-alanine and alanine; alpha-aminobutyric acid, gamma-aminobutyric acid and beta-aminoisobutyric acid; isoleucine, leucine and hydroxyproline; 3-methylhistidine and 1-methylhistidine;
the 41D/13C/15N marked isotope amino acid 2 group isomers comprise: [2H4] -beta-alanine and [2H4] -alanine; [2H6] -gamma-aminobutyric acid and [2H6] -alpha-aminobutyric acid.
The method does not use an ion pair reagent of perfluorocarboxylic acids, and the reagent can cause pollution of the whole LC-MS and is not easy to clean in mass spectrometry, so that instrument signals are reduced, noise is enhanced, the detection sensitivity of a negative ion compound is influenced for a long time, and the ion pair reagent is not required to be used in mass spectrometry.
The method adopts a non-derivatization pretreatment method, the derivatization pretreatment step is complex, the production is difficult, the stability of the derivatization reagent is poor, the cost is high, and the development of a cheap non-derivatization method with high sensitivity and stability is an important direction in amino acid research.
According to a preferred embodiment of the invention, the sample is a serum, plasma, urine sample or dried blood spot sample.
Further, preferably, the pretreatment step is:
a serum/plasma/urine sample treatment: 50 mu L of serum/plasma/urine sample is put into a 1.5mL centrifuge tube, added with methanol containing 10 mu L of internal standard mixed solution, 200ul of 10MG/ML DTT and 0.1% of FA, evenly mixed by vortex, and centrifuged for 10min at 14000rpm at 4 ℃. Transferring the supernatant into a centrifuge tube of 200 mu L to 2ml, drying by using nitrogen at 40 ℃, redissolving 100 mu L of complex solution (0.1% formic acid, 10mM ammonium formate and 70% acetonitrile water), mixing uniformly by using a vortex, shaking for 3min, centrifuging for 10min at 4 ℃ and 14000rpm, taking the supernatant and filtering by using a 0.2 mu m filter membrane to obtain a sample to be detected;
b, processing a dried blood spot sample: (ii) a Three round holes with the diameter of 3.2mM are punched on the dried blood spots, 5 mu L of internal standard mixed solution and 150ul of 60% methanol aqueous solution containing 0.1% FA and 10MG/ML DTT are added, the mixture is oscillated and incubated for 30min at 40 ℃, supernatant is transferred, nitrogen is blown at 40 ℃, the mixture is dried, then the sample is re-dissolved in 50ul of re-solution (0.1% formic acid, 10mM ammonium formate and 70% acetonitrile) after drying, the mixture is oscillated and incubated for 10min at 35 ℃, and the mixture is centrifuged and transferred to a 96-well filter plate for filtration, so that a sample to be tested is obtained.
Preferably, the C18 Column of the liquid chromatography is an ACQUITY UPLC BEH Amide Column,1.7 μm,2.1mm X50 mm, Column temperature: 45 ℃; the flow rate is 0.3 mL/min; sample introduction amount: 3 μ L.
According to a preferred embodiment of the present invention, the concentrations of the isotopically labeled amino acids in the internal standard mixture are as follows: d5-glycine was 400. mu.M, 13C6,15N 4-argininosuccinic acid was 16. mu.M, and the concentration of the remaining 39 isotopically labeled internal standards was 80. mu.M.
Preferably, the eight concentrations of mixed standard solutions are prepared by adding a correction wire matrix into a mixed standard working solution for dilution, the correction wire matrix is a substitute matrix of 2% BSA + PBS, and the dilution times corresponding to the eight concentrations of mixed standard solutions are 2, 4, 8, 20, 80, 160, 800 and 1600 times respectively; in the mixed standard working solution, lysine (Lys), histidine (His), arginine (Arg), alanine (Ala), glutamic acid (Glu), isoleucine (Ile), phenylalanine (Phe), serine (Ser), valine (Val), threonine (Thr), tyrosine (Tyr), asparagine (Asn), tryptophan (Trp), glutamine (Gln), methionine (Met), aspartic acid (Asp), glycine (Gly), leucine (Leu), proline (Pro), cysteine (Cys), phosphoethanolamine (PEtN), gamma-aminobutyric acid (GABA), taurine (Tau), 3-aminoisobutyric acid (bAib), ethanolamine (EtN), ornithine (Orn), anserine (Ans), carnosine (Car), citrulline (Cit), hydroxyproline (Hyp), 1-methylhistidine (1MHis), 3-methylhistidine (3 is), Hydroxylysine (Hylys), homocitrulline (Hcit), homocysteine (Hcy), alpha-aminoadipic acid (Aad), diaminobutyric acid (Abu), sarcosine (Sar), phosphoserine (P-Ser), beta-alanine (beta-Ala), kynurenine (Kyn), argininosuccinic acid (Asa), cystathionine (Cth) at concentrations of 6400. mu.M, 2400. mu.M, 6400. mu.M, 4800. mu.M, 4000. mu.M, 6400. mu.M, 3200. mu.M, 7200. mu.M, 4000. mu.M, 800. mu.M, 6400. mu.M, 4000. mu.M, 12800. mu.M, 4000. mu.M, 4800. mu.M, 800. mu.M, 1600. mu.M, 800. mu.M, 4000. mu.M, 800. mu.M, 800 mu, 800. mu.M, 800. mu.M.
Preferably, the specific conditions of mass spectrometry are: adopting a multi-ion reaction monitoring mode of positive ion electric spray ionization, namely atomizing: 50kPa, heating gas: 55kPa, air curtain air: 35kPa, spray voltage: 5500V, desolventizing temperature: at 550 ℃.
The invention also discloses a kit for simultaneously determining 43 amino acids without using an ion pair reagent and non-derivatization, which comprises:
1) isotopic internal standard: D8-L-lysine hydrochloride, 13C 6-L-histidine hydrochloride, D7-arginine, D3-alanine, D5-glutamic acid, D10-L-isoleucine, D5-phenylalanine, D3-L-serine, D8-valine, D2-L-threonine, D6-tyrosine, D3-L-asparagine, D3-L-tryptophan, D5-glutamine, D3-methionine, D3-aspartic acid, D2-glycine, D3-leucine, D3-L-proline, 13C 3-L-cysteine, D4-phosphorylethanolamine, D6-gamma-aminobutyric acid, D4-taurine, D3-3-aminoisobutyric acid hydrochloride, D4623-L-proline, 13C 2-ethanolamine, D7-ornithine, D4-L-anserine, D4-L-carnosine, D2-L-citrulline, D4-L-hydroxyproline, D3-1-methyl-L-histidine, D3-L-homocitrulline, D4-homocysteine, D3-alpha-aminoadipic acid, D6-L-2-aminobutyric acid, D3-sarcosine, 13C3, 15N-L-phosphoserine, D4-beta-alanine, D4-L-kynurenine, 13C6,15N 4-arginosuccinic acid, D4-cystathionine;
2) mixed standard solutions of eight concentrations;
the mixed standard solutions with the eight concentrations are prepared by adding mixed standard working solutions into a correction line matrix, wherein the correction line matrix is a substitute matrix of 2% BSA + PBS, and the dilution times corresponding to the mixed standard solutions with the eight concentrations are respectively 2, 4, 8, 20, 80, 160, 800 and 1600 times; in the mixed standard working solution, lysine (Lys), histidine (His), arginine (Arg), alanine (Ala), glutamic acid (Glu), isoleucine (Ile), phenylalanine (Phe), serine (Ser), valine (Val), threonine (Thr), tyrosine (Tyr), asparagine (Asn), tryptophan (Trp), glutamine (Gln), methionine (Met), aspartic acid (Asp), glycine (Gly), leucine (Leu), proline (Pro), cysteine (Cys), phosphoethanolamine (PEtN), gamma-aminobutyric acid (GABA), taurine (Tau), 3-aminoisobutyric acid (Aibb), ethanolamine (EtN), ornithine (Orn), anserine (Ans), carnosine (Car), citrulline (Cit), hydroxyproline (Hyp), 1-methylhistidine (1MHis), 3-methylhistidine (3MHis), Hydroxylysine (Hylys), homocitrulline (Hcit), homocysteine (Hcy), alpha-aminoadipic acid (Aad), diaminobutyric acid (Abu), sarcosine (Sar), phosphoserine (P-Ser), beta alanine (beta-Ala), kynurenine (Kyn), argininosuccinic acid (Asa), cystathionine (Cth) at concentrations of 6400, 2400, 6400, 4800, 4000, 6400, 3200, 7200, 4000, 800, 6400, 4000, 800, 12800, 4000, 4800, 800, 4800, 800, 2400, 1600, 800, 4000, 800 μ M, respectively;
3) mobile phase:
mobile phase A: water containing 0.1% formic acid +10mM ammonium formate + 20% acetonitrile; mobile phase B: containing 0.1% formic acid +10mM ammonium formate + 90% acetonitrile in water;
4) four levels of quality control substances, wherein the concentration of each amino acid added in the four levels of quality control substances is consistent with the concentration of the third, fourth, sixth and seventh gradient concentrations in eight gradient concentrations of the mixed standard solution, and the target value is determined by detection.
The invention has the beneficial effects that:
the invention establishes a method for simultaneously detecting 43 amino acids and 41D/13C/15N labeled isotope amino acids in a sample by optimizing a sample pretreatment method and ultra-high performance liquid chromatography-mass spectrometry conditions, nearly eliminates the influence of matrix effect by full internal standard quantification, simultaneously exists qualitative and quantitative ion pairs, can carry out accurate qualitative and quantitative analysis on 43 indexes, and is a detection method with simple sample treatment, high flux and reliable result.
The research on amino acids is the most important loop in metabonomics, and the research on amino acids in science is relatively extensive and systematic at present. Amino acids are a dynamic balance system in vivo, many amino acids are mutually related, the full-spectrum amino acid level of patients with tumors, severe cases, postoperative patients, hepatopathy patients, diabetes patients, cardiovascular diseases and prostatic cancer patients is rapidly detected and evaluated for nutritional health, and clinical nutrition supplement can be guided by combining amino acid metabonomics analysis. Meanwhile, through systematic analysis of the full-spectrum amino acid detection result, the clinical significance and intervention regulation suggestion of the detection result can be prompted, and reference is provided for further diagnosis and analysis of doctors and dieticians; in addition, the full-spectrum amino acid detection by the mass spectrometry technology can also provide reference for the fields of nutritional health product development, environmental detection and the like. The extraction method used in the invention can remove sample impurities and reduce matrix effect, and has simple and rapid operation and accurate detection result.
The method takes the relative retention time and the qualitative ion pairs of various amino acids as qualitative bases, and takes standard products to make a standard curve for quantification, wherein 41 amino acid internal standards of 2D/13C/15N labeled isotopes are used as internal standards for quantification, so that the influence of matrix effect is eliminated. Meanwhile, the method applies four levels of quality control quality inspection methods to ensure the accuracy and effectiveness of the detection result.
The invention realizes the purpose of simultaneously detecting 43 amino acids and 41D/13C/15N isotope-labeled amino acids in a sample by applying the liquid chromatography-tandem mass spectrometry technology for the first time, and the method has the advantages of simple and convenient operation, quick analysis, high flux and low cost, and can realize the real-time monitoring of the level of the amino acids of a human body.
Drawings
FIG. 1 is a graph of LC-MS/MS collected TICs for the determination of internal standards of 43 amino acids and 41 amino acid isotopes by the method of the invention.
FIG. 2 is a chromatogram of ILE, LEU, HYP 3 isomers and internal standards of corresponding isotopes determined by the method of the present invention.
FIG. 3 is a chromatogram of ALA, beta-ALA, SAR 3 isomers and corresponding isotope internal standards determined by the method of the present invention.
FIG. 4 is a chromatogram of the method of the present invention for measuring GABA, bAib and ABU 3 isomers and internal standards of corresponding isotopes.
FIG. 5 is a chromatogram of the 1MHIS and 3MHIS 2 isomers and corresponding internal isotope standards determined by the method of the present invention.
FIG. 6 is a chromatogram of Leu, ILE, Val, which are the abnormal indicators detected by the method of the present invention for determining a maple syrup urine disease positive sample.
FIG. 7 shows the abnormality index detected by the method of the present invention in the case of assaying argininosuccinic aciduria positive samples
Chromatogram of ASA, Cit.
FIG. 8 is a chromatogram of Phe, which is an abnormal indicator detected by a positive sample for phenylalanine hydroxylase deficiency determined by the method of the present invention.
FIG. 9 is a chromatogram of Cit, an abnormal index detected by the method of the present invention, in the case of assaying a positive sample for citrullinemia.
In FIGS. 2 to 9, the solid-filled peaks are the actual sample detection peaks or the peaks of the non-isotopically labeled amino acids, and the non-solid-filled peaks are the peaks of the isotopic internal standard.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
Examples
(1) Preparing a sample to be tested:
a serum/plasma/urine sample treatment: 50 mu L of serum/plasma/urine sample is put into a 1.5mL centrifuge tube, added with methanol containing 10 mu L of internal standard mixed solution and 20mg/mLDTT of 200ul of 0.1 percent FA, evenly mixed by vortex, and centrifuged for 10min at 14000rpm at 4 ℃. Transferring the supernatant into a centrifuge tube of 200 mu L to 2ml, drying by using nitrogen at 40 ℃, redissolving 100 mu L of complex solution (0.1 percent formic acid, ammonium formate and 70 percent acetonitrile water), mixing uniformly by using a vortex, shaking for 3min, centrifuging for 10min at 4 ℃ and 14000rpm, taking the supernatant and filtering by using a 0.2 mu m filter membrane to obtain a sample to be detected;
b, dry blood spot sample treatment: punching three round holes with the diameter of 3.2mm on the dried blood spots, adding 5 mu L of internal standard mixed solution and 150ul of 60% MEOH solution containing 0.1% FA and 10mg/mL DTT, oscillating and incubating for 30min at 40 ℃, transferring supernatant, blowing nitrogen at 40 ℃, re-dissolving a sample in 50ul of re-solution (0.1% formic acid, ammonium formate and 70% acetonitrile water) after drying, oscillating and incubating for 10min at 35 ℃, centrifuging and transferring to a 96-well filter plate for filtering to obtain a sample to be detected;
(2) ultra-high performance liquid chromatography-mass spectrometry detection
The sample to be detected enters a chromatographic column for separation through a gradient elution mode, and the liquid chromatogram reference condition is as follows:
a chromatographic column: c18 Column, ACQUITY UPLC BEH Amide Column,1.7 μm,2.1mm 100 mm
Column temperature: 45 deg.C
Sample introduction volume: 3 μ l
Flow rate: 400 mul/min
Mobile phase A: 0.1% formic acid +10mM ammonium formate + 20% acetonitrile in water
Mobile phase B: 0.1% formic acid +10mM ammonium formate + 90% acetonitrile in water
Gradient elution conditions:
0-3.25min, 95% B; changing from 95% B to 55% B in 3.25-6.25 min; gradually changing from 55% B to 50% B for 6.25-6.5 min; changing from 50% B to 0% B gradually for 6.5-6.8 min; 6.8-7.3min, 0% B; 7.31-7.8min, 20% B; changing from 20% B to 95% B in 7.8-8 min; 8-10min, flow rate 0.3mL/min, 95% B.
Mass spectrum parameter reference conditions:
adopting a multi-ion reaction monitoring mode of positive ion electric spray ionization, namely atomizing: 50 kPa; heating gas: 55 kPa; air curtain air: 35 kPa; spraying voltage: 5500V; the temperature of the desolvation: 550 ℃;
MRM mass spectrum parameters:
(3) calculation results
Preparing a standard working solution: 43 amino acids-lysine (Lys), histidine (His), arginine (Arg), alanine (Ala), glutamic acid (Glu), isoleucine (Ile), phenylalanine (Phe), serine (Ser), valine (Val), threonine (Thr), tyrosine (Tyr), asparagine (Asn), tryptophan (Trp), glutamine (Gln), methionine (Met), aspartic acid (Asp), glycine (Gly), leucine (Leu), proline (Pro), cysteine (Cys), phosphoethanolamine (PEtN), gamma-aminobutyric acid (GABA), taurine (Tau), 3-aminoisobutyric acid (bAib), ethanolamine (EtN), ornithine (Orn), anserine (Ans), carnosine (Car), citrulline (Cit), hydroxyproline (Hyp), 1-methylhistidine (1MHis), 3-methylhistidine (3MHis), Hydroxylysine (Hylys), homocitrulline (Hcit), homocysteine (Hcy), alpha-aminoadipic acid (Aad), diaminobutyric acid (Abu), sarcosine (Sar), phosphoserine (P-Ser), beta-alanine (beta-Ala), kynurenine (Kyn), argininosuccinic acid (Asa), cystathionine (Cth) at concentrations of 6400. mu.M, 2400. mu.M, 6400. mu.M, 4800. mu.M, 4000. mu.M, 6400. mu.M, 3200. mu.M, 7200. mu.M, 4000. mu.M, 800. mu.M, 6400. mu.M, 4000. mu.M, 12800. mu.M, 4000. mu.M, 4800. mu.M, 800. mu.M, 1600. mu.M, 800. mu.M, 4000. mu.M, 800 mu.M, 800. mu.M, and 800. mu.M, formulation protocol of standard working solution:
the standard working solution is contained in a brown bottle and stored at-20 ℃ for later use.
The isotope labeled internal standard substance is prepared into mixed internal standard liquid by adopting the concentrated stock dissolving process which is the same as that of the corresponding amino acid in the standard working solution, wherein in the mixed internal standard liquid, the concentration of D5-glycine is 400 mu M, the concentration of 13C6, the concentration of 15N 4-argininosuccinic acid is 16 mu M, and the concentration of the rest 39 isotope labeled internal standards is 80 mu M.
The calibration points containing eight concentration levels of the mixed standard solutions prepared by adding standard working solutions to the PBS surrogate matrix were used to make the standard curves, the concentrations of which are shown in the table below. Preparing a standard curve by using the mixed standard solutions with eight concentrations, wherein the concentration ratio of the standard solution to the internal standard is taken as an X axis, and the peak area ratio of the standard solution to the internal standard is taken as a Y axis; and carrying out linear regression analysis to obtain a regression equation, substituting the peak areas of the corresponding amino acids into the regression equation, and respectively calculating the concentrations of the amino acids in the serum/plasma, the blood spots and the urine samples.
And (3) data analysis: the linearity measured by the method of the invention is as follows:
the quality control product comprises four levels of low-medium concentration quality control serum, is prepared by adding mixed amino acid working solution into normal serum/plasma, blood spots and urine samples, the concentration of each added amino acid is consistent with the third, fourth, sixth and seventh gradient concentrations in eight gradient concentrations of the mixed standard solution, and the target value is determined by detection.
The result of the detection
The method is compared with a chromosystems MASSCHECK-AAA analysis method (through CE 0123-IVD authentication) in a correct degree, wherein the reference range of MASSCHECK-QC (MC-QC) is within +/-20% deviation of a target value, and the measured values of other concentration points and other indexes are within a given target range except that the ASA low point is limited by the sensitivity of an instrument. As shown in the table below. As shown in FIG. 1, LC-MS/MS collected TIC plots of 43 amino acids and 41 amino acid isotope internal standards were determined using the method of the present invention. Referring to FIG. 2, the chromatogram of ILE, LEU, HYP 3 isomers and internal standards of the corresponding isotopes is determined by the method of the present invention. Referring to FIG. 3, the chromatogram of ALA, beta-ALA and SAR 3 isomers and internal standards of corresponding isotopes is determined by the method of the present invention. Referring to FIG. 4, the chromatogram of GABA, bAib and ABU 3 isomers and internal standards of corresponding isotopes was determined by the method of the present invention. Referring to FIG. 5, chromatograms of 1MHIS and 3MHIS 2 isomers and corresponding internal isotope standards were determined using the method of the present invention. As shown in FIG. 6, the chromatogram of Leu, ILE, Val, which are abnormal indicators detected in a maple syrup urine positive sample by the method of the present invention, is shown. As shown in FIG. 7, the method of the present invention is used to determine the positive arginyl succinyl uricosuric acid sample, and the chromatogram of ASA and Cit, which are the abnormal indicators, is detected. As shown in FIG. 8, the chromatogram of Phe, which is an abnormal indicator, was obtained by assaying a positive sample for phenylalanine hydroxylase deficiency by the method of the present invention. As shown in FIG. 9, the chromatogram of Cit, which is an abnormal index detected by the method of the present invention, is used for determining a positive sample of citrullinemia.
The study simultaneously determined 43 amino acids in serum/plasma, blood spot and urine samples by LC-MS/MS method. The method can detect the peak time and the ion pair of the target object at the same time, has high sensitivity, and can greatly avoid the interference of cross reaction. Meanwhile, 41 internal standards are used for quantification by an internal standard method, so that the influence of matrix effect is eliminated. The method is characterized in that complex derivatization treatment is not carried out, ion pair reagents polluting instruments are not used, and the method is high in detection sensitivity, strong in specificity, simple in pretreatment process and capable of quickly and simultaneously completing separation and detection of 43 amino acids.
The present invention is not limited to the above-mentioned preferred embodiments, and any other products in various forms can be obtained by anyone in the light of the present invention, but any changes in the shape or structure thereof, which have the same or similar technical solutions as those of the present application, fall within the protection scope of the present invention.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (9)
1. A method for simultaneously determining 43 amino acids without using an ion-pair reagent and non-derivatization, comprising the steps of:
1) pretreating a sample, and adding an internal standard mixed solution in the pretreatment process to obtain a sample to be detected;
2) carrying out qualitative and internal standard quantitative detection on 43 amino acids in a sample by using a UPLC-MS/MS method, wherein the internal standard quantitative detection comprises the following steps: preparing a standard curve by using the mixed standard solutions with eight concentrations, wherein the concentration ratio of the standard solution to the internal standard is taken as an X axis, and the peak area ratio of the standard solution to the internal standard is taken as a Y axis; carrying out linear regression analysis to obtain a regression equation, substituting the peak area of the corresponding amino acid into the regression equation, and calculating the concentration of the amino acid in the sample;
wherein the liquid phase chromatographic conditions are as follows: a chromatographic column: c18 column, mobile phase a: water containing 0.1% formic acid +10mM ammonium formate + 20% acetonitrile, mobile phase B: containing 0.1% formic acid +10mM ammonium formate + 90% acetonitrile in water; gradient elution conditions: 0-3.25min, 95% B; changing from 95% B to 55% B in 3.25-6.25 min; gradually changing from 55% B to 50% B in 6.25-6.5 min; changing from 50% B to 0% B gradually for 6.5-6.8 min; 6.8-7.3min, 0% B; 7.31-7.8min, 20% B; changing from 20% B to 95% B in 7.8-8 min; 8-10min, flow rate 0.3mL/min, 95% B;
the mass spectrum adopts a multi-ion reaction monitoring mode of positive ion electrospray ionization;
the 43 amino acids are: lysine (Lys), histidine (His), arginine (Arg), alanine (Ala), glutamic acid (Glu), isoleucine (Ile), phenylalanine (Phe), serine (Ser), valine (Val), threonine (Thr), tyrosine (Tyr), asparagine (Asn), tryptophan (Trp), glutamine (Gln), methionine (Met), aspartic acid (Asp), glycine (Gly), leucine (Leu), proline (Pro), cysteine (Cys), phosphoethanolamine (PEtN), gamma-aminobutyric acid (GABA), taurine (Tau), beta-aminoisobutyric acid (bAib), ethanolamine (EtN), ornithine (Orn), anserine (Ans), carnosine (Car), citrulline (Cit), hydroxyproline (Hyp), 1-methylhistidine (1MHis), 3-methylhistidine (3MHis), Hydroxylysine (Hylys), homocitrulline (Hcit), homocysteine (Hcy), alpha-aminoadipic acid (Aad), alpha-aminobutyric acid (Abu), sarcosine (Sar), phosphoserine (P-Ser), beta alanine (beta-Ala), kynurenine (Kyn), argininosuccinic acid (Asa), cystathionine (Cth);
the internal standard mixed solution comprises 41 kinds of amino acid internal standards which are respectively as follows: D8-L-lysine hydrochloride, 13C 6-L-histidine hydrochloride, D7-arginine, D3-alanine, D5-glutamic acid, D10-L-isoleucine, D5-phenylalanine, D3-L-serine, D8-valine, D2-L-threonine, D6-tyrosine, D3-L-asparagine, D3-L-tryptophan, D5-glutamine, D3-methionine, D3-aspartic acid, D5-glycine, D3-leucine, D3-L-proline, 13C 3-L-cysteine, D4-phosphorylethanolamine, D6-gamma-aminobutyric acid, D4-taurine, D3-3-aminoisobutyric acid hydrochloride, D4623-L-proline, 13C 2-ethanolamine, D7-ornithine, D4-L-anserine, D4-L-carnosine, D2-L-citrulline, D4-L-hydroxyproline, D3-1-methyl-L-histidine, D3-L-homocitrulline, D4-homocysteine, D3-alpha-aminoadipic acid, D6-L-2-aminobutyric acid, D3-sarcosine, 13C3, 15N-L-phosphoserine, D4-beta-alanine, D4-L-kynurenine, 13C6,15N 4-arginosuccinic acid, and D4-cystathionine.
2. The method of claim 1, wherein: the sample is serum, plasma, urine sample or dry blood spot sample; the pretreatment steps are as follows:
for serum, plasma or urine sample pretreatment: putting 50 mu L of sample into a 1.5mL centrifuge tube, adding methanol containing 10 mu L of internal standard mixed solution, 200ul of 10mg/mL DTT and 0.1% FA, uniformly mixing by vortex, and centrifuging for 10min at the temperature of 4 ℃ and the rpm of 14000; transferring the supernatant into a centrifuge tube of 200 mu L to 2ml, drying by blowing nitrogen at 40 ℃, redissolving 100 mu L of redissolution which is 0.1 percent formic acid, 10mM ammonium formate and 70 percent acetonitrile aqueous solution, uniformly mixing by vortex, shaking for 3min, centrifuging for 10min at 4 ℃ and 14000rpm, taking the supernatant and filtering by a 0.2 mu m filter membrane to obtain a sample to be detected;
for dry blood spot samples: punching three round holes with the diameter of 3.2mM on the dried blood spots, adding 5 mu L of internal standard mixed solution and 150ul of 60% methanol aqueous solution containing 0.1% FA and 10mg/mL DTT, oscillating and incubating for 30min at 40 ℃, transferring supernatant, blowing nitrogen at 40 ℃, re-dissolving the sample in 50ul of complex solution after drying, wherein the complex solution is 0.1% formic acid, 10mM ammonium formate and 70% acetonitrile aqueous solution, oscillating and incubating for 10min at 35 ℃, centrifuging and transferring to a 96-well filter plate for filtering, and obtaining the sample to be detected.
3. The method of claim 1, wherein: the C18 Column of liquid chromatography adopts ACQUITY UPLC BEH Amide Column,1.7 μm,2.1mm X50 mm, Column temperature: 45 ℃; the flow rate is 0.4 mL/min; sample introduction amount: 3 μ L.
4. The method of claim 1, wherein: the concentration of each isotope labeled amino acid in the internal standard mixed solution is as follows: d5-glycine was 400. mu.M, 13C6,15N 4-argininosuccinic acid was 16. mu.M, and the concentration of the remaining 39 isotopically labeled internal standards was 80. mu.M.
5. The method of claim 1, wherein: the mixed standard solutions with the eight concentrations are prepared by adding mixed standard working solutions into a correction line matrix, wherein the correction line matrix is a substitute matrix of 2% BSA + PBS, and the dilution times corresponding to the mixed standard solutions with the eight concentrations are respectively 2, 4, 8, 20, 80, 160, 800 and 1600 times; in the mixed standard working solution, lysine (Lys), histidine (His), arginine (Arg), alanine (Ala), glutamic acid (Glu), isoleucine (Ile), phenylalanine (Phe), serine (Ser), valine (Val), threonine (Thr), tyrosine (Tyr), asparagine (Asn), tryptophan (Trp), glutamine (Gln), methionine (Met), aspartic acid (Asp), glycine (Gly), leucine (Leu), proline (Pro), cysteine (Cys), phosphoethanolamine (PEtN), gamma-aminobutyric acid (GABA), taurine (Tau), 3-aminoisobutyric acid (bAib), ethanolamine (EtN), ornithine (Orn), anserine (Ans), carnosine (Car), citrulline (Cit), hydroxyproline (Hyp), 1-methylhistidine (1MHis), 3-methylhistidine (3 is), Hydroxylysine (Hylys), homocitrulline (Hcit), homocysteine (Hcy), alpha-aminoadipic acid (Aad), diaminobutyric acid (Abu), sarcosine (Sar), phosphoserine (P-Ser), beta-alanine (beta-Ala), kynurenine (Kyn), argininosuccinic acid (Asa), cystathionine (Cth) at concentrations of 6400. mu.M, 2400. mu.M, 6400. mu.M, 4800. mu.M, 4000. mu.M, 6400. mu.M, 3200. mu.M, 7200. mu.M, 4000. mu.M, 800. mu.M, 6400. mu.M, 4000. mu.M, 12800. mu.M, 4000. mu.M, 4800. mu.M, 800. mu.M, 1600. mu.M, 800. mu.M, 4000. mu.M, 800. mu.M, 800 mu, 800. mu.M, 800. mu.M.
6. The method of claim 1, wherein: the specific conditions of the mass spectrum are as follows: adopting a multi-ion reaction monitoring mode of positive ion electric spray ionization, namely atomizing: 50kPa, heating gas: 55kPa, air curtain air: 35kPa, spray voltage: 5500V, desolventizing temperature: at 550 ℃.
7. The method of claim 1, wherein:
the mass spectrum parameters of each amino acid are:
8. a kit for simultaneously determining 43 amino acids without using an ion pair reagent and non-derivatization, comprising:
1) isotope internal standard mixed solution: D8-L-lysine hydrochloride, 13C 6-L-histidine hydrochloride, D7-arginine, D3-alanine, D5-glutamic acid, D10-L-isoleucine, D5-phenylalanine, D3-L-serine, D8-valine, D2-L-threonine, D6-tyrosine, D3-L-asparagine, D3-L-tryptophan, D5-glutamine, D3-methionine, D3-aspartic acid, D2-glycine, D3-leucine, D3-L-proline, 13C 3-L-cysteine, D4-phosphorylethanolamine, D6-gamma-aminobutyric acid, D4-taurine, D3-3-aminoisobutyric acid hydrochloride, D4623-L-proline, 13C 2-ethanolamine, D7-ornithine, D4-L-anserine, D4-L-carnosine, D2-L-citrulline, D4-L-hydroxyproline, D3-1-methyl-L-histidine, D3-L-homocitrulline, D4-homocysteine, D3-alpha-aminoadipic acid, D6-L-2-aminobutyric acid, D3-sarcosine, 13C3, 15N-L-phosphoserine, D4-beta-alanine, D4-L-kynurenine, 13C6,15N 4-arginosuccinic acid, D4-cystathionine; wherein D5-glycine is 400 μ M, 13C6,15N 4-argininosuccinic acid is 16 μ M, and the concentrations of the other 39 isotope labeling internal standards are all 80 μ M;
2) mixed standard solutions of eight concentrations;
the mixed standard solutions with the eight concentrations are prepared by adding a correction wire matrix into a mixed standard working solution for dilution, wherein the correction wire matrix is a substitute matrix of 2% BSA + PBS, and the dilution times corresponding to the mixed standard solutions with the eight concentrations are respectively 2, 4, 8, 20, 80, 160, 800 and 1600 times; in the mixed standard working solution, lysine (Lys), histidine (His), arginine (Arg), alanine (Ala), glutamic acid (Glu), isoleucine (Ile), phenylalanine (Phe), serine (Ser), valine (Val), threonine (Thr), tyrosine (Tyr), asparagine (Asn), tryptophan (Trp), glutamine (Gln), methionine (Met), aspartic acid (Asp), glycine (Gly), leucine (Leu), proline (Pro), cysteine (Cys), phosphoethanolamine (PEtN), gamma-aminobutyric acid (GABA), taurine (Tau), 3-aminoisobutyric acid (Aibb), ethanolamine (EtN), ornithine (Orn), anserine (Ans), carnosine (Car), citrulline (Cit), hydroxyproline (Hyp), 1-methylhistidine (1MHis), 3-methylhistidine (3MHis), Hydroxylysine (Hylys), homocitrulline (Hcit), homocysteine (Hcy), alpha-aminoadipic acid (Aad), diaminobutyric acid (Abu), sarcosine (Sar), phosphoserine (P-Ser), beta alanine (beta-Ala), kynurenine (Kyn), argininosuccinic acid (Asa), cystathionine (Cth) at concentrations of 6400, 2400, 6400, 4800, 4000, 6400, 3200, 7200, 4000, 800, 6400, 4000, 800, 12800, 4000, 4800, 800, 4800, 800, 2400, 1600, 800, 4000, 800 μ M, respectively;
3) mobile phase:
mobile phase A: containing 0.1% formic acid +10mM ammonium formate + 20% acetonitrile in water; mobile phase B: containing 0.1% formic acid +10mM ammonium formate + 90% acetonitrile in water;
4) four levels of quality control.
9. A kit according to claim 8, characterized in that it comprises: in the four levels of quality control, the concentration of each amino acid added is consistent with the third, fourth, sixth and seventh gradient concentrations in eight gradient concentrations of the mixed standard solution, and the target value is determined by detection.
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