Liquid chromatography-tandem mass spectrometry method for determining multiple amino acids

Abstract

The invention discloses a liquid chromatography-tandem mass spectrometry method for determining various amino acids, which comprises the following steps: using an octadecyl chain and fluorocarbon chain mixed bonded silica gel chromatographic column, and matching with a fluorine-containing compound mobile phase for retention and separation, the method greatly improves the chromatographic retention of the amino acids, improves the peak pattern, and improves the detection sensitivity. According to the method, the separation degree between isomers is improved, the detection speed is increased, meanwhile, more stable reproducibility is obtained, the detection linear range completely covers clinical normal and abnormal values, and the method can be used for accurate quantitative analysis of amino acid in a clinical blood sample.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry, and in particular relates to an amino acid analysis method using liquid chromatography tandem mass spectrometry (LC-MS / MS). Background Art

[0002] Amino acids are the basic building blocks of protein molecules in biological organisms and an important material basis for maintaining homeostasis. They are one of the important substances in the body's metabolism and have a wide range of biological functions and important physiological significance. Defects in related proteins and enzymes involved in amino acid metabolism in the human body or various pathological conditions can lead to abnormal amino acid metabolism and changes in the body's amino acid levels. Therefore, detecting the amino acid content in biological samples is of great significance, providing auxiliary basis for the exploration of clinical analysis biomarkers, explaining the mechanism of the disease, and diagnosing related diseases. Therefore, continuously improving the accuracy and work efficiency of amino acid analysis in biological samples and reducing detection costs are of great significance for the clinical detection and promotion of amino acids.

[0003] At present, commonly used analytical methods include amino acid analyzer determination, high performance liquid chromatography, gas chromatography, capillary electrophoresis, liquid chromatography tandem mass spectrometry (LC-MS / MS), etc. In recent years, LC-MS / MS has become an important tool for analyzing amino acid content in biological samples due to its high sensitivity, high selectivity and high throughput analysis, and is particularly suitable for application scenarios that require high sensitivity and simultaneous analysis of multiple components.

[0004] Chromatographic retention and separation of amino acids in complex biological samples are the main problems to be solved by LC-MS / MS detection technology. The overall polarity of amino acid molecules is relatively large. The use of HILIC hydrophilic columns can increase chromatographic retention. However, HILIC columns have limited separation and retention capabilities for multiple species and isomers, and the reproducibility of the method is poor. 18 , T3, etc.) have stable performance and a wide pH tolerance range, so they are more widely used in the separation of various amino acids and their isomers. However, due to the high polarity of amino acids themselves, reversed-phase chromatography can usually only retain and separate most amino acids at a high water phase mobile phase ratio, and isomers with very similar structures are still difficult to distinguish. In addition, peak elution at a high water phase mobile phase ratio is not conducive to the ionization of amino acids in mass spectrometry, which may lead to an enhanced matrix effect.

[0005] At present, there are two main methods to improve the chromatographic separation of amino acids. One is chemical derivatization, which adds a group to the amino acid to increase its hydrophobicity and enhance its retention on the reversed-phase column. However, the derivatization method generally requires heating reaction and waiting, which is cumbersome, has many influencing factors, and is time-consuming. In addition, the derivatization reagent usually has a certain contamination on the mass spectrometer and may produce additional matrix effects. The other is ion pair chromatography, in which ion pair reagents are added to the mobile phase to interact with amino acids through ionic bonds to form ion pairs, and then the hydrophobic chains of the ion pair reagents and the hydrophobic chains of the stationary phase of the reversed-phase column form intermolecular forces, thereby increasing the retention and separation of amino acids. Perfluorocarboxylic acid anionic surfactants are often used in ion pair LC-MS / MS detection of amino acids due to their low surface energy and boiling point, such as trifluoroacetic acid, heptafluorobutyric acid, nonafluoropentanoic acid, tridecafluoroheptanoic acid and perfluorooctanoic acid. Generally, the longer the carbon chain of the perfluorocarboxylic acid, the weaker the acidity, the stronger the retention ability, and the higher the price. However, stronger retention does not necessarily mean better. Too strong retention can also cause problems such as tailing, residue, long equilibrium time, and even incomplete elution of hydrophobic amino acids. Only with appropriate retention and elution capabilities can multiple amino acids be detected quickly and specifically.

[0006] In amino acid ion-pair chromatography, compared with the various choices of ion-pair reagents, the stationary phase is less selective, and the classic reversed-phase C 18 For example, Zoppa M et al. "Method for the quantification of underivatized amino acids on dry blood spots from newborn screening by HPLC-ESI-MS / MS" used Discovery C 18The chromatographic column (50 mm × 2.1 mm × 5 μm) was used to separate 40 kinds of amino acids. Porous graphite carbon columns can also be used for the detection of amino acids because of their stronger hydrophobicity and better acid resistance as stationary phases. For example, Le A et al. "A rapid, sensitive method for quantitative analysis of underivatized amino acids by liquid chromatography-tandemmass spectrometry (LC-MS / MS)" used a Hypercarb column (4.6 mm ID × 50 mm, 3μm, Thermo Fisher Scientific) to separate 33 kinds of amino acids. However, whether it is a reverse phase column or a graphite carbon column, complex two-dimensional chromatography or multiple chromatographic separations are required to obtain an overall separation scheme from polar to non-polar amino acids and multiple isomeric amino acids. Summary of the invention

[0007] The main purpose of the present invention is to solve the many problems existing in the prior art of amino acid detection and to provide a rapid, efficient and highly sensitive liquid chromatography-tandem mass spectrometry analysis and detection method for multiple amino acids. To achieve the above purpose, the technical solution adopted by the present invention is: Provided is a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry, wherein the mobile phase of the method contains fluoride as an ion pair reagent, preferably the fluoride is selected from one or more of nonafluoropentanoic acid, tridecafluoroheptanoic acid (TDFHA) and perfluorooctanoic acid, and the stationary phase is selected from octadecyl chain C 18 And fluorocarbon chain FC mixed bonded phase chromatographic column.

[0008] Furthermore, the present invention provides a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry, wherein the mobile phase of the method is selected from one or more of a methanol-water solution containing tridecafluoroheptanoic acid (TDFHA), an acetonitrile-water solution containing tridecafluoroheptanoic acid (TDFHA), a methanol-isopropanol-water solution containing tridecafluoroheptanoic acid (TDFHA), an acetonitrile-isopropanol-water solution containing tridecafluoroheptanoic acid (TDFHA), or a methanol-acetonitrile-isopropanol-water solution containing tridecafluoroheptanoic acid (TDFHA).

[0009] Furthermore, the present invention provides a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry, wherein the mobile phase of the method comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is selected from a methanol-water solution containing tridecafluoroheptanoic acid (TDFHA), and the preferred volume percentage of methanol is 40-50%; the mobile phase B is selected from a methanol-isopropanol-water solution containing or not containing tridecafluoroheptanoic acid (TDFHA), and the preferred volume percentage of methanol in the methanol-isopropanol-water is 80-90%, the volume percentage of isopropanol is 5-15%, and the balance is water.

[0010] Furthermore, the present invention provides a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry, wherein the concentration of tridecafluoroheptanoic acid (TDFHA) in mobile phase A of the method is 0.5-5 mM, preferably 0.8-3 mM, and more preferably 1-2 mM.

[0011] Furthermore, the present invention provides a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry, wherein the method uses CNW E-Gen AAA as a chromatographic column (preferably the chromatographic column model is 3.0*100 mm, 3 μm), and the stationary phase is octadecyl chain C 18 And fluorocarbon chain FC mixed bonded phase silica gel.

[0012] Furthermore, the present invention provides a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry, wherein the tandem mass spectrometry uses electrospray ionization to perform quantitative detection of the substances to be detected.

[0013] Furthermore, the present invention provides a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry, wherein the liquid chromatography adopts gradient elution: the starting ratio is 0% B, 0~1 min; 0% B, 1~2 min; 20% B, 2~3 min; 20% B, 3~4 min; 60% B, 4~5 min; 60% B, 5~5.1 min; 0% B, 5.1~7 min; column temperature: 25~40°C; flow rate: 0.6~1 mL / min; injection volume: 5~30 μL; detection time: 7 min.

[0014] Furthermore, the present invention provides a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry, wherein the mass spectrometry conditions are positive ion scanning; curtain gas: 35 psi; collision gas: high; nebulizer gas: 65 psi; heating gas: 65 psi; ion source voltage: 5500 V; and ion source temperature: 550°C.

[0015] Furthermore, the present invention provides a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry, the method comprising the steps of adding a precipitant to the blood or a blood sample, taking the supernatant, adding a solvent to dilute it, and then injecting the sample.

[0016] Furthermore, the present invention provides a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry, wherein the precipitant is selected from a mixed solvent of methanol-acetonitrile, and the volume ratio of the methanol-acetonitrile is 1:(0.5-2).

[0017] Furthermore, the present invention provides a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry, wherein the multiple amino acids are selected from one or more of aspartic acid, proline, asparagine, glutamine, serine, glutamic acid, citrulline, threonine, glycine, tyrosine, alanine, methionine, γ-aminobutyric acid, valine, kynurenine, tryptophan, phenylalanine, alloisoleucine, isoleucine, leucine, histidine, ornithine, arginine, asymmetric dimethylarginine, symmetric dimethylarginine, lysine, hydroxyproline, and taurine.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention uses a fluorinated compound as an ion pair reagent to react with an octadecyl chain C 18 When used with CNW E-Gen AAA columns with fluorocarbon chain FC mixed bonded phase, the electronegative fluorine atoms of the stationary phase FC chain can interact with fluoride-containing ion pair reagents to enhance dipole-dipole or hydrogen bonding to retain additional analytes and increase the retention of polar compounds. It is especially suitable for the separation of halogenated compounds and their isomers, which is more effective than ordinary C 18 More selective. The isomers of amino acids and interfering substances are well separated by chromatography, and the method has better specificity. The chromatographic retention and separation principle of this method has not been reported before, and it is highly novel and can be extended to the separation and detection of the same type of difficult-to-retain compounds.

[0019] 2. The ion pair reagent of the fluorine-containing compound used in the present invention and the bonded phase FC of the stationary phase both contain F atoms, and the two are more compatible. The method can achieve chromatographic equilibrium in a shorter time. Rapid equilibrium and efficient separation greatly shorten the chromatographic operation speed, so that 28 kinds of amino acids can be retained and eluted within 7 minutes in one injection, with short detection time and high throughput.

[0020] 3. The mobile phase of the present invention has a high proportion of organic phase, and the solubility and stability of fluorine-containing compounds therein are good, so the method has better reproducibility, peaks appear under high organic phase, amino acids are fully ionized, and sensitivity is significantly improved, so the present invention has no obvious matrix effect.

[0021] 4. The sample pretreatment of the present invention is simple, without the steps of drying and reorganization, and has a high recovery rate, good accuracy and precision. The method can be combined with a 96-well plate to achieve full automation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The following is an LC-MS / MS graph of 28 kinds of amino acids involved in the patent of the present invention, including (1-taurine, 2-aspartic acid, 3-proline, 4-asparagine, 5-glutamine, 6-serine, 7-hydroxyproline, 8-glutamic acid, 9-citrulline, 10-threonine, 11-glycine, 12-tyrosine, 13-alanine, 14-methionine, 15-kynurenine, 16-valine, 17-γ-aminobutyric acid, 18-tryptophan, 19-phenylalanine, 20-alloleucine, 21-isoleucine, 22-leucine, 23-histidine, 24-ornithine, 25-arginine, 26-asymmetric dimethylarginine, 27-symmetric dimethylarginine, 28-lysine).

[0023] Figure 2 LC-MS / MS chart comparing the separation of leucine and its isomers between different chromatographic columns under the same mobile phase. The chromatographic conditions are: A. E-Gen AAA 3×100 mm 3μm, B. Hypercarb3×100 mm 3μm, C. Kinetex polar C18, phenomenex3×100 mm 2.6 μm; 1-hydroxyproline, 2-alloleucine, 3-isoleucine, 4-leucine. DETAILED DESCRIPTION

[0024] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0025] Example 1 (1) Plasma sample pretreatment Take 50 μL of the plasma sample to be tested, add 300 μL of precipitant (methanol:acetonitrile = 1:1, containing internal standard) to a 96-well collection plate, shake at 1200 rpm for 5 min, transfer to a precipitation plate, place in a 96-well positive pressure extraction device for pressure filtration, transfer 50 μL of the clarified liquid, add 450 μL of distilled water, shake at 1500 rpm for 1 min, and then inject for analysis.

[0026] (2) Liquid phase conditions

[0027] (3) Mass spectrometry conditions

[0028] (4) Amino acid mass spectrometry parameters Serial number Analyte name Abbreviations Precursor ion (Da) Product ion (Da) Declustering voltage(V) Collision energy (V) 1 Taurine Tau 126.0 108.0 40 13 2 Aspartic acid Asp 134.1 74.1 30 23 3 Proline Pro 116.1 70.1 30 58 4 Asparagine Asn 133.1 116.1 25 14 5 Glutamine Gln 147.069 84.069 30 52 6 Serine Ser 106.1 60.1 38 28 7 Hydroxyproline Hyp 132.1 86.1 40 45 8 Glutamate Glu 148.1 56.1 40 49 9 Citrulline Cit 176.1 113.1 32 20 10 Threonine Thr 120.1 74.1 30 30 11 Glycine Gly 76.1 30.1 28 25 12 Tyrosine Tyr 182.1 136.1 30 27 13 Alanine Ala 90.1 44.1 39 35 14 Methionine Met 150.1 104.1 30 18 15 Kynurenine Kyn 209.1 94.1 30 11 16 Valine Val 118.1 72.1 30 37 17 GABA GABA 104.1 87.1 30 13 18 Tryptophan Trp 205.1 146.1 30 32 19 Phenylalanine Phe 166.1 120.1 30 38 20 Alloisoleucine Allo-Ile 132.1 86.1 30 35 21 Isoleucine Ile 132.1 86.1 30 30 22 Leucine Leu 132.1 86.1 30 25 23 Histidine His 156.1 110.1 33 43 24 Ornithine Orn 133.1 70.1 30 50 25 Arginine Arg 175.1 116.1 30 30 26 Asymmetric dimethylarginine ADMA 203.2 46.2 34 40 27 Symmetric dimethylarginine SDMA 203.2 172.2 28 16 28 Lysine Lys 147.106 84.106 30 55 (5) Amino acid isotope internal standard mass spectrometry parameters: Serial number Analyte name Abbreviations Precursor ion (Da) Product ion (Da) Declustering voltage(V) Collision energy (V) 1 Taurine-d4 Tau-d4 130.0 48.0 40 24 2 Aspartic acid-d3 Asp-d3 137.1 91.1 30 15 3 Proline-d3 Pro-d3 119.1 73.1 30 23 4 Asparagine-13C4 Asn-13C4 137.1 90.1 25 13 5 Glutamine-13C4 Gln-d5 152.1 89.1 30 22 6 Serine-13C3 Ser-13C3 109.1 62.1 38 15 7 Hydroxyproline-d3 Hyp-d3 135.1 71.1 40 28 8 Glutamic acid-13C515N Glu-13C515N 154.1 89.1 40 22 9 Citrulline-d7 Cit-d7 183.1 120.1 32 21 10 Threonine-2H515N Thr-2H515N 126.1 80.1 30 15 11 Glycine-13C15N Gly-13C15N 78.1 32.1 28 25 12 Tyrosine-d4 Tyr-d4 186.1 140.1 30 15 13 Alanine-D4 Ala-d4 94.1 48.1 39 16 14 Methionine-d3 Met-d3 153.1 107.1 30 15 15 Kynurenine-13C6 Kyn-13C6 215.1 100.1 30 11 16 Valine-d8 Val-d8 126.11 80.1 30 15 17 GABA-d6 GABA-d6 110.1 93.1 30 13 18 Tryptophan-d5 Trp-d5 210.1 151.1 30 23 19 Phenylalanine-13C515N Phe-13C515N 171.1 125.1 30 15 20 Allo-isoleucine-13C6 Allo-Ile-13C6 138.1 91.1 30 35 21 Isoleucine-13C6 Ile-13C6 138.1 91.1 30 15 22 Leucine-d3 Leu-d3 135.1 89.1 30 15 23 His-13C6 His-13C6 162.1 115.1 33 19 24 Ornithine-d7 Orn-d7 140.1 77.1 30 25 25 Arginine-13C6 Arg-13C6 181.3 121.1 30 20 26 Asymmetric dimethylarginine-d6 ADMA-d6 209.2 52.2 34 40 27 Symmetric dimethylarginine-d6 SDMA-d6 209.2 175.2 28 16 28 Lysine-d4 Lys-d4 151.1 88.1 30 25 (6) Linear range of amino acids Serial number Amino acid name Abbreviations Linear range (unit is μmol / L, units marked with "*" are nmol / L) 1 Taurine Tau 7.04－704.22 2 Aspartic acid Asp 2.40－204.42 3 Proline Pro 10.42－1042.30 4 Asparagine Asn 4.11－411.22 5 Glutamine Gln 45.04－4503.56 6 Serine Ser 9.42－940.24 7 Hydroxyproline Hyp 1.10－110.12 8 Glutamate Glu 4.76－476.18 9 Citrulline Cit 2.48－248.30 10 Threonine Thr 12.72－1272.41 11 Glycine Gly 27.98－2797.50 12 Tyrosine Tyr 3.93－392.96 13 Alanine Ala 22.32－2231.56 14 Methionine Met 1.53－153.27 15 Kynurenine* Kyn* 54.52－5452.35 16 Valine Val 15.88－1588.99 17 GABA* GABA* 42.39－4235.40 18 Tryptophan Trp 3.04－305.34 19 Phenylalanine Phe 6.21－620.68 20 Alloisoleucine allo-Ile 7.85－785.23 21 Isoleucine Ile 5.08－507.59 22 Leucine Leu 5.23－522.53 23 Histidine His 4.23－423.27 24 Ornithine Orn 7.83－783.07 25 Arginine Arg 4.01－400.69 26 Asymmetric dimethylarginine* ADMA* 9.45－944.84 27 Symmetric dimethylarginine* SDMA* 15.82－1582.20 28 Lysine Lys 8.05－805.25 (7) Accuracy, precision, recovery and matrix effect of the 28 amino acids involved in this invention

[0029] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected; the scope of the present invention to be protected is defined by the attached claims and their equivalents.

Claims

1. A method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry, wherein the mobile phase of the method contains fluoride as an ion pair reagent, preferably the fluoride is selected from one or more of nonafluoropentanoic acid, tridecafluoroheptanoic acid (TDFHA) and perfluorooctanoic acid, and the stationary phase is selected from octadecyl chain C 18 And fluorocarbon chain FC mixed bonded phase chromatographic column.

2. The method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry according to claim 1, characterized in that The mobile phase of the method is selected from one or more of a methanol-water solution containing tridecafluoroheptanoic acid (TDFHA), a methanol-isopropanol-water solution containing tridecafluoroheptanoic acid (TDFHA), or a methanol-water solution and a methanol-isopropanol-water solution containing tridecafluoroheptanoic acid (TDFHA).

3. The method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry according to claim 1, characterized in that The mobile phase of the method comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is selected from a methanol-water mixed solution containing tridecafluoroheptanoic acid (TDFHA), and the preferred volume percentage of methanol is 40-50%; the mobile phase B is selected from a methanol-isopropanol-water solution containing or not containing tridecafluoroheptanoic acid (TDFHA), and the preferred volume percentage of methanol in the methanol-isopropanol-water is 80-90%, the volume percentage of isopropanol is 5-15%, and the balance is water.

4. The method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry according to claim 3, characterized in that The concentration of tridecafluoroheptanoic acid (TDFHA) in the mobile phase A of the method is 0.5-5 mM, preferably 0.8-3 mM, and more preferably 1-2 mM.

5. The method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry according to claim 1, characterized in that The method uses CNW E-Gen AAA as a chromatographic column (preferably, the chromatographic column model is 3.0*100 mm, 3 μm), and the stationary phase filler of the chromatographic column is octadecyl chain C 18 And fluorocarbon chain FC mixed bonded phase silica gel.

6. The method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry according to claim 1, characterized in that The tandem mass spectrometer uses electrospray ionization to perform quantitative detection on the substance to be tested.

7. The method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry according to claim 1, characterized in that The ion pair chromatography adopts gradient elution: the starting ratio is 0%B, 0-1 min; 0%B, 1-2 min; 20%B, 2-3 min; 20% B, 3-4 min; 60%B, 4~5 min; 60%B, 5~5.1 min; 0%B; 5.1-7 min, 0% B; Column temperature: 25~40℃; flow rate: 0.6~1 mL / min; injection volume: 5~30 μL; detection time: 7 min.

8. The method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry according to claim 1, characterized in that The mass spectrometry condition is to use positive ion scanning; Curtain gas: 35 psi; Collision gas: high; Atomizing gas: 65psi; Heating gas: 65 psi; ion source voltage: 5500 V; ion source temperature: 550 °C.

9. The method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry according to claim 1, characterized in that The method comprises the steps of adding a precipitant to the blood or a blood sample, taking the supernatant, adding a solvent to dilute it, and then injecting the sample.

10. The method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry according to claim 9, characterized in that The precipitant is selected from a mixed solvent of methanol and acetonitrile, and the volume ratio of the methanol to acetonitrile is 1:(0.5-2).

11. The method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry according to claim 1, characterized in that The multiple amino acids are selected from one or more of aspartic acid, proline, asparagine, glutamine, serine, glutamic acid, citrulline, threonine, glycine, tyrosine, alanine, methionine, γ-aminobutyric acid, valine, kynurenine, tryptophan, phenylalanine, alloisoleucine, isoleucine, leucine, histidine, ornithine, arginine, asymmetric dimethylarginine, symmetric dimethylarginine, lysine, hydroxyproline, and taurine.

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