A liquid chromatography-tandem mass spectrometry method for determining multiple amino acids

Abstract

The present invention discloses a liquid chromatography tandem mass spectrometry method for determining multiple amino acids. The method includes using a chromatographic column of silica gel bonded with a mixed octadecyl chain and fluorocarbon chain, and using a mobile phase containing a fluorine compound for retention and separation. This method greatly improves the chromatographic retention of amino acids, improves the peak shape, increases the separation degree between isomers, speeds up the detection speed, and at the same time obtains more stable reproducibility. The detection linear range completely covers clinical normal and abnormal values, and can be used for precise quantitative analysis of amino acids in clinical blood samples.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry, and particularly 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 that make up the protein molecules of biological organisms and an important material basis for maintaining homeostasis. They are one of the important substances in the body's metabolism, with a wide range of biological functions and important physiological significance. Defects in the related proteins and enzymes involved in amino acid metabolism in the human body or various pathological states will lead to abnormal amino acid metabolism and changes in the amino acid levels in the body. Therefore, detecting the amino acid content in biological samples is of great significance, providing an auxiliary basis for the exploration of clinical analysis biomarkers, the interpretation of disease mechanisms, and the diagnosis of related diseases. Therefore, continuously improving the accuracy and working efficiency of amino acid analysis in biological samples and reducing the detection cost are of great significance for the clinical detection and popularization of amino acids.

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

[0004] The chromatographic retention and separation of amino acids in complex biological samples are the most important problems to be solved by LC-MS / MS detection technology. The overall polarity of amino acid molecules is relatively large. Using a HILIC hydrophilic column can increase chromatographic retention. However, the HILIC chromatographic column has limited separation and retention capabilities for multiple varieties and isomers, and the reproducibility of the method is poor. Reversed-phase chromatographic columns (such as C 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 large polarity of amino acids themselves, reversed-phase chromatography usually requires a high proportion of aqueous mobile phase to achieve the retention and separation of most amino acids, and it is still difficult to distinguish isomers with very similar structures. In addition, eluting at a high proportion of aqueous mobile phase is not conducive to the ionization of amino acids in the mass spectrometry, which may lead to an increase in matrix effect.

[0005] At present, there are mainly two methods for improving amino acid chromatographic separation. One is chemical derivatization, which attaches a group to the amino acid to increase its hydrophobicity and enhance its retention on the reversed-phase chromatographic column. However, the derivatization method generally requires heating reactions and waiting, with cumbersome operations, many influencing factors, and long time consumption. Moreover, the derivatization reagents usually have a certain degree of contamination to the mass spectrometer and may produce additional matrix effects. The other is ion-pair chromatography, where an ion-pair reagent is added to the mobile phase to interact with the amino acid through ionic bonds to form an ion pair, and then the hydrophobic chain of the ion-pair reagent forms an intermolecular force with the hydrophobic chain of the stationary phase of the reversed-phase chromatographic column, thereby increasing the retention and separation of amino acids. Perfluorocarboxylic acid anionic surfactants are often used in the 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, etc. 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, it is not that the stronger the retention is, the better. Too strong retention will also cause problems such as tailing, residue, long equilibration time, and even incomplete elution of hydrophobic amino acids. Appropriate retention and elution abilities are required to quickly and specifically detect various amino acids.

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

[0007] The main object of the present invention is to solve various problems existing in the detection of amino acids in the prior art, and to provide a liquid chromatography-tandem mass spectrometry analysis and detection method for multiple amino acids with high speed, high efficiency and high sensitivity; To achieve the above object, the technical solution adopted by the present invention is:

[0008] Provide 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, and 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 F-C mixed-bonded phase chromatographic column.

[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 is selected from one or more of a methanol-aqueous solution containing tridecafluoroheptanoic acid (TDFHA), an acetonitrile-aqueous solution containing tridecafluoroheptanoic acid (TDFHA), a methanol-isopropanol-aqueous solution containing tridecafluoroheptanoic acid (TDFHA), an acetonitrile-isopropanol-aqueous solution containing tridecafluoroheptanoic acid (TDFHA), or a methanol-acetonitrile-isopropanol-aqueous solution containing tridecafluoroheptanoic acid (TDFHA).

[0010] Furthermore, the present invention provides a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry. The mobile phase of the method includes mobile phase A and mobile phase B. Mobile phase A is selected from a methanol-aqueous solution containing tridecafluoroheptanoic acid (TDFHA), and preferably the volume percentage content of methanol is 40-50%; mobile phase B is selected from a methanol-isopropanol-aqueous solution containing or not containing tridecafluoroheptanoic acid (TDFHA), and preferably the volume percentage content of methanol in methanol-isopropanol-water is 80-90%, the volume percentage content of isopropanol is 5-15%, and the balance is water.

[0011] Furthermore, the present invention provides a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry. 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.

[0012] Furthermore, the present invention provides a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry. The method uses CNW E-Gen AAA as the chromatographic column (preferably the chromatographic column model is 3.0*100mm, 3 μm), and the stationary phase is octadecyl chain C 18 and fluorocarbon chain F-C mixed-bonded silica gel.

[0013] Furthermore, the present invention provides a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry. The tandem mass spectrometry uses electrospray ionization for quantitative detection of the substance to be measured.

[0014] Furthermore, the present invention provides a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry. The liquid chromatography uses 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.

[0015] Furthermore, the present invention provides a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry. The mass spectrometry conditions are positive ion scanning; curtain gas: 35 psi; collision gas: high; nebulizing gas: 65 psi; heating gas: 65 psi; ion source voltage: 5500 V; ion source temperature: 550 °C.

[0016] Further, the present invention provides a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry. The method includes the steps of adding a precipitating agent to blood or a blood sample, and then taking the supernatant, diluting it with a solvent, and injecting it for analysis.

[0017] Further, the present invention provides a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry. The precipitating agent is selected from a mixed solvent of methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 1:(0.5 - 2).

[0018] Further, the present invention provides a method for detecting multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry. 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.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The present invention uses a fluorine-containing compound as an ion pair reagent in combination with a CNW E-Gen AAA chromatographic column with a bonded phase containing a mixed bond of octadecyl chain C 18 and fluorocarbon chain F-C. The electronegative fluorine atoms of the fixed-phase F-C chain can undergo enhanced dipole-dipole or hydrogen bond interactions with the fluorine-containing ion pair reagent to additionally retain the analyte, increasing the retention of polar compounds. It is particularly suitable for the separation of halogenated compounds and their isomers, and is more selective than ordinary C 18 . Good chromatographic separations are achieved among the amino acid isomers and interfering substances, and the specificity of the method is better. The chromatographic retention and separation principle of this method have not been reported, and it has high novelty and can be extended to the separation and detection of the same type of difficult-to-retain compounds.

[0021] 2. Both the ion pair reagent of the fluorine-containing compound and the bonded phase F-C of the stationary phase used in the present invention contain F atoms, and their compatibility is better. The method can reach chromatographic equilibrium in a shorter time. The rapid equilibrium and efficient separation greatly shorten the chromatographic running speed, enabling the retention and elution of 28 amino acids to be completed within 7 minutes in one injection. The detection time is short and the throughput is high.

[0022] 3. The proportion of the organic phase in the mobile phase of the present invention is high, and the solubility and stability of the fluorine-containing compound in it are good. Therefore, the method has better reproducibility. When eluting under a high organic phase, the amino acids are fully ionized, and the sensitivity is significantly improved. Therefore, the present invention has no obvious matrix effect.

[0023] 4. The sample pretreatment of the present invention is simple, without steps such as drying and recombination. It has a high recovery rate, good accuracy and precision. The method can be combined with a 96-well plate to achieve full-automatic processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the LC-MS / MS diagram of 28 amino acids involved in the present invention patent for the examples, where (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-alloisoleucine, 21-isoleucine, 22-leucine, 23-histidine, 24-ornithine, 25-arginine, 26-asymmetric dimethylarginine, 27-symmetric dimethylarginine, 28-lysine).

[0025] Figure 2 It is the LC-MS / MS diagram for comparing the resolution between leucine and its isomers among different chromatographic columns under the same mobile phase. The chromatographic conditions are as follows: A. E-Gen AAA 3×100 mm 3μm, B. Hypercarb 3×100 mm 3μm, C. Kinetex polar C18, phenomenex 3×100 mm 2.6 μm; 1-hydroxyproline, 2-alloisoleucine, 3-isoleucine, 4-leucine. DETAILED DESCRIPTION OF THE INVENTION

[0026] To make the technical means, creative features, achieved purposes and functions of the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments.

[0027] Example 1

[0028] (1) Pretreatment of plasma samples

[0029] Take 50 μL of the plasma sample to be tested, add 300 μL of the precipitant (methanol:acetonitrile = 1:1, containing internal standard) to a 96-well collection plate, shake at 1200 rpm for 5 min, transfer it to a precipitation plate, place it in a 96-well positive pressure extraction device for pressure filtration, then pipette 50 μL of the clear liquid and add 450 μL of distilled water, shake at 1500 rpm for 1 min and then inject it for analysis.

[0030] (2) Liquid phase conditions

[0031]

[0032] (3) Mass spectrometry conditions

[0033]

[0034] (4)Amino acid mass spectrometry parameters

[0035] Serial number Analyte name English abbreviation Parent ion (Da) Product ion (Da) Declustering potential (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 Glutamic acid 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 γ-Aminobutyric acid 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

[0036] (5)Amino acid isotope internal standard mass spectrometry parameters:

[0037] Serial number Analyte name English abbreviation Parent ion (Da) Product ion (Da) Declustering potential (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 γ-Aminobutyric acid-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 Alloisoleucine-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 Histidine-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

[0038] (6)Linear range of amino acids

[0039] Serial number Amino acid name English abbreviation Linear range (unit: μmol / L, marked with "*" unit: 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 Glutamic acid 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 γ-Aminobutyric acid* 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

[0040] (7)Accuracy, precision, recovery rate and matrix effect of 28 amino acids involved in the present invention

[0041]

[0042] The above has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed; the scope of protection claimed by the present invention is defined by the appended 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 tridecafluoroheptanoic acid as an ion pair reagent; The multiple amino acids are selected from 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; The method uses a chromatographic column with a stationary phase packing of a mixed-bonded silica gel of octadecyl chain C 18 and fluorocarbon chain F-C; The mobile phase of the method includes mobile phase A and mobile phase B. Mobile phase A is selected from a methanol - water mixed solution containing tridecafluoroheptanoic acid, and the volume percentage content of methanol is 40 - 50%; Mobile phase B is selected from a methanol - isopropanol - water solution, and in the methanol - isopropanol - water, the volume percentage content of methanol is 80 - 90%, the volume percentage content of isopropanol is 5 - 15%, and the balance is water; The ion pair chromatography uses gradient elution, and the elution program is: 。 2. The detection method for multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry according to claim 1, wherein The concentration of tridecafluoroheptanoic acid in mobile phase A of the method is 0.5 - 5 mM.

3. The detection method for multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry according to claim 1, wherein The concentration of tridecafluoroheptanoic acid in mobile phase A of the method is 0.8 - 3 mM.

4. The detection method for multiple amino acids in blood based on ion pair chromatography - tandem mass spectrometry according to claim 1, wherein The concentration of tridecafluoroheptanoic acid in mobile phase A of the method is 1 - 2 mM.

5. The detection method of 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 the chromatographic column.

6. The detection method for multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry according to claim 5, wherein The model of the chromatographic column is 3.0 * 100 mm, 3 μm.

7. The detection method for multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry according to claim 1, wherein The tandem mass spectrometry uses electrospray ionization for quantitative detection of the substance to be measured.

8. The detection method for multiple amino acids in blood based on ion pair chromatography - tandem mass spectrometry according to claim 1, characterized in that The column temperature of the ion pair chromatography: 25 - 40 °C; flow rate: 0.6 - 1 mL / min; injection volume: 5 - 30 μL; detection time: 7 min.

9. The detection method for multiple amino acids in blood based on ion pair chromatography-tandem mass spectrometry according to claim 1, characterized in that The method includes the step of adding a precipitant to the blood sample, taking the supernatant, diluting it with a solvent and then injecting it.

10. The detection method for 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 - acetonitrile, and the volume ratio of methanol - acetonitrile is 1:(0.5 - 2).

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