Method for simultaneously determining five amino acid neurotransmitters in biological matrix based on two-dimensional liquid chromatography-ultraviolet derivatization method

A two-dimensional liquid chromatography, amino acid technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problem of difficult separation of multi-component endogenous substances, achieve simplified pretreatment process, large sample volume, high resolution effects

Pending Publication Date: 2022-03-01
HUNAN AGRICULTURAL UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0004] In view of the defects in the prior art, the purpose of the present invention is to provide a method for qualitatively quantifying, rapidly analyzing, and efficiently separating five kinds of amino acid neurotransmitters in different animal biological matrices by two-dimensional liquid chromatography. General methods in the prior art are difficult to separate multi-component endogenous substances and defects affected by endogenous matrix

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  • Method for simultaneously determining five amino acid neurotransmitters in biological matrix based on two-dimensional liquid chromatography-ultraviolet derivatization method
  • Method for simultaneously determining five amino acid neurotransmitters in biological matrix based on two-dimensional liquid chromatography-ultraviolet derivatization method
  • Method for simultaneously determining five amino acid neurotransmitters in biological matrix based on two-dimensional liquid chromatography-ultraviolet derivatization method

Examples

Experimental program
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Embodiment 1

[0042] A two-dimensional liquid chromatography-ultraviolet derivatization method for the simultaneous determination of five amino acid neurotransmitters in blood and brain tissue:

[0043] 1. Targets: L-glutamic acid, L-aspartic acid, glycine, taurine and γ-aminobutyric acid.

[0044] 2. Test reagents: Ultrapure water is Milli-Q ultrapure water system; acetonitrile and methanol are chromatographically pure; ammonia water, phosphoric acid, potassium tetraborate, NBD-F, disodium hydrogen phosphate and sodium dihydrogen phosphate are analytical grade solvents.

[0045] 3. Standard solution: L-glutamic acid, L-aspartic acid, glycine, taurine and γ-aminobutyric acid standard stock solution: Accurately weigh the standard respectively, add L-glutamic acid and L-day Aspartic acid was prepared into a 10mmol / L stock solution, glycine, taurine and γ-aminobutyric acid were all prepared into a 100mmol / L stock solution, stored at -80°C, with a shelf life of 3 months.

[0046] 4. Standard w...

Embodiment 2

[0069] Quantitative analysis of blood and brain tissue samples

[0070] 1. The target is the same as in Example 1

[0071] 2, test reagent is the same as embodiment 1

[0072] 3, standard solution is the same as embodiment 1

[0073] 4. The standard working solution is the same as in Example 1

[0074] 5, instrument and material are the same as embodiment 1

[0075] 6. Biological sample pretreatment method

[0076] 6.1. Pretreatment of plasma and serum samples: 100 μL of pig plasma, sheep plasma and rat serum were transferred into 2 mL tubes with a pipette, and then 1 mL of acetonitrile saline mixture (750:250, v / v) was added. Spin in a refrigerated centrifuge at 4°C for 30s, and centrifuge at 14500r / min for 10min. Take 100 µL of the supernatant for derivatization.

[0077] 6.2. Pretreatment of brain tissue samples: Weigh 1 g of pig and rat brain tissue and 0.5 ml of frozen normal saline (0.9%) into 2 ml centrifuge tubes, homogenize in a tissue homogenizer for 1 min, wei...

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Abstract

The invention discloses a method for simultaneously determining five amino acid neurotransmitters in a biological matrix based on a two-dimensional liquid chromatography-ultraviolet derivatization method, which comprises the following steps: carrying out pre-column derivatization pretreatment on a biological sample to obtain a solution to be detected, detecting the solution to be detected through a two-dimensional liquid chromatography system, and quantitatively analyzing five amino acid neurotransmitter targets. The method realizes simultaneous and rapid detection of multiple components by two-dimensional liquid chromatography, has the advantages of large sample introduction volume, high resolution, good peak shape, no influence by other endogenous amines and the like, and is suitable for qualitative and quantitative detection of aspartic acid, glutamic acid, glycine, taurine and gamma-aminobutyric acid in endogenous biological samples.

Description

technical field [0001] The invention relates to a method for detecting amino acid neurotransmitters in animal biological matrices, in particular to a two-dimensional liquid chromatography system using pre-column derivatization combined with ultraviolet detection to simultaneously detect aspartic acid, glutamic acid, The invention discloses a method for five kinds of amino acids including glycine, taurine and gamma-aminobutyric acid, belonging to the field of analysis technology. Background technique [0002] At present, the research on amino acids has been extensive, and the focus is often on the common amino acids in the human body. The research on amino acid neurotransmitters in animals is not extensive. Amino acid neurotransmitters are generally considered to include excitatory amino acids (glutamate, aspartate) and inhibitory amino acids (glycine, taurine, and gamma-aminobutyric acid) (M,P.; Q,L.; acta , K.R.J.A.c., Review of recent advances in analytical techniques for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/34G01N30/86
CPCG01N30/02G01N30/06G01N30/34G01N30/8675G01N2030/062G01N2030/067
Inventor 刘兆颖赵雪娇孙志良
Owner HUNAN AGRICULTURAL UNIV
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