Method for directly detecting amino acid in biological tissue uniform sample by adopting high performance liquid chromatography-tandem mass spectrometry

A liquid chromatography mass spectrometry, biological tissue technology, applied in the field of detection of amino acids in uniform samples of biological tissue, can solve the problems of limited amino acid types, inability to determine amino acids, and high detection costs, and achieves low sample dosage, wide amino acid concentration range, The effect of high detection sensitivity

Active Publication Date: 2017-10-27
EXPERIMENTAL RES CENT CHINA ACAD OF CHINESE MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The Accq·tag method is currently widely used as an amino acid analysis method. It needs to purchase reagent packs and kits, and the cost is high. A

Method used

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  • Method for directly detecting amino acid in biological tissue uniform sample by adopting high performance liquid chromatography-tandem mass spectrometry
  • Method for directly detecting amino acid in biological tissue uniform sample by adopting high performance liquid chromatography-tandem mass spectrometry
  • Method for directly detecting amino acid in biological tissue uniform sample by adopting high performance liquid chromatography-tandem mass spectrometry

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preparation example Construction

[0060] In the preparation or preparation method of the extract disclosed by the present invention, the specific implementation of using formic acid and ammonium formate as buffer components is exemplarily described, as other buffers that meet the disclosure requirements of the present invention, such as acetic acid and ammonium acetate buffer system, or other buffer systems, the same method can also be used to prepare the extract.

[0061] Further, as the extraction solution disclosed in the present invention, the content of acetonitrile directly affects the extraction efficiency and detection sensitivity, for example, its content can be selected between 50%-95%, or between 60-90%, or between 70-90%. Between 85%.

[0062] The selection of the extract can be optimized according to the properties of the biological tissue sample. For example, an extract of the following components can be prepared:

[0063] (1) 50% acetonitrile aqueous solution, comprising 0.1% formic acid and 1...

Embodiment 1

[0086] Testing of serum samples.

[0087] 1. Experimental equipment

[0088]

[0089] 2. Reagents

[0090]Standards: Glycine, β-Alanine, Sarcosine, Alanine, γ-Aminobutyric acid, Serine, Valine, Threonine, Taurine, Leucine, Isoleucine, trans-4-Hydroxy- L-Proline), Asparaginate, Ornithine, Lysine, Methionine, L-2-Aminoadipic acid, Phenylalanine, 1-Methyl-histidine, 3-Methyl-histidine, Arginine, Citrulline (Citrulline), Tryptophan (Tryptophan) and Folic acid (Folic acid) were all purchased from Sigma Company in the United States, and Glutamine (Glutamine) and Histidine (Histidine) were purchased from TCI Company in Canada.

[0091] Acetonitrile and formic acid: mass spectrometry grade, Fisher Company, USA; ammonium formate: Sigma Company, USA; experimental water: prepared by Milli-Q system ultrapure water purification system.

[0092] 3. Sample

[0093] Serum samples come from Sprague-Dawley rats, collected and provided according to the requirements of the uniform sample...

Embodiment 2

[0139] Urine sample testing.

[0140] 1. Experimental equipment

[0141] With embodiment 1.

[0142] 2. Reagents

[0143] Glycine, β-Alanine, Sarcosine, Alanine, γ-Aminobutyric acid, Serine, Histamine ( Histamine), Valine, Taurine, Leucine, Isoleucine, trans-4-Hydroxy-L-Proline , Asparaginate, Ornithine, Homocysteine, Lysine, Glutamic Acid, Methionine, L-2 Aminoadipic acid (L-2-Aminoadipic acid), phenylalanine (Phenylalanine), arginine (Arginine), citrulline (Citrulline), tryptophan (Tryptophan), folic acid (Folic acid) are all purchased from American sigma company, glutamine (Glutamine) was purchased from Canadian TCI company.

[0144]Acetonitrile and formic acid: mass spectrometry grade, Fisher Company, USA; ammonium formate: Sigma Company, USA; experimental water: prepared by Milli-Q system ultrapure water purification system.

[0145] 3. Sample

[0146] Urine samples came from Sprague-Dawley rats, collected and provided according to the standardized and reasonable o...

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Abstract

The invention discloses a method for directly detecting amino acid in a biological tissue uniform sample by adopting high performance liquid chromatography-tandem mass spectrometry. The method comprises the step of treating a sample with an extracting solution, wherein the extracting solution is composed of a liquid chromatogram moving phase, including acetonitrile and buffer solution; the concentration of acetonitrile is 50-95%; the buffer solution is composed of formic acid with concentration of 0.1%-0.3% and ammonium formate with concentration of 1-10mM or is composed of acetic acid with concentration of 0.1%-0.3% and ammonium acetate in concentration of 1-10mM; the pH of the extracting solution is within 3-5.5. The method disclosed by the invention can be used for directly detecting the biological tissue uniform sample, such as, blood, urine and cerebrospinal fluid; the sample treating method is simple; the sample dosage is less and can be as low as 4mu l; the detection sensitivity is high and can reach up to pg/ml; the detectable amino acid concentration range is wide; the matrix interference is less; the detection accuracy is high; up to 29 amino acids can be simultaneously detected and the detection time is short.

Description

technical field [0001] The application of the invention belongs to the field of instrumental analysis, and specifically relates to a method for directly detecting amino acids in uniform samples of biological tissues by using liquid chromatography mass spectrometry. Background technique [0002] The detection and analysis of amino acids is an important research method in the fields of life science, food science, and clinical medicine, so the research and improvement of amino acid analysis methods are of great significance. The amino acid analysis method was first automated in 1958, and then gradually developed, and the sensitivity, accuracy, and degree of automation were gradually improved. [0003] In 2008, Boogers et al combined UPLC with Accq·tag (Waters Company) online derivatization method to analyze the hydrolyzed amino acids in casein and serum albumin. [0004] The Accq·tag method is currently widely used as an amino acid analysis method. It needs to purchase reagent...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
Inventor 郭娜雷燕范斌闫寒
Owner EXPERIMENTAL RES CENT CHINA ACAD OF CHINESE MEDICAL SCI
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