Method for quantitatively detecting free amino acid in clinical sample

A quantitative detection method, free amino acid technology, applied in the field of analysis and detection, can solve problems such as easy contamination of instruments, difficulty in obtaining, matrix interference, etc., and achieve the effects of reducing detection interference, avoiding contamination of instruments, and strong specificity

Inactive Publication Date: 2017-01-25
武汉生物技术研究院有限公司
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  • Application Information

AI Technical Summary

Problems solved by technology

Direct detection of matrix interference is serious, affecting the accuracy of quantification
The derivatization method can be divided into the derivatization of the amino group and the derivation of the carboxyl group: for the derivatization of the amino group, the detection sensitivity is high and the specificity is good, but the derivatization reagent is complicated and difficult to obtain; for the carboxyl group derivation, n-butanol is commonly used, but existing The detection method is not separated by the chromatographic column during the detection, and the sample is directly injected, the matrix interference is serious, and the instrument is easy to be polluted, and the deuterated amino acid is used as the internal standard, and the cost is high

Method used

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  • Method for quantitatively detecting free amino acid in clinical sample
  • Method for quantitatively detecting free amino acid in clinical sample
  • Method for quantitatively detecting free amino acid in clinical sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Sample handling:

[0023] ①Drop the blood extracted from the human body on cardboard, and dry overnight at room temperature;

[0024] ②Use a hole puncher to punch blood spots with a diameter of about 3.0mm, place them in a 1.5mL centrifuge tube, and vortex extract with 200μL formic acid / methanol (0.5%, v / v) solution for 30min;

[0025] ③ Take 4 μL of the extract in step ② and blow dry, add 50 μL of 37wt% HCl / n-BuOH (1:4, v / v) solution, derivatize in a water bath at 75°C for 30 min, centrifuge and dry;

[0026] ④ Reconstitute in 200 μL methanol / water (5%, v / v) (containing 2 ng / mL deuterated internal standard), separate on a C18+ chromatographic column, and detect by mass spectrometry.

[0027] The deuterated internal standards are: deuterated n-butanol derived amino acid standard (amino acid standard purchased from Sigma, USA), deuterated n-butanol derived acylcarnitine (acylcarnitine standard purchased from U.S. PerkinElmer) . Derivation conditions were the same as f...

Embodiment 2

[0042] Sample handling:

[0043] ① Add 200 μL of formic acid / methanol (0.5%, v / v) solution to 10 μL of urine, vortex for 1 min, centrifuge, take the supernatant, and blow dry;

[0044] ② Add 50 μL of 37wt% HCl / n-BuOH (1:4, v / v) solution, derivatize in a water bath at 75°C for 30 minutes, centrifuge and blow dry;

[0045] Reconstitute in 200 μL of methanol / water (5%, v / v) (containing 2 ng / mL deuterated internal standard), separate on a C18+ chromatographic column, and detect by mass spectrometry. Separation conditions and mass spectrometry parameters are the same as in Example 1.

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Abstract

The invention discloses a method for quantitatively detecting a free amino acid in a clinical sample, and belongs to the field of analysis and detection. According to the method, an acidic methanol solution is used for extracting the free amino acid in the clinical sample, then the property of the amino acid is changed through alcohol derived carboxyl, and deuteration agent derived amino acid serves as an internal standard instead of deuteration amino acid to be favorable for chromatographic separation and quantitative mass spectrometric detection. The operation of the method is simple, the amino is basically separated within a short period of time, the interference of a matrix is greatly reduced, the cost is low, the specificity is good, and the method is suitable for quantitative detection of the free amino acid in the clinical sample.

Description

technical field [0001] The invention relates to a quantitative detection method for free amino acids in clinical samples, belonging to the field of analysis and detection. Background technique [0002] Amino acid is one of the three major nutrients of the living body, and is also a necessity for life activities. It can provide the nutrients needed for metabolism, ensure the growth, development, and reproduction of the body, and promote the development of the brain and intelligence. Amino acids play a vital role in energy metabolism, signal pathway conduction, nerve conduction, and lipid transport. They exist in two forms in the body, one is in the blood in the free form, and the other is in the protein in the form of binding. Under normal conditions, the content of amino acids in the blood is in a balanced state. When the body is abnormal, it will lead to metabolic disorders of amino acids in the blood. Amino acid metabolism defects caused by congenital deficiency of amino...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/04G01N30/06G01N30/34G01N30/72G01N30/88G01N2030/045G01N2030/067G01N2030/8813G01N2030/8822
Inventor 陈希冯钰锜刘欢杨帆李思思刘宜子王凯张晗蕊魏侠李遥力
Owner 武汉生物技术研究院有限公司
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