A Rapid Quantitative Analysis Method for 48 Amino Acids

A technology of amino acid and aminobutyric acid, which is applied in the direction of analysis materials, measuring devices, material separation, etc., can solve the problems of poor separation ability of isomers, inability to detect amino acids, and many test steps, so as to improve accuracy and repeatability performance, improve derivatization efficiency, and improve detection throughput

Active Publication Date: 2022-03-04
GUANGZHOU WOMEN AND CHILDRENS MEDICAL CENTER
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  • Abstract
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Problems solved by technology

However, each of these methods has some defects, which limit their popularization and application in clinical laboratories: the IEX method has a long data collection time (>120min) and cannot separate important biomarkers such as methionine and homocitrulline disadvantages; the OPA / FMOC-HPLC method can only analyze more than 20 common amino acids at a time, and the data acquisition time is faster than the IEX method, but it takes 30 minutes for a sample, and the ability to resist matrix interference and isomer separation is poor, resulting in The specificity of the method is not high; the iTraq-LC-MS / MS method has the advantages of rapidity and sensitivity, but the iTraq reagent used in the method is expensive, and the method is sensitive to sulfur-containing amino acids, such as methionine, cystine, The detection of cysteine, homocysteine, homocysteine, etc. has the disadvantages of poor repeatability and low recovery (<80%)
[0005] In the early 1990s, some scholars proposed to use chloroformyl ester derivatization-gas chromatography-mass spectrometry to analyze amino acids. This method significantly improved the sensitivity and detection throughput of the method, but this method could not detect amino acids containing guanidine groups, such as citrulline. Acid, homocitrulline, arginine, etc., and this type of compound is of great significance in the screening and diagnosis of urea cycle disorders (amino acid metabolism disorders)
The EZ:Faast derivatization-HPLC-MS / MS method introduced by Phenomenex to detect plasma amino acids is a method developed on the basis of the above-mentioned chloroformyl ester derivatization pretreatment. This method can analyze guanidino amino acids, but The pretreatment process uses solid-phase extraction to extract amino acids and then conducts derivatization reactions, which requires a large amount of plasma (100 μL plasma), many test steps, and cumbersome operations, which affects the recovery rate and accuracy of the method; secondly, Phenomenex EZ: Pyridine and chloroform in Faastt derivatization reagents are highly toxic
Thirdly, Phenomenex EZ:Faast only uses 3 compounds as internal standards, which cannot analyze the differences in the extraction and derivatization reactions of amino acids with different molecular structures during the sample pretreatment process, and the matrix effect and ion suppression effect in the data collection process. Absolute quantification of all analytes cannot be performed by calibrating for differences caused by

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  • A Rapid Quantitative Analysis Method for 48 Amino Acids
  • A Rapid Quantitative Analysis Method for 48 Amino Acids
  • A Rapid Quantitative Analysis Method for 48 Amino Acids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Example 1 A method for rapid quantitative analysis of 48 amino acids

[0114] 1. Reagents

[0115]Standards of pipemidic acid (purity≥97.0%) and α-aminobutyric acid (purity≥99.0%) were purchased from Anpu Company (Shanghai, China), and other standard amino acids, trihydroxypropylphosphine and ammonium formate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chloroformyl propyl ester, 3-picoline, isooctane and n-propanol were purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). 13 C 3 - Propanol, D 3 -Methionine and D 4 - Cystathionine was purchased from Cambridge Isotope Laboratories (Andover, MA, USA).

[0116] Solution preparation

[0117] Amino acid standard stock solution: Accurately weigh each amino acid standard, dissolve with ultrapure water or 1mol / L HCl solution to volume.

[0118] Standard 1: Accurately weigh the amino acid standard as shown in Table 2, add water or 1mL of 1mol / L hydrochloric acid aqueous solution to dissolve, and prepare a...

Embodiment 2

[0163] Example 2 A kit for detecting amino acid content by derivatization HPLC-MS / MS method

[0164] 1. Composition

[0165] Derivatization reagent kits, internal standard kits, and standards

[0166] Wherein, the combination of derivatization reagents is: derivatization reagent A and derivatization reagent B prepared in Example 1;

[0167] The combination of internal standard solution is: internal standard solution A and internal standard solution B prepared in Example 1;

[0168] The standard products are: standard product 1, standard product 2 and standard product 3.

[0169] 2. How to use

[0170] (1) Preparation of calibrator

[0171] According to the method of Example 1, calibrator 1 to calibrator 7 were prepared.

[0172] (2) Sample pretreatment

[0173] With embodiment 1.

[0174] (3) Sample data collection

[0175] With embodiment 1.

Embodiment 4

[0176] Embodiment 4 detects clinical sample

[0177] 1. Test samples

[0178] The plasma of volunteers without abnormal amino acid metabolism and patients with neonatal cholestasis syndrome was selected for detection.

[0179] 2. Detection method

[0180] As described in Example 1.

[0181] 3. Test results

[0182] Test results such as figure 2 shown. The content of methionine, citrulline and lysine in the plasma of patients with neonatal cholestasis syndrome was significantly increased, and the plasma amino acid profile of volunteers without abnormal amino acid metabolism was normal.

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Abstract

The invention discloses a method for rapid quantitative analysis of 48 kinds of amino acids. The invention provides a derivatization reagent combination for quantitative analysis and detection of amino acid content by a derivatization HPLC-MS / MS method, including a derivatization reagent A and a derivatization reagent B; wherein, the derivatization reagent A is n-propanol and 3-methanol The mixed solution of base pyridine, the mixed solution of dichloromethane, chloroformyl propyl ester and isooctane as the derivatization reagent B. And further established the stable isotope internal standard combination, sample pretreatment method and liquid chromatography-tandem mass spectrometry method based on the derivatization method. The derivatization reagent proposed by the present invention has high derivatization efficiency and low reagent toxicity; the method requires less sample volume, simple, fast, good repeatability, and low detection cost; and the absolute quantitative analysis of 48 amino acids is realized in only 22 minutes , which greatly shortens the analysis time and improves the detection throughput; it has great popularization and application value.

Description

technical field [0001] The invention relates to the technical field of biological detection, more specifically, to a method for rapid quantitative analysis of 48 kinds of amino acids. Background technique [0002] Amino acids are a class of organic compounds containing amino and carboxyl functional groups. They are the basic constituent units and metabolites of proteins, and are an important class of biomarkers. The analysis of amino acid content in human body fluids (including blood, urine, cerebrospinal fluid, and interstitial fluid) can be used for screening and auxiliary diagnosis of amino acid metabolism disorders and evaluation and monitoring of human nutritional status. [0003] Amino acid metabolism disorders often lead to changes in the levels of multiple amino acids, which poses a challenge to traditional analysis methods. The ELISA method that analyzes one compound at a time is completely unsuitable for the analysis of such compounds. In addition, the presence of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/89
CPCG01N30/89
Inventor 彭敏芝刘丽
Owner GUANGZHOU WOMEN AND CHILDRENS MEDICAL CENTER
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