Method of rapidly and quantitatively analyzing 48 amino acids

Abstract

The invention discloses a method of rapidly and quantitatively analyzing 48 amino acids. A derivatization reagent combination for quantitatively analyzing and detecting an amino acid content by a derivatization HPLC-MS / MS method is provided, and comprises a derivatization reagent A and a derivatization reagent B; the derivatization reagent A is a mixed solution of n-propanol and 3-methylpyridine;and the derivatization reagent B is a mixed solution of dichloromethane, chloroformyl propyl ester and isooctane. Furthermore, a stable isotope internal standard solution combination, a sample pretreatment method and a liquid chromatography-tandem mass spectrometry method based on the derivatization method are established. The derivatization reagent proposed by the invention has high derivatization efficiency and low toxicity; the method requires fewer samples, the processing is simple and quick, the repeatability is good, and the detection cost is low; absolute quantitative analysis on 48 amino acids is realized by only 22 minutes, the analysis time is greatly shortened, and the detection throughput is improved; and great promotion and application value are achieved.

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...

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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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