Alpha-ketoglutarate content determination reagent kit and method based on HPLC (high performance liquid chromatography)

Abstract

The invention discloses an alpha-ketoglutarate content determination reagent kit and a method based on HPLC (high performance liquid chromatography). The reagent kit comprises a reagent I, a reagent II, a reagent III, a reagent IV and an alpha-ketoglutarate standard, wherein the reagent I comprises hydrochloric acid, EDTA (ethylene diamine tetraacetic acid) and water; the reagent II comprises NaOHand water; the reagent III comprises phenylhydrazine hydrochloride and water; the reagent IV comprises dipotassium phosphate, monopotassium phosphate and water. The method comprises the steps of generating an absorption substance under ultraviolet by a reaction of alpha-ketoglutarate and a derivation agent, and after performing separation by the high performance liquid chromatography, determiningan alpha-ketoglutarate content through an ultraviolet detector. The kit and the method can detect a microgram level content and are high in sensitivity; a sample pretreatment process is simple and easy to operate; the manpower is saved; an extracting solution comprises simple ingredients; only aqueous solution extraction is required; the cost is low; and compared with a biological immunodetectionmethod, the required reagents have obvious price advantages, and are nontoxic basically, safe and environmentally friendly.

Description

technical field [0001] The invention belongs to the field of life sciences, and in particular relates to a high-performance liquid chromatography (HPLC)-based assay kit and method for alpha-ketoglutarate content. Background technique [0002] α-Ketoglutarate is an important metabolic intermediate in the microbial tricarboxylic acid cycle, and is a key node connecting intracellular carbon-nitrogen metabolism. Its position in the cycle is after isocitrate and before succinyl-CoA. At this site, anaplerosis can produce α-ketoglutarate through the transamination of glutamate, thereby achieving the purpose of replenishing this intermediate metabolite. [0003] At present, the methods for detecting α-ketoglutarate in the market can be divided into three categories, spectroscopic method, biological immunoassay technology and high performance liquid phase detection technology. The principle of spectrometry is to use the absorption value of α-ketoglutaric acid derivatives at specific...

AI Technical Summary

Problems solved by technology

The principle of spectrometry is to use the absorption value of α-ketoglutaric acid derivatives at specific wavelengths for approximate content detection. Generally, it is very susceptible to the influence of complex impurities in the system, pigments, etc., and the content of the test is prone to high content

The method of biological immunoassay is susceptible to cross-reaction interference, and secondly, the antibody is not universal and takes a long time to prepare. The whole time period is very long and the price is expensive.

Chromatographic technology has developed rapidly in recent years, and the application of detection of small molecules in organisms has become more and more extensive. However, there are few reports on the lack of detection technology based on high performance liquid chromatography (HPLC) for the detection of α-ketoglutarate in the market. Therefore need a kind of high performance liquid chromatography α-ketoglutarate content detection method that sample pretreatment is convenient and the loss rate is higher