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Method for screening superoxide anion scavenging agent by coupling ultrahigh-performance liquid chromatography with mass spectrum

An ultra-high performance liquid phase, superoxide anion technology, applied in the field of analytical chemistry, can solve the problems of difficult detection, short superoxide anion life, false negatives, etc., to avoid false positive and false negative results, high accuracy, and sample detection. fast effect

Active Publication Date: 2011-07-20
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Existing methods for screening superoxide anion scavengers and detecting and quantifying superoxide anions mainly contain electron self-selection resonance method (YASUKO NODA, TAKAO KANEYUKI, AKITANEMORI, AND LESTER PACKER, J.Agric.Food Chem.50(2002), 166 -171), chemiluminescence (hahanara Banu, Gillian M.Greenway, R.Alan Wheatley, Analytica Chimica Acta 541 (2005) 91-97), ultraviolet detection (Bayer W F, FridovichI, 161 (1987) 559-566) , these methods are convenient and fast, and can meet the screening requirements to a certain extent, but false positive and false negative results are prone to occur in spectral screening, and electron spin resonance technology is relatively short in the detection of superoxide anion due to the short lifetime of superoxide anion. difficulty

Method used

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  • Method for screening superoxide anion scavenging agent by coupling ultrahigh-performance liquid chromatography with mass spectrum
  • Method for screening superoxide anion scavenging agent by coupling ultrahigh-performance liquid chromatography with mass spectrum
  • Method for screening superoxide anion scavenging agent by coupling ultrahigh-performance liquid chromatography with mass spectrum

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Embodiment 1

[0075] The method for superoxide anion scavengers screened by ultra-high performance liquid chromatography and mass spectrometry provided by the invention may further comprise the steps:

[0076]The ultra-high-performance liquid chromatography refers to: compared with high-performance liquid chromatography, it has ultra-high resolution, ultra-high speed and ultra-high sensitivity;

[0077] The superoxide anion scavenger is a natural product extract or a monomer; the superoxide anion scavenger described in this embodiment is a monomeric catechin;

[0078] (1) Preparation of superoxide anion generating system buffer

[0079] Enzymatic reaction is one of the main sources of producing superoxide anion in organisms. The superoxide anion generation system used in the present invention is xanthine oxidase-xanthine enzymatic reaction system, and xanthine oxidase catalyzes the substrate xanthine to produce superoxide anion;

[0080] The superoxide anion generating system buffer solut...

Embodiment 2

[0122] The superoxide anion scavenger described in the present embodiment is genistin;

[0123]The sample is as follows: the total reaction volume is 200 microliters, xanthine oxidase at a final concentration of 100 nanomoles / liter is incubated at 37°C for 30 minutes, and genistin is added at a final concentration of 20 micromoles / liter at a final concentration of 100 nanomoles / liter. Xanthine in micromole / liter, 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2,4-disulfobenzene)-2H- at a final concentration of 25 micromole / liter Sodium tetrazolium salt, react at 37°C for 5 minutes, add 800 microliters of methanol to terminate the reaction, add internal standard sodium tauroursodeoxycholate with a final concentration of 1 micromol / liter as a sample; for ultra-high performance liquid Determination by phase chromatography and mass spectrometry;

[0124] All the other are with embodiment 1;

[0125] According to the detection procedure of Example 1, the scavenging rate I of the 20 micr...

Embodiment 3

[0127] The superoxide anion scavenger described in the present embodiment is berberine;

[0128] The sample is as follows: the total reaction volume is 200 microliters, xanthine oxidase at a final concentration of 100 nanomoles / liter is incubated at 37°C for 30 minutes, and berberine with a final concentration of 20 micromoles / liter is added at a final concentration of 100 nanomoles / liter. Xanthine in micromole / liter, 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2,4-disulfobenzene)-2H- at a final concentration of 25 micromole / liter Sodium tetrazolium salt, react at 37°C for 5 minutes, add 800 microliters of methanol to terminate the reaction, add internal standard sodium tauroursodeoxycholate with a final concentration of 1 micromol / liter as a sample; for ultra-high performance liquid Determination by phase chromatography and mass spectrometry;

[0129] All the other are with embodiment 1;

[0130] According to the detection procedure of Example 1, it is detected and calculated t...

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Abstract

The invention provides a method for screening a superoxide anion scavenging agent by coupling ultrahigh-performance liquid chromatography with a mass spectrum. The method comprises the following steps of: analyzing the scavenging rate of a natural product extract or monomer on superoxide anions and detecting and quantifying the content of superoxide anions by detecting the change of 2-(4-iodbenzene)-3-(4-nitrobenzene)-5-(2,4-disulfobenzoic)-2H-tetrazole sodium salt serving as a trapping agent before and after reaction with superoxide anions. A method for coupling ultrahigh-performance liquid chromatography with the mass spectrum is applied to screening of the superoxide anion scavenging agent, so that the dosage of a sample is small, detection is quick and accurate and a relevant coefficient in a linear equation is up to 0.998; and the mass spectrum is used for detecting in specific to the mass-charge ratio of a compound, so that high accuracy and high specificity are achieved, false positive and false negative results in spectrometry screening are avoided, and the problem of difficulty in detecting caused by short service lives of the superoxide anions in an electronic spin resonance technology is solved. The method is used for screening the superoxide anion scavenging agent and detecting and quantifying the superoxide anions.

Description

field of invention [0001] The invention belongs to the field of analytical chemistry and relates to a method for screening superoxide anion scavengers by ultra-high performance liquid chromatography and mass spectrometry, in particular to a method for screening superoxide anion scavengers by ultra high performance liquid chromatography and mass spectrometry. technical background [0002] With the development of free radical medicine, it has been recognized that many diseases are related to free radicals. The body produces superoxide anion free radicals during the metabolic process. If the excess superoxide anion free radicals are not removed in time, they will accumulate in the body to produce cytotoxicity, destroy the structure and function of the biofilm, and cause various diseases. Therefore, antioxidant drugs play an important role in anti-aging and prevention and treatment of various related diseases. Natural medicine contains a variety of different antioxidant active ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/72
Inventor 刘志强刘舒邢俊鹏宋凤瑞郑重刘淑莹
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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