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A kind of assay method of acrolein dna adduct in saliva

A method for measuring acrolein, which is applied in the field of physical and chemical testing of saliva samples, and achieves the effects of high sensitivity, easy operation, and reduced consumption

Active Publication Date: 2018-02-27
CHINA NAT TOBACCO QUALITY SUPERVISION & TEST CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, the simultaneous analysis of α-Acr-dG and γ-Acr-dG in saliva has not been reported

Method used

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  • A kind of assay method of acrolein dna adduct in saliva
  • A kind of assay method of acrolein dna adduct in saliva
  • A kind of assay method of acrolein dna adduct in saliva

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0033] 1. Instruments and reagents:

[0034] AB SCIEX triple quadrupole tandem mass spectrometer (Applied Biosystems, California, USA), Agilent 1200 high performance liquid chromatography (Agilent, USA), NanoDrop 2000 ultra-micro spectrophotometer (Thermo Fisher Scientific, USA), constant temperature incubator ( Thermo Fisher Scientific Corporation of the United States), nitrogen concentration and drying instrument (Bitezi Company, Sweden), Milli-Q water purification system (Merck Group, Germany), Analyst 1.5.1 data acquisition and processing software.

[0035]Deoxyribonuclease Ⅰ, alkaline phosphatase (New England Biotechnology Co., Ltd., USA), phosphodiesterase Ⅰ, ammonium acetate, formic acid, and calf thymus DNA (Sigma Corporation, USA), methanol (Deoksan Reagent Co., Ltd., South Korea), Strata-X polymer column (33 μm, 30 mg / 1mL, Guangdong Philome Scientific Instruments Co., Ltd.), Oragene DNA saliva collector (OG-500, DNA Genotek, Canada). Standard α-Acr-dG, γ-Acr-dG and ...

example 2

[0041] Example 2: As described in Example 1, the method established in this study was used to detect acrolein-DNA adducts in 5 saliva samples (each sample was detected three times in parallel). Calculate the concentration of DNA adducts in each sample based on the standard curve and peak area, according to the formula:

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Abstract

A determination method of an acrolein-DNA adduct in saliva is a determination method of alpha-Acr-dG and gamma-Acr-dG in DNA, and concretely comprises the following steps: collecting saliva through adopting an OG-500 saliva collecting tube, carrying out DNA extraction on the saliva, carrying out enzyme hydrolysis on the obtained DNA solution, and sequentially adding a stable isotope internal standard substance and DNA hydrolase; and carrying out Strata-X column solid phase extraction on the obtained hydrolysate, collecting the obtained eluate, blowing the eluate with nitrogen at room temperature until the eluate is dry, re-dissolving the dried eluate in 100[mu]L of a 30% methanol-water solution, introducing the obtained new solution to an LC-MS / MS system, and accurately detecting the content levels of the alpha-Acr-dG and gamma-Acr-dG. The stable isotope is adopted as an internal standard quantitative analysis substance in order to reduce the error caused by a pretreatment process, and tandem mass spectrometry well improves the selectivity, the accuracy and the sensitivity of the method. The method well improves the separation degree of target objects through selecting a chromatographic column and optimizing the eluting conditions.

Description

technical field [0001] The invention belongs to the technical field of physical and chemical testing of saliva samples, and mainly relates to the technical field of determination of α-Acr-dG and γ-Acr-dG adducts in saliva DNA, specifically a liquid chromatography-tandem mass spectrometer (LC - MS / MS) for the determination of major acrolein-DNA adducts in saliva. Background technique [0002] Acrolein is a kind of active α, β-unsaturated aldehyde, which widely exists in environmental substrates and is mainly produced by incomplete combustion of organic matter, such as industrial waste gas, cigarette smoke, motor vehicle exhaust and cooking fume. Acrolein has two electrophilic groups, which can react with nucleophilic substances in the body without the metabolism of the body, such as bases in DNA, to produce DNA adducts. The most studied is the addition of acrolein to guanine (Acr-dG), that is, 6-hydroxy-1,N 2 -propanol-2'-guanine (α-Acr-dG) and 8-hydroxy-1,N 2 -propanol-2'...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02
CPCG01N30/02G01N2030/027
Inventor 胡清源陈欢侯宏卫刘鲁娟张小涛朱欣潮张森
Owner CHINA NAT TOBACCO QUALITY SUPERVISION & TEST CENT
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