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Determination method of formaldehyde-DNA adducts in saliva

A determination method and adduct technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., to reduce consumption, improve selectivity, accuracy and sensitivity, and improve the effect of chromatographic separation process

Inactive Publication Date: 2016-09-14
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 HOMe-dA and HOMe-dG in saliva has not been reported

Method used

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  • Determination method of formaldehyde-DNA adducts in saliva
  • Determination method of formaldehyde-DNA adducts in saliva
  • Determination method of formaldehyde-DNA adducts in saliva

Examples

Experimental program
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Effect test

example 1

[0035] 1. Instruments and reagents:

[0036]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.

[0037] Deoxyribonuclease I, Alkaline Phosphatase (New England Biotechnology, USA), Phosphodiesterase I, Ammonium Acetate, Formic Acid, and Calf Thymus DNA (Sigma, USA), NaBH 3 CN (Shanghai Aladdin Reagent Company), Methanol (Deoksan Reagent Company, South Korea), Strata-X polymer column (33 μm, 30 mg / 1mL, Guangdong Philomen Scientific Instrument Co., Ltd.), Oragene DNA saliva collector (OG-500, DNA Genotek, Canada). Standard ...

example 2

[0043] Example 2: As described in Example 1, the method established in this study was used to detect formaldehyde-DNA adducts in 5 saliva samples (each sample was tested 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

Provided is a determination method of formaldehyde-DNA adducts in saliva, namely the determination method of HOMe-dA and HOMe-dG in the saliva; the determination method includes the steps: collecting the saliva by using an OG-500 saliva collection tube, carrying out DNA extraction on the saliva, carrying out enzyme hydrolysis of the DNA solution, and successively adding a stable isotope internal standard, a reducing agent NaBH3CN and a DNA hydrolase; and carrying out solid-phase extraction of the hydrolysate with a Strata-X small column, collecting an eluted liquid, nitrogen-blowing to be dried at room temperature, redissolving in a methanol aqueous solution, introducing into an LC-MS / MS system, analyzing, and accurately detecting the content levels of Me-DA and Me-dG. The stable isotope is used as an internal standard quantitative analysis material, so the error caused by the sample pretreatment process can be reduced, and the selectivity, accuracy and sensitivity of the method can be improved by tandem mass spectrometry. Through selection and optimization of the chromatographic column and the elution conditions, the chromatographic separation process is relatively improved, the time of chromatographic analysis is shortened, and the consumption amount of an organic solvent is decreased.

Description

technical field [0001] The invention belongs to the technical field of physical and chemical testing of saliva samples, and mainly relates to N in saliva 6 -Hydroxymethyl-adenine (HOMe-dA) and N 2 - The technical field of determination of hydroxymethyl-guanine (HOMe-dG) DNA adducts, specifically a liquid chromatography-tandem mass spectrometer (LC-MS / MS) for the determination of formaldehyde-DNA adducts in saliva Methods. Background technique [0002] Formaldehyde is a pollutant widely present in environmental substrates, such as industrial production, motor vehicle exhaust and interior decoration, etc. Formaldehyde has human genotoxicity and is listed as a class I carcinogen by the International Agency for Research on Cancer (IARC). Active aldehyde groups can attack nucleophilic groups on DNA to form DNA adducts, where N 6 -Hydroxymethyl-adenine (HOMe-dA) and N 2 -Hydroxymethyl-guanine (HOMe-dG) is the major formaldehyde-DNA adduct. These two structural forms are unsta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
CPCG01N30/02G01N2030/027
Inventor 胡清源陈欢侯宏卫刘鲁娟韩书磊朱贝贝陈建
Owner CHINA NAT TOBACCO QUALITY SUPERVISION & TEST CENT
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