液相色谱-串联质谱法同时检测DNA中3-甲基腺嘌呤和3-乙基腺嘌呤  

Simultaneous Determination of N3-methyladenine and N3-ethyladenine in DNA by Liquid Chromatography-Tandem Mass Spectrometry

作　　者：田永峰[1,2] 侯宏卫[1] 张小涛[1,2] 刘勇[2] 王安[2] 胡清源[1] 

机构地区：[1]国家烟草质量监督检验中心,郑州450001 [2]中国科学院安徽光学精密机械研究所,合肥230001

出　　处：《分析化学》2014年第8期1200-1204,共5页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金资助项目(Nos.21277174;21347002)~~

摘　　要：利用阳离子交换固相萃取柱（Waters Oasis MCX）富集净化DNA样品,建立了液相色谱串联质谱（LC-MS/MS）同时检测DNA中3-甲基腺嘌呤（N3-MeA）和3-乙基腺嘌呤（N3-EtA）的方法。采用氘代-3-甲基腺嘌呤（d3-N3-MeA）和氘代-3-乙基腺嘌呤（d5-N3-EtA）为内标;进样量3μL,分析时间为13 min;亲水相互作用色谱柱（Waters XBridge HILIC）进行液相分离,流动相为10 mmol/L甲酸铵-乙腈溶液（5∶95,V/V,pH=4.0）,流速250μL/min;质谱条件：电喷雾离子源,多反应监测正离子扫描方式;电喷雾电压：5500 V,雾化气：369 Pa,气帘气：185 Pa,电离温度：400℃,驻留时间：40 ms。本方法对N3-MeA和N3-EtA的检出限分别为0.043和0.007μg/L,方法回收率为87.8%~103.0%。采用本方法检测了卷烟烟气粒相物暴露的DNA中N3-MeA和N3-EtA含量。结果表明,卷烟烟气粒相物暴露后的小牛胸腺DNA中3-甲基腺嘌呤和3-乙基腺嘌呤可被本方法定量检出。A liquid chromatography-tandem mass spectrometry （ LC-MSMS ） method has been developed for the simultaneous determination of N3-methyladenine （ N3-MeA ） and N3-ethyladenine （ N3-EtA ） in calf thymus DNA. The DNA samples has been purified and enriched by cation exchange cartridge （ Waters Oasis MCX） . d3-N3-MeA and d5-N3-EtA were used as isotope internal standard. The DNA samples were injected with autosampler. The injected volume was 3 μL and analysis time was 13 min. The sample separation was carried out on hydrophilic interaction chromatograph （ Waters XBridge HILIC ） with 10 mmol/L ammonium formate-acetonitrile （5：92, V/V, pH=4. 0） as mobile phase. The flow rate was set at 250 μL/min. Mass spectrometry was performed by electrospray ionization （ ESI ） with multi-reactions monitoring （ MRM ） . The optimized operation conditions of MS were as follows： nebulizer gas 369 Pa; curtain gas 185 Pa, turbo ionspray temperature 400 ℃, ionspray voltage 5500 V, dwell time 40 ms. The limits of detection were 0. 043 and 0. 007 μg/L for N3-MeA and N3-EtA, respectively. The recoveries were between 87. 8% and 103. 0%for N3-MeA and N3-EtA. This method was successfully applied to the determination of N3-MeA and N3-EtA in calf thymus DNA by cigarette smoke condensate （ CSC） exposure. This method is appropriate for routine analysis and accurate quantification of N3-MeA and N3-EtA by CSC exposure.

关 键 词：3-甲基腺嘌呤 3-乙基腺嘌呤 液相色谱串联质谱 DNA 

分 类 号：O657.63[理学—分析化学]