Measurement of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine in DNA and human urine by high performance liquid chromatography-electrospray tandem mass spectrometry
Measurement of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine in DNA and human urine by high performance liquid chromatography-electrospray tandem mass spectrometry
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DOI：
10.1016/s0891-5849(01)00462-2
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发表时间：
2001-04-01
影响因子：
7.4
通讯作者：
Poulsen, HE
中科院分区：
文献类型：
--
作者：
Weimann, A;Belling, D;Poulsen, HE
建立了高效液相色谱-串联质谱法测定DNA和尿液中8-氧-2‘-脱氧鸟苷和8-氧-2’-脱氧腺苷的方法。对于尿样，除了加入缓冲液和内标物，然后重新溶解含有8-氧-2‘-脱氧鸟苷的沉淀物和离心步骤，然后将样品注入高效液相色谱柱上，不进行样品制备。8-氧代-2‘-脱氧鸟苷和8-氧代-2’-脱氧腺苷的检出限约为0.3 nm，对应于7.5fmol进样。长时间分析，即50个样品，只需最小的灵敏度损失即可进行分析。人体尿样中8-氧代-2‘-脱氧鸟苷的排泄浓度在1-100 NM之间，8-氧代-2’-脱氧腺苷在0.3 NM以下。在小牛胸腺中，每10(6)个未经修饰的碱基可以检测到大约1个氧化的鸟苷和腺苷。8-氧-2‘-脱氧鸟苷的含量很高，每10(6)2’-脱氧鸟苷有30个，8-氧-2‘-脱氧腺苷的含量达到或低于检测限。这些结果表明，高效液相色谱-串联质谱仪是一种分析组织中氧化DNA修饰以及分析尿中氧化核苷酸排泄以确保最小伪影形成的高度灵敏和特异的方法。(C)2001年爱思唯尔科学公司。
A method for the determination of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine in DNA and urine by High Performance Liquid Chromatography (HPLC)-Tandem Mass Spectrometry is described. For the urine samples there is no sample preparation except for addition of buffer and internal standards followed by redissolvation of precipitate containing 8-oxo-2'-deoxyguanosine and a centrifugation step before the samples are injected onto the HPLC column. The detection Limit for 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine is approximately 0.3 nM corresponding to 7.5 fmol injected. Long runs, that is, > 50 samples, can be analyzed with only minimal loss of sensitivity. The concentrations excreted into urine samples from humans are between 1 and 100 nM for 8-oxo-2'-deoxyguanosine and below 0.3 nM for 8-oxo-2'-deoxyadenosine. In calf thymus DNA levels down to about 1 oxidized guanosine and adenosine per 10(6) unmodified bases can be detected. High levels of 8-oxo-2'-deoxyguanosine were found, 30 per 10(6) 2'-deoxyguanosine, levels of 8-oxo-2'-deoxyadenosine are at or below the detection limit. These findings indicate that High Performance Liquid Chromatography-Tandem Mass Spectrometry is a highly sensitive and specific method for analysis of oxidative DNA modifications in tissue as well as for analysis of excretion of oxidized nucleotides into urine that ensures a minimum artifact formation. (C) 2001 Elsevier Science Inc.