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.
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.
Department of Clinical Pharmacology Q7642, Rigshospitalet, Copenhagen, Denmark.
A
A
Weimann
D
D
Belling
H E
HE
Poulsen
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D002021:Buffers; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis*; D003839:Deoxyadenosines / Q000032:analysis* / Q000652:urine; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000652:urine; D006801:Humans; D006868:Hydrolysis; D013058:Mass Spectrometry; D012680:Sensitivity and Specificity; D013950:Thymus Gland / Q000737:chemistry
D002021:Buffers; D003839:Deoxyadenosines; C078251:2'-deoxy-7,8-dihydro-8-oxoadenosine; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D003849:Deoxyguanosine