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

Abstract

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.