DETERMINATION OF 8-OXOGUANINE IN DNA BY GAS-CHROMATOGRAPHY MASS-SPECTROMETRY AND HPLC-ELECTROCHEMICAL DETECTION - OVERESTIMATION OF THE BACKGROUND LEVEL OF THE OXIDIZED BASE BY THE GAS-CHROMATOGRAPHY MASS-SPECTROMETRY ASSAY

Two analytical methods, one involving the combined use of reverse-phas e HPLC and electrochemical detection (HPLC-EC) and one involving a mas s spectrometric detection after gas chromatography separation (GC/MS), were developed for the detection of 8-oxoguanine in DNA. In order to obtain quantitative results, 2,6-diamino-8-oxopurine, whose chemical s tructure and electrochemical response are very similar to 8-oxoguanine , has been employed as an internal standard in the HPLC-EC assay. In t he case of the GC/MS method, an isotopically stable (M + 4) 8-oxoguani ne has been employed as an internal standard. Both methods are able to detect approximately 1 modification per 10(6) DNA bases. The backgrou nd level of 8-oxoguanine in DNA as determined by GC/MS is approximatel y 50-fold higher than that determined by the HPLC-EC assay. The discre pancy between the two methods is due to an artifactual oxidation of gu anine during the derivatization reaction as demonstrated by using pure guanine. The amount of 8-oxoguanine in guanine, determined by GC/MS, increases linearly with the time of derivatization, indicating that an oxidation occurs during the silylation reaction. Derivatization under nitrogen atmosphere reduces but does not suppress the artifactual oxi dation. The amount of 8-oxoguanine in DNA, quantified by GC/MS, is com parable to that obtained by HPLC-EC when 8-oxoguanine is prepurified b y HPLC or by immunoaffinity chromatography, prior to the silylation re action. The artifactual formation of 8-oxoguanine during the derivatiz ation reaction may explain, at least in part, why the values reported for 8-oxoguanine determination by GC/MS are generally about 1 order of magnitude higher than that determined by HPLC-EC. Prepurification of 8-oxoguanine from guanine is recommended in order to obtain reliable r esults by GC/MS which may be compared to HPLC-EC.