A novel HPLC procedure for the analysis of 8-oxoguanine in DNA

The chromatographic quantitation of 8-oxoguanine adducts in DNA is widespread in the literature, although results obtained by HPLC of 8-oxodeoxyguanosine do not always agree with levels determined by GC-MS. To help explain this discrepancy, here we describe a novel procedure for the analysis of 8-oxoguanine adducts in DNA. Although it proved difficult to directly quantitate 8-oxoguanine in the presence of high levels of endogenous guanine using conventional reversed-phase HPLC, a simple preincubation of DNA acid hydrolysates with guanase allowed such analyses. The assay relied on our observation that 8-oxoguanine was not a substrate for guanase, and on sensitive electrochemical detection. The limit of detection for 8-oxoguanine was 5 nM or 250 fmol on column. Using this procedure, the background level of 8-oxoguanine in commercially available calf thymus DNA was 0.4 nmol/mg DNA or 3.2 mol/105 mol guanine.