Suppression of adventitious formation of 8-oxoguanine(TMS)4 from guanine during trimethylsilylation

GC/MS quantitation of 8-oxoguanine derived from DNA may yield artificially high values when trimethylsilylation is used as the technique to form a volatile derivative. Significant quantities of the tetrakis trimethylsilyl-derivative of 8-oxoguanine may be formed from guanine during the derivatization reaction at elevated temperatures. We have screened eight antioxidants in an attempt to identify a substance that will reduce the spurious formation of this product. Whereas several compounds were active, N-phenyl-1-naphthylamine was the most effective agent. Moreover, this compound was sufficiently basic to act as a catalyst for the derivatization reaction. Our data, which are based on the use of an isotopically labeled internal standard, show that the level of the tetrakis trimethylsilyl derivative of 8-oxoguanine remained constant as a function of derivatization heating time when N-phenyl-1-naphthylamine was added to a reaction in which guanine was present. In contrast, the level in control samples increased linearly with reaction time indicating formation of 8-oxoGua during derivatization. The level of 8-oxoguanine was measured in both a synthetic oligodeoxynucleotide containing a single 8-oxo-2'-deoxyguanosine moiety and commercially available calf thymus DNA using this technique.