Molecular dosimetry of 7-alkyl- and O6-alkylguanine in DNA by electrochemical detection.

A methodology is described for the quantitation of 7-alkyl- and O6-alkylguanine in DNA isolated from experimental animals exposed to alkylating agents. Following purification, the DNA is hydrolysed under acid conditions after which 7-alkyl- and O6-alkylguanine are separated from unmodified bases by HPLC using a strong cation exchange column. The fractions containing the methylated purines are subsequently analyzed by HPLC using a reverse phase column coupled to an electrochemical detector (amperometric). This method allows the detection of 10-20 fmoles 7-alkyl- and O6-alkylguanine, when pure markers are analyzed. In practice, the detection limit is 0.5 adducts per 10(6) nucleotides for the methylated and 1 adduct per 10(6) nucleotides for the ethylated form of 7-alkyl- and O6-alkylguanine using 25 micrograms DNA.