Determination of N7-(2-hydroxyethyl)guanine by HPLC with electrochemical detection

A method has been developed for the determination of N7-(2-hydroxyethyl)guanine (N7-EtOHGua) via HPLC with electrochemical detection (EC). N7-EtOHGua is the major base adduct formed in DNA upon exposure to ethylene oxide. N7-EtOHGua, released from DNA, was separated from the unmodified nucleobases by chromatography on a reversed-phase column. For electrochemical detection, an amperometric detector cell was used with a glassy carbon working electrode, set at 1.35 V relative to an Ag/AgCl reference electrode. With purified N7-EtOHGua a linear dose-response relation was observed in the range between 0.11 and 13 pmol. The signal-to-noise ratio during analysis of 0.11 pmol N7-EtOHGua was about 8 to 1. Determination of adducts in a series of DNA samples treated with 0.16–10 mM ethylene oxide showed a linear dose-dependent increase in the level of N7-modifications. For DNA samples, the detection limit of this HPLC-EC analysis is 1 N7-EtOHGua per 6 × 10⁶ nucleotides.