Microscale Solid-Phase Extraction and HPLC Separation of Purine Deoxyribonucleoside Monophosphate Adducts of Benzo[a]pyrene Diol Epoxide

Abstract

Benzo[a]pyrene, a polycyclic aromatic hydrocarbon, is a potent mutagen and genotoxic compound. Activation of benzo[a]pyrene by oxidative metabolism in cells leads to the formation of highly reactive (±) anti benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxides, which bind covalently to DNA bases, in particular to the nucleophilic N2 atom of guanine and the N6 atom of adenine. Here we have investigated the recovery of eight different enantiomers of purine deoxyribonucleoside monophosphate benzo[a]pyrene diol epoxide adducts after a microscale solid-phase extraction (SPE). Microscale SPE was carried out using a pipette tip with a bed of C18 chromatograph packing material. We also tested the ability of microscale SPE to isolate adducts derived from the in vitro reaction of calf thymus DNA with (±) anti benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide. The recoveries of benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide monophosphate adduct standards were good, varying between 65% and 92%, except for the trans BPDE-dGMP adduct derived from the (−) enantiomer of benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide, which presumably became decomposed to tetrols during the purification process. After using a neutral condition during the SPE treatment, the decomposition to tetrols of the trans BPDE-dGMP adduct could be avoided and the recovery increased to 79%.