Simultaneous detection of five different 2-hydroxyethyl-DNA adducts formed by ethylene oxide exposure, using a high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry assay

A method has been developed for the simultaneous detection and quantitation of five different (2-hydroxyethyl)-DNA adducts (HE-DNA adducts) that could be formed as a result of exposure to ethylene oxide (EO). In addition to the major N7-HE-guanine (N7-HEG) adducts this assay can also measure the less prevalent but potentially more biologically significant N1-HE-2’-deoxyadenosine (N1-HEdA), O6-HE-2’-deoxyguanosine (O6-HEdG), N6-HE-2’-deoxyadenosine (N6-HEdA) and N3-HE-2’-deoxyuridine adducts (N3-HEdU). The method involves the isolation of HE-adducts from the unmodified nucleosides by either neutral thermal hydrolysis or enzymatic digestion, followed by HPLC purification, before detection and quantification by liquid-chromatography tandem mass spectrometry (LC-MS/MS) using selective reaction monitoring (SRM). The limits of detection were in the range 0.5 – 25 fmoles for each individual adduct, making this one of the most sensitive assays available for the detection of N7-HEG. To illustrate the possible applications of the assay, it has been employed in the measurement of endogenous/background and EO-induced HE-adducts in a variety of DNA samples.