Immunoaffinity/gas chromatography/high-resolution mass spectrometry method for the detection of N²,3-ethenoguanine

Etheno adducts are formed after exposure to a number of carcinogens, including vinyl chloride, as well as endogenously as a result of lipid peroxidation. A sensitive and selective assay for N²,3-ethenoguanine (εGua) was developed using immunoaffinity (IA) columns made with polyclonal antibodies to eGua followed by gas chromatography/electron capture negative chemical ionization/high-resolution mass spectrometry (GC/ECNCI/HRMS) analysis of its pentafluorobenzyl derivative. These IA columns were specific for εGua and did not bind guanine, deoxyguanosine, 1,N⁶-ethenoadenine, or 1,N²-ethenoguanine. The level of recovery of standards from the IA columns was 107 ± 7% and throughout the entire method (using nucleoside enzymatic digestion) with or without DNA was 72 ± 6%. Four different hydrolysis/digestion procedures were compared, nucleoside enzymatic (EZ), neutral thermal hydrolysis (NT), formic acid hydrolysis (FA), and HCl hydrolysis. All hydrolysis methods with subsequent IA chromatography produced linear standard curves with r² values of 0.999 or better. The level of εGua in chloroethylene oxide-treated calf thymus DNA (CEO-ctDNA) was 38 ± 2, 42 ± 3, and 49 ± 2 fmol of εGua/μg of DNA using EZ, NT, and FA, respectively. These numbers remained consistent when the amount of DNA processed was doubled or tripled. These numbers were comparable to the previously published value of 55 ± 8 fmol of εGua/μg of DNA for the same DNA using HCl hydrolysis, cation exchange cleanup, and LC/MS analysis [Yen, T. Y., et al. (1996) J. Mass Spectrom. 31, 1271-1276]. Additionally, HCl hydrolysis of rat liver DNA from control and vinyl fluoride-exposed rats gave similar εGua results when compared to those from enzymatic digestion using this method. This method gave a detection limit of 5 εGua adducts/10⁸ normal dGuo nucleosides in 150 μg of DNA using EZ and somewhat lower detection limits using NT and HCl hydrolysis. The method is more sensitive and selective than previously used methods for the quantitation of this adduct.