GENERAL-METHOD FOR DETERMINING ETHYLENE-OXIDE AND RELATED N-7-GUANINE DNA-ADDUCTS BY GAS CHROMATOGRAPHY-ELECTRON CAPTURE MASS-SPECTROMETRY

A 112-mu g sample of DNA was spiked with 103 pg of N-7-(2'-hydroxyethyl)guanine and 100 pg of N-7-(2'-hydroxyethyl-d4)guanine, the internal standard. The sample was subjected to the following sequence of steps: heating at 100 degrees C, precipitation of the DNA with HCl, reaction with nitrous acid to form the corresponding xanthines, reaction twice with pentafluorobenzyl bromide (first to derivatize NH, then OH), solid-phase extraction on silica and detection by gas chromatography-electron capture mass spectrometry. The absolute, overall yield of final product for both the analyte and internal standard was 9.7%. Conveniently, the three chemical reactions are conducted sequentially in the same vial and, aside from a washing step, are separated only by evaporations. Corresponding N-7-guanine methyl, phenyl and styrene oxide adducts were detected at about the 50-ng level by the procedure, to indicate the generality of the method.