Quantitative detection ofN7-(2-hydroxyethyl)guanine adducts in DNA using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Abstract

High‐performance liquid chromatography (HPLC) was combined with electrospray ionization tandem mass spectrometry (ESI‐MS/MS) to develop a sensitive and selective method for the quantitative measurement of N⁷‐(2‐hydroxyethyl)guanine (N⁷‐HEG) adducts in DNA obtained from ethylene oxide‐exposed biological samples. Selected reaction monitoring (SRM) was used as the detection mode while the fragmentation product ion at m/z 152 generated from the precursor protonated N⁷‐HEG (m/z 196) was monitored. The detection limits for N⁷‐HEG were estimated by twofold serial dilution and determined to be 4 fmol in neat standard solution and 16 fmol when a matrix effect is considered. When the mass spectrometer was operated in the selected ion monitoring mode using only the first quadrupole (without MS/MS function), the detection limits increased to 128 fmol and 1 pmol (when matrix effect is considered), respectively. A good linear correlation (R² = 0.999) was observed for signal intensities obtained by injecting 16 fmol–33 pmol of N⁷‐HEG into the HPLC/ESI‐MS/MS system. Hep G2 cells were incubated for 8 h with medium containing various concentrations of ethylene oxide (ranging from 0.05 to 5.0 mM). A dose–response relationship was established, indicating that the adduct formation increases with the exposure level. The method shows potential, although the detection limit needs to be lowered for practical applications, for use in monitoring N⁷‐HEG formation in other biological systems. Copyright © 2001 John Wiley & Sons, Ltd.