Quantitative Analysis of the DNA Adduct N2,3- Ethenoguanine Using Liquid Chromatography Electrospray Ionization Mass Spectrometry

Abstract

The need for specificity and sensitivity in the analysis of DNA adducts has led the development of GC/MS methods. Such methods require chemical derivatization (i.e. silylation, electrophore labelling), which can also bring its own sets of problems, including the production of artifacts, interferences and sample to sample variability in derivatization. To obviate such problems, a liquid chromatographic/electrospray ionization mass spectrometric (LC/ESI-MS) method was developed to quantify N2,3-ethenoguanine (εGua), a promutagenic DNA adduct of vinyl chloride exposure. The response of εGua to isotopically labelled internal standard [13C4]εGua was linear (r2 = 0.999) and reproducible from 0.027 to 0.538 pmol μl-1. We obtained an accuracy of 86±14% by analyzing chloroethylene oxide (CEO)-treated calf thymus DNA enriched with authentic εGua. The analysis of CEO-treated calf thymus DNA samples not enriched with authentic εGua provided a precision of 15%. The detection limits with a signal-to-noise ratio (S/N) 2.5:1 were obtained in the determination of authentic εGua at 5 fmol per injection. The detection limit obtained in the routine analysis of the biological samples was 50 fmol εGua with S/N = 3:1. The applicability of the method was established by determining εGua in rats treated with CEO by portal vein injection and an unexposed human liver. It was observed that the concentration of εGua in the rat livers increased with increase in dose and was inversely related to the time after, CEO exposure. This trend suggests rapid repair of the adduct in rat livers. In the human liver DNA sample, εGua was quantitated at 0.06±0.01 pmol mg-1 DNA.