LC/MS/MS Method for the Quantitation of trans-2-Hexenal-Derived Exocyclic 1,N2-Propanodeoxyguanosine in DNA

Abstract

trans-2-Hexenal is an α,β-unsaturated aldehyde to which humans are exposed daily in small amounts. Hexenal has demonstrated mutagenicity and genotoxicity in vitro and reacts with deoxyguanosine to form diastereomeric hexenal-derived exocyclic 1,N²-propanodeoxyguanosine (Hex-PdG) adducts. A highly sensitive and specific method for the measurement of Hex-PdG in DNA has not previously been available. An LC/MS/MS assay for the quantitation of Hex-PdG, using [¹³C₄¹⁵N₂]Hex-PdG as an internal standard, was developed, to assess binding of hexenal to DNA. Samples were purified prior to analysis by centrifuge filtration and solid phase extraction and analyzed by LC/MS/MS in the selected reaction monitoring (SRM) mode (SRM m/z 366.2 → 250.2 for Hex-PdG; SRM m/z 372.2 → 256.2 for [¹³C₄¹⁵N₂]Hex-PdG). Recovery of standards was 89% or greater, and quantitation was unaffected by the addition of increasing concentrations of calf thymus DNA (ctDNA). The limit of quantitation, determined in samples of 200 μg of ctDNA spiked with analyte standard, was 0.015 fmol/μg DNA, which corresponds to ∼5 Hex-PdG/10⁹ unmodified nucleotides. Hex-PdG was detected in ctDNA treated with 0.021 μM, 0.21 μM, or 2.1 mM hexenal but not in untreated DNA. Furthermore, Hex-PdG was not detected in DNA exposed to reactive oxygen species-mediated deoxyribose attack and lipid peroxidation, which resulted in a significant increase in the malondialdehyde-derived pyrimido[1,2-a]purin-10(3H)one. Hex-PdG was not detected in DNA of untreated rat liver, but Hex-PdG in hexenal-treated calf thymus DNA was quantifiable when spiked into the rat liver DNA at 0.035 or 0.35 fmol/μg DNA. These data indicate that Hex-PdG is formed following hexenal treatment and that this method is suitable for in vitro or in vivo assessment of Hex-PdG formation.