trans-2-Hexenal is an MG SRC="/images/gifchars/alpha.gif" BORDER=0>,mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-unsaturated aldehyde to which humans are exposed daily in small amounts.Hexenal has demonstrated mutagenicity and genotoxicity in vitro and reacts with deoxyguanosine toform diastereomeric hexenal-derived exocyclic 1,N2-propanodeoxyguanosine (Hex-PdG) adducts. A highlysensitive 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 centrifugefiltration and solid phase extraction and analyzed by LC/MS/MS in the selected reaction monitoring(SRM) mode (SRM m/z 366.2 mages/entities/rarr.gif"> 250.2 for Hex-PdG; SRM m/z 372.2 mages/entities/rarr.gif"> 256.2 for [13C415N2]Hex-PdG).Recovery of standards was 89% or greater, and quantitation was unaffected by the addition of increasingconcentrations of calf thymus DNA (ctDNA). The limit of quantitation, determined in samples of 200mages/entities/mgr.gif">g of ctDNA spiked with analyte standard, was 0.015 fmol/mages/entities/mgr.gif">g DNA, which corresponds to ~5 Hex-PdG/109 unmodified nucleotides. Hex-PdG was detected in ctDNA treated with 0.021 mages/entities/mgr.gif">M, 0.21 mages/entities/mgr.gif">M, or2.1 mM hexenal but not in untreated DNA. Furthermore, Hex-PdG was not detected in DNA exposed toreactive oxygen species-mediated deoxyribose attack and lipid peroxidation, which resulted in a significantincrease in the malondialdehyde-derived pyrimido[1,2-a]purin-10(3H)one. Hex-PdG was not detected inDNA of untreated rat liver, but Hex-PdG in hexenal-treated calf thymus DNA was quantifiable whenspiked into the rat liver DNA at 0.035 or 0.35 fmol/mages/entities/mgr.gif">g DNA. These data indicate that Hex-PdG is formedfollowing hexenal treatment and that this method is suitable for in vitro or in vivo assessment of Hex-PdG formation.