Detection of 1,N(2)-propano-2'-deoxyguanosine adducts in genomic DNA by ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry in combination with stable isotope dilution

Abstract

Crotonaldehyde (Cro) is one of widespread and genotoxic α,β-unsaturated aldehydes and can react with the exocyclic amino group of 2'-deoxyguanosine (dG) in genomic DNA to form 1,N(2)-propano-2'-deoxyguanosine (ProdG) adducts. In this study, two diastereomers of high purity were prepared, including non-isotope and stable isotope labeled ProdG adducts, and exploited stable isotope dilution-based calibration method. By taking advantage of synthesized ProdG standards, we developed a sensitive ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) method for accurate quantification of two diastereomers of ProdG adducts. In addition to optimization of the UHPLC separation, ammonium bicarbonate (NH4HCO3) was used as additive in the mobile phase for enhancing the ionization efficiency to ProdG adducts and facilitating MS detection. The limits of detection (LODs, S/N=3) and the limits of quantification (LOQs, S/N=10) are estimated about 50 amol and 150 amol, respectively. By the use of the developed method, both diastereomers of ProdG adducts can be detected in untreated human MRC5 cells with a frequency of 2.4-3.5 adducts per 10(8) nucleotides. Crotonaldehyde treatment dramatically increases the levels of ProdG adducts in human MRC5 in a concentration-dependent manner.

Keywords: 1,N(2)-propano-2′-deoxyguanosine adducts (ProdG adducts); Crotonaldehyde (Cro); Ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI–MS/MS).