Detection of 1,N2-propano-2′-deoxyguanosine adducts in genomic DNA by ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry in combination with stable isotope dilution

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 (NH 4 HCO 3 ) 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.