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A sensitive and accurate stable isotope dilution UHPLC–MS/MS method was developed and validated for the detection and quantification of ProdG adducts in human urine, a surrogate for the ProdG adducts in genomic DNA of human. A specific solid phase extraction (SPE) approach was established for selective enrichment of urinary ProdG adducts and elimination of urinary matrix facilitating the coupled MS/MS detection. The recovery of the method is estimated about 84.8–107.2%, and the precision are about 0.8–3.6% for intraday and 2.8–10.0% for interday. Due to that the matrix effect is efficiently eliminated by SPE pretreatment, the limits of detection (LODs, S/N = 3) and quantification (LOQs, S/N = 10) are decreased to 100 and 300 amol for urinary ProdG adducts, respectively. By coupling the developed SPE pretreatment with the UHPLC–MS/MS analysis, ProdG adducts were accurately quantified in healthy human urine. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]