Analysis of 3,N4-ethenocytosine in DNA and in human urine by isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry

The promutagenic etheno DNA adducts have been detected in tissue DNA of rodents and humans from various exogenous and endogenous sources. While other etheno DNA adducts have been detected and quantified by isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS), similar analysis for 3,N-4-ethenocytosine (epsilon Cyt) has not been available. In this report, a GC/NICI/MS assay was developed for detection and quantification of epsilon Cyt in DNA and in human urine samples. The stable isotope of epsilon Cyt with 7 mass units higher than the normal epsilon Cyt was synthesized and used as internal standard of the assay. The adduct-enriched fraction of DNA hydrolysate was derivatized with pentafluorobenzyl bromide before GC/NICI/MS analysis with selective ion monitoring at [M - 181](-) fragments of pentafluorobenzylated epsilon Cyt and its isotope analogue. One femtogram (S/N > 40) of pentafluorobenzylated epsilon Cyt was detected when injected on column with selective ion monitoring mode. The limit of quantification for the entire assay was 7.4 fmol of epsilon Cyt, which was approximately one thousand times lower than that of the HPLC/fluorescence assay for the nucleoside 3,3/N-4-etheno-2'-deoxycytidine in DNA. Analysis of chloroacetaldehyde-treated calf thymus DNA by both GC/NICI/MS and HPLC/fluorescence methods provided similar adduct levels and thus verified the assay. This GC/NICI/MS method was used for analysis of epsilon Cyt in two smokers' urine samples and the average level of epsilon Cyt was 101 +/- 17 pg/mL/g of creatinine. Thus, quantification of epsilon Cyt in DNA and in urine by this highly specific and ultrasensitive isotope dilution GC/NICI/MS assay may facilitate research on the role of epsilon Cyt in carcinogenesis and in cancer development.