Analysis of 3,N(4)-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 (epsilonCyt) has not been available. In this report, a GC/NICI/MS assay was developed for detection and quantification of epsilonCyt in DNA and in human urine samples. The stable isotope of epsilonCyt with 7 mass units higher than the normal epsilonCyt 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 epsilonCyt and its isotope analogue. One femtogram (S/N > 40) of pentafluorobenzylated epsilonCyt was detected when injected on column with selective ion monitoring mode. The limit of quantification for the entire assay was 7.4 fmol of epsilonCyt, which was approximately one thousand times lower than that of the HPLC/fluorescence assay for the nucleoside 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 epsilonCyt in two smokers' urine samples and the average level of epsilonCyt was 101 +/- 17 pg/mL/g of creatinine. Thus, quantification of epsilonCyt in DNA and in urine by this highly specific and ultrasensitive isotope dilution GC/NICI/MS assay may facilitate research on the role of epsilonCyt in carcinogenesis and in cancer development.
Chen
H J
HJ
Department of Chemistry, National Chung Cheng University, 160 San-Hsing, Ming-Hsiung, Chia-Yi 62142, Taiwan. chehjc@ccunix.ccu.edu.tw
Lin
T C
TC
Hong
C L
CL
Chiang
L C
LC
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003596:Cytosine / Q000031:analogs & derivatives* / Q000652:urine*; D004247:DNA / Q000737:chemistry*; D018736:DNA Adducts / Q000652:urine*; D005453:Fluorescence; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006801:Humans; D007553:Isotope Labeling
D018736:DNA Adducts; C051143:3,N(4)-ethanocytosine; D003596:Cytosine; D004247:DNA