QUANTITATIVE-DETERMINATION OF CYCLOBUTANE THYMINE DIMERS IN DNA BY STABLE ISOTOPE-DILUTION MASS-SPECTROMETRY

In order to understand the role of UV-induced DNA lesions in biologica l processes such as mutagenesis and carcinogenesis, it is essential to detect and quantify DNA damage in cells, In this paper we present a n ovel and both highly selective and sensitive assay using capillary gas chromatography (GC) combined with mass spectrometry (MS) for the dete ction and accurate quantitation of a major product of UV-induced DNA d amage (cis-syn cyclobutadithymine). Quantitation of the cyclobutane th ymine dimer was achieved by the use of an internal standard in the for m of a stable H-2-labeled analogue. Both isotopically labeled and nonl abeled dimers were prepared directly from their corresponding monomers , Each was identified as their trimethylsilyl ether derivative by GC-M S. Calibration plots were obtained for known quantities of both nonlab eled analyte and internal standard. Quantitation of cis-syn cyclobutad ithymine was demonstrated in DNA exposed to UVC radiation over a dose range of 0 to 3500 J m(-2). Under the conditions used, the limit of de tection was found to be 20-50 fmol on column (equivalent to 0.02-0.05 nmol dimer per mg DNA), The results of the present study indicate that capillary CC-MS is an ideally suited technique for selective and sens itive quantification of cis-syn cyclobutadithymine in DNA and hence UV -induced DNA damage.