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Quantitative Determination of Cyclobutane Thymine Dimers in DNA by Stable Isotope‐Dilution Mass Spectrometry
Author(s) -
Podmore Ian D.,
Cooke Marcus S.,
Herbert Karl E.,
Lunec Joseph
Publication year - 1996
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1996.tb02463.x
Subject(s) - chemistry , mass spectrometry , chromatography , thymine , detection limit , dna , cyclobutane , analyte , dna damage , isotope dilution , dimer , quantitative analysis (chemistry) , analytical chemistry (journal) , biochemistry , organic chemistry , ring (chemistry)
In order to understand the role of UV‐induced DNA lesions in biological processes such as mutagenesis and carcinogenesis, it is essential to detect and quantify DNA damage in cells. In this paper we present a novel and both highly selective and sensitive assay using capillary gas chromatography (GC) combined with mass spectrometry (MS) for the detection and accurate quantitation of a major product of UV‐induced DNA damage ( cissyn cyclobutadithymine). Quantitation of the cyclobutane thymine dimer was achieved by the use of an internal standard in the form of a stable 2 H‐labeled analogue. Both isotopically labeled and nonlabeled dimers were prepared directly from their corresponding monomers. Each was identified as their trimethylsilyl ether derivative by GC‐MS. Calibration plots were obtained for known quantities of both nonlabeled analyte and internal standard. Quantitation of cis‐syn cyclobutadithymine 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 detection 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 GC‐MS is an ideally suited technique for selective and sensitive quantification of cis‐syn cyclobutadithymine in DNA and hence UV‐induced DNA damage.