| Abstract: |
Formamidopyrimidines, 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), are among major lesions in DNA generated by hydroxyl radical attack, UV radiation or photosensitization in vitro and in vivo. FapyAde and FapyGua exist in living cells at detectable background levels and are formed by exposure of cells to DNA-damaging agents. Numerous prokaryotic and eukaryotic DNA glycosylases exist for the repair of formamidopyrimidines by base excision repair pathway in cells indicating their biological significance. Moreover, they are premutagenic lesions, albeit to different extents, revealing a possible role in disease processes. Methodologies using gas chromatography/mass spectrometry (GC/MS) with capillary columns have been developed to accurately measure FapyAde and FapyGua in DNA in vitro and in vivo. Stable isotope-labeled analogues of these compounds have been synthesized and are commercially available to be used as internal standards for quantification. GC/MS with isotope-dilution provides excellent sensitivity and selectivity for positive identification and accurate quantification, and has widely been applied in the past to the measurement of formamidopyrimidines under numerous experimental conditions. This article reports on the details of this GC/MS methodology. |
