Chemiluminescent detection of thymine hydroperoxides by high-performance liquid chromatography

High-performance liquid chromatography (HPLC) with chemiluminescence detection was used to detect thymine hydroperoxides, which are expected to occur in biological tissues as primary products in free radical-mediated DNA damage. The thymine hydroperoxides were chemically prepared by the acid-catalysed hydrogen peroxide oxidation of thymine and 5-hydroxymethyluracil and their hydroperoxide products were identified as trans- or cis-5-chloro-6-hydroperoxy-5,6-dihydrothymine and 5-hydroperoxymethyluracil, respectively, by means of NMR, IR and mass spectrometry. The results confirmed that these thymine hydroperoxides are specifically detectable at picomole levels by the described HPLC method with high reproducibility.