Quantitative HPLC-UV Analysis of DNA Lesions

HPLC-UV analysis, cleanup and enrichment of the samples were performed on a 4.6 mm × 150 mm Luna C18 (2) 100 Å column (5 μ min particle size, Phenomenex) loaded with a pre-column C18 (2) cartridge, on an Agilent 1100 HPLC-UV system (Agilent, USA). The gradient program used an eluent composed by 2 mM ammonium formate (solvent A), acetonitrile (solvent B), and methanol (solvent C) (Table S5). The time windows used for the collection of fractions containing the lesions are reported in Table S6 (Figure S11). The collected fractions were freeze-dried, pooled, freeze-dried again, and redissolved in 50 μL ddH₂O before LC-MS/MS analysis. The quantification of the dA, dG, dC, and Thy was based on their absorbance at 260 nm (Cui et al., 2013 (link)). The same analytical protocol was used also for the quantification of the normal 2′-deoxyribonucleosides in the enzymatic digestion studies.