Quantitation of uracil in rodent diet

Feeding of high levels of uracil to laboratory rodents results in the formation of calculi in the lumen of the urinary bladder. This urolithiasis stimulates cellular proliferation in the bladder and has been used in studies of two-stage carcinogenesis. Quantitation of uracil in rodent diet was achieved by extraction from the diet with ammonium hydroxide. The extract was applied to a strong anion-exchange solid-phase extraction column. Uracil is not retained on this matrix which adsorbs the majority of contaminants in the extract. The uracil was quantitated by HPLC on an ODS microbore column (100 x 2 mm internal diameter) eluted at 0.5 ml/min with 200 mM KH2PO4, pH 3.5, at 30 degrees C. Three structurally related pyrimidine bases, cytosine, uracil, and thymine, showed increasing retention on this column/solvent combination, thereby demonstrating selectivity of the analysis. Recovery of uracil was 76-90% with lower values observed when dietary levels of uracil were in excess of 4.5%.