Batch separation of uracil derivatives from urine.

Commercial mixed-bed deionizing resins have been modified by conversion to the sulfonic acid and acetate forms and used to prepare extracts of urine that contains uracil derivatives (pKa greater than 8). A rapid batch extraction process, requiring less than 15 min is described for the first time that can be used either for rapid spectrophotometric screening of altered levels of these uracil derivatives or for accurate measurement of uracil and pseudouridine by high-performance liquid chromatography on reversed-phase columns using 0.002 M ammonium formate (pH 6) in water as the solvent. The spectrophotometric assay is based on the spectral shift of uracil derivatives when neutral aqueous solutions are made alkaline. After the batch extraction, the factor for conversion of absorbancy change to nmol is 0.0046 per nmol/ml for uracil and 0.0035 per nmol/ml for pseudouridine. Pseudouridine levels in two test urines were found to be 24 and 19.3 nmol/mumol of creatinine, which are consistent with published values of 17.6-24.