A high-performance liquid chromatographic method for the determination of pseudouridine and uric acid in native human urine and ultrafiltrated serum

Abstract

A simple and reliable HPLC method for quantitative determination of pseudouridine and uric acid in human urine and serum using a cation-exchange resin is described. This method is straightforward (12 runs of urine samples per day since the sample is only diluted into buffer and then chromatographed), sensitive, and highly reproducible. The column is stable over long periods (∼3 months of uninterrupted use at a time; it is thereafter easily restored to the original state). Mean excretion values for pseudouridine (in μmol/mmol creatinine) are 26.4 ± 3.1 (17 female adults), 23.8 ± 2.5 (12 male adults), 164.7 ± 32.2 (37 male preterm infants); mean values for uric acid (μmol/mmol creatinine) are, respectively, 310.3 ± 90.5, 278.2 ± 56.1, and 1108 ± 314. Human serum is deproteinized by pressure ultrafiltration in microcollodion bags with a nominal exclusion molecular weight of 12,400 and then put directly onto the HPLC column. The complete procedure takes 4 h.