Determination of pseudouridine in human urine and serum by high-performance liquid chromatography with post-column fluorescence derivatization.

A selective and sensitive method for the determination of pseudouridine in human urine and serum is described. The method is based on high-performance liquid chromatography with post-column fluorescence derivatization. Pseudouridine and 5-fluorouridine (internal standard) in a 10-fold diluted urine sample or a deproteinized serum sample are separated on a reversed-phase column (TSK gel ODS-80) with isocratic elution and successively subjected to derivatization involving periodate oxidation followed by fluorescence reaction with meso-1,2-bis(4-methoxyphenyl)ethylenediamine. The detection limit for pseudouridine is 4 pmol in a 100-microliters injection volume.