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Abstract
A procedure is presented for the direct and simultaneous determination of ascorbic acid (AA), isoascorbic acid (IA), and uric acid (UA) in human plasma by paired-ion reversed-phase high-performance liquid chromatography. An Ultrasphere ODS (C18) column is used with a pH 5.25 mobile phase containing 0.04M sodium acetate, 0.005M tetrabutylammonium phosphate, and 0.2 mg/mL disodium EDTA. Plasma samples preserved with an equal volume of 10% metaphosphoric acid are diluted 10-fold with mobile phase and filtered through 0.2 micron filters. The injection volume is 10 uL. Detection of AA, IA, and UA is by amperometry using a glassy carbon electrode and Ag/AgCl reference electrode. The applied potential is +0.6 volt and the sensitivity setting is 100 nAmps. As little as 0.25 ng of each component can be detected at this setting and the electrode response is linear over the AA, IA, and UA ranges encountered in human plasma.