Optimal conditions for the simultaneous ion-pairing HPLC determination of L-ascorbic, dehydro-L-ascorbic, D-ascorbic,...

The present report deals with the optimization of a reversed-phase high-performance liquid chromatography (RP-HPLC) method useful for a simultaneous determination of L-ascorbic, isoascorbic, dehydro-L-ascorbic, and uric acids. For this purpose, we investigated (a) the stability of all solutes and especially of L-ascorbic acid under various conditions, (b) the resolution of components of interest by the addition of a positively charged ion-pairing reagent in the mobile phase of different pH levels, and (c) the optimal detection conditions of ascorbate-related compounds. The results demonstrated that L-ascorbic acid remains stable in refrigerated buffer solution of pH 5.0 containing EDTA. A mobile phase of pH 5.0 containing 5 mM cetyltrimethylammonium bromide as an ionic pair offers optimal resolution. The combined use of an ultraviolet (UV) detector and of an electrochemical detector is preferable for the determination of the solutes after reduction of dehydro-L-ascorbic acid to L-ascorbic acid. However, for the direct determination of dehydro-L-ascorbic acid, a previous detection of contents by a UV detector at 232 nm is recommended. The investigation reported here may facilitate future biological studies associated with the determination of L-ascorbic acid and related compounds.