Optimal conditions for the simultaneous ion-pairing HPLC determination of L-ascorbic, dehydro-L-ascorbic, D-ascorbic, and uric acids with on-line ultraviolet absorbance and electrochemical detection

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 electro-chemical 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. (C) 1998 Academic Press.