A NON-ION-PAIRING HPLC METHOD FOR MEASURING NEW FORMS OF ASCORBATE AND ASCORBIC-ACID

A new high-performance liquid-chromatography (HPLC) method separates vitamins L-ascorbic acid (C-1) and L-ascorbyl-2-sulfate (C-2) Previous HPLC reverse phase methods utilized ion-pairing reagents and resulted in co-elution with other substances. Vitamin C-1 preceded C-2 in retention time. In this new method, which deletes the ion-pairing reagent, vitamin C-2 precedes the elution of vitamin C-1. This new direct method allows for a better separation from other conflicting metabolites and constituents of extraction mixtures. In addition, this method provides a way of identifying ascorbyl-2-monophosphate (C-3), a form of vitamin C that may be found in animal diets. Examples will be shown of HPLC profiles of extractives from tissue and diets known to contain these new forms of vitamin C.