Ascorbic acid (AA) functions as an antiascorbutic agent and is a cofactor in a variety of enzymatic processes. As antioxidant it is used in pharmaceutical manufacturing and in the food industry. When exposed to oxidant stress, AA is reversibly converted to dehydroascorbic acid (DHA); then DHA is degraded to 2,3-diketogulonic acid and to other inactive species. AA and DHA are difficult to quantify due to their instability and their highly polar character. In contrast to AA, DHA has a weak UV absorption. In order to increase the sensitivity for DHA, derivatization prior or after the chromatographic separation is convenient. The present communication describes the development and validation of a reversed-phase ion pair high-performance liquid chromatography method for the separation and quantitation of DHA, AA and paracetamol. The UV-absorptivity of the DHA was enhanced by precolumn derivatization with 4,5-dimethyl-1,2-phenylenediamine. The derivatization reaction was carried out under mild conditions (10 min at ambient temperature in the dark) and the resulting DHA-quinoxaline derivative was monitored at 360 nm. The chromatographic separations were performed on a Phenomenex Synergi 4u Hydro-RP (150 x 4.6 mm) under isocratic elution conditions, using hexadecyltrimethylammonium bromide as ion-pairing reagent in the mobile phase. The validated method was successfully tested on pharmaceuticals.
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