Simultaneous determination of ascorbic acid and dehydroascorbic acid by reversed-phase ion-pair HPLC with pre-column derivatisation.

A method is described for the separation and quantitation of ascorbic acid and dehydroascorbic acid by reversed-phase ion-pair high-performance liquid chromatography, following pre-column derivatisation of dehydroascorbic acid with 1,2-phenylenediamine. Use of tridecylammonium formate or hexadecyltrimethylammonium bromide as ion-pairing reagents in the mobile phase produced good retention for ascorbic acid and excellent resolution between ascorbic acid and the derivatised dehydroascorbic acid. The UV absorptivity of dehydroascorbic acid was greatly enhanced by dervatisation, permitting its simple determination in foodstuffs. A change of detector wavelength from 348 nm (for the dehydroascorbic acid derivative) to 290 nm (for ascorbic acid) was employed for the analysis of solutions containing a five-fold excess of ascorbic acid over dehydroascorbic acid, using the the same detector sensitivity (0.1 a.u.f.s.) for each. The method has been applied to the analysis of orange juice