Enzymatically validated liquid chromatographic method for the determination of ascorbic and dehydroascorbic acids in fruit and vegetables

A liquid chromatographic method has been described for the determination of total vitamin C, ascorbic acid (AA) and dehydroascorbic acid (DHAA) in fruits and vegetables. The complete separation of AA and DHAA could be achieved on a C18 column using 0.2 M KH2PO4 (pH adjusted to 2.4 with H3PO4) as the mobile phase at a flow-rate of 0.5 ml/min. Since the detection sensitivity was poor for DHAA even at 210 nm, it was estimated as the difference between the total AA after DHAA reduction and AA content of the original sample, using dithiothreitol (DTT) as the precolumn reductant. The reaction times for the complete conversion of DHAA to AA at room temperatures were 150, 120, 90 and 75 min for 1, 2, 4 and 8 mmol DTT per mmol of DHAA, respectively. The percentage recovery ranged from 81.7 to 105.9. AA contents of some selected fruits and vegetables were analyzed comparatively by liquid chromatography and enzymatic assay to validate the method.