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 fru its and vegetables. The complete separation of AA and DHAA could be achieve d on a C-18 column using 0.2 M KH2PO4 (pH adjusted to 2.4 with H3PO4) as th e mobile phase at a flow-rate of 0.5 ml/min. Since the detection sensitivit y was poor for DHAA even at 210 nm, it was estimated as the difference betw een 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, 1 20, 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 se lected fruits and vegetables were analyzed comparatively by liquid chromato graphy and enzymatic assay to validate the method. (C) 2000 Elsevier Scienc e B.V All rights reserved.