Abstract
A highly sensitive method for the simultaneous determination of ascorbate (AsA), dehydroascorbate (DHA), 2,3-diketogulonate (2,3-DKG), carbamyl ascorbate (CAA) and carbamylated dehydroascorbate derivative (CDA) was developed by high-performance liquid chromatography with post-column derivatization. The successful separation of these substances was achieved by an adsorption chromatography using poly(ethylene glycol) copolymer as a packing material in the separation column. For the detection, each substance was boiled with benzamidine in alkaline solution, producing fluorescence products. Both CAA and CDA were alkaline-labile, degrading to AsA and 2,3-DKG, so that these carbamylated derivatives could be detected in a similar manner as AsA and 2,3-DKG, respectively. The detection limits for quantitative determination of these substances were less than 0.5 μ M, and the coefficients of variation of the peak areas were in the range of 2.2 to 2.8%. The usefulness and practicability of the present method were verified by application to the determination of these substances in plant leaves soaked in 0.5 M sodium cyanate solution.
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