Validation of a micromethod for determining oxidized and reduced vitamin C in plasma by HPLC-fluorescence

An HPLC micro-method with fluorescence detection has been developed to determine total vitamin C (vit C) and dehydroascorbic acid (DHA) concentrations in human plasma samples. This method is based on the rapid, specific reaction of DHA with dimethyl-o-phenylenediamine (DMPD) to form a fluorescent quinoxaline derivative that is quantified by HPLC in less than 5 minutes. The method was assessed with reference to the direct 2,4-dinitrophenylhydrazine (DNPH) colorimetric method. They were well correlated (r3 = 0.879), but the DMPD-HPLC method had the limit of detection 6 times lower than the standard method and the relative error for a vitamin standard was 10 times better than that of the standard method. The plasma DHA to total vit C ratio varied from 10 to 60%, depending on sample processing. Plasma that were immediately analysed contained 10% DHA whatever the subject's age; frozen deproteinized samples kept 1 week (-67 degrees C) had 20%, and blood samples kept for one hour at room temperature before treatment had up to 60% DHA. The ratio in capillary samples taken from the finger was 11-42%. This rapid, specific and very sensitive micro-method is well suited to routine measurements of plasma vit C.