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) conc entrations in human plasma samples. This method is based on the rapid, specific reaction of DHA with dimethyl-o-phenylene-diamine (DMPD) to form a fluorescent quinoxaline derivative that is quantified by HPLC i n less than 5 minutes. The method was assessed with reference to the d irect 2,4-dinitrophenyl-hydrazine (DNPH) colorimetric method. They wer e well correlated (r(2) = 0.879), but the DMPD-HPLC method had the lim it of detection 6 times lower than the standard method and the relativ e error for a vitamin standard was 10 times better than that of the st andard method. The plasma DHA to total vit C ratio varied from 10 to 6 0%, depending on sample processing. Plasma that were immediately analy sed contained 10% DHA whatever the subject's age; frozen deproteinized samples kept 1 week (-67 degrees C) had 20%, and blood samples kept f or one hour at room temperature before treatment had up to 60% DHA. Th e ratio in capillary samples taken from the finger was 11-42%. This ra pid, specific and very sensitive micro-method is well suited to routin e measurements of plasma vit C.