Quantitative Estimation of Dehydroascorbic Acid and Ascorbic Acid by High-Performance Liquid Chromatography—Application to Human Milk, Plasma, and Leukocytes

A ‘high-performance’ liquid chromatographic (HPLC) method for quantitation of dehydroascorbic acid and ascorbic acid and its application to protein-free human milk, blood plasma and leukocytes (buffy layer) is described. In the method, DL-homocysteine was used to convert dehydroascorbic acid quantitatively to ascorbic acid that was measured by reversed phase liquid chromatography. Fresh human milk was found to contain ascorbic acid 54.3±6.5 mg/1 (mean±SEM; n=4) and dehydroascorbic acid 21. 0±9.1 mg/1 (mean±SEM, n=4) when stored at +4°C. The concentration of both forms of ascorbic acid was found to detoriate in similar ratios during storage at +4°C, and pasteurization considerably increased the loss of vitamin C. After pasteurization the milk contained ascorbic acid 8.6±3.4 mg/1 (mean±SEM, n=4) and dehydroascorbic acid 6.6±2.4 mg/1 (mean±SEM, n=4). In plasma the dehydroascorbic acid content (0.16±0.03 mg/1, mean±SEM, n=23) was lower than that of ascorbic acid (9.96±0.75 mg/1, mean±SEM, n=23). The ascorbic acid concentration in the leukocyte mixtures was 0.21±0.04 mg/109 cells (mean±SEM, n=10) and dehydroascorbic acid concentration 0.09±0.03 mg/109 cells (mean±SEM, n=8). A statistically significant (r=0.599, p<0.05) correlation was established between the concentrations of ascorbic acid in plasma and leukocytes.