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

Abstract

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/l (mean .+-. SEM; n=4) and dehydroascorbic acid 21.0 .+-. 9.1 mg/l (mean .+-. SEM, n=4) when stored at +4.degree. C. The concentration of both forms of ascorbic acid was found to deterioriate in similar ratios during storage at +4.degree. C, and pasteurization considerably increased the loss of vitamin C. After pasteurization the milk contained ascorbic acid 8.6 .+-. 3.4 mg/l (mean .+-. SEM, n=4) and dehydroascorbic acid 6.6 .+-. 2.4 mg/l (mean .+-. SEM, n=4). In plasma the dehydroascorbic acid content (0.16 .+-. 0.03 mg/l, mean .+-. SEM, n=23) was lower than that of ascorbic acid (9.96 .+-. 0.75 mg/l, 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 .+-. 0.3 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.