[Determination of ascorbic acid in blood serum using high-performance liquid chromatography and its correlation with...

Insufficient supply of the populations of the Czech and Slovak Republics with vitamin C is connected with the occurrence of some diseases due to modern civilization. It is therefore necessary to obtain reliable data about the supply of the population with this most important antioxidant of blood plasma. In order to establish a simple, rapid and at the same time specific determination of vitamin C in serum, the present authors have introduced, on the basis of foreign data, the HPLC method of determination of ascorbic acid on reverse phases with UV-detection (245 nm), using 3.7 mM KH2PO4 (pH = 4) as the mobile phase. Very good separation of ascorbic acid from serum (Fig. 1) was obtained. The detection limit is 3.25 pmol of ascorbic acid. Parallelly with the chromatographic determinations, ascorbic acid was determined in 235 volunteers also by the spectrophotometric (colorimetric) method employing 2,4-dinitrophenylhydrazine. Both methods correlate very well (Fig. 2): correlation coefficient, r = 0.93, the level of probability p < 0.000001. The yield (Table 1) is 98.6% and 97.6% for HPLC, or 105.4% and 111.4% for the colorimetric method. The reproducibility of both methods gives coefficients of variation of 3.6% and 2.9%. The described HPLC method is sufficiently precise, specific and suitable for the determination of the levels of ascorbic acid in serum.