Comparison between three fluorometric micromethods for determination of vitamin A in serum

Three common micromethods for vitamin A1 determination were applied to blood, and compared with each other. Methods under investigation were: silicic acid column technique described by Pollack et al [16], HPLC procedure published by de Ruyter and de Leenheer [6, 7] and correction formula method according to Thompson et al [20]. The serum levels of vitamin A were measured in 51 fasting individuals. The average overall agreement was 104% and 116% of the HPLC value for the silicic acid column technique and for the correction formula method, respectively. The mean values obtained by the three methods do not differ significantly. Using these techniques simultaneously, however, in some cases serum values for the same sample show wide differences. Although vitamin A is usually present in biological samples both as retinol and as retinyl esters, the simple correction formula method does not distinguish between them. Their separate estimation, however, can be achieved by different chromatographic procedures. The HPLC method therefore used offers several advantages over the described silicic acid column technique. This technique includes fluorescence detection and provides a rapid and simple estimation of retinol and retinyl palmitate levels using only 180 microliter serum. For these reasons, the HPLC assay is suggested for routine determination of vitamin A in blood.