Fluorimetric assay of 1 alpha,25-dihydroxyvitamin D3 in human plasma

The first fluorimetric method for assaying plasma 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is described. Lipid extracts from human plasma were successively purified by a Bond Elut NH2 column and a normal-phase HPLC column, and the 1,25-(OH)2D3 fractions obtained were fluorescence-labelled with 4-[2-(6,7-dimethoxy-4-methtyl-3-oxo-3,4-dihydroquinoxalyl)et hyl]-1, 2,4-triazoline-3,5-dione (DMEQ-TAD). The fluorescent adducts of 1,25-(OH)2D3 were analyzed by HPLC on a reversed-phase column, after extensive elimination of the degradation products of excess reagents, by a Bond Elut PSA column followed by a normal-phase HPLC column. The intra- and inter-assay coefficients of variation were within 10% in both assays. The analytical recoveries of standard 1,25-(OH)2D3 added to plasma were quantitative. The present fluorimetric assay was compared with the established radioreceptor assay (RRA) used in measuring plasma 1,25-(OH)2D3 concentration. The regression analysis of the data afforded a relationship y = 1.049x - 2.657 and a correlation coefficient (r) of 0.900.