MULTIPLE FLUOROMETRIC ASSAY OF 3 MAJOR VITAMIN-D-3 METABOLITES (25-HYDROXYVITAMIN D-3, 24,25-DIHYDROXYVITAMIN D-3 AND 1,25-DIHYDROXYVITAMIND-3) IN PLASMA AND ITS APPLICATION TO CLINICAL-STUDIES

We have developed a fluorometric method for routine analyses of 25-hyd roxyvitamin Dg (25-OHD3) and 24,25- and 1,25-dihydroxyvitamin D-3 [24, 25- and 1,25-(OH)(2)D-3] in human plasma. Lipid extracts from plasma ( 2.5 mi) were purified using a Sep-Pak silica cartridge followed by nor mal-phase HPLC, and each of the three metabolites was assayed in separ ate lines. Each metabolite fraction was treated with fluorescent dieno phile (DMEQ-TAD) and quantified on reversed-phase HPLC after appropria te cleanup steps. Labeled 25-OHD3 was quantified directly, 24,25-(OH)( 2)D-3 was analyzed after cleanup by a Bond Elut PSA cartridge and 1,25 -(OH)(2)D-3 was quantified after purification by a Bond Elut PSA cartr idge and normal-phase HPLC. In the assay of 25-OHD3, we demonstrated t he usefulness of a non-radioactive internal standard for the fluoromet ric assay. The multiple fluorometric assay was successfully applied to the clinical monitoring of vitamin D-3 therapy in a vitamin D-deficie nt patient with metabolic bone disease.