Multiple fluorometric assay of three major vitamin D-3 metabolites (25-hydroxyvitamin D-3, 24,25-dihydroxyvitamin D-3 and 1,25-dihydroxyvitamin D-3) in plasma and its application to clinical studies

We have developed a fluorometric method for routine analyses of 25-hydroxyvitamin 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 normal-phase HPLC, and each of the three metabolites was assayed in separate lines. Each metabolite fraction was treated with fluorescent dienophile (DMEQ-TAD) and quantified on reversed-phase HPLC after appropriate 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 cartridge and normal-phase HPLC. In the assay of 25-OHD3, we demonstrated the usefulness of a non-radioactive internal standard for the fluorometric assay. The multiple fluorometric assay was successfully applied to the clinical monitoring of vitamin D-3 therapy in a vitamin D-deficient patient with metabolic bone disease.