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
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We have developed a fluorometric method for routine analyses of 25-hydroxyvitamin D<sub>3</sub> (25-OHD<sub>3</sub>) and 24, 25- and 1, 25-dihydroxyvitamin D<sub>3</sub> [24, 25- and 1, 25-(OH)<sub>2</sub>D<sub>3</sub>] in human plasma. Lipid extracts from plasma (2.5ml) 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-OHD<sub>3</sub> was quantified directly, 24, 25-(OH)<sub>2</sub>D<sub>3</sub> was analyzed after cleanup by a Bond Elut PSA cartridge and 1, 25-(OH)<sub>2</sub>D<sub>3</sub> was quantified after purification by a Bond Elut PSA cartridge and normal-phase HPLC. In the assay of 25-OHD<sub>3</sub>, 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<sub>3</sub> therapy in a vitamin D-deficient patient with metabolic bone disease.
- Analytical Sciences
Analytical Sciences 13(2), 255-261, 1997-04-10
The Japan Society for Analytical Chemistry