Simultaneous determination of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D, and 1,25-dihydroxyvitamin D in plasma or serum.
We describe a simultaneous assay for the principal vitamin D metabolites: 25-hydroxyvitamin D, 24-25-dihydroxyvitamin D, and 1,25-dihydroxyvitamin D. Special attention has been paid to simplification of the extensive extraction and purification procedures used in previously described simultaneous assays. All three metabolites were isolated with a single extraction step, followed by only one gradient liquid-chromatographic procedure. For final quantitation we used competitive protein binding assays, involving readily available binding proteins and commercially purchased tritiated vitamin D metabolites. Concentrations in the plasma of healthy subjects (mean age, 27 years), sampled during December were 51 (SD 17) nmol/L, 4.1 (SD 1.3) nmol/L, and 124 (SD 26) pmol/L for 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D and 1,25-dihydroxyvitamin D, respectively. Intra- and interassay CVs for the three metabolites were 4.4 and 3.9%, 6.7 and 8.0%, and 7.0 and 4.8%, respectively.
D015650:24,25-Dihydroxyvitamin D 3; D015652:25-Hydroxyvitamin D 2; D000328:Adult; D001667:Binding, Competitive; D002117:Calcitriol / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004100:Dihydroxycholecalciferols / Q000097:blood*; D004872:Ergocalciferols / Q000031:analogs & derivatives* / Q000097:blood; D006801:Humans; D006887:Hydroxycholecalciferols / Q000097:blood*; D012016:Reference Values
D004100:Dihydroxycholecalciferols; D004872:Ergocalciferols; D006887:Hydroxycholecalciferols; D015652:25-Hydroxyvitamin D 2; D015650:24,25-Dihydroxyvitamin D 3; D002117:Calcitriol