Determination of 1,25-dihydroxyvitamin D3 in plasma using thin-layer chromatography and modified competitive protein...

A simplified assay for calcitriol in plasma samples, using high-performance thin-layer chromatography (HPTLC) followed by competitive protein binding assay ( CPBA ), is described. For quantitative separation of femtomol concentrations of calcitriol from other metabolites of vitamin D, HPTLC can be used instead of column chromatography on Sephadex LH-20 and HPLC on silica gel. Since the CPBA with duodenal cytosol from chickens as a binding protein and [3H]1,25-dihydroxycholecalciferol as radioligand is highly sensitive, detection limit 10 fmol per assay, the volume of plasma sample can be reduced to 1 ml.