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An improved method is described which permits the simultaneous determination of 25-hydroxyvitamin D [25-(OH)D], 24,25-dihydroxyvitamin D [24,25-(OH)2D] and 1,25-dihydroxyvitamin D [1,25-(OH)2D] in milliliters of human serum. Methodological improvements enabled a rapid and almost complete extraction of the three metabolites from serum and omission of adding labeled internal standards to each serum sample for the calculation of individual recoveries. Commercially available stable chick embryo intestinal mucosa cytosol preparation made the troublesome preparation of cytosol receptor for 1,25-(OH)2D unnecessary. The procedure involves saturation of serum with ammonium carbonate and extraction with methanol/ethyl acetate, followed by separation of 25-(OH) D from the dihydroxy metabolites of vitamin D by Sephadex LH-20 column chromatography and further separation of the dihydroxy metabolites into 24,25-(OH)2D and 1,25-(OH)2D by high-pressure liquid chromatography. This is followed by individual determination of each metabolite by competitive protein-binding assay or radioreceptor assay.
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