An improved competitive protein binding assay for 1,25-dihydroxyvitamin D

A practical procedure for the routine analysis of blood levels of 1,25-dihydroxyvitamin D has been developed. 1,25-Dihydroxyvitamin D was extracted from serum with methylene chloride:methanol. The concentration of acidic lipids in the extract was reduced with an alkaline wash. The serum extract was chromatographed on Sephadex LH-20, which was extracted with methanol prior to use in the columns. The chromatography was aided by a device which simultaneously collected and evaporated the eluates. High pressure liquid chromatography to further purify 1,25-dihydroxyvitamin D was found to be unnecessary. These modifications resulted in negligible 1,25-dihydroxyvitamin D values (