Purification of plasma vitamin D metabolites for radioimmunoassay

The ability of four solvent systems to extract tritiated 25-hydroxy vitamin D₃, 24,25-dihydroxy vitamin D₃, 25,26-dihydroxy vitamin D₃ and 1α,25-dihydroxy vitamin D₃ from plasma was compared. Diethyl ether gave the highest yield of metabolites and lowest yield of “lipid” materials, the dihydroxylated metabolites were more readily extracted than 25-hydroxy vitamin D₃. Following extraction with ether the vitamin D metabolites could be separated, without prior purification, on a 6.2 mm × 250 mm Zorbax-Sil high pressure liquid chromatography (HPLC) column, a simplification of previous methods. Plasma 1α,25-dihydroxy vitamin D levels measured after purification by this method were not significantly different from those obtained by an established, more complex method.