Liquid-Solid Extraction of Vitamin D3Metabolites from Plasma for Analysis by HPLC, GC/MS and Protein Binding Techniques

Abstract

Liquid-solid extraction of vitamin D₃ metabolites from human plasma using octadecylsilane bonded silica has been studied. Steroid-protein interactions were minimized by diluting the plasma (or serum) with two volumes of saline and passing the solution through the sorbent at 64°C. Highly purified secosteroid fractions were obtained by washing with aqueous methanol, drying the sorbent in situ with a stream of nitrogen for one minute and eluting with mixtures of hexane/chloroform. Recoveries of vitamin D₃ metabolites were essentially quantitative. Applications to the rapid analysis of 25-hydroxy- and 1α, 25-dihydroxy-vitamin D in plasma by high-performance liquid chromatography, gas chromatography-mass spectrometry or by a receptor protein assay are reported.