Isolation and quantitation of endogenous vitamin D and its physiologically important metabolites in human plasma by high pressure liquid chromatography

We have developed methodology for the precise measurement of vitamin D, 25-OHD, 24,25-(OH)₂D and 1α,25-(OH)₂D in relatively small quantities (3-5 ml) of normal human plasma. It involves initial extraction with methanol-methylene chloride and separation of phases by centrifugation in polypropylene tubes, followed by discontinuous flow gradient chromatography on LH-20. These procedures accomplish the removal of interfering lipophilic substances and excellent resolution of vitamin D metabolites with good recovery (94%). Further separation of metabolites from interfering U.V. absorbing lipophilic substances is achieved by HPLC using isocratic conditions in normal and reverse phase systems. The U.V. absorbing peak areas eluting from HPLC in the positions corresponding to standard vitamin D, 25-(OH)D and 24,25-(OH)₂D are reproducibly quantitated by automated integration. 1α,25-(OH)₂D Is measured by ligand binding assay using intestinal cytosol because plasma concentrations of this metabolite are too low for optical detection. Values in normal human plasma obtained in late summer are: vitamin D = 15.2 ± 1.8 ng/ml; 25-OHD = 28.6 ± 2.1 ng/ml; 24,25-(OH)₂D = 2.1 ± 0.5 ng/ml; 1α,25-(OH)₂D = 0.035 ± 0.003 ng/ml (±S.D.). The methodology described or modification thereof should provide a formidable tool in the investigation of the regulation of vitamin D metabolism in vivo in both physiologic and pathologic states.