Analysis of 25-hydroxy vitamin D3 in plasma by high-performance liquid chromatography

A specific high-performance liquid chromatographic (HPLC) procedure for the determination of 25-(OH) vitamin D3 (25-OH-D) in plasma is described. The organic solvent extract from plasma is first purified on an HPLC silica column and then chromatographed on a reversed-phase column in a high-performance liquid chromatograph fitted with an ultraviolet detector (254 nm); quantitation from plasma samples containing as low as 2 ng/ml of 25-OH-D is reported. Endogenous substances do not interfere with the assay. The determination of vitamin 25-OH-D concentrations in plasma in normal subjects is described.