A Method for Simultaneous Determination of 25-Hydroxyvitamin D₂ and 25-Hydroxyvitamin D₃ in Human Plasma by Using Two Steps of High-Performance Liquid Chromatography

Abstract

A method for simultaneous determination of 25-hy droxyvitamin D₂ (25-OH-D₂) and 25-hydroxyvitamin D₃ (25-OH-D₃) in human plasma has been developed by using two steps of high-performance liquid chromatography (HPLC). Lipids extracted from 0.5ml of human plasma were first subjected to the preparative HPLC using a Nucleosil 5C₁₈ column (reversed-phase type) and a 25-OH-D fraction containing 25-OH D₂ and 25-OH-D₃ was separated. The separated fraction was subsequently subjected to the analytical HPLC using a Zorbax SIL column (straight phase type). Since the peaks corresponding to 25-OH-D₂ and 25-OH-D₃ were clearly separated from one another on the chromatogram of the analytical HPLC, the metabolites could be simultaneously determined by estimating the respective peak heights. When the fractions corresponding to the respective peaks were separately collected by repeatedly applying rather large quantities of human plasma and were subjected to gas chromatography-mass spectrometry (GC-MS), they were identified as containing 25-OH-D₂ and 25-OH-D₃, respectively. The proposed method was applied to plasma samples of human adults taking 400 I.U./day of vitamin D₂ for 8 weeks and the values were 22.5±8.1 ng/ml for 25-OH-D₃ and 11.5±1.8ng/ml for 25-OH-D₂ (mean±S. D.), respectively.