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.

A method for simultaneous determination of 25-hydroxyvitamin 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 0.51 ml 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 +/- 1.8 ng/ml for 25-OH-D2 (mean +/- S.D.), respectively.