Abstract
As a standard compounds in studying the metabolism of vitamin D_2, 25-hydroxyvitamin D_2 (25-OH-D_2) bound to vitamin D binding protein (DBP) was isolated and purified from rabbit plasma. A plasma sample obtained from a rabbit which received 500,000 IU of vitamin D_2 was applied to a Sephadex G-200 column, and an eluate containing 25-OH-D_2 bound to DBP was collected and lyophilized. The lyophilized protein fraction was saponified. The resulting unsaponifiable matter was separated and applied to HPLC using a Zorbax SIL column as a stationary phase and 5.5% isopropanol in n-hexane as a mobile phase. The purified compound was identified as 25-OH-D_2 by HPLC, UV spectrum and GC-MS, and confirmed to be useful as as anthentic standard for HPLC assay of 25-OH-D_2. The yield of the purified 25-OH-D_2 was about 30μg from 55ml of rabbit plasma.
