Isolation and purification of in vivo-generated 25-hydroxyvitamin D2 by preparative high-performance liquid chromatography.
In order to obtain a standard compound of 25-hydroxyvitamin D2 (25-OH-D2), a method for isolating in vivo-generated 25-OH-D2 from the blood of rats or rabbits was established by using several steps of preparative high-performance liquid chromatography (HPLC). When the unsaponifiable matter of the plasma obtained from rats or rabbits receiving a large dose of vitamin D2 was applied to the preparative HPLC using a Zorbax SIL column, a peak denoted as peak X was observed on the chromatogram. Since the peak X was thought to be due to 25-OH-D2 from the experiments of time course and dose-response, it was purified by subjecting it to successive preparative HPLC using several kinds of columns. From the results of ultraviolet (UV) absorption spectrum, gas chromatography-mass spectrometry (GC-MS) and mass chromatography, the purified peak X compound was confirmed to be 25-OH-D2. The proposed method for isolating in vivo-generated 25-OH-D2 is very convenient, because the time to perform each HPLC is very short though several steps of HPLC are used.
D000818:Animals; D002112:Calcifediol; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004872:Ergocalciferols / Q000378:metabolism; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006887:Hydroxycholecalciferols / Q000096:biosynthesis / Q000097:blood* / Q000302:isolation & purification; D008297:Male; D011817:Rabbits; D051381:Rats; D013056:Spectrophotometry, Ultraviolet
D004872:Ergocalciferols; D006887:Hydroxycholecalciferols; D002112:Calcifediol