Quantitation of the main metabolites of vitamin D in a single serum sample. I. Extraction, separation and purification of metabolites.
A method for extraction, separation and purification of the main serum metabolites of vitamin D from a single serum sample is described. The method involved extraction of serum by diethylether and separation and purification of vitamin D, 25-OHD and the dihydroxymetabolites 24,25-(OH)2D, 25,26-(OH),2D and 1,25-(OH)2D by elution in three steps from a short open silicic acid column. The eluted vitamin D metabolites were further separated and purified by high pressure liquid chromatography (HPLC). The HPLC systems described separated the D2 and D3 forms of vitamin D, 25-OHD, 1,25-(OH)2D, and probably also 24,25-(OH)2D and 25,26-(OH)2D. The metabolites were purified by the methods described for further quantitation by UV-absorption or competitive protein binding assays, and were found to be homogenous on re-chromatography with different HPLC systems. Good recoveries were obtained for all the metabolites.
D015650:24,25-Dihydroxyvitamin D 3; D002112:Calcifediol; D002117:Calcitriol; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004100:Dihydroxycholecalciferols / Q000097:blood; D006801:Humans; D006887:Hydroxycholecalciferols / Q000097:blood; D012824:Silicic Acid; D014807:Vitamin D / Q000097:blood* / Q000302:isolation & purification; D014812:Vitamin K / Q000378:metabolism
D004100:Dihydroxycholecalciferols; D006887:Hydroxycholecalciferols; D014812:Vitamin K; D012824:Silicic Acid; D014807:Vitamin D; D015650:24,25-Dihydroxyvitamin D 3; D002117:Calcitriol; D002112:Calcifediol; C026032:25,26-dihydroxycholecalciferol