Vitamin D is found in foods, these include vitamin D2 (calciferol), which is found in mushrooms, and vitamin D3 (cholecalciferol), which is found in meat, egg, and fish. Other active compounds include the metabolites 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3.
Vitamin D in foods is typically present at trace levels and it is difficult to separate from contaminants. Pretreatment prior to analysis involves hydrolysis to remove lipids, followed by solvent extraction. The resulting extract is separated by preparative LC for further purification and then analyzed by HPLC. Automated preparative purification was performed using a PLC561 preparative HPLC system.
In this analysis, a two step separation was made to isolate vitamins D2 and D3, the first step used preparative LC normal-phase adsorption mode, and the second step HPLC reversed-phase partition mode. By combining these two techniques it was possible to to isolate and detect both vitamin D2 and vitamin D3 with excellent results.