Rapid high-performance liquid chromatographic method for the estimation of toxicological levels of 25-hydroxycholecalciferol (25-OH D3) in human serum

A reversed-phase HPLC method for determination of 25-hydroxycholecalciferol (25-OH D3) in serum samples is described. The method involves extraction of 25-OH D3 with hexane, followed by differential resolubilization in acetonitrile. The sample is then applied directly on the C18 bonded phase column of the liquid chromatograph, and developed in a acetonitrile-water solvent system. In the range 12.5–400 ng, a linear relationship was observed between detector response and the amount of 25-OH D3 placed on the column. Also, a very good correlation was observed between the amounts of 25-OH D3 added to serum samples, and the calculated amounts. The method can be used for screening for toxicological levels of 25-OH D3 in patients treated with high doses of vitamin D3.