Liquid Chromatographic Cleanup and Determination of Low Levels of Vitamin D3 in Sheep Plasma

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Abstract
A liquid chromatographic (LC) method is described for the determination of vitamin D3 in sheep plasma. Samples are extracted by one of 2 different methods, depending on the concentration of vitamin D> The samples are purified by using either a Sep-Pak silica cartridge or a small alumina column, followed by additional cleanup on a Metalsorb LC column. Final analysis was carried out on a 5 μm C18 column using a radial compression separation system with an acetonitrile-methanol solvent system. Vitamin D3 was completely resolved from any interfering compounds in the plasma; total run time was less than 15 min, using a variable wavelength detector set at 264 nm. The method was successfully applied to samples at levels of 1-10 ng added vitamin D3/mL sheep plasma, with recoveries in the range 90-97%.