Determination of Low Levels of Vitamin D3 in Animal Feeds, Using Sephadex LH-20 and Normal Phase High Pressure Liquid Chromatography

Abstract

Abstract A procedure has been developed for the purification and quantification of vitamin D3 at levels of 4000 down to 500 IU/kg from a variety of animal feeds. The method involves initial extraction with dichloromethane followed by filtration through a Sep-Pak cartridge. The D3 fraction from the Sep-Pak is further purified by using a 45 × 1.6 cm Sephadex LH-20 column followed by a Partisil-10 PAC cleanup column. Final analysis is performed by high pressure liquid chromatography (HPLC) using a LiChrosorb NH2 10 μm column and a variable wavelength UV detector set at 264 nm. The detection response curve for a vitamin D3 standard was linear over the range 5 to 100 ng, using both peak area and peak height measurements; the minimum detectable amount was 5 ng. The average percent recovery of vitamin D3 in animal feeds was 94.16 ± 5.2%.