Liquid chromatographic determination of vitamin D in fortified milk

A method for the determination of vitamins D2 + D3 in fortified milk is described. Vitamins D2 and D3 are extracted from the saponified sample and converted to isotachysterols with antimony trichloride. The isotachysterols are quantitated using liquid chromatography with ultraviolet detection at 301 nm, which is the absorption maximum. At this wavelength other materials present in the sample do not interfere with the analysis of isotachysterols and therefore a cleanup step is avoided. Recoveries of vitamin D added to skim milk were 98.1% (SD 5.3), 96.7% (SD 3.3), and 96.0% (SD 5.1) for samples fortified with 200, 400, and 600 IU/quart, respectively. For whole milk, recoveries were 102.0% (SD 6.5) and 97.1% (SD 3.5) in samples fortified with vitamin D equivalent to 200 and 400 IU/quart, respectively. The detection limit for vitamin D is 40 IU/quart.