A method for routine estimation of vitamin D activity in human and bovine milk

To estimate the antirachitic activity of human and bovine milk, we developed a modern biochemical method for determining vitamin D metabolites in milk. Vitamin D metabolites were assayed from milk whey and from whole milk. Milk whey yielded poor recovery of both endogenous and added vitamin D, suggesting a marked loss of vitamin D activity to milk fat during homogenization and separation of the milk whey. A method for assaying the vitamin D metabolites in whole milk involves 1) lipid extraction, 2) cold methanol and ether precipitation, 3) alkaline backwash to reduce the amount of interfering lipids, 4) an efficient reverse-phase preparative purification, 5) an additional silica purification for vitamin D, 6) an analytical high-performance liquid chromatography, and 7) separate sensitized protein-binding assays for vitamin D and 25-hydroxyvitamin D. The method for whole milk resulted in good recovery of added vitamin D, and levels of assayed metabolites and their calculated antirachitic activity agreed well with earlier reports, that is, about 10-50 IU of vitamin D activity per liter.