Vitamin D Testing in Food

Prior to the discovery of vitamin D, rickets was a bone illness that frequently occurred in children, which is still seen today in developing countries. In his experiments with rickets-induced dogs, Sir Edward Mellanby discovered that cod liver oil contained some type of anti-rachitic factor that cured their condition. The effects of this fat soluble component were studied further by a number of researchers and the component was later named vitamin D.

Vitamin D is actually a group of fat-soluble prohormones consisting of several metabolites and analogues and is an essential component for human and animal health. Vitamin D helps regulate calcium and phosphorus levels in the blood, helps maintain the immune system, plays a significant role in maintaining cellular growth and activity, functions as an anti-inflammatory agent, and may help prevent and treat specific types of cancers.

There are two major forms of vitamin D: D2 (ergocalciferol) and D3 (cholecalciferol).

Vitamin D must undergo two hydroxylation steps within the body to convert to its physiologically active form. The first hydroxylation step occurs in the liver and produces 25-hydroxyvitamin D3 (calcidiol). The second hydroxylation occurs in the kidneys to produce 1α,25-dihydroxyvitamin D3 (calcitriol). While D2 and D3 appear to have similar biological activity in humans and rats, only D3 is biologically active in poultry.

The Institute of Medicine of the National Academies, Food and Nutrition Board, has established minimum daily requirements and upper limits for vitamin D. The minimum daily requirements for vitamin D are: 5µg (200 IU) per day for infants and most adults, 10µg (400 IU) per day for adults between the ages of 50 and 70 and 10µg (600 IU) per day for adults over the age of 70. The upper limits for vitamin D are 25µg (1000 IU) per day for infants and 50µg (2000 IU) per day for all other age groups.

Samples tested for vitamin D are saponified with ethanolic KOH solution containing antioxidants and the vitamin D is then extracted into an ether solution. This ether phase is evaporated to dryness, reconstituted in heptane and cleaned on a solid phase extraction column. The eluted fraction is evaporated to dryness under nitrogen and reconstituted in the preparative mobile phase. This solution is then further purified on a preparative HPLC, using a silica column, and the fraction containing the vitamin D collected. Again the fraction is dried under nitrogen and reconstituted in methanol.