Vitamin D exists in two forms: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Vitamin D3 is produced in the body after sun exposure and is also the form used in most fortified foods. Dietary supplements may contain either vitamin D2 or D3. Measurement of vitamin D in foods and dietary supplements has primarily been performed by liquid chromatography (LC) with UV absorbance detection. Current methodology tends to suffer from a number of limitations, including the common use of vitamin D2 as an internal standard for vitamin D3, and poor chromatographic resolution of vitamin D species from each other or from other sample components. New chromatographic stationary phases with enhanced selectivity for steroid-type molecules may improve the separation of vitamin D species. Liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (LC/MS/MS), with stable isotope-labeled internal standards for D2 and D3, provide improvements in measurement accuracy and robustness.
In addition to vitamin D, a vitamin D metabolite, 25-hydroxyvitamin D (or 25(OH)D) is also found in some foods, such as beef, beef liver, chicken liver, and eggs. Studies indicate that 25(OH)D may have 5 times the bioactivity of vitamin D, and therefore foods containing even low levels of 25(OH)D may contribute significantly to overall vitamin D status. With the new interest in levels of 25-hydroxyvitamin D in food, efforts for method development are now focused on the measurement of endogenous levels of vitamins D2, D3, 25(OH)D2, and 25(OH)D3 in unfortified foods. The focus is on developing an LC-MS/MS method for the simultaneous determination of all the species of interest.