Development of Methods for Measurement of Vitamin D and its Metabolites in Foods, Fortified Foods, and Dietary Supplements

Summary

Vitamin D is a fat-soluble vitamin that is essential for bone health. Vitamin D deficiency is associated with muscle weakness and osteoporosis and can contribute to an increased risk of falls and fractures. Sources of vitamin D include sun exposure and food. Very few foods have naturally occurring levels of vitamin D that have a meaningful impact on vitamin D intake. However, fortified foods, such as milk, margarine, and cereals, do provide a portion of the recommended intake of vitamin D. Dietary supplements can also serve as a source of vitamin D. Accurate measurement of the levels of vitamin D in these products is essential for assessing dietary intake of vitamin D and in correlating vitamin D intake with overall health.

Description

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.

Major Accomplishments

• Values assigned for vitamin D₂ and D₃ levels in 5 fortified and 3 unfortified food materials.
• Values assigned for 25(OH)D₃ in 4 unfortified food materials.
• ID-LC-MS/MS method developed for determination of vitamin D and metabolites from endogenous food matrices using a derivatization approach.

Additional Technical Details

NIST has developed new analytical methodology based on a derivatization approach followed by LC-MS/MS for vitamins D₂, D₃, 25(OH)D₂, and 25(OH)D₃ in foods, fortified foods, and dietary supplements.  The validity of NIST’s approach has been externally confirmed through a robust comparison with other trusted methodologies [Roseland et al. J. Agric. Food Chem. 2016], and has been used to assign values for vitamins D₂, D₃, and 25(OH)D₃ in 8 food and dietary supplement SRMs.

Associated Product(s)

• • SRM 1546a Meat Homogenate (Vitamin D₃, 25(OH)D₃)
  • SRM 1549a Whole Milk Powder (Vitamin D₃, 25(OH)D₃)
  • SRM 1577c Bovine Liver (25(OH)D₃)
  • SRM 1845a Whole Egg Powder (Vitamin D₃, 25(OH)D₃)
  • SRM 1849a Infant/Adult Nutritional Formula I (Vitamin D₃)
  • SRM 1869 Infant/Adult Nutritional Formula II (Vitamin D₂, Vitamin D₃)
  • SRM 3235 Soy Milk (Vitamin D₂)
  • SRM 3280 Multivitamin/Multielement Tablets (Vitamin D₂)
  • SRM 3532 Calcium-Containing Solid Oral Dosage Form (Vitamin D₃)