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


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.


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), with stable isotope-labeled internal standards for D2 and D3, might also provide improvements in measurement accuracy and robustness. To date, however, LC/MS has not been thoroughly investigated for measurement of vitamin D in foods and dietary supplements.

Additional Technical Details:

NIST is developing new analytical methodology based on LC/MS for vitamin D2 and vitamin D3 in foods, fortified foods, and dietary supplements. Chromatographic stationary phases with enhanced selectivity for steroid-type compounds have been evaluated as part of the method development effort. These phases include biphenyl, pentafluorophenyl, and cholesterol columns, as well as others. Sample preparation techniques also need to be investigated because the isolation of vitamin D from matrices having high fat content can be problematic. Solid-phase extraction (SPE) shows some promise for reducing the time required for sample preparation as well as minimizing potential interferences but has not been studied in detail. Stable isotope-labeled D2 and D3 have been obtained and will be utilized as internal standards as part of the method development. Once preliminary method development has been completed, the methodology will be evaluated for possible value assignment of vitamin D in existing Standard Reference Materials (SRMs) or in new SRMs as they are developed.

Major Accomplishments:

  • A variety of liquid chromatographic stationary phases have been evaluated for the separation of vitamin D2 and D3. Preference has been given to phases that are compatible with LC/MS eluent systems.
  • Preliminary investigations have been performed on isolation of vitamin D from food matrices using solid-phase extraction or other techniques

Start Date:

October 1, 2007

End Date:


Lead Organizational Unit:




Lacey C. Brent
Catherine A. Rimmer
Karen W. Phinney


General Information:
Karen Phinney
301-975-4457 Telephone

100 Bureau Drive, M/S 8392
Gaithersburg, MD 20899-8392