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Development of Methods for Measurement of Vitamin D and its Metabolites 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) 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.

Additional Technical Details:

NIST is developing new analytical methodology based on LC/MS and LC/MS/MS for vitamins D2, D3, 25(OH)D2, and 25(OH)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, as well as columns with solid-core particles and smaller particle sizes. These phases include biphenyl, pentafluorophenyl, and cholesterol columns, as well as others.

Preliminary sample preparation work on unfortified foods indicates that saponification followed by solvent extraction or solvent extraction alone are most promising for extraction of vitamin D and metabolites from unfortified foods. Solid-phase extraction is being investigated as a mechanism to increase recovery of vitamin D and metabolites. The combination of low endogenous levels and the complex food matrix requires the use of derivatization, which greatly reduces LC/MS/MS background. Current work is focused on optimizing the separation to eliminate interferences from the derivatizing agent, identifying the best MRM transitions for quantitation, and optimizing the extraction methods for existing SRMs. Once preliminary method development has been completed, the methodology will be evaluated for possible value assignment of vitamin D and its metabolites in existing SRMs or in new SRMs as they are developed.

Major Accomplishments:

  • Certification of vitamin D2 and D3 levels in SRM 3280 Multivitamin/Multielement Tablets and SRMs 1849 and 1849a Infant/Adult Nutritional Formula, respectively, have been completed.
  • Preliminary investigations have been performed on isolation of vitamin D and metabolites from endogenous food matrices using solid-phase extraction and derivatization

Start Date:

October 1, 2007

End Date:


Lead Organizational Unit:

Melissa M. Phillips
Phone: 301-975-4134

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

Stephen A. Wise
Phone: 301-975-3112

100 Bureau Drive, M/S 8390
Gaithersburg, MD 20899-8390