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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 occurred 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. 


Illustration showing a triangle with 9 sectors representing different levels of fat, protein, and carbohydrate in foods, with points indicating food matrix Standard Reference Materials.

Composition of food matrix Standard Reference Materials mapped within the nine sectors of the food triangle. 

Credit: NIST

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 Publications 

1. Austad, J., Barrett, B., Betz, J. M., Burdette, C. Q., Don, C., Erickson, A., Griffiths, J., Hildreth, J. B., Ingle, P., Jennens, M., Ji, D., Joseph, G., Kennedy, D. C., Mudge, E., Noestheden, M., Meaney, M., Phinney, C. S., Rettinger, M., Richards, L. D., Rimmer, C. A., Sarma, N., Sauza, B., Schaneberg, B. T., Solyom, A. M., Sullivan, D. M., Szpylka, J., Traub, J., Walters, D. L., Woods, J. P., Yang, J., Yoo, S. J., Zhang, T., Zhang, W., Zhang, Y., Zhu, J., Zielinski, G., and Coates, S. G., "AOAC SMPR 2015 (R) 016 Standard Method Performance Requirements (SMPRs) for Determination of Vitamin D in Dietary Supplement Finished Products and Ingredients," Journal of Aoac International, 100, 1182-1184 (2017). 

2. Burdette, C. Q., "Technical Note: Determination of Cholecalciferol (Vitamin D-3) in Standard Reference Material 3532 Calcium-Containing Solid Oral Dosage Form," Journal of Aoac International, 100, 1304-1307 (2017). 

3. Wise, S. A., Tai, S. S. C., Burdette, C. Q., Camara, J. E., Bedner, M., Lippa, K. A., Nelson, M. A., Nalin, F., Phinney, K. W., Sander, L. C., Betz, J. M., Sempos, C. T., and Coates, P. M., "Role of the National Institute of Standards and Technology (NIST) in Support of the Vitamin D Initiative of the National Institutes of Health, Office of Dietary Supplements," Journal of Aoac International, 100, 1260-1276 (2017). 

4. Roseland, J. M., Patterson, K. Y., Andrews, K. W., Phillips, K. M., Phillips, M. M., Pehrsson, P. R., Dufresne, G. L., Jakobsen, J., Gusev, P. A., Savarala, S., Nguyen, Q. V., Makowski, A. J., Scheuerell, C. R., Larouche, G. P., Wise, S. A., Harnly, J. M., Williams, J. R., Betz, J. M., and Taylor, C. L., "Interlaboratory Trial for Measurement of Vitamin D and 25-Hydroxyvitamin D [25(OH)D] in Foods and a Dietary Supplement Using Liquid Chromatography-Mass Spectrometry," Journal of Agricultural and Food Chemistry, 64, 3167-3175 (2016). 

5. Sander, L. C., Bedner, M., Duewer, D. L., Lippa, K. A., Phillips, M. M., Phinney, K. W., Rimmer, C. A., Schantz, M. M., Sharpless, K. E., Tai, S. S. C., Thomas, J. B., Wise, S. A., Wood, L. J., Betz, J. M., and Coates, P. M., "The development and implementation of quality assurance programs to support nutritional measurements," Analytical and Bioanalytical Chemistry, 405, 4437-4441 (2013). 

6. Sharpless, K. E., Gill, L. M., Margolis, S. A., Wise, S. A., and Elkins, E., "Preparation of Standard Reference Material 2383 (Baby Food Composite) and use of an interlaboratory comparison exercise for value assignment of its nutrient concentrations," Journal of Aoac International, 82, 276-287 (1999). 


Created March 28, 2009, Updated October 31, 2023