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Development of Standard Reference Materials for Vitamin D Metabolites


The prevalence of vitamin D deficiency/insufficiency within the general population is an area of concern. Vitamin D status is generally assessed by measuring levels of the metabolite 25-hydroxyvitamin D (25(OH)D) in serum or plasma. A number of studies have reported discrepancies between assays used to measure 25(OH)D. This variability calls into question the ability of 25(OH)D assays to accurately identify individuals affected by vitamin D deficiency, and there have been calls for standardization of vitamin D measurements. NIST has developed Standard Reference Materials (SRMs) for vitamin D metabolite calibration solutions and for vitamin D metabolites in human serum. SRM 2972a 25-Hydroxyvitamin D Calibration Solutions and SRM 972a Vitamin D Metabolites in Frozen Human Serum will help to ensure the reliable calibration and validation, respectively, of assays for determining these metabolites.


Vitamin D plays an important role in bone health, and recent evidence suggests that vitamin D deficiency may also be associated with an increased risk of certain types of cancer. The two major forms of vitamin D are vitamin D2 and vitamin D3. The most widely used indicator of vitamin D status is the measurement of the metabolite 25-hydroxyvitamin D (25(OH)D) in either serum or plasma. Because circulating 25(OH)D can arise from hydroxylation of either vitamin D2 or D3, measurement of total 25(OH)D (both 25(OH)D2 and 25(OH)D3) is essential for accurate assessment of vitamin D status. A number of studies, including interlaboratory comparisons, have reported discrepancies between the results of assays used to measure 25(OH)D. NIST has been working with the National Institutes of Health’s Office of Dietary Supplements to develop SRMs for vitamin D in serum. SRM 972a provides a mechanism to assess the accuracy of clinical assays for vitamin D in serum and serves as an adjunct to the quality assurance program for 25(OH)D measurements. This SRM should help to promote correct identification of those affected by vitamin D deficiency while also reducing the likelihood of unnecessary treatment.

Additional Technical Details:

SRM 972a Vitamin D Metabolites in Frozen Human Serum consists of four pools of fresh-frozen serum. Each pool has a different levels of 25(OH)D2 and 25(OH)D3. Level 4 was fortified with 3-epi-25(OH)D3, a metabolite that may be found at significant levels in samples from both infants and adults. NIST designed the SRM to pose similar analytical challenges to those encountered in patient samples. In order to value assign the four serum pools, NIST has developed isotope dilution LC-MS and LC-MS/MS methodology that incorporates stable isotope labeled internal standards for both 25(OH)D2 and 25(OH)D3. For mass spectrometry-based methods, the presence of C-3 epimers of 25(OH)D2 and 25(OH)D3 can present problems because they have identical masses and fragmentation patterns. Although the physiological significance of these C-3 epimers remains unclear, it is necessary to resolve these isomers chromatographically to avoid errors in quantitation when mass spectrometric detection is used. The Certificate of Analysis for SRM 972a includes values for 25(OH)D2, 25(OH)D3, and 3-epi-25(OH)D3.

SRM 2972a 25-Hydroxyvitamin D Calibration Solutions consists of four separate ethanolic solutions of vitamin D metabolites: a high and low level 25-hydroxyvitamin D3, and a single level of each 25-hydroxyvitamin D2 and 3-epi-25-hydroxyvitamin D3. SRM 2972a is intended to calibrate assays that are used for the determination of these metabolites and to provide traceability to the SI unit of mass.

Major Accomplishments:

  • Isotope dilution LC-MS and LC-MS/MS methodology for 25(OH)D has been developed; the LC-MS/MS method is a Reference Measurement Procedure recognized by the Joint Committee on Laboratory Medicine
  • SRM 972a and SRM 2972a are available for purchase
  • A companion material, SRM 2973 Vitamin D Metabolites in Human Serum – High Level, has also been prepared and is currently being characterized
SRM 972 Vitamin D in Human Serum

Start Date:

October 1, 2005

End Date:


Lead Organizational Unit:


Associated Products:

SRM 972a Vitamin D Metabolites in Frozen Human Serum
SRM 2972a 25-Hydroxyvitamin D Calibration Solutions

Associated Publications: 

Click on title in reference to view paper:

Phinney, K.W., Bedner, M., Tai, S.S.-C. , Vamathevan, V.V., Sander, L.C., Sharpless, K.E., Wise, S.A., Yen , J.H., Schleicher, R.L., Chaudhary-Webb, M., Pfeiffer, C.M., Betz, J.M., Coates, P.M., Picciano, M.F., Development and Certification of a Standard Reference Material for Vitamin D Metabolites in Human Serum, Anal. Chem., 84 (2), 956–962, 2012.

Bedner, M., Phinney, K.W., Development and Comparison of Three Liquid Chromatography-Atmospheric Pressure Chemical Ionization/Mass Spectrometry Methods for Determining Vitamin D Metabolites in Human Serum, J. Chromatogr. A, 1240, 132-139, 2012.

Tai , S.S.-C., Bedner, M., Phinney, K.W., Development of a Candidate Reference Measurement Procedure for the Determination of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in Human Serum Using Isotope-Dilution Liquid Chromatography/Tandem Mass Spectrometry, Anal. Chem., 82, 1942-1948, 2010.

Phinney, K.W., Development of a Standard Reference Material for Vitamin D in Serum, Am. J. Clin. Nutr., 88(2), 511S-512S, Suppl. S, 2008.


Mary Bedner
Phone: 301-975-4142