Development of a Standard Reference Material for Vitamin D in Human Serum

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 (25OHD) in serum or plasma. A number of studies have reported discrepancies between assays used to measure 25OHD. This variability calls into question the ability of 25OHD assays to accurately identify individuals affected by vitamin D deficiency, and there have been calls for standardization of vitamin D measurements. NIST has developed a Standard Reference Material (SRM) for vitamin D metabolites in human serum. SRM 972 Vitamin D in Human Serum will help ensure that individuals affected by vitamin D deficiency are correctly identified and receive appropriate treatment.

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 (25OHD) in either serum or plasma. Because circulating 25OHD can arise from hydroxylation of either vitamin D₂ or D₃, measurement of total 25OHD (both 25OHD₂ and 25OHD₃) 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 25OHD. NIST has been working with the National Institutes of Health’s Office of Dietary Supplements to develop a Standard Reference Material for vitamin D in serum. SRM 972 will provide a mechanism to assess the accuracy of clinical assays for vitamin D in serum and can serve as an adjunct to quality assurance programs for 25OHD measurements. This SRM should help to promote correct identification of those affected by vitamin D deficiency while also reducing the likelihood of unnecessary treatment.

The reference material, SRM 972 Vitamin D in Human Serum, consists of four pools of fresh-frozen serum. Each pool has a different level of 25OHD₂, 25OHD₃, or both. One pool also contains 3-epi-25OHD₃, a metabolite that may be found at significant levels in samples from infants. 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 which incorporates stable isotope labeled internal standards for both 25OHD₂ and 25OHD₃. For mass spectrometry-based methods, the presence of C-3 epimers of 25OHD₂ and 25OHD₃ can present problems because they have identical masses and fragmentation patterns. Although the physiological significance of these C-3 epimers remains unclear, we found that it was necessary to resolve these isomers chromatographically to avoid errors in quantitation when mass spectrometric detection is used. The Certificate of Analysis for SRM 972 includes values for 25OHD₂, 25OHD₃, and 3-epi-25OHD₃.

Major Accomplishments:

• Isotope dilution LC/MS methodology for 25OHD has been developed, and LC-MS/MS methodology is currently in development    
• SRM 972 is available.
• A companion material, SRM 2972 25-Hydroxy-Vitamin D₂ and -D₃ Calibrant Solutions has also been prepared.