Development of Reference Materials to Support Accuracy in Measurement of 25-Hydroxyvitamin D
Tue 4:00 PM - Track 1: Vitamin D
Karen Phinney
*Karen W. Phinney, *Mary Bedner>, *Lane C. Sander, *Katherine E. Sharpless, *Susan S.-C. Tai, #Veronica V. Vamathevan, and *Stephen A. Wise.

*Analytical Chemistry Division, National Institute of Standards and Technology, Gaithersburg, MD 20899
#National Measurement Institute, Pymble NSW 2073, Australia
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. Because circulating 25OHD can arise from hydroxylation of either vitamin D2 or D3, measurement of both 25OHD2 and 25OHD3 is essential for accurate assessment of vitamin D status.

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 a need exists for standardization of vitamin D measurements.

NIST has recently developed a Standard Reference Material (SRM) for vitamin D metabolites in human serum. 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 present at significant levels in infants. These four pools are intended to pose the same analytical challenges as those that might be encountered during routine measurements of 25OHD. In order to value assign these materials, NIST developed isotope-dilution LC/MS and LC/MS/MS methodology for the analytes of interest. For mass spectrometry-based methods, chromatographic resolution of C-3 epimers of 25(OH)D2 and 25(OH)D3 is essential to their determination because epimers have identical masses and fragmentation patterns. Although the physiological significance of these C-3 epimers remains unclear, failure to account for their presence in clinical samples may be a source of biases that are sometimes observed between immunological and mass spectrometric based assays.

NIST has also recently introduced SRM 2972 25-Hydroxyvitamin D2 and D3 Calibration Solutions. This SRM consists of two separate ethanolic solutions, one each for 25OHD2 and 25OHD3 at known concentrations. The concentration of the 25OHD species is these solutions is approximately 10 times higher than that normally found in serum so that dilutions can be made to prepare a calibration curve. Both SRM 972 and SRM 2972 were developed in collaboration with the National Institutes of Health’s Office of Dietary Supplements.