Development and certification of a standard reference material for vitamin D metabolites in human serum

Abstract

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health's Office of Dietary Supplements (NIH-ODS), has developed a Standard Reference Material (SRM) for the determination of 25-hydroxyvitamin D [25(OH)D] in serum. SRM 972 Vitamin D in Human Serum consists of four serum pools with different levels of vitamin D metabolites and has certified and reference values for 25(OH)D(2), 25(OH)D(3), and 3-epi-25(OH)D(3). Value assignment of this SRM was accomplished using a combination of three isotope-dilution mass spectrometry approaches, with measurements performed at NIST and at the Centers for Disease Control and Prevention (CDC). Chromatographic resolution of the 3-epimer of 25(OH)D(3) proved to be essential for accurate determination of the metabolites.

Figure 1

Selected ion chromatograms (LC-MS) for vitamin D metabolites and their labeled internal standards in Level 3 of SRM 972. Experimental details are provided in the text.

Figure 2

LC-MS/MS multiple reaction monitoring chromatograms for Level 4 of SRM 972. Measurements of 25(OH)D₃ and 3-epi-25(OH)D₃ were made using a Zorbax CN column (A) and measurements of 25(OH)D₂ were made using a Zorbax XDB-C₁₈ column (B). Further experimental details are provided in the text.