Evaluation of 3-epi-25-hydroxyvitamin D3 cross-reactivity in the Roche Elecsys Vitamin D Total protein binding assay

Background: Presence of the 3-epi-25-hydroxyvitamin D-3 [3-epi-25(OH)D-3] metabolite affects accurate determination of 25(OH)D-3 by most routine liquid chromatographytandem mass spectrometry (LC-MS/MS) methods and to an unknown extent in present immuno- and protein binding assays. We studied 3-epi-25(OH)D-3 cross-reactivity in a competitive protein binding (CPB) assay (Roche Elecsys). Methods: Neonatal samples, containing up to 58% of 3-epi-25(OH)D-3 were used for measurement by the CPB assay and by an LC-MS/MS method separating 25(OH)D-3 and 3-epi-25(OH)D-3. Analytical recovery was also studied by addition of exogenous 3-epi-25(OH)D-3. Results: The CPB assay showed approximately 51% cross-reactivity to 3-epi-25(OH)D-3 at exogenous addition. In contrast, there was minimal 3-epi-25(OH)D-3 recognition by the CPB assay when present as the natural endogenous metabolite. Conclusions: The automated CPB assay displays minimal 3-epi-25(OH)D-3 cross-reactivity in samples containing significant concentrations of endogenous 3-epi-25(OH)D-3. Exogenous 3-epi-25(OH)D-3 added to human serum or plasma seems to behave different from endogenous presence, and caution is warranted when using samples spiked with vitamin D metabolites for testing analytical specificity or external quality assurance in immuno-or protein binding assays.