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

Background: Presence of the 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3] metabolite affects accurate determination of 25(OH)D3 by most routine liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods and to an unknown extent in present immuno- and protein binding assays. We studied 3-epi-25(OH)D3 cross-reactivity in a competitive protein binding (CPB) assay (Roche Elecsys).

Methods: Neonatal samples, containing up to 58% of 3-epi-25(OH)D3 were used for measurement by the CPB assay and by an LC-MS/MS method separating 25(OH)D3 and 3-epi-25(OH)D3. Analytical recovery was also studied by addition of exogenous 3-epi-25(OH)D3.

Results: The CPB assay showed approximately 51% cross-reactivity to 3-epi-25(OH)D3 at exogenous addition. In contrast, there was minimal 3-epi-25(OH)D3 recognition by the CPB assay when present as the natural endogenous metabolite.

Conclusions: The automated CPB assay displays minimal 3-epi-25(OH)D3 cross-reactivity in samples containing significant concentrations of endogenous 3-epi-25(OH)D3. Exogenous 3-epi-25(OH)D3 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.

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http://dx.doi.org/10.1515/cclm-2013-0702DOI Listing

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