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.
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.
Authors: Konstantinos Makris; Harjit P Bhattoa; Etienne Cavalier; Karen Phinney; Christopher T Sempos; Candice Z Ulmer; Samuel D Vasikaran; Hubert Vesper; Annemieke C Heijboer Journal: Clin Chim Acta Date: 2021-03-10 Impact factor: 6.314
Authors: Yeow-Kuan Chong; Chi-Chun Ho; Shui-Yee Leung; Susanna K P Lau; Patrick C Y Woo Journal: Comput Struct Biotechnol J Date: 2018-08-28 Impact factor: 7.271
Authors: Jos P M Wielders; Graeme F Carter; Heike Eberl; Gary Morris; Heinz Jürgen Roth; Christian Vogl Journal: J Clin Lab Anal Date: 2014-08-17 Impact factor: 2.352