Abstract
BackgroundThe implementation of mass spectrometry to measure serum 25-hydroxyvitamin D [25(OH)D] concentrations has led to concerns regarding the measurement and reporting of the C3-epimer of 25-hydroxyvitamin D3 [3-epi-25(OH)D3], for which there is a near-total lack of data regarding its clinical significance. MethodsWe developed a chromatographic method to resolve (>90%) 3-epi-25(OH)D3 from 25(OH)D3 using a pentafluorophenyl propyl chromatographic column. Using LC–MS/MS, we determined the serum concentrations of 25(OH)D3 and 3-epi-25(OH)D3 in 626 patients aged 3days to 94years undergoing routine vitamin D testing. ResultsComparison between DiaSorin RIA and the new LC–MS/MS method for total 25(OH)D had acceptable agreement. Our data indicate an increase in 25(OH)D3 rather than a reduction in epimer concentration. An average of 3.3ng/ml of 3-epi-25(OH)D3 was detected in adolescents and adults. Inclusion of 3-epi-25(OH)D3 in the total 25(OH)D3 concentration resulted in 9% (<1year) and 3% (1 to 94years) potential misclassification of patients as vitamin D sufficient. ConclusionsThe new LC–MS/MS method is capable of chromatographically separating 25(OH)D3 and 3-epi-25(OH)D3. It was used to confirm that the contribution of 3-epi-25OHD3 to total 25OHD3 concentrations decreases with age in infants and is detectable in adults.
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