Abstract
BACKGROUND: The 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 D(3) [3-epi-25(OH)D(3)], for which there is a near-total lack of data regarding its clinical significance. METHODS: We developed a chromatographic method to resolve (>90%) 3-epi-25(OH)D(3) from 25(OH)D(3) using a pentafluorophenyl propyl chromatographic column. Using LC-MS/MS, we determined the serum concentrations of 25(OH)D(3) and 3-epi-25(OH)D(3) in 626 patients aged 3 days to 94 years undergoing routine vitamin D testing. RESULTS: Comparison 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)D(3) rather than a reduction in epimer concentration. An average of 3.3 ng/ml of 3-epi-25(OH)D(3) was detected in adolescents and adults. Inclusion of 3-epi-25(OH)D(3) in the total 25(OH)D(3) concentration resulted in 9% (<1 year) and 3% (1 to 94 years) potential misclassification of patients as vitamin D sufficient. CONCLUSIONS: The new LC-MS/MS method is capable of chromatographically separating 25(OH)D(3) and 3-epi-25(OH)D(3). It was used to confirm that the contribution of 3-epi-25OHD(3) to total 25OHD(3) concentrations decreases with age in infants and is detectable in adults.