Full-scan Mass Spectral Evidence for 3-epi-25-hydroxyvitamin D₃ in Serum of Infants and Adults

Background: Since the introduction of liquid chromatography-mass spectrometry (LC/MS) for assessing vitamin D status, it has been shown that the C-3 epimer can account for a significant proportion of circulating 25-hydroxyvitamin D₃ (25OHD₃) concentrations in infants. However, some question whether monitoring a single MS transition at a chromatographic retention time typical for 3-epi-25OHD₃ sufficiently warrants conclusions about the identity of the substance generating the signal. Therefore, we aimed to substantiate the evidence for 3-epi-25OHD₃ in infants by collision induced dissociation (CID)-MS/MS product ion scans. A second objective was mass spectrometric investigation of the presence and prevalence of the 3-epi metabolite in serum from adults.

Methods: Serum samples from six infants and 32 adults were studied using an ultra performance LC/tandem MS (UPLC/MS/MS) method designed to separate the 3-epi-25OHD₃ from 25OHD₃. Samples were submitted to liquid/liquid extraction and Sephadex LH-20 fractionation, prior to column-switching UPLC with MS/MS recording of CID product ion spectra of the [M+H](+) precursor ion. The respective standards were analyzed under identical UPLC/MS/MS conditions for comparison.

Results: In the chromatograms of all samples, two peaks eluted with retention characteristics and spectra closely matching those observed for the 25OHD₃ and the 3-epi standards. The percentage of the 3-epi metabolite relative to 25OHD₃ in infants ranged from 15% to 41%, and in adults from 2.5% to 17%.

Conclusions: This preliminary finding suggests that the prevalence of 3-epi-25OHD₃ in serum of infants is considerable, and that even in adults the concentrations of this form should not be neglected.