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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

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Full-scan mass spectral evidence for 3-epi-25-hydroxyvitamin D3 in serum of infants and adults

Hedwig C.M. Stepman1 / An Vanderroost1 / Dietmar Stöckl1 / 1

1Laboratory for Analytical Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium

Corresponding author: Linda M. Thienpont, Laboratory for Analytical Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium Phone: +32 9 264 81 29, Fax: +32 9 264 81 98

Citation Information: Clinical Chemistry and Laboratory Medicine. Volume 49, Issue 2, Pages 253–256, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: 10.1515/CCLM.2011.050, December 2010

Publication History

Published Online:


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.

Keywords: 3-epi-25-hydroxyvitamin D3; 25-hydroxyvitamin D; product ion spectrum

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