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Endocrine Abstracts (2015) 37 GP11.05 | DOI: 10.1530/endoabs.37.GP.11.05

ECE2015 Guided Posters Calcium, Vitamin D and Bone (1) (9 abstracts)

Determination of reference values for serum total 1,25-dihydroxyvitamin D3 using an extensively validated 2D ID-UPLC–MS/MS method

Niek Dirks 1 , Frans Martens 1 , Dirk Vanderschueren 3 , Jaak Billen 3 , Mariette Ackermans 2 , Erik Endert 2 , Marinus Blankenstein 1 & Annemieke Heijboer 1


1VU University Medical Center, Amsterdam, The Netherlands; 2Academic Medical Center, Amsterdam, The Netherlands; 3KU Leuven, Leuven, Belgium.


1,25-dihydroxyvitamin D3, the hormonally active metabolite of vitamin D3, tightly controls calcium blood levels. An increase of 1,25-dihydroxyvitamin D3 leads to an increase in calcium concentration with calcium originating from various resources, including bone tissue. To assess a patient’s vitamin D status the precursor metabolite 25-hydroxyvitamin D3 is determined. However, measurement of 1,25-dihydroxyvitamin D is required when disorders of 1α-hydroxylation, extrarenal 1α-hydroxylation, or vitamin D receptor defects are suspected. The aim of this study was to determine reference values for 1,25-dihydroxyvitamin D3 using a 2D ID-UPLC–MS/MS method.

The 2D ID-UPLC–MS/MS method (Xevo TQ-S tandem quadrupole mass spectrometer and an Acquity UPLC system (Waters)), able to measure picomolar concentrations of 1,25-dihydroxyvitamin D3 in human serum, was extensively validated with regard to sensitivity, specificity, and robustness. Intra-assay variation was <5% and inter-assay variation was <7.5% over the whole dynamic range. Limit of quantitation was 3.4 pmol/l. Our method correlated well with a published 1,25-dihydroxyvitamin D3 LC–MS/MS method (Vanderschueren et al. J Clin Endocrinol Metab 2013) (r=0.98) and with the average 1,25-dihydroxyvitamin D results of the vitamin D External Quality Assessment Scheme (DEQAS) determined with LC–MS/MS (r=0.93).

Reference values determined in 96 plasma samples of healthy volunteers (46 women and 50 men, aged 20–70 years) were 59–159 pmol/l (non-parametric 95% CI). The female part of the reference group showed a statistically significant decrease of 1,25-dihydroxyvitamin D3 concentration with age. The presence of significantly higher average 1,25-dihydroxyvitamin D3 levels in premenopausal women taking oral anticonceptive pills compared to postmenopausal women suggests that this effect is estrogen-related.

In conclusion, we described a 2D ID-UPLC–MS/MS method able to measure 1,25-dihydroxyvitamin D with a high sensitivity and precision. In addition, references values for 1,25-dihydroxyvitamin D were established using this method.

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