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

To assess a patient's vitamin D status the precursor metabolite 25-hydroxyvitamin D can be determined. However, measurement of 1,25-dihydroxyvitamin D is required when disorders of 1a-hydroxylation, extrarenal 1a-hydroxylation, or vitamin D receptor defects are suspected. The aim of this study was to determine reference values for 1,25-dihydroxyvitamin D3 and D2 using a 2D ID-UPLC-MS/MS method. The LC-MS/MS method, able to measure picomolar concentrations of both 1,25-dihydroxyvitamin D3 and D2 in human serum, was extensively validated. Intra-assay variations were <5% and 8.5% and <7.5% and 11%, for 1,25-dihydroxyvitamin D3 and D2, respectively, over the whole dynamic range (3.1-376 and 3.1-652pmol/L). Limit of quantitation was 3.4pmol/L for both compounds. Our method correlated well with a published LC-MS/MS method (r=0.87) and with the average 1,25-dihydroxyvitamin D3 results of the vitamin D External Quality Assessment Scheme (DEQAS) determined with LC-MS/MS (r=0.93). Reference ranges, determined in 96 plasma samples of healthy volunteers were 59-159pmol/L and <17pmol/L for respectively 1,25-dihydroxyvitamin D3 and D2. The female part of the reference group showed a statistically significant decrease of 1,25-dihydroxyvitamin D3 concentrations with age. The presence of significantly higher average 1,25-dihydroxyvitamin D3 levels in premenopausal women taking oral contraceptive pills compared to postmenopausal women suggests that this effect is estrogen-related, as estrogens lead to a higher vitamin D binding protein. The major finding of the present study is a reference interval of 59-159pmol/L for 1,25-dihydroxyvitamin D3 determined with a highly sensitive and precise LC-MS/MS method