title: Determination of human reference values for serum total 1,25-dihydroxyvitamin D using an extensively validated 2D ID-UPLC-MS/MS method.
abstract: 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 D3and D2using a 2D ID-UPLC-MS/MS method.
  
  
  
  The LC-MS/MS method, able to measure picomolar concentrations of both 1,25-dihydroxyvitamin D3and D2in human serum, was extensively validated. Intra-assay variations were <5% and 8.5% and <7.5% and 11%, for 1,25-dihydroxyvitamin D3and 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 D3results 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 D3and D2. The female part of the reference group showed a statistically significant decrease of 1,25-dihydroxyvitamin D3concentrations with age. The presence of significantly higher average 1,25-dihydroxyvitamin D3levels 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 D3determined with a highly sensitive and precise LC-MS/MS method.
authors.affiliation: Department of Clinical Chemistry, Endocrine Laboratory, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Niek F
authors.initials: NF
authors.lastname: Dirks
authors.affiliation: Department of Clinical Chemistry, Endocrine Laboratory, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Frans
authors.initials: F
authors.lastname: Martens
authors.affiliation: Department of Clinical and Experimental Medicine KU Leuven, Laboratory of Clinical and Experimental Endocrinology, Leuven, Belgium; Clinical Department of Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Dirk
authors.initials: D
authors.lastname: Vanderschueren
authors.affiliation: Department of Clinical and Experimental Medicine KU Leuven, Laboratory of Clinical and Experimental Endocrinology, Leuven, Belgium; Clinical Department of Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Jaak
authors.initials: J
authors.lastname: Billen
authors.affiliation: Clinical Department of Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium; Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium.
authors.forename: Steven
authors.initials: S
authors.lastname: Pauwels
authors.affiliation: Department of Clinical Chemistry, Laboratory of Endocrinology, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Mariëtte T
authors.initials: MT
authors.lastname: Ackermans
authors.affiliation: Department of Clinical Chemistry, Laboratory of Endocrinology, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Erik
authors.initials: E
authors.lastname: Endert
authors.affiliation: Department of Internal Medicine, Endocrinology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Martin den
authors.initials: MD
authors.lastname: Heijer
authors.affiliation: Department of Clinical Chemistry, Endocrine Laboratory, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Marinus A
authors.initials: MA
authors.lastname: Blankenstein
authors.affiliation: Department of Clinical Chemistry, Endocrine Laboratory, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: a.heijboer@vumc.nl.
authors.forename: Annemieke C
authors.initials: AC
authors.lastname: Heijboer
mesh_terms: D000328:Adult; D000368:Aged; D002117:Calcitriol / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004872:Ergocalciferols / Q000097:blood*; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D017698:Postmenopause / Q000097:blood; D017697:Premenopause / Q000097:blood; D012016:Reference Values; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014807:Vitamin D / Q000031:analogs & derivatives / Q000097:blood; D014815:Vitamins / Q000097:blood*; D055815:Young Adult
chemical_list: D004872:Ergocalciferols; D014815:Vitamins; D014807:Vitamin D; C003564:1,25-dihydroxyergocalciferol; C097949:1,25-dihydroxyvitamin D; D002117:Calcitriol
keywords: 1,25-dihydroxyvitamin D; Comparison; LC–MS/MS; Reference values