title: Analysis and quantification of vitamin D metabolites in serum by ultra-performance liquid chromatography coupled to tandem mass spectrometry and high-resolution mass spectrometry--a method comparison and validation.
abstract: BACKGROUND
  The aim of the work was to develop and validate a method for the quantification of vitamin D metabolites in serum using ultra-high-pressure liquid chromatography coupled to mass spectrometry (LC/MS), and to validate a high-resolution mass spectrometry (LC/HRMS) approach against a tandem mass spectrometry (LC/MS/MS) approach using a large clinical sample set.
  
  
  METHODS
  A fast, accurate and reliable method for the quantification of the vitamin D metabolites, 25-hydroxyvitamin D2 (25OH-D2) and 25-hydroxyvitamin D3 (25OH-D3), in human serum was developed and validated. The C3 epimer of 25OH-D3 (3-epi-25OH-D3) was also separated from 25OH-D3. The samples were rapidly prepared via a protein precipitation step followed by solid-phase extraction (SPE) using an HLB μelution plate. Quantification was performed using both LC/MS/MS and LC/HRMS systems.
  
  
  RESULTS
  Recovery, matrix effect, inter- and intra-day reproducibility were assessed. Lower limits of quantification (LLOQs) were determined for both 25OH-D2 and 25OH-D3 for the LC/MS/MS approach (6.2 and 3.4 µg/L, respectively) and the LC/HRMS approach (2.1 and 1.7 µg/L, respectively). A Passing & Bablok fit was determined between both approaches for 25OH-D3 on 662 clinical samples (1.11 + 1.06x). It was also shown that results can be affected by the inclusion of the isomer 3-epi-25OH-D3.
  
  
  CONCLUSIONS
  Quantification of the relevant vitamin D metabolites was successfully developed and validated here. It was shown that LC/HRMS is an accurate, powerful and easy to use approach for quantification within clinical laboratories. Finally, the results here suggest that it is important to separate 3-epi-25OH-D3 from 25OH-D3.
authors.affiliation: Clinical Chemistry, University Hospital of Lausanne, CHUV (Centre Hospitalier Universitaire Vaudois), Route du Bugnon 46, 1011, Lausanne, Switzerland.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Bruce
authors.forename: Bertrand
authors.initials: B
authors.lastname: Rochat
authors.forename: Alexandre
authors.initials: A
authors.lastname: Béguin
authors.forename: Benoît
authors.initials: B
authors.lastname: Pesse
authors.forename: Idris
authors.initials: I
authors.lastname: Guessous
authors.forename: Olivier
authors.initials: O
authors.lastname: Boulat
authors.forename: Hugues
authors.initials: H
authors.lastname: Henry
mesh_terms: D015652:25-Hydroxyvitamin D 2 / Q000097:blood*; D002112:Calcifediol / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D016014:Linear Models; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D015652:25-Hydroxyvitamin D 2; D002112:Calcifediol