title: A new quantitative LC tandem mass spectrometry assay for serum 25-hydroxy vitamin D.
abstract: BACKGROUND
  The accurate measurement of 25-hydoxy vitamin D (25OH-D) in serum has been a challenge for many years. We developed a liquid chromatography tandem mass spectrometry (LC Tandem MS) assay for the quantitative determination of 25OH-D(2) and 25OH-D(3) in serum. The new method was compared with two widely used commercially available immunoassays.
  
  
  METHODS
  Sample preparation involved protein precipitation with acetonitrile containing deuterated forms of the target species as internal standards. An API 5000 mass spectrometer coupled with a photoionization source was used for quantitation. The performance of the new LC Tandem MS assay was compared with a radioimmunoassay (RIA, Diasorin) and a chemiluminescence immunoassay (ECLIA, Roche Diagnostics), analysing serum obtained from 152 individuals.
  
  
  RESULTS
  Using 100 μl of serum, the LC Tandem MS assay had a limit of quantitation of 1.3 nmol/L for both 25OH-D(2) and 25OH-D(3) with a linear response between 1.3 and 625 nmol/L and accuracy of between 95 and 124%. Intra- and inter-assay precision were ≤7% and ≤4%, respectively. Measurement of 25OH-D levels in 152 serum samples gave run averages of 71, 56 and 62 nmol/L for LC Tandem MS, ECLIA and RIA, respectively. Correlations between the various methods were: LC Tandem MS vs. RIA: r=0.931; LC Tandem MS vs. ECLIA: r=0.784; RIA vs. ECLIA: r=0.787. The LC Tandem MS method had a positive proportional bias of 26% over the RIA, whereas the ECLIA showed variable differences.
  
  
  CONCLUSIONS
  The new LC Tandem MS assay is accurate and precise at physiologically relevant 25OH-D concentrations, and compares favourably with the RIA. In contrast, the ECLIA shows variable bias with the other assays tested.
authors.affiliation: Bone Research Program, ANZAC Research Institute, The University of Sydney, Sydney, Australia. markusherr@aol.com
authors.forename: Markus
authors.initials: M
authors.lastname: Herrmann
authors.forename: Tim
authors.initials: T
authors.lastname: Harwood
authors.forename: Olivia
authors.initials: O
authors.lastname: Gaston-Parry
authors.forename: Dorothy
authors.initials: D
authors.lastname: Kouzios
authors.forename: Tang
authors.initials: T
authors.lastname: Wong
authors.forename: Anna
authors.initials: A
authors.lastname: Lih
authors.forename: Mark
authors.initials: M
authors.lastname: Jimenez
authors.forename: Margaret
authors.initials: M
authors.lastname: Janu
authors.forename: Markus J
authors.initials: MJ
authors.lastname: Seibel
mesh_terms: D015652:25-Hydroxyvitamin D 2 / Q000097:blood*; D001774:Blood Chemical Analysis / Q000379:methods*; D002112:Calcifediol / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
chemical_list: D015652:25-Hydroxyvitamin D 2; D002112:Calcifediol