title: Variability in the analysis of 25-hydroxyvitamin D by liquid chromatography-tandem mass spectrometry: the devil is in the detail.
abstract: BACKGROUND
  Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly used in clinical laboratories for the analysis of 25-hydroxyvitamin D (25OHD), but measurement is not straightforward. Importantly, LC-MS/MS is not a single technique: variables in sample preparation, chromatography and ionisation/fragmentation should each be considered.
  
  
  METHODS
  We analysed results from a survey organised by the international Vitamin D External Quality Assessment Scheme (DEQAS), to determine the influence of such variables on the results for two DEQAS distributions.
  
  
  RESULTS
  65 laboratories returned questionnaires. 346 (57%) individual results were from laboratories using electrospray ionisation (ESI), and 259 (43%) from laboratories using atmospheric pressure chemical ionisation (APCI). Although the mean ratio of results was not significantly different between ESI and APCI (P=0.5828), there was greater variation (P<0.0001) in results obtained by laboratories using ESI. Greater variation (P<0.05) was also observed between results from laboratories monitoring non-specific water-loss transitions. Only 3 laboratories (5%) could resolve the isobaric metabolite 3-epi-25OHD(3) from 25OHD(3).
  
  
  CONCLUSIONS
  There are many variables to consider when using LC-MS/MS, including assay standardisation/calibration, chromatography and MS conditions. MS/MS alone cannot distinguish isobaric metabolites such as 3-epi-25OHD(3). Interference can also occur if non-specific transitions are used. Laboratories should always subscribe to an EQA scheme for 25OHD analysis.
authors.affiliation: Department of Clinical Biochemistry, King's College Hospital NHS Foundation Trust, London, UK. lewis.couchman@nhs.net
authors.forename: Lewis
authors.initials: L
authors.lastname: Couchman
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Benton
authors.forename: Cajetan F
authors.initials: CF
authors.lastname: Moniz
mesh_terms: D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D019411:Clinical Laboratory Techniques / Q000592:standards; D003625:Data Collection; D006801:Humans; D011786:Quality Control; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods / Q000592:standards; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000097:blood
chemical_list: D014807:Vitamin D; C104450:25-hydroxyvitamin D