title: Overestimation of 25-hydroxyvitamin D3 by increased ionisation efficiency of 3-epi-25-hydroxyvitamin D3 in LC-MS/MS methods not separating both metabolites as determined by an LC-MS/MS method for separate quantification of 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum.
abstract: BACKGROUND
  An LC-MS/MS method was developed for simultaneous quantification of 25-hydroxyvitamin D3 (25(OH)D3), 3-epi-25(OH)D3, and 25(OH)D2 in human serum.
  
  
  METHODS
  Sample preparation consisted of protein precipitation followed by off-line SPE. Calibration curves for each vitamin D metabolite were constructed in phosphate-buffered saline with 60 g/L albumin including its corresponding stable isotope labelled (SIL) internal standard. A pentafluorophenyl (PFP) analytical column was used to resolve 25(OH)D3 from 25(OH)D2 and 3-epi-25(OH)D3, followed by SRM registration using positive ESI-MS/MS. Accuracy was assessed from measurement of samples with NIST reference method procedure (RMP) assigned values. The PFP LC-MS/MS method was compared to an in-house C18 column LC-MS/MS method, not resolving 25(OH)D3 from 3-epi-25(OH)D3, using adult and newborn samples.
  
  
  RESULTS
  Intra-assay and inter-assay coefficients of variation were less than 4% and 7.5%, respectively for all three vitamin D metabolites; lower limits of quantification were 1, 1 and 2 nmol/L and linearity of methods were 1-500, 1-200 and 2-500 nmol/L for 25(OH)D3, 3-epi-25(OH)D3 and 25(OH)D2, respectively. The PFP LC-MS/MS method showed minimal bias to the NIST RMP. Method comparison revealed that in the C18 LC-MS/MS method, the 3-epi-25(OH)D3 concentration is overestimated inadvertently not only from co-elution of both analytes, but also by an additional 30-40% higher ionisation efficiency of 3-epi-25(OH)D3 when compared to 25(OH)D3.
  
  
  CONCLUSIONS
  This accurate LC-MS/MS method allows the simultaneous measurement of 25(OH)D3, 3-epi-25(OH)D3, and 25(OH)D2 in human serum. Due to increased ionisation efficiency, the contribution of the 3-epi-25(OH)D3 metabolite to the total 25(OH)D3 concentration is significantly overestimated in MS methods that do not resolve 3-epi-25(OH)D3 from 25(OH)D3 and may compromise its use in infant samples known to have significant amounts of 3-epi-25(OH)D3.
authors.affiliation: Department of Clinical Chemistry, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands. Electronic address: j.v.d.ouweland@cwz.nl.
authors.forename: Johannes M W
authors.initials: JM
authors.lastname: van den Ouweland
authors.affiliation: Department of Clinical Chemistry, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands.
authors.forename: Antonius M
authors.initials: AM
authors.lastname: Beijers
authors.affiliation: Department of Clinical Chemistry, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands.
authors.forename: Henny
authors.initials: H
authors.lastname: van Daal
mesh_terms: D015652:25-Hydroxyvitamin D 2 / Q000097:blood* / Q000737:chemistry* / Q000378:metabolism; D000328:Adult; D002112:Calcifediol / Q000031:analogs & derivatives / Q000097:blood* / Q000737:chemistry* / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D007231:Infant, Newborn; 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
keywords: 25-hydroxyvitamin D; 3-epi-25-hydroxyvitamin D(3); C(3)-epimer; Ionisation behaviour; LC–MS/MS; MS response factor