title: Enhanced 3-epi-25-hydroxyvitamin D3 signal leads to overestimation of its concentration and amplifies interference in 25-hydroxyvitamin D LC-MS/MS assays.
abstract: BACKGROUND
  3-epi-25-hydroxyvitamin D3 (3-epi-25OHD3) interferes in most liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for 25-hydroxyvitamin D (25OHD). The clinical significance of this is unclear, with concentrations from undetectable to 230 nmol/L reported. Many studies have quantified 3-epi-25OHD3 based on 25OHD3 calibrators or other indirect methods, and we speculated that this contributes to the observed variability in reported 3-epi-25OHD3 concentrations.
  
  
  METHODS
  We compared continuous MS/MS infusions of 3-epi-25OHD3 and 25OHD3 solutions, spiked both analytes into the same serum matrix and analysed patient samples to assess the effect of three different quantitation methods on 3-epi-25OHD3 concentration. Experiments were performed on an LC-MS/MS system using a phenyl column which does not resolve 3-epi-25OHD3, and a modified method utilizing a Zorbax SB-CN column that chromatographically resolves 3-epi-25OHD3 from 25OHD3.
  
  
  RESULTS
  A greater 3-epi-25OHD3 signal, compared with 25OHD3, was observed during equimolar post-column continuous infusion of analyte solutions, and following analysis of a serum pool spiked with both analytes. 3-epi-25OHD3 signal enhancement was dependent on mobile phase composition. Compared with 3-epi-25OHD3 calibrators, indirect quantitation methods resulted in up to 10 times as many samples having 3-epi-25OHD3 concentrations ≥ 10 nmol/L, and an approximately fourfold increase in the maximum observed 3-epi-25OHD3 concentration to 95 nmol/L.
  
  
  CONCLUSIONS
  Enhanced 3-epi-25OHD3 signal leads to overestimation of its concentrations in the indirect quantitation methods used in many previous studies. The enhanced signal may contribute to greater interference in some 25OHD LC-MS/MS assays than others. We highlight that equimolar responses cannot be assumed in LC-MS/MS systems, even if two molecules are structurally similar.
authors.affiliation: Department of Clinical Biochemistry, University College London Hospitals NHS Foundation Trust, London, UK.
authors.forename: N
authors.initials: N
authors.lastname: Flynn
authors.forename: F
authors.initials: F
authors.lastname: Lam
authors.forename: A
authors.initials: A
authors.lastname: Dawnay
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001774:Blood Chemical Analysis / Q000379:methods*; D002648:Child; D002675:Child, Preschool; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D008875:Middle Aged; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry; D055815:Young Adult
chemical_list: D014807:Vitamin D; C104450:25-hydroxyvitamin D
keywords: 25-hydroxyvitamin D; 3-epi-25-hydroxyvitamin D3; LC-MS/MS; epimer; ionization