title: Simultaneous quantification of four vitamin D metabolites in human serum using high performance liquid chromatography tandem mass spectrometry for vitamin D profiling.
abstract: OBJECTIVE
  For quantification of 25-hydroxyvitamin D(3) (25OH-D(3)), 25-hydroxyvitamin D(2) (25OH-D(2)), 3-epi-25-hydroxyvitamin D(3) (3-epi-25OH-D(3)) and 24R,25-dihydroxyvitamin D(3) (24R,25(OH)(2)-D(3)) in human serum a high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed and validated.
  
  
  METHODS
  After protein precipitation further purification is achieved with on-line sample preparation using a reversed phase (RP) C-4 column. Chromatographic separation is realized by a RP-column with core shell material and pentafluorophenyl (PFP) selectivity. Atmospheric pressure chemical ionization in the positive ion mode with multi-reaction monitoring is used for analyte detection.
  
  
  RESULTS
  Baseline separation of the analytes is achieved below 10 min. The method is linear over the range 4.0-265.3 nmol/L for 25OH-D(3), 3.9-183.6 nmol/L for 25OH-D(2), 2.0-133.8 nmol/L for 3-epi-25OH-D(3) and 2.8-129.9 nmol/L for 24R,25(OH)(2)-D(3) (r(2)>0.998). The limit of quantification is 4.0 nmol/L for 25OH-D(3), 3.9 nmol/L for 25OH-D(2), 2.0 nmol/L for 3-epi-25OH-D(3) and 2.8 nmol/L for 24R,25(OH)(2)-D(3). The CVs for the intra-day and inter-day precision are <5% and <4%, respectively. Metabolite levels for a set of 50 human serum samples have been determined and resulted in the detection of considerable amounts of 3-epi-25OH-D(3) and 24R,25(OH)(2)-D(3).
  
  
  CONCLUSIONS
  This highly specific HPLC-MS/MS method is suitable for vitamin D profiling. There is a correlation between 25OH-D(3) and 24R,25(OH)(2)-D(3). Serum concentration of 24R,25(OH)(2)-D(3) increases disproportionally with increasing concentration of 25OH-D(3).
authors: Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany. silvia.baecher@med.uni-muenchen.de
authors: Silvia
authors: S
authors: Baecher
authors: Andreas
authors: A
authors: Leinenbach
authors: Jo Anne
authors: JA
authors: Wright
authors: Stephan
authors: S
authors: Pongratz
authors: Uwe
authors: U
authors: Kobold
authors: Roland
authors: R
authors: Thiele
mesh_terms: D015652:25-Hydroxyvitamin D 2 / Q000097:blood* / Q000302:isolation & purification; D001774:Blood Chemical Analysis / Q000592:standards*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006887:Hydroxycholecalciferols / Q000097:blood* / Q000302:isolation & purification; D057230:Limit of Detection; D012015:Reference Standards; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000592:standards*
chemical_list: D006887:Hydroxycholecalciferols; D015652:25-Hydroxyvitamin D 2