title: Quantification of 1α,25-dihydroxy vitamin D by immunoextraction and liquid chromatography-tandem mass spectrometry.
abstract: BACKGROUND
  1α,25-dihydroxy vitamin D [1,25(OH)(2)D] is the active metabolite of vitamin D. Antibody-based detection methods lack specificity, but when combined with isotope dilution/ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry, immunoextraction provides an attractive method for 1,25(OH)(2)D. We developed a method for simultaneous quantification of 1,25(OH)(2)D(2) and 1,25(OH)(2)D(3) with a 4.6-min instrument cycle time. Results are available 36 h after sample preparation begins.
  
  
  METHODS
  Sample preparation consisted of protein precipitation, immunoextraction with solid-phase anti-1,25(OH)(2)D antibody, and derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione. Analytes were resolved using reversed-phase UPLC and quantified using positive ion electrospray ionization-tandem mass spectrometry. We used hexadeuterated 1,25(OH)(2)D(3) and 1,25(OH)(2)D(2) as internal standards and performed method comparisons against the DiaSorin RIA and an LC-MS/MS method available at a reference laboratory.
  
  
  RESULTS
  1,25(OH)(2)D(3) intraassay and interassay imprecision was 5.6% and 8.0% (120 pmol/L) and 8.7% and 13% (48 pmol/L). Limits of detection and quantification were 1.5 pmol/L and 3.0 pmol/L, respectively. 1,25(OH)(2)D(2) intraassay and interassay imprecision was 8.7% and 11% (186 pmol/L) and 11% and 13% (58 pmol/L). Limits of detection and quantification were both 1.5 pmol/L. Comparison with RIA had a proportional bias of 0.75, constant bias of -4.1, and Pearson correlation (r(2)) of 0.31. Comparison with a reference LC-MS/MS assay had a proportional bias of 0.89, constant bias of 3.7, and r(2) of 0.88.
  
  
  CONCLUSIONS
  Protein precipitation with antibody-based extraction is effective for sample preparation before LC-MS/MS analysis of derivatized 1,25(OH)(2)D. This method appears to have improved specificity over a clinically used RIA with low imprecision and limits of detection.
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, WA 98195, USA.
authors.forename: Frederick G
authors.initials: FG
authors.lastname: Strathmann
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Laha
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Hoofnagle
mesh_terms: D000906:Antibodies; D002117:Calcitriol / Q000097:blood* / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods; D004872:Ergocalciferols / Q000097:blood* / Q000276:immunology; D006801:Humans; D007201:Indicator Dilution Techniques; D057230:Limit of Detection; D012015:Reference Standards; D012016:Reference Values; D052616:Solid Phase Extraction / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D000906:Antibodies; D004872:Ergocalciferols; C003564:1,25-dihydroxyergocalciferol; D002117:Calcitriol