title: Quantitation of 1α,25-dihydroxyvitamin D by LC-MS/MS using solid-phase extraction and fixed-charge derivitization in comparison to immunoextraction.
abstract: BACKGROUND
  The objective of this study was to present analysis of 1α,25-dihydroxyvitamin D (DHVD) by solid-phase extraction (SPE) using fixed-charge derivitization extraction to enhance ionization for liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in comparison to traditional immunoextraction (IE) techniques.
  
  
  METHODS
  Full analytical validation of both a SPE and IE LC-MS/MS assay was performed, and included accuracy, intra- and inter-assay precision, limit of detection and limit of quantitation. Performance of these two assays was compared with reference laboratory IE LC-MS/MS testing.
  
  
  RESULTS
  This SPE LC-MS/MS assay demonstrated similar performance to the IE LC-MS/MS assay validated simultaneously. Intra-assay precision for low (12 pg/mL), medium (25 pg/mL) and high (60 pg/mL) control samples was 7.2%, 13.7% and 11.3% for DHVD2, respectively, and 9.1%, 5.9% and 8.9% for DHVD3. The inter-assay precision was 11.6%, 10.3% and 3.9% for DHVD2 and 10.6%, 7.0% and 5.6% for DHVD3. The limit of detection was 1.9 and 2.7 pg/mL for DHVD2 and DHVD3, and limit of quantitation was 4 pg/mL for both DHVD2 and DHVD3. Comparison to a reference LC-MS/MS assay showed excellent correlation (slope 0.936, R2=0.996, -0.2% bias).
  
  
  CONCLUSIONS
  The study demonstrated comparability of the SPE-LC-MS/MS assay for analysis of DHVD and offers an attractive option for assessment of vitamin D status as an alternative to traditional IE techniques.
authors.forename: Nancy
authors.initials: N
authors.lastname: Chan
authors.forename: Erin J
authors.initials: EJ
authors.lastname: Kaleta
mesh_terms: D001774:Blood Chemical Analysis / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D052616:Solid Phase Extraction / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors; D014807:Vitamin D / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D014807:Vitamin D