title: Preanalytical evaluation of serum 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 measurements using LC-MS/MS.
abstract: BACKGROUND
  Vitamin D testing is increasing worldwide. Although immunoassays are still widely used in Japan for the measurement of serum 25-hydroxyvitamin D (25OHD) as an indicator of vitamin D status, development of a simple and high-throughput MS-based method is still needed for routine use in clinical laboratories.
  
  
  METHODS
  We designed a method using a triple quadrupole mass spectrometer equipped with a two-step separation approach that used the Aria TLX-2 HPLC system in the selected reaction monitoring mode. Analytical performance of the system and effects of various preanalytical factors were tested.
  
  
  RESULTS
  High-throughput quantitative analysis of 25OHD3 and D2 at 15 samples/h was achieved using 25 μl of serum/plasma. Intra- and inter-assay CVs for 25OHD3 were 5% and 7%, respectively. Limit of detection for 25OHD3 was 0.31 ng/ml. No significant effects were seen for clotting time, repeated freeze-thaw cycles, anti-coagulants and possible interfering substances. A good correlation (r(2)=0.947) was found between the present system and the DiaSorin radioimmunoassay. Serum 25OHD3 levels in apparently healthy Japanese subjects were 25.5±9.8 ng/ml for men and 20.9±7.1 ng/ml for women.
  
  
  CONCLUSIONS
  This high-throughput LC-MS/MS 25-OHD assay has the potential to be used as a routine clinical laboratory assay for assessing vitamin D status.
authors.affiliation: Clinical Proteomics Research Center, Chiba University Hospital, Chiba, Japan.
authors.forename: Asuka
authors.initials: A
authors.lastname: Mochizuki
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Kodera
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Saito
authors.forename: Mamotu
authors.initials: M
authors.lastname: Satoh
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Sogawa
authors.forename: Motoi
authors.initials: M
authors.lastname: Nishimura
authors.forename: Masanori
authors.initials: M
authors.lastname: Seimiya
authors.forename: Masayuki
authors.initials: M
authors.lastname: Kubota
authors.forename: Fumio
authors.initials: F
authors.lastname: Nomura
mesh_terms: D015652:25-Hydroxyvitamin D 2 / Q000097:blood*; D001774:Blood Chemical Analysis / Q000379:methods*; D002112:Calcifediol / Q000097:blood*; D002853:Chromatography, Liquid; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008297:Male; D012015:Reference Standards; D053719:Tandem Mass Spectrometry
chemical_list: D015652:25-Hydroxyvitamin D 2; D002112:Calcifediol