Routine Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Measurement of the 25-Hydroxy Metabolites of Vitamins D^sub 2^ and D^sub 3

Measurement of 25-hydroxyvitamin D2 and D3 (25-OH D2 and D3) is essential for investigating vitamin D deficiency. Competitive binding techniques are unable to distinguish between the 2 metabolites and suffer from interference from other hydroxy metabolites of vitamin D. We used isotope-dilution liqu...

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Bibliographic Details
Published in:Clinical chemistry (Baltimore, Md.) Md.), 2005-09, Vol.51 (9), p.1683
Main Authors: Maunsell, Zoë, Wright, Dennis J, Rainbow, Sandra J
Format: Article
Language:English
Subjects:
Ionization
Liquid chromatography
Mass spectrometry
Metabolites
Vitamin D
Vitamins
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Summary:Measurement of 25-hydroxyvitamin D2 and D3 (25-OH D2 and D3) is essential for investigating vitamin D deficiency. Competitive binding techniques are unable to distinguish between the 2 metabolites and suffer from interference from other hydroxy metabolites of vitamin D. We used isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for routine determination of 25-OH D2 and D3 with a stable-isotope-labeled internal standard (IS). Serum samples (100 microL) were denatured with methanol-propanol containing IS, vortex-mixed, extracted into hexane, and dried under nitrogen. The reconstituted extract was chromatographed on a BDS C8 HPLC column, and the metabolites and IS were detected by electrospray ionization MS/MS in multiple-reaction monitoring mode. 25-OH D2 and D3 and the IS nearly coeluted, whereas 1alpha-hydroxyvitamin D3 was separated; total run time was 8 min. The interassay CVs for 25-OH D2 were 9.5% and 8.4% at 52 and 76 nmol/L, respectively, and for 25-OH D3 were 5.1% and 5.6% at 55 and 87 nmol/L, respectively. The detection limit of the present method was
ISSN:0009-9147
1530-8561