Development and optimization of an LC-MS/MS-based method for simultaneous quantification of vitamin D2, vitamin D3, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3

Jiri Adamec, Amber Jannasch, Jianjie Huang, Emily Hohman, James C. Fleet, Munro Peacock, Mario G. Ferruzzi, Berdine Martin, Connie M. Weaver

Research output: Contribution to journalArticle

48 Scopus citations

Abstract

Simultaneous and accurate measurement of vitamin D and 25-hydroxyvitamin D in biological samples is a barrier limiting our ability to define "optimal" vitamin D status. Thus, our goal was to optimize conditions and evaluate an LC-MS method for simultaneous detection and quantification of vitamin D2, vitamin D3, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in serum. Extraction and separation of vitamin D forms were achieved using acetone liquid-liquid extraction and by a reversed phase C8 column, respectively. Detection was performed on a triple quadrupole tandem mass spectrometer (QQQ-MS/MS) equipped with atmospheric pressure photo ionization source. The LOQs for all analytes tested were 1 ng/mL for hydroxylated molecules and 2 ng/mL for the parent vitamin Ds. RSD at lower LOQ (2 ng/mL) and in medium (80 ng/mL) and high (200 ng/mL) quality control samples did not exceed 20 and 15% CV, respectively. Accuracy of the method for determination of hydroxylated molecules was also validated using National Institutes of Standards and Technology standard samples and found to be in the range of 90.9-111.2%. In summary, a sensitive and reproducible method is reported for simultaneous quantification of vitamin D2, vitamin D3, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D 3 molecules in biological samples.

Original languageEnglish (US)
Pages (from-to)11-20
Number of pages10
JournalJournal of Separation Science
Volume34
Issue number1
DOIs
StatePublished - Jan 1 2011

Keywords

  • Cholecalciferol
  • Ergocalciferol
  • LC-MS/MS
  • Vitamin D

ASJC Scopus subject areas

  • Analytical Chemistry
  • Filtration and Separation

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