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

45 Citations (Scopus)

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

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25-Hydroxyvitamin D 2
Ergocalciferols
Calcifediol
Cholecalciferol
Vitamins
Vitamin D
Molecules
Photoionization
Liquids
Mass spectrometers
Ion sources
Acetone
Atmospheric pressure
Quality control

Keywords

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

ASJC Scopus subject areas

  • Analytical Chemistry
  • Filtration and Separation

Cite this

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

In: Journal of Separation Science, Vol. 34, No. 1, 01.2011, p. 11-20.

Research output: Contribution to journalArticle

Adamec, Jiri ; Jannasch, Amber ; Huang, Jianjie ; Hohman, Emily ; Fleet, James C. ; Peacock, Munro ; Ferruzzi, Mario G. ; Martin, Berdine ; Weaver, Connie M. / 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. In: Journal of Separation Science. 2011 ; Vol. 34, No. 1. pp. 11-20.
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