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

J Sep Sci. 2011 Jan;34(1):11-20. doi: 10.1002/jssc.201000410. Epub 2010 Nov 16.

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 D(2) , vitamin D(3) , 25-hydroxyvitamin D(2) and 25-hydroxyvitamin D(3) 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 D(2) , vitamin D(3) , 25-hydroxyvitamin D(2) and 25-hydroxyvitamin D(3) molecules in biological samples.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 25-Hydroxyvitamin D 2 / analysis*
  • Calcifediol / analysis*
  • Cholecalciferol / analysis*
  • Chromatography, Liquid / instrumentation
  • Chromatography, Liquid / methods*
  • Chromatography, Liquid / standards
  • Ergocalciferols / analysis*
  • Humans
  • Molecular Structure
  • Reference Standards
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Tandem Mass Spectrometry / instrumentation
  • Tandem Mass Spectrometry / methods*
  • Tandem Mass Spectrometry / standards

Substances

  • Ergocalciferols
  • Cholecalciferol
  • 25-Hydroxyvitamin D 2
  • Calcifediol