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

Authors

Jiri Adamec, University of Nebraska-LincolnFollow
Amber Jannasch, Bindley Bioscience Center, Purdue University, West Lafayette, IN
Jianjie Huang, Department of Foods and Nutrition, Purdue University, West Lafayette, IN
Emily Hohman, Department of Foods and Nutrition, Purdue University, West Lafayette, IN
James C. Fleet, Department of Foods and Nutrition, Purdue University, West Lafayette, IN
Munro Peacock, Department of Medicine, Indiana University Indianapolis, IN
Mario G. Ferruzzi, Department of Foods and Nutrition, Purdue University, West Lafayette, IN
Berdine Martin, Department of Foods and Nutrition, Purdue University, West Lafayette, IN
Connie M. Weaver, Department of Foods and Nutrition, Purdue University, West Lafayette, IN

Date of this Version

2011

Citation

J Sep Sci. 2011 January ; 34(1): 11–20.

Comments

Open Access. Used by Permission.

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 D₃ molecules in biological samples.