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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.

Journal of separation science (2010-12-21)
Jiri Adamec, Amber Jannasch, Jianjie Huang, Emily Hohman, James C Fleet, Munro Peacock, Mario G Ferruzzi, Berdine Martin, Connie M Weaver
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
25-Hydroxyvitamin D3 (6,19,19-d3), ≥97 atom % D, ≥98% (CP)
Sigma-Aldrich
25-Hydroxyvitamin D3 (6,19,19-d3) solution, 5 μg/mL in ethanol, 97 atom % D, 98% (CP)

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