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  • Analysis and quantification of vitamin D metabolites in serum by ultra-performance liquid chromatography coupled to tandem mass spectrometry and high-resolution mass spectrometry--a method comparison and validation.

Analysis and quantification of vitamin D metabolites in serum by ultra-performance liquid chromatography coupled to tandem mass spectrometry and high-resolution mass spectrometry--a method comparison and validation.

Rapid communications in mass spectrometry : RCM (2012-12-15)
Stephen J Bruce, Bertrand Rochat, Alexandre Béguin, Benoît Pesse, Idris Guessous, Olivier Boulat, Hugues Henry
ABSTRACT

The aim of the work was to develop and validate a method for the quantification of vitamin D metabolites in serum using ultra-high-pressure liquid chromatography coupled to mass spectrometry (LC/MS), and to validate a high-resolution mass spectrometry (LC/HRMS) approach against a tandem mass spectrometry (LC/MS/MS) approach using a large clinical sample set. A fast, accurate and reliable method for the quantification of the vitamin D metabolites, 25-hydroxyvitamin D2 (25OH-D2) and 25-hydroxyvitamin D3 (25OH-D3), in human serum was developed and validated. The C3 epimer of 25OH-D3 (3-epi-25OH-D3) was also separated from 25OH-D3. The samples were rapidly prepared via a protein precipitation step followed by solid-phase extraction (SPE) using an HLB μelution plate. Quantification was performed using both LC/MS/MS and LC/HRMS systems. Recovery, matrix effect, inter- and intra-day reproducibility were assessed. Lower limits of quantification (LLOQs) were determined for both 25OH-D2 and 25OH-D3 for the LC/MS/MS approach (6.2 and 3.4 µg/L, respectively) and the LC/HRMS approach (2.1 and 1.7 µg/L, respectively). A Passing & Bablok fit was determined between both approaches for 25OH-D3 on 662 clinical samples (1.11 + 1.06x). It was also shown that results can be affected by the inclusion of the isomer 3-epi-25OH-D3. Quantification of the relevant vitamin D metabolites was successfully developed and validated here. It was shown that LC/HRMS is an accurate, powerful and easy to use approach for quantification within clinical laboratories. Finally, the results here suggest that it is important to separate 3-epi-25OH-D3 from 25OH-D3.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
25-Hydroxyvitamin D2, ≥98.0% (HPLC)
Supelco
25-Hydroxyvitamin D2 solution, 50 μg/mL in ethanol, ampule of 1 mL, certified reference material, Cerilliant®
Supelco
25-Hydroxyvitamin D2 solution, 5 μg/mL in ethanol, ampule of 1 mL, certified reference material, Cerilliant®

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