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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

Authors

  • Stephen J. Bruce,

    1. Clinical Chemistry, University Hospital of Lausanne, CHUV (Centre Hospitalier Universitaire Vaudois), Lausanne, Switzerland
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  • Bertrand Rochat,

    1. Quantitative Mass Spectrometry Facility [qMSF], BH-18-228, CHUV (Centre Hospitalier Universitaire Vaudois), Lausanne, Switzerland
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  • Alexandre Béguin,

    1. Clinical Chemistry, University Hospital of Lausanne, CHUV (Centre Hospitalier Universitaire Vaudois), Lausanne, Switzerland
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  • Benoît Pesse,

    1. Clinical Chemistry, University Hospital of Lausanne, CHUV (Centre Hospitalier Universitaire Vaudois), Lausanne, Switzerland
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  • Idris Guessous,

    1. Community Prevention Unit, University Institute of Social and Preventive Medicine (IUMSP University Hospital of Lausanne, CHUV (Centre Hospitalier Universitaire Vaudois), Lausanne, Switzerland
    2. Unit of Population Epidemiology, Division of Primary Care Medicine, Department of Community Medicine, Primary Care and Emergency Medicine, Geneva University Hospitals, Geneva 14, Switzerland
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  • Olivier Boulat,

    1. Clinical Chemistry, University Hospital of Lausanne, CHUV (Centre Hospitalier Universitaire Vaudois), Lausanne, Switzerland
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  • Hugues Henry

    Corresponding author
    • Clinical Chemistry, University Hospital of Lausanne, CHUV (Centre Hospitalier Universitaire Vaudois), Lausanne, Switzerland
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H. Henry, Clinical Chemistry, University Hospital of Lausanne, CHUV (Centre Hospitalier Universitaire Vaudois), Route du Bugnon 46, 1011 Lausanne, Switzerland.

E-mail: Hugues.Henry@chuv.ch

Abstract

RATIONALE

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.

METHODS

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.

RESULTS

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.

CONCLUSIONS

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. Copyright © 2012 John Wiley & Sons, Ltd.

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