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Licensed Unlicensed Requires Authentication Published by De Gruyter November 2, 2021

Comparison of two LC-MS/MS methods for the quantification of 24,25-dihydroxyvitamin D3 in patients and external quality assurance samples

Sieglinde Zelzer ORCID logo, Caroline Le Goff, Stéphanie Peeters, Chiara Calaprice, Andreas Meinitzer, Dietmar Enko, Walter Goessler, Markus Herrmann and Etienne Cavalier ORCID logo



In-house developed liquid-chromatography mass spectrometry (LC-MS/MS) methods are used more and more frequently for the simultaneous quantification of vitamin D metabolites. Among these, 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) is of clinical interest. This study assessed the agreement of this metabolite in two validated in-house LC-MS/MS methods.


24,25(OH)2D3 was measured in 20 samples from the vitamin D external quality assurance (DEQAS) program and in a mixed cohort of hospital patients samples (n=195) with the LC-MS/MS method at the Medical University of Graz (LC-MS/MS 1) and at the University of Liège (LC-MS/MS 2).


In DEQAS samples, 24,25(OH)2D3 results with LC-MS/MS 1 had a proportional bias of 1.0% and a negative systemic difference of −0.05%. LC-MS/MS 2 also showed a proportional bias of 1.0% and the negative systemic bias was −0.22%. Comparing the EQA samples with both methods, no systemic bias was found (0.0%) and the slope was 1%. The mean difference of 195 serum sample measurements between the two LC-MS/MS methods was minimal (−0.2%). Both LC-MS/MS methods showed a constant bias of 0.31 nmol/L and a positive proportional bias of 0.90%, respectively.


This study is the first to assess the comparability of 24,25(OH)2D3 concentrations in a mixed cohort of hospitalized patients with two fully validated in-house LC-MS/MS methods. Despite different sample preparation, chromatographic separation and ionization, both methods showed high precision measurements of 24,25(OH)2D3. Furthermore, we demonstrate the improvement of accuracy and precision measurements of 24,25(OH)2D3 in serum samples and in the DEQAS program.

Corresponding author: Prof. Markus Herrmann, MD, FRCPA, Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Auenbruggerplatz 15 8036 Graz, Austria, Phone: 0043 316 385 13145, Fax: 0043 316 385 13430, E-mail:


This study is part of the dissertation of the first author Zelzer S.

  1. Research funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

  2. Author contribution: Conceptualization, SZ, MH and EC; Data curation, SZ and EC; Formal analysis, SZ, CL and EC; Methodology, SZ, AM, SP and CC; Project administration, AM, DE, MH and EC; Supervision, CLP, SP, WG and HM; Writing – original draft, SZ and MH; Writing – review and editing, CLG, SP, CC, AM, DE, WG and EC. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  3. Competing interests: The authors declared that there is no conflict of interest.

  4. Informed consent: Not applicable.

  5. Ethical approval: The ethical approval of this study was provided by the Ethical Committee of the Johannes Kepler University Linz (Linz, Austria) (Ref: C-108-16) and was carried out in accordance to the guidelines laid down in the Declaration of Helsinki 1964, and amended at the 59th World Medical Assembly, Seoul, Korea, October 2008.


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Received: 2021-07-13
Revised: 2021-08-27
Accepted: 2021-09-29
Published Online: 2021-11-02
Published in Print: 2022-01-26

© 2021 Walter de Gruyter GmbH, Berlin/Boston

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