Analytical and Bioanalytical Chemistry

, Volume 410, Issue 16, pp 3835–3846 | Cite as

Improved LC-MS/MS method for the quantification of hepcidin-25 in clinical samples

  • Ioana M. Abbas
  • Holger Hoffmann
  • María Montes-Bayón
  • Michael G. Weller
Research Paper


Mass spectrometry-based methods play a crucial role in the quantification of the main iron metabolism regulator hepcidin by singling out the bioactive 25-residue peptide from the other naturally occurring N-truncated isoforms (hepcidin-20, -22, -24), which seem to be inactive in iron homeostasis. However, several difficulties arise in the MS analysis of hepcidin due to the “sticky” character of the peptide and the lack of suitable standards. Here, we propose the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces after testing several types of vials for the preparation of stock solutions and serum samples for isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions with the aim of developing a LC-MS/MS method for the sensitive and reliable quantification of hepcidin-25 in serum samples. A chromatographic separation based on usual acidic mobile phases was compared with a novel approach involving the separation of hepcidin-25 with solvents at high pH containing 0.1% of ammonia. Both methods were applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with good correlation of the results. Finally, we recommend a LC-MS/MS-based quantification method with a dynamic range of 0.5–40 μg/L for the assessment of hepcidin-25 in human serum that uses TFA-based mobile phases and silanized glass vials.

Graphical abstract

Structure of hepcidin-25 (Protein Data Bank, PDB ID 2KEF).


Hepcidin-25 Liquid chromatography Tandem mass spectrometry ID-LC-MS/MS Silanization Basic solvent Alkaline mobile phase Adsorption Peptide losses Recovery Validation 



The authors would like to thank Dr. Andreas Lehmann for instrumentation support in the LC-MS/MS analysis.

Funding information

I.M.A. was supported by the SALSA (School of Analytical Sciences Adlershof) program funded by DFG (German Research Foundation).

Compliance with ethical standards

The experiments with human serum were conducted with commercially available materials purchased from Dunn Labortechnik, Asbach, Germany.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2018_1056_MOESM1_ESM.pdf (454 kb)
ESM 1 (PDF 454 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Ioana M. Abbas
    • 1
    • 2
  • Holger Hoffmann
    • 3
    • 4
  • María Montes-Bayón
    • 5
  • Michael G. Weller
    • 1
  1. 1.Division 1.5 Protein AnalysisFederal Institute for Materials Research and Testing (BAM)BerlinGermany
  2. 2.School of Analytical Sciences AdlershofHumboldt-Universität zu BerlinBerlinGermany
  3. 3.Division 1.8 Environmental AnalysisFederal Institute for Materials Research and Testing (BAM)BerlinGermany
  4. 4.Department of ChemistryHumboldt-Universität zu BerlinBerlinGermany
  5. 5.Department of Physical and Analytical ChemistryUniversity of OviedoOviedoSpain

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