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