Improved LC-MS/MS method for the quantification of hepcidin-25 in clinical samples
- 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 an LC-MS/MS method for the sensitive and reliable quantification of hepcidin-25 in serum samples. AMass 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 an 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 an 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.…
Autor*innen: | Ioana M. AbbasORCiD, Holger Hoffmann, M. Montes-Bayón, Michael G. WellerORCiD |
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Dokumenttyp: | Zeitschriftenartikel |
Veröffentlichungsform: | Verlagsliteratur |
Sprache: | Englisch |
Titel des übergeordneten Werkes (Englisch): | Analytical and Bioanalytical Chemistry |
Jahr der Erstveröffentlichung: | 2018 |
Organisationseinheit der BAM: | 1 Analytische Chemie; Referenzmaterialien |
1 Analytische Chemie; Referenzmaterialien / 1.5 Proteinanalytik | |
1 Analytische Chemie; Referenzmaterialien / 1.8 Umweltanalytik | |
Verlag: | Springer Nature |
Verlagsort: | Heidelberg |
Jahrgang/Band: | 410 |
Ausgabe/Heft: | 16 |
Erste Seite: | 3835 |
Letzte Seite: | 3846 |
DDC-Klassifikation: | Naturwissenschaften und Mathematik / Chemie / Analytische Chemie |
Freie Schlagwörter: | Adsorption; Basic solvent; Chronic kidney disease; HPLC; Iron disorders; Liquid chromatography; Mass spectrometry; Metrology; Mobile phase; Peptide analysis; Peptide losses; QC; Quality control; Recovery; Round robin exercise; Silanization; Validation |
Themenfelder/Aktivitätsfelder der BAM: | Chemie und Prozesstechnik |
Chemie und Prozesstechnik / Chemische Charakterisierung und Spurenanalytik | |
DOI: | 10.1007/s00216-018-1056-0 |
ISSN: | 1618-2642 |
ISSN: | 1618-2650 |
Verfügbarkeit des Dokuments: | Datei im Netzwerk der BAM verfügbar ("Closed Access") |
Datum der Freischaltung: | 01.06.2018 |
Referierte Publikation: | Ja |
Datum der Eintragung als referierte Publikation: | 01.06.2018 |