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

Abstract

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).

Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR

“…This is based on an LC-MS/MS quantification method developed in our laboratory for the quantification of hepcidin-25 using mobile phases containing 0.1% ammonia (pH 11) [32]. For chromatographic separation of the obtained metal species, a neutral or basic pH of the mobile phases is required for complex stability.…”

Use of high‐pH (basic/alkaline) mobile phases for LC–MS or LC–MS/MS bioanalysis

“…Alternatively, ammonium hydroxide can be added to ammonium formate or ammonium acetate solution (5–50 m m ) to raise the pH to a targeted pH value. Since basic mobile phases can absorb CO 2 from the ambient air or lose CO 2 at pH < 8.5 (mainly for ammonium bicarbonate‐based buffer), the pH of basic mobile phases may change after extended storage/use at ambient room temperature, especially when the buffer or ammonium hydroxide concentrations are low (Abbas, Hoffmann, Montes‐Bayón, & Weller, ; Heinig, Wirz, Yuan, Tingler, & Mylott, ; Hess, Höfner, & Wanner, ; Liu, Li, Xu, Nasser, & Heidbreder, ; Snyder, Kirkland, & Dolan, ). Hence, basic mobile phases should be prepared more often and better protected from ambient air than acidic ones.…”

“…Finally, since peptide– or protein–metal or inhibitor complexes are usually less stable in acidic mobile phases, the use of basic mobile phases may prove to be critical in the investigation of peptide– or protein–metal or inhibitor complexes (Abbas et al, ; Shi, Greig, Solowiej, & Murray, ).…”

“…Despite the many advantages, there are still some cautions, which one should be aware of when using high‐pH mobile phases. First of all, in comparison with acidic mobile phases, the pH of basic mobile phases may change/drift more easily owing to the gradual absorption or loss of carbon dioxide and/or evaporation of buffering components, which can lead to a greater shift in the retention time and/or sensitivity change from run to run or even within a run (Abbas et al, ; Heinig et al, ; Hess et al, ; Liu et al, ; Snyder et al, ). Ammonium bicarbonate was reported to leave white residues on curtain plates (Amundsen et al, ).…”

Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR

“…We introduced a RP-HPLC (reversed-phase high-performance liquid chromatography) separation step complementary to MS detection for a sensitive and robust analysis of the species resulting from the complexation of Hep-25 with copper. This is based on an LC-MS/MS quantification method developed in our laboratory for the quantification of hepcidin-25 using mobile phases containing 0.1% ammonia (pH of 11) [44]. For chromatographic separation of the obtained metal species, a neutral or basic pH of the mobile phases is required for complex stability.…”