Analysis of hepcidin, a key peptide for Fe homeostasis, viasulfur detection by capillary liquid chromatography-inductively coupled plasma mass spectrometry

Abstract

Since its discovery the role of hepcidin as key regulator of iron homeostasis has been stressed by many authors. This peptide hormone of 25 amino acids, out of which 8 are cysteines, holds promise as a novel biomarker in iron metabolism disorders. In this work, we illustrate the progress of a new method for the analysis of hepcidin via sulfur detection using inductively coupled plasma mass spectrometry (ICP-MS) after capillary liquid chromatography for separation of the species. Three different ICP-MS-based strategies have been evaluated to overcome S polyatomic interferences: (1) a collision/reaction cell instrument with Xe as collision gas; (2) the monitoring of SO⁺ by adding O₂ to the reaction cell and (3) a double focusing system (DF-ICP-MS). The latter one provided best limits of detection for S (7 ng mL⁻¹) and good precision and accuracy to monitor S isotope ratios so it was used for hepcidin determination in urine samples by online isotope dilution. Quantitative recoveries of the peptide standard (101.7 ± 1.4%) are obtained with the proposed setup after controlling the column temperature (50 °C) and using the X-skimmer cone. Different sample clean-up procedures were studied in order to apply the developed quantitative methodology to urine samples. Multidimensional (dialysis + solid phase extraction) procedures provided best results yielding a 12-fold preconcentration factor. The obtained extracts were analyzed simultaneously by the developed capLC-ICP-MS setup and also by capLC-ESI-q-TOF for confirmation purposes. The results obtained revealed that ESI-q-TOF detection is more suitable for hepcidin determination in urine samples regarding both selectivity and sensitivity.