Validation of hepcidin quantification in plasma using LC-HRMS and discovery of a new hepcidin isoform

Background: Hepcidin, a 25 amino acid peptide, plays an important role in iron homeostasis. Some hepcidin truncated peptides have antibiotic effects. Results: A new analytical method for hepcidin determination in human plasma using LC–HRMS operating in full-scan acquisition mode has been validated. The extraction consists of protein precipitation and a drying reconstitution step; a 2.1 x 50 mm (idxL) C18 analytical column was used. Detection specificity, stability, accuracy, precision and recoveries were determined. The LOQ/LOD were 0.25/0.1 nM, respectively. More than 600 injections of plasma extracts were performed, allowing evaluation of the assay robustness. Hepcidin-20, hepcidin-22 and a new isoform, hepcidin-24, were detected in patients. Conclusion: The data underscore the usefulness of LC–HRMS for in-depth investigations related to hepcidin levels and pathways.