Hepcidin-25 has attracted much attention ever since its discovery in 2001. It is widely recognized that this liver produced peptide hormone plays a major role in the regulation of iron levels in mammals and can reveal important clinical information about several pathological states in patients suffering from iron-related disorders [1].
With the aim to tackle the current difficulties in hepcidin quantification and improve the status of this promising biomarker in the clinical field, we developed a rapid and robust analytical strategy for the quantification of hepcidin-25 in human samples based on HPLC-MS/MS (QqQ) to be implemented in routine laboratories. The novelty of the method is the use of special HPLC vials to avoid adsorptive losses due to the basic character of the peptide that causes interaction with the silanol groups of the vial’s glass surface. Up to 90% decrease in the MS/MS signal was observed, when commercial HPLC vials were used, while vials treated with 3-(2-aminoethylamino)propylmethyl-dimethoxysilane or 1H,1H,2H,2H-perfluorooctyltriethoxysilane, leading to no significant losses in the dynamic range of physiological hepcidin-25 mean serum levels (10-20 µg/L).
Careful analytical validation was performed for determining the reproducibility, repeatability, limit of quantification (0.5 µg/L) and linearity (0.5-40 µg/L) of the method. Serum samples from 9 healthy volunteers were analyzed with a median hepcidin-25 level of 3.3 µg/L, comparable to results reported in the literature [2,3].
References
[1] T. Ganz, Blood., 2011, 117, 4425.
[2] A. M. Butterfield et al, Clin Chem, 2010, 56, 1725.
[3] A.T. Murphy et al, Blood, 2007, 110, 1048.