Quantification of hepcidin using matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry

Abstract

Hepcidin is known to be a key systemic iron-regulatory hormone which has been demonstrated to be associated with a number of iron disorders.Hepcidin concentrations are increased in inflammation and suppressed in hemochromatosis. In view of the role of hepcidin in disease, its potential as a diagnostic tool in a clinical setting is evident. This study describes the development of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay for the quantitative determination of hepcidin concentrations in clinical samples. A stable isotope labeled hepcidin was prepared as an internal standard and a standard quantity was added to urine samples. Extraction was performed with weak cation-exchange magnetic nanoparticles. The basic peptides were eluted from themagnetic nanoparticles using a matrix solution directly onto a target plate and analyzed byMALDITOF MS to determine the concentration of hepcidin. The assay was validated in charcoal stripped urine, and good recovery (70-80%)was obtained, aswere limit of quantitation data (5nmol/L), accuracy (RE <10%), precision (CV <21%), within -day repeatability (CV <13%) and between-day repeatability (CV <21%). Urine hepcidin levels were 10nmol/mmol creatinine in healthy controls, with reduced levels in hereditary hemochromatosis (P<0.000005) and elevated levels in inflammation (P<0.0007). In summary a validated method has been developed for the determination of hepcidin concentrations in clinical samples. © 2009 John Wiley & Sons, Ltd.