Quantification of hepcidin using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
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 the magnetic nanoparticles using a matrix solution directly onto a target plate and analyzed by MALDI-TOF MS to determine the concentration of hepcidin. The assay was validated in charcoal stripped urine, and good recovery (70-80%) was obtained, as were limit of quantitation data (5 nmol/L), accuracy (RE <10%), precision (CV <21%), within -day repeatability (CV <13%) and between-day repeatability (CV <21%). Urine hepcidin levels were 10 nmol/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.
Bansal
Sukhvinder S
SS
King's College London, Pharmaceutical Sciences Division 150 Stamford Street, Waterloo, London SE1 9NH, UK. sukhi.bansal@kcl.ac.uk
Halket
John M
JM
Fusova
Jane
J
Bomford
Adrian
A
Simpson
Robert J
RJ
Vasavda
Nisha
N
Thein
Swee Lay
SL
Hider
Robert C
RC
D000328:Adult; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000652:urine; D005260:Female; D064451:Hepcidins; D006801:Humans; D008297:Male; D008875:Middle Aged; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D055815:Young Adult
D023181:Antimicrobial Cationic Peptides; C578702:HAMP protein, human; D064451:Hepcidins