Quantification of hepcidin using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry |
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Authors: | Sukhvinder S Bansal John M Halket Jane Fusova Adrian Bomford Robert J Simpson Nisha Vasavda Swee Lay Thein Robert C Hider |
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Institution: | 1. King's College London, Pharmaceutical Sciences Division 150 Stamford Street, Waterloo, London SE1 9NH, UK;2. King's College London, Nutritional Sciences Division, 150 Stamford Street, Waterloo, London SE1 9NH, UK;3. King's College London, Institute of Liver Studies, King's College Hospital, London SE5 9RS, UK;4. King's College London, Division of Gene and Cell Based Therapy, James Black Centre, London SE5 9NU, UK;5. King's College London, Department of Hematological Medicine, Kings College Hospital, London SE5 9RS, UK |
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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 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. Copyright © 2009 John Wiley & Sons, Ltd. |
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