Development of a mass spectrometry diagnostic assay to monitor hepcidin levels in hemochromatosis patients

Hepcidin has been identified as the principle regulatory peptide essential for iron homeostasis. Expression of this cysteine-rich peptide is up-regulated by inflammation and iron overload, whereas hypoxia, iron deficiency and erythropoiesis suppress hepcidin expression. The quantitative analysis of hepcidin in bodily fluids will provide an insight into the pathogenesis of disorders of iron metabolism such as hereditary hemochromatosis. A mass spectrometry-based approach was taken to detect and quantify urinary hepcidin as urine is readily available and collection of this sample type is non-invasive. Samples were first purified using micro solid phase extraction (μSPE), followed by matrix assisted laser desorption/ionization-orthogonal-time-of-flight mass spectrometry (MALDI-TOF-MS) with inclusion of an internal standard for the quantitative analysis of unlabelled urinary hepcidin. Monoisotopic resolution of hepcidin-25 was observed with the MALDI-TOF-MS and extraction recovery of more than 70% was achieved. Spot-to-spot variations of less than 3.5% RSD and intra- and inter-day precision of less than 9.5% RSD was observed. In our study, average hepcidin-25 levels of 1.7 nmol/mmol creatinine were observed in healthy subjects, compared to an average of 1.1 nmol hepcidin-25/mmol creatinine in hemochromatosis patients. In one of the patients, the hepcidin level was below the detection limit (1.25 nM). From the stability experiments of hepcidin-25, it is recommended that urine samples be stored at -80oC and undergo minimal freeze-thaw cycles. It is also advised that extraction be carried out within 3 hours of thawing. Preliminary analysis using LC-ESI-IT-MS showed multiple charge states of hepcidin-25. However, a variation of less than 5% in hepcidin-25 concentration was observed when the same sample was analyzed with both LC-MS and MALDI-TOF-MS, and thus they can be used to counter-validate each other. In summary, a validated method has been developed for the quantification of unlabelled urinary hepcidin-25 which can be used to investigate the levels of hepcidin-25 in iron-related disorders