Quantitation of hepcidin in serum using ultra-high-pressure liquid chromatography and a linear ion trap mass spectrometer.

Hepcidin is a peptide hormone that functions as a key regulator of mammalian iron metabolism. Biological levels are increased in end-stage renal disease and during inflammation but suppressed in hemochromatosis. Thus hepcidin levels have diagnostic importance. This study describes the development of an analytical method for the quantitative determination of the concentration of hepcidin in clinical samples. The fragmentation of hepcidin was investigated using triple quadrupole and linear ion trap mass spectrometers. A standard quantity of a stable isotopically labelled hepcidin internal standard was added to serum samples. Extraction was performed by protein precipitation and weak cation-exchange magnetic nanoparticles. Chromatography was carried out on sub 2 microm particle stationary phase, using ultra-high-pressure liquid chromatography and a linear ion trap for quantitation. The lower limit of quantitation was 0.4 nmol/L with less than 20% accuracy and precision. The mean hepcidin concentration in sera for controls was 4.6 +/- 2.7 nmol/L, in patients with sickle cell disease, 7.0 +/- 8.9 nmol/L; in patients with end-stage renal disease, 30.5 +/- 15.7 nmol/L; and patients with penetrant hereditary hemochromatosis, 1.4 +/- 0.8 nmol/L.