Quantification of Hepcidin-25 in Human Serum by Isotope Dilution Micro-HPLC–Tandem Mass Spectrometry

Abstract

To the Editor:Hepcidin-25, a liver-produced peptide hormone, was initially isolated from human urine and blood ultrafiltrate. Hepcidin-25 is thought to be the central regulator of iron metabolism (1 ). Iron deficiency is associated with low hepcidin-25 concentrations and anemia of chronic disease with high concentrations, but the true diagnostic value of hepcidin-25 is still under investigation. A recently published review (2 ) stated that only semiquantitative methods for comparative studies based on mass spectrometry (MS) have been used for the determination of hepcidin in serum and urine.Isotope-dilution MS is generally accepted as yielding high analytical specificity and accuracy. We report an isotope-dilution-micro-HPLC-tandem MS (MS/MS) method that allows the quantification of hepcidin-25 present at less than nanomole-per-liter concentrations.During sample preparation, hepcidin-25 undergoes strong nonspecific binding to surfaces. A stable isotope-labeled internal standard helps to compensate for any matrix effects. Micro-HPLC-MS/MS with monolithic capillary columns ensures highest resolution and limits of quantification (3 ).Native hepcidin-25 (M r 2789) was purchased from Bachem AG. Blood samples were collected and anonymized in-house according to the Roche Diagnostics policy, and informed consent was obtained from all sample donors; results were not used for regulatory purposes.Calibrators with concentrations of 0.1-100.0 nmol/L were prepared from hepcidin-25. For sample preparation we added 5 L concentrated formic acid and 50 L internal standard solution (1450 nmol/L in water, 0.04% acetic acid) to 45 L human serum or calibrator. Samples were ultrafiltered with a Microcon Ultracel YM-10 filter (Millipore). The flow-through was transferred into HPLC vials. A Thermo Electron Quantum-Ultra triple-quadrupole mass spectrometer, Dionex Ultimate 3000 micro-HPLC, and PS-DVB Monolithic 200-m internal diameter ϫ 5 cm column (P/N 161409) were used for online micro-HPLC-MS/MS. A 2-L sample was injected. The mobile phases were (eleunt A) 1% formic acid/0.025% trifluoroacetic acid in water and (eleunt B) 1% formic acid/0.025% trifluoroacetic acid in acetonitrile, flow rate 3 L/min, with a linear gradient from 0% to 80% eleunt B during 7 min and then held at 80% eleunt B until minute 11.We used microelectrospray ionization in the positive mode; recorded selected reaction-monitoring transitions were m/z 930.83 1145.5 and m/z 935.531152.6, collision energy 33 V, and argon collision gas 2.0 mTorr. Carryover was excluded by blank injections. Samples were kept in long-term storage at Ϫ80°C and were stable for at least 2 days at 6°C. Processed samples were stable for 48 h at 6°C. We observed no ion suppression attributable to changing elution conditions and monitoring-signal intensities; postcolumn infusion ex-

Hepcidin in Human Iron Disorders: Diagnostic Implications

“…In addition, Ganz et al (29 ) developed a competitive (c)ELISA for human serum hepcidin. Other groups have also reported reliable hepcidin assays that can be divided in 3 main methodologies: (a) MS (85)(86)(87)(88)(89)(90)(91)(92)(93)(94)(95); (b) immunochemical assays, comprising cRIA (96,97 ), cELISA (13,29,98,99 ), and a 2-site ELISA (100 ); and (c) a ligand-binding assay (43 ) ( Table 1). Of the currently available commercial immunochemical research kits for serum hepcidin, we found the RIA and enzyme-immunoassay kits of Bachem (purchased November 2009 and August 2010, respectively) to be suitable to differentiate between hepcidin concentrations in serum samples of controls and patients with various iron disorders, whereas the bioactive hepcidin kit of DRG Instruments, (purchased October 2009) gave similar concentrations for all samples and could not discriminate between iron metabolism disorders (unpublished results).…”

A fully human anti-hepcidin antibody modulates iron metabolism in both mice and nonhuman primates

“…16 Mass spectroscopic assays [17][18][19][20] and some immunoassays 21 using polyclonal Abs have been shown to reliably detect hepcidin but have limits in terms of cost, ease of transfer to a hospital laboratory setting, or scalability and reproducibility for assays involving polyclonal Abs. For this reason, we and others have developed sandwich enzyme-linked immunosorbent assays (ELISAs) using monoclonal Abs to detect hepcidin.…”

Results of the first international round robin for the quantification of urinary and plasma hepcidin assays: need for standardization

“…Some methods use an internal standard, either hepcidin analogs or bioactive hepcidin-25 synthesized with stable isotopes. [3][4][5][6][7][8][9][10] Recently, immunochemical (IC) assays for hepcidin-25 have also been developed, which comprise of competitive radioimmunoassays (RIA) 11 and enzyme-linked immunosorbent assays (ELISA). 12,13 Currently there is no reference method for hepcidin measurements.…”