Serum Hepcidin-25 Quantification by WCX-TOF MS

Serum Hep-25 was measured in the Netherlands by a combination of weak cation exchange chromatography and time-of-flight mass spectrometry (WCX-TOF MS) [36 (link)]. An internal standard (synthetic heavy Hep-25 stable isotope +40; custom made Peptide International Inc.) was used for quantification [37 (link)]. Peptide spectra were generated on a Microflex LT matrix-enhanced laser desorption/ionisation TOF MS platform (Bruker Daltonics, Bremen, Germany). The lower detection limit of this method was 0.5 nM; average coefficients of variation were 2.8% (intra-run) and 6.4% (inter-run) [37 (link)]. The median reference level of serum Hep-25 in adults is 4.5 nM for men (reference range <0.5–<14.7 nM), 2.0 nM for premenopausal women (<0.5–<12.3 nM), and 4.9 nM (<0.5–15.6 nM) for postmenopausal women. These reference levels for the WCX-TOF MS method are derived from those of a previously published ELISA method, based on the regression line between the ELISA and WCX-TOF MS results obtained for the same samples from patients without hepcidin isoforms [36 (link),38 (link)].