A high-performance liquid chromatography-tandem mass spectrometry method for the quantification of hepcidin in human plasma

Abstract

Aims We have developed and validated a high-performance liquid chromatography tandem-mass spectrometry method (HPLC-MS/MS) for the measurement of human plasma hepcidin-25. Methods HPLC-MS/MS analysis was performed using on-line solid phase extraction, electrospray ionisation and selected reaction monitoring. Quantification was by isotope dilution. Analytical performance was assessed in terms of linearity, accuracy and precision. Results Analysis time was 7min/sample. The method was linear over the range of 5–100 ng/mL (r2>0.998, n = 14;8). Inter- and intra-day accuracy and precision, using quality controls at 15, 40 and 80 ng/mL, were 95.4–106.2% and n = 14;8), respectively. The lower limit of quantification was 5 ng/mL with an inter-day accuracy and precision of 105% and 9.8%, respectively. A dilution protocol (1/5) using a quality control sample at 250 ng/mL showed acceptable inter- and intra-day analytical performance. Hepcidin-25 is stable in plasma when stored at room temperature for up to 2 hours. Moderate increases (9–20%) in hepcidin concentrations were observed in plasma samples stored for 83 days at both –20 and –70 degrees. Discussion We report a robust, precise and automated mass-spectrometry based assay for the measurement of plasma hepcidin-25. Further investigations into sample handling and storage conditions will be carried out.