A Rapid and Sensitive LC–MS–MS Method for Determination of Hepcidin-25 in Human Serum, and Measurement of its Diurnal Rhythm for Healthy Subjects

Hepcidin is a recently discovered small defensin-like peptide hormone which is a key regulator of systemic iron homeostasis. Serum and urine hepcidin levels may be of diagnostic importance for iron-related disorders. We have developed a sensitive and selective ultra-performance liquid chromatographic–tandem mass spectrometric (UPLC–MS–MS) method for quantitative determination of the concentration of hepcidin in clinical samples. Solid-phase extraction was used to isolate the analyte from biological matrices, and the extracts were injected on to a Waters Acquity UPLC BEH C 18 column with gradient elution. Detection was performed by triple-quadrupole tandem mass spectrometry in multiple reaction monitoring mode with positive electrospray ionization. This UPLC–MS–MS method was validated by use of a recently introduced validation strategy based on accuracy profiles. Accuracy, precision, and linearity were good in the concentration range 0.50–500 ng mL −1 . The lower limit of quantification was 0.50 ng mL −1 serum. A diurnal rhythm of serum hepcidin-25 levels was found by standardizing the sampling time from 07:30 on one day to 07:30 on the third day for eight healthy volunteers. The results indicated that circulating hepcidin-25 concentrations are different between genders and for different diurnal rhythms.