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Blood, 1 August 2007, Vol. 110, No. 3, pp. 1048-1054.
Prepublished online as a Blood First Edition Paper on April 13, 2007; DOI 10.1182/blood-2006-11-057471.
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Submitted November 13, 2006
Accepted April 3, 2007
Quantitation of hepcidin from human and mouse serum using liquid chromatography tandem mass spectrometry
Anthony T Murphy*, Derrick R Witcher, Peng Luan, and Victor J Wroblewski
Department of Drug Disposition Bioproducts, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN, United States
Department of Biotechnology Discovery Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN, United States
* Corresponding author; email: atm{at}lilly.com.
The hepatic peptide hormone, hepcidin, is considered the central regulator of iron metabolism. Characterizing the circulating levels of this peptide is critical to understanding its role in the development of clinically relevant syndromes, such as anemia of inflammation/chronic disease, and may provide insight into potential clinical interventions. While quantitative methods have been published for the determination of urinary hepcidin and serum prohepcidin, no definitive methods have been published for the determination of hepcidin in serum. In this report, we describe a quantitative method for the determination of both human and mouse hepcidin in serum and plasma. The method employs protein precipitation and solid phase extraction followed by liquid chromatographic separation and tandem mass spectrometry detection. The method has a quantitative range of 0.25 to 500 ng/mL serum for mouse hepcidin and 1 to 500 ng/mL serum for human hepcidin. The method utilizes small sample volumes (50 µL for mice and 100 µL for humans) and 96-well formats for rapid sample processing. The method was used to establish baseline serum and plasma concentrations of hepcidin in normal C57Bl/6 mice and healthy human volunteers.
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