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J Pharmacol Toxicol Methods. 2009 May-Jun;59(3):171-80. doi: 10.1016/j.vascn.2009.02.004. Epub 2009 Feb 28.

Development of a method for the sensitive and quantitative determination of hepcidin in human serum using LC-MS/MS.

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1
Pharmacokinetics & Drug Metabolism, Amgen, Inc., Thousand Oaks, CA 91320, USA.

Abstract

INTRODUCTION:

Hepcidin, a 25-amino acid peptide hormone, plays a crucial regulatory role in iron metabolism. Elevated hepcidin has been observed in response to inflammation and is speculated to be a causative factor in inflammatory anemia due to induction of functional iron deficiency. Hepcidin has been suggested as a biomarker of anemia of inflammation. An accurate assessment of human serum hepcidin is critical to understand its role in anemia.

METHODS:

An LC-MS/MS method was developed to quantify hepcidin in human serum using chemically synthesized hepcidin as a standard and stable isotope labeled hepcidin as internal standard. Rabbit serum was used as a surrogate matrix for standards due to the presence of endogenous hepcidin in human serum. The method was validated to FDA criteria for bioanalytical assays.

RESULTS:

The calibration curve was validated over the range of 2.5 to 500 ng/mL. Hepcidin was stable in serum for at least 16 h at room temperature, 90 days at -60 to -80 degrees C, and after three F/T cycles. Interday accuracy (% RE) and precision (%CV) were -11.2% and 5.6%, respectively at the LLOQ, and less than +/-7.0% and 9.2%, respectively for higher concentrations. The mean accuracy of quality control samples (5.00, 15.0, 100 and 400 ng/mL) in 21 analytical batches was between -0.7 and +2.1%, with mean precision between 5.1% and 13.4%. Hepcidin was below 2.5 ng/mL in 31 of 60 healthy subjects, while the mean concentration was less than 10 ng/mL. Sepsis and chronic kidney disease patients had mean serum concentrations of 252 ng/mL (n=16, median 121 ng/mL) and 99 ng/mL (n=50, median 68 ng/mL), respectively.

CONCLUSIONS:

A fully validated LC-MS/MS method has been described for the determination of hepcidin in human serum. This method was applied to the determination of hepcidin in over 1200 human samples.

PMID:
19258043
DOI:
10.1016/j.vascn.2009.02.004
[Indexed for MEDLINE]
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