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J Pharmacol Toxicol Methods. 2015 Jan-Feb;71:110-9. doi: 10.1016/j.vascn.2014.09.008. Epub 2014 Oct 2.

Development of LC-MS methods for quantitation of hepcidin and demonstration of siRNA-mediated hepcidin suppression in serum.

Author information

1
Barnett Institute, Northeastern University, Boston, MA 02115, United States.
2
Alnylam Pharmaceuticals, Inc., Cambridge, MA 02138, United States.
3
Barnett Institute, Northeastern University, Boston, MA 02115, United States. Electronic address: si.wu@neu.edu.
4
Barnett Institute, Northeastern University, Boston, MA 02115, United States. Electronic address: b.karger@neu.edu.

Abstract

INTRODUCTION:

A requisite step in developing a therapeutic to modulate the levels of hepcidin is the development of a quantitative method for measuring the concentration of serum hepcidin.

METHODS:

To this end, an LC-MS method, based on selected reaction monitoring (SRM) with a triple quadrupole MS and an isotopically labeled hepcidin as internal standard, was developed to measure hepcidin in mouse and monkey sera.

RESULTS:

Initially, 40 normal cynomolgus monkeys and 40 normal mice were studied to determine the normal endogenous levels of hepcidin, and an average of 50ng/mL was found in the monkeys and 46ng/mL in the mice. Next, experiments were conducted where an siRNA, targeting hepcidin, was administered to cynomolgus monkeys, resulting in effective hepcidin reduction (inhibition rate) of 87% after 24h and 74% after 48h, demonstrating to effectively reduce serume level of hepcidin.

CONCLUSIONS:

For better sensitivity, especially for the low volumes available for mouse sera, a second LC-MS method, based on parallel reaction monitoring (PRM) using a Orbitrap MS was developed and shown to be at least 10 fold lower in detection limits (or consumption of serum volume) than the SRM approach.

KEYWORDS:

Hepcidin; Pharmacodynamics; Pharmacokinetics; Quantitation; SRM and PRM mass spectrometry; siRNA

PMID:
25281793
DOI:
10.1016/j.vascn.2014.09.008
[Indexed for MEDLINE]

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