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

作者：Li Siyang; Nakayama Tomoko; Akinc Akin; Wu Shiaw Lin^*; Karger Barry L

来源：Journal of Pharmacological and Toxicological Methods, 2015, 71: 110-119.

DOI：10.1016/j.vascn.2014.09.008

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

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 LCMS 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 50 ng/mL was found in the monkeys and 46 ng/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 24 h and 74% after 48 h, 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.

• 出版日期2015-2
• 单位东北大学