Development of a Novel LC/MS/MS Extraction Assay for Galanthamine in Guinea Pig Plasma and its Application to Nerve Agent Countermeasures

Abstract

Galanthamine Hydrobromide (GAL HBr), approved material for treatment of mild to moderate Alzheimer’s disease, is a centrally-acting reversible Acetylcholinesterase Inhibitor (AChEI) that is currently under evaluation as a therapeutic countermeasure against organophosphorus G- and V-Series nerve agents, which can induce rapid lethality in guinea pigs and humans. It has been shown that upon combination with Atropine (ATR) and pyridine-2Aldoxime Methochloride (2-PAM), a single dose of GAL administered before or soon after the acute exposure to a lethal dose of organophosphorus compounds can safely counteract toxicity in guinea pigs. To that end a new sample preparation extraction method analysis assay has been developed to enable future high-throughput, reproducible, and sensitive assays to quantitate galanthamine in guinea pig plasma. Samples were prepared with Diphenhydramine Hydrochloride (DPH HCl) internal standard and recovered with a 10 min liquid-liquid trichloromethane extraction. Samples were analyzed with a reversed phase liquid chromatographic column interfaced to a triple quadrupole mass spectrometer (LC/MS/MS) operating in the positive ion Multiple Reaction Monitoring (MRM) Turbo Ionspray mode. Precursor to product ion (M+H)+ transitions of 288-to-213 m/z and 256-to-167 m/z for GAL and DPH were observed, respectively. Sample run times of 1.50 min were achieved. Overall extraction method development proved to be acceptable and rugged.