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

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.