Sensitive liquid chromatographic method for the determination of a specific M1 agonist, LY246708, an investigational agent with potential for the treatment of Alzheimer's disease, in human plasma

Abstract A reversed-phase HPLC method is reported for the determination of a new M1 agonist, LY246708, in human plasma. The compound and an internal standard were extracted from plasma with hexane at basic pH. The organic extract was evaporated to dryness and the residue was reconstituted with mobile phase [0.5% diethylamine (pH 3, adjusted with phosphoric acid)—acetonitrile (70:30, v/v)]. The analytes were separated from endogenous substances on a Zorbax CN column; the effluent was monitored by measuring its absorbance at 296 nm. The limit of quantification was determined as 1.5 ng/ml and the response was linear from 1.5 to 20 ng/ml. Validation studies showed the method to be both repeatable and reproducible. Its robustness was demonstrated by transfer between analytical laboratories and continued use in support of pharmacokinetic studies and therapeutic monitoring of the compound.