HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ASSAY FOR XANOMELINE, A SPECIFIC M-1 AGONIST, AND ITS METABOLITE IN HUMAN PLASMA

A reversed-phase high-performance liquid chromatographic assay (HPLC) was utilized for monitoring xanomeline (LY246708/NNC 11-0232) and a me tabolite, desmethylxanomeline, in human plasma. Xanomeline, desmethylx anomeline and internal standard were extracted from plasma with hexane at basic pH. The organic solvent extract was evaporated to dryness wi th nitrogen and the dried residue was reconstituted with 0.2 M HCl-met hanol (50:50, v/v). A Zorbax CN 150 x 4.6 mm I.D., 5-mu m column and m obile phase consisting of 0.5% (5 ml/l) triethylamine (TEA) adjusted t o pH 3.0 with concentrated orthophosphoric acid-tetrahydrofuran (THF) (70:30, v/v) produced consistent resolution of analytes from endogenou s co-extracted plasma components. Column effluent was monitored at 296 nm/0.008 a.u.f.s. and the assay limit of quantification was 1.5 ng/ml . A linear response of 1.5 to 20 ng/ml was sufficient to monitor plasm a drug/metabolite concentrations during clinical trials. HPLC assay va lidation as well as routine assay quality control (QC) samples indicat ed assay precision/accuracy was better than +/-15%.