Simultaneous Determination of Donepezil and Its Three Metabolites in Human Plasma Using LC-MS-MS.

Abstract

A simple and selective assay for the determination of donepezil (Aricept(®)) and its three metabolites including 6-O-desmethyl (M1), 5-O-desmethyl (M2) and N-oxide (M6) metabolites in human plasma was developed and validated using liquid chromatography with tandem mass spectrometry. An analog of donepezil was used as an internal standard (IS) for all the analytes. The analytes and the IS were extracted from plasma by solid-phase extraction. The analytes were chromatographically separated on Cadenza CD-C18 column with gradient elution, then detected with electrospray positive ionization in multiple reaction monitoring mode. The established method showed linearity ranging 0.5-100 ng/mL for donepezil and 0.2-40 ng/mL for all three metabolites and was fully validated in accordance with bioanalytical guidelines. Selectivity, clear peak separation and no carryover were ensured for all the analytes. The intra- and inter-batch reproducibility assessments demonstrated that accuracy and precision were within the acceptance criteria. Minimal matrix effects and consistent extraction recovery were noted. Stability assessment demonstrated that all the analytes were stable for at least 184 days at -20°C. Assay of post-dose samples also showed clear peak separation of the analytes, indicating successful clinical application.