Simultaneous Determination of Donepezil and its Two Metabolites in Human Serum by Liquid Chromatography Coupled With Mass Spectrometry

Abstract

Alzheimer’s disease is a progressive neurodegenerative disease. Its incidence increases with aging of population and currently ranks among the most frequent dementia. Donepezil is indicated for the treatment of Alzheimer’s type dementia and acts as a centrally acting reversible acetyl cholinesterase (AChE) inhibitor. It is metabolized by CYP 450 isoenzymes 2D6 and 3A4 in the liver. The main metabolite, 6-O-desmethyldonepezil, has been reported to inhibit AChE to the same extent as donepezil. AGNP-TDM Expert Group recommended their routine therapeutic monitoring. We developed a new chromatographic method coupled with mass spectrometric detection to determination of donepezil, 5-O-desmethyldonepezil and 6-O-desmethyldonepezil in the human serum. Analysis was performed on a Waters UPLC H-class system (Waters, Milford MA, USA) in combination with XEVO TQD triple quadrupole (Micromass, Manchester, UK). Separation was made on BEH C18 column, using a mobile phase gradient (water: acetonitrile: ammonium acetate), time of analysis 7 min. Precipitation of serum proteins was performed with a precipitation reagent. The method was validated by the FDA rules in terms of repeatability, intermediate precision accuracy, matrix effect and stability. The coefficients of variation were found in the range from 2.77% to 12.04%, a recovery between 93.98 - 107.90% and the bias in the range from -10% to -3%. The presented method is applicable to routine therapeutic monitoring of donepezil and its two metabolites in the serum with simple sample preparation.