Monitoring Plasma Levels of Donepezil, 5-O-Desmethyl-Donepezil, 6-O-Desmethyl-Donepezil, and Donepezil-N-Oxide by a Novel HPLC Method in Patients With Alzheimer Disease

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In humans, donepezil (D) is metabolized to 5-O-desmethyl-donepezil (5DD), 6-O-desmethyl-donepezil (6DD), and donepezil-N-oxide (DNox). Although 6DD and DNox are pharmacologically active, the activity of 5DD is unknown. At present, no routine methods are available to detect D and its 3 metabolites simultaneously. In this study, a novel high-performance liquid chromatography method was developed and applied to a population of patients with Alzheimer disease on stable treatment with the drug.


Liquid–liquid extraction from plasma was accomplished by means of a solvent mixture of n-hexane/dichloromethane/ethylacetate (45:40:15) after sample alkalinization. Disopyramide was the internal standard. After evaporation, the residue was reconstituted in 200 μL of mobile phase (acetonitrile 85%:1% acetic acid 15%) and 50 μL was injected into the high-performance liquid chromatography column (X-Terra, RP8; flow: 1 mL/min). Photometric and fluorimetric detectors were used in tandem, to maximize the sensitivity of fluorescent compounds (D, 5DD, and DNox) and also to reveal nonfluorescent compounds (6DD and internal standard).


The method was linear in the 10–100 ng/mL concentration range. Imprecision (coefficient of variation) varied between 3.2% and 12.6% and inaccuracy (% mean absolute error) between 1.3% and 13.3%, depending on the compound, concentration, and detection mode. The quantitation limits were 0.1–0.3 ng/mL for fluorescent compounds and 1.2–4.3 ng/mL for photometric compounds. D, 5DD, 6DD, and DNox through concentrations were measured in 54 patients with Alzheimer disease on treatment with D (10 mg q.d.). No interfering peaks by endogenous compounds or coadministered drugs were noted. Plasma concentrations were quite variable among patients (D: 10–106 ng/mL; 5DD: 0.07–2.8 ng/mL; 6DD: 1.2–36 ng/mL; DNox: 0.5–45.4 ng/mL). Of note, in 6 patients, the plasma concentrations of the 2 active metabolites (6DD and DNox) were higher than those of the parent drug.


The above method proved to be suitable for therapeutic drug monitoring and may be useful in ascertaining the real contribution of metabolites to the therapeutic effects of donepezil.

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