Determination of donepezil, an acetylcholinesterase inhibitor, in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantification of donepezil, a centrally and selectively acting acetylcholinesterase inhibitor, in human plasma. After sample alkalinization with 0.5 ml of NaOH (0.1 M), the test compound was extracted from 1 ml of plasma using isopropanol–hexane (3:97, v/v). The organic phase was back-extracted with 75 μl of HCl (0.1 M) and 50 μl of the acid solution was injected into a C18 STR ODS-II analytical column (5 μm, 150×4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.02 M, pH 4.6), perchloric acid (6 M) and acetonitrile (59.5:0.5:40, v/v) and was delivered at a flow-rate of 1.0 ml/min at 40 °C. The peak was detected using a UV detector set at 315 nm, and the total time for a chromatographic separation was ∼8 min. The method was validated for the concentration range 3–90 ng/ml. Mean recoveries were 89–98%. Intra- and inter-day relative standard deviations were less than 7.3 and 7.6%, respectively, at the concentrations ranging from 3 to 90 ng/ml. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it could be successfully applied for pharmacokinetic studies and therapeutic drug monitoring.