Quantitative determination of donepezil in human plasma by liquid chromatography/tandem mass spectrometry employing an automated liquid–liquid extraction based on 96-well format plates: Application to a bioequivalence study

An automated high-throughput liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was developed for quantitative determination of donepezil in human plasma. 150 μL of plasma samples were placed in 2.2 mL 96-deepwell plates and both donepezil and loratadine (IS) were extracted from human plasma by liquid–liquid extraction (LLE), using hexane as the organic solvent. Robotic liquid handling workstations were employed for all liquid transfer and solution preparation steps and resulted in a short sample preparation time. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated and reconstituted in a small volume of reconstitution solution. The method developed, includes a sample analysis performed by reversed phase LC–MS/MS, with positive ion electrospray ionization, using multiple reaction monitoring (MRM). The chromatographic run time was set for 2.0 min with a flow rate of 0.7 mL/min in a C18 analytical column. The method was significantly sensitive, specific, accurate and precise for the determination of donepezil in human plasma and had the shortest run time. The curve was proven to be linear for the concentration range of 0.1–100 ng/mL. After validation, the method was applied to the rapid and reliable quantitative determination of donepezil in a bioequivalence study after per os administration of a 5 mg donepezil tablet.