Simultaneous determination of donepezil, 6-O-desmethyl donepezil and spinosin in beagle dog plasma using liquid chromatography-tandem mass spectrometry and its application to a drug-drug interaction study

Spinosin, which is traditionally used for sedation and sleep disorders, has recently shown potential effects in alleviating memory loss. As spinosin is the main bioactive component in a standardized dried 50% ethanol extract of the seeds of Zizyphus jujuba var. spinosa, a Phase lib clinical trial is ongoing, in Korea for the combination of the above extract formulated in a tablet (DHP1401 tablet) with donepezil hydrochloride (Aricept (R) tablet) in patients with mild to moderate Alzheimer's disease. Therefore, to promote safety and efficacy evaluations, a reliable method for the simultaneous detection and analysis of the two drugs is needed. Toward this end, in this study, we established and validated a rapid and sensitive LC-MS/MS method for the simultaneous determination of donepezil, its pharmacologically active metabolite 6-O-desmethyl donepezil, and spinosin in beagle dog plasma (50 mu L). After optimization of the system, we used methanol for simple protein precipitation. Chromatographic separation was performed using a Phenomenex Luna C-18 column (100 x 2.0 mm, 3 mu m) with a mobile phase consisting of 0.1% formic acid in acetonitrile-0.1% formic acid in distilled water (2:8, v/v) at a flow rate of 0.65 mL/min. All analytes were detected and quantified in selected reaction monitoring mode. All calibration curves showed good linearity (r >= 0.9965) over the concentration range of 0.02-20, 0.02-10, and 0.5-250 ng/mL for donepezil, for 6-O-desmethyl donepezil, and spinosin, respectively. This validated method was then successfully applied to a pharmacokinetic study in beagle dogs with no evidence for potential drug-drug interactions between DHP1401 and donepezil hydrochloride. This information and optimized assay can be useful for the anticipated co-administration of these two drugs in clinical settings. (C) 2019 Elsevier B.V. All rights reserved.