The developed method was validated as per the current international regulatory guidelines on bioanalytical method validation. The method can be readily

applicable for usage during the bioequivalence evaluation of various generic formulations for submission as part of abbreviated new drug applications. Donepezil reference standard was procured as a gift sample from a signaling pathway Pharma company and HPLC grade methanol, acetonitrile were commercially procured and all other chemicals were of analytical grade. 0.01 N hydrochloric acid was prepared by diluting 0.1 ml of hydrochloric acid to 1000 ml in a volumetric flask with milli Q water. Mixture of dichloromethane and hexane was prepared by mixing one part of dichloromethane and four parts of hexane. 1% formic acid was prepared by adding 10 ml of formic acid to a 1000 ml volumetric flask and made up the volume with milli Q water and similarly 0.1% formic acid solution was prepared by adding 1 ml of formic acid to a 1000 ml volumetric flask and made up the volume with milli Q water. 50% methanol was prepared by mixing 500 ml of methanol and 500 ml of water in a reagent bottle. Rinsing solution which

is used for auto sampler wash was prepared by mixing 0.1% formic acid and methanol in the ratio of 80:20. Mobile phase consisting of 0.1% learn more formic acid and methanol mixture (70:30) was prepared by mixing 700 ml of 0.1% formic acid with 300 ml of methanol. Donepezil and donepezil D7 stock solutions were prepared at a concentration of 0.1 mg/ml

by dissolving in 0.01 N hydrochloric acid solution and the stock solutions were stored in the refrigerator. Spiking solutions of donepezil for the preparation of calibration standards and quality control samples were prepared in mobile phase and spiked in to the plasma at the ratio of 1:50. The calibration curve from 50 to 25,000 pg/ml was generated using ten calibration standards at the Edoxaban concentrations of 50 pg/ml (STD 1), 100 pg/ml (STD 2), 200 pg/ml (STD 3), 500 pg/ml (STD 4), 2500 pg/ml (STD 5), 5000 pg/ml (STD 6), 10,000 pg/ml (STD 7), 15,000 pg/ml (STD 8), 20,000 pg/ml (STD 9), 25,000 pg/ml (STD 10). The quality control samples were prepared at the concentrations of 50 pg/ml (LLOQQC), 150 pg/ml (LQC), 9000 pg/ml (MQC) and 18,000 pg/ml (HQC). The bulk spiked calibration standards and quality control samples were stored in the freezer. Internal standard dilution was prepared at a concentration of 3000 pg/ml using mobile phase. Donepezil from the plasma was extracted using liquid–liquid extraction technique. Plasma aliquot of 0.3 ml (300 μl) was added to the polypropylene tube containing 50 μl of internal standard dilution and vortexed the tubes. 0.5 ml of 1 N sodium hydroxide solution was added and vortexed for thorough mixing. To vortexed sample added 5 ml of dichloromethane and hexane mixture and tumble the tubes for about 10–15 min.