All samples described above were quantified using fresh calibration curve and compared to freshly prepared quality control samples at the same concentration level. Liquid chromatography coupled with the mass spectrometer (LC–MS/MS) has now become a universally acceptable technique for the estimation of drugs from the biological fluids as part of bioequivalence evaluations. Donepezil and internal

standard were scanned in the positive mode for the parent ion and reproducible daughter ion and the m/z ratio of 380.2/91.2 and 387.3/98.2 respectively were selected for donepezil and internal standard. The quantification was performed in Multiple Reaction Monitoring (MRM) selleck products mode in analyst software. The compound specific mass spectrometric parameters are optimized to produce the reproducible responses for the analyte and internal

standard. Chromatographic conditions are optimized to achieve good resolution and symmetric peak shape for the analyte at the lower level of quantification. The chromatographic conditions like flow rate (1.0 ml/min) Everolimus datasheet and column (C18 column) conditions were also optimized with the runtime of 4 min. The analyte and internal standard were quantified at 1.8 min. Other conditions are optimized for the reproducible quantification method. Liquid–liquid extraction technique was chosen for the simple and cost effective extraction procedure and the conditions are optimized to yield cleaner extract of the sample to avoid the quantification issues with the LCMSMS. Protein precipitation with acetonitrile was tried but the recovery was found to be low. Organic solvent mixture consisting of dichloromethane and hexane was yielded good recovery and better chromatography compared to individual solvents. Sample volume of 300 μl was optimized to have the sensitivity and quantifiable

and acceptable peak shape at the lower limit of quantification of 50 pg/ml. Lesser sample volumes are also attempted but the peak shape and response at the lower limit of quantification are not acceptable Thiamine-diphosphate kinase with respect to signal to noise ratio. The quality control samples were prepared at the concentrations specified in the bioanalytical method validation guidelines. The LOQQC was prepared at approximately same concentration of lowest calibration standard. The LQC was prepared at the concentration less than three times of lowest calibration standard. MQC concentration was prepared at approximately 35% of the highest calibration standard. HQC concentration was prepared at the concentration of approximately 70% of the highest calibration standard. The LCMSMS method was selective for the intended analyte since the quantification is based on the mass to charge ratio of parent as well as product ion in MRM transition mode which are selective and specific.