DEVELOPMENT AND VALIDATION OF BIOANALYTICAL METHOD FOR ESTIMATION OF ITOPRIDE HYDROCHLORIDE USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH UV DETECTION IN RABBIT PLASMA

Abstract

Development and validation of simple, sensitive, rapid and precise bioanalytical method for estimation of Itopride hydrochloride in bulk and rabbit plasma. An isocratic elution was achieved using Rubitas C18 column (250×4.6mm, 5µm) and Jasco UV 2075 plus detector. The mobile phase composed of Acetonitrile: Methanol (60:40 V/V) with flow rate 1min/ml , retention time was 4.571 min and eluents were detected at 258 nm. The method was validated according to ICH guidelines and showed good compliance. The calibration curve was linear for concentration range 100-1200ng/ml for standard Itopride hydrochloride (R²= 0.9989) and Itopride hydrochloride in spiked plasma (R²= 0.9922).The accuracy and precision at all the tested quality control levels (LQC:200ng/ml , MQC:600ng/ml, HQC:1000ng/ml) were found to be within accepted limit (% CV less than 15%). % recovery at LQC, MQC and HQC was found to be 93.87%, 96.81% and 97.07% respectively. Carry over study indicate the response of sample was found below the LLOQ. Itopride hydrochloride stability was performed for short term, long term, freeze thaw and stock solution stability using two concentration levels (LQC and HQC).% mean stability was observed to be within accepted limits (85-115%). Results confirmed developed method was accurate, precise and specific. This method was suitable for pharmacokinetic study.