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Development and Validation of Reverse Phase High Performance Chromatography Method for Determination of Olanzapine in Microsample Rat Plasma: Application to Preclinical Pharmacokinetic Study Pervaiz, Fahad; Ahmad, Mahmood; Minhas, Muhammad Usman & Sohail, Muhammad Abstract Purpose:To develop a sensitive and validated reverse phase-high performance liquid chromatographic (RP-HPLC) method for quantification of olanzapine in micro-sample of rat plasma using UV detection. Methods: A single oral dose of olanzapine (7 mg/kg) was given to overnight fasted rats (n = 6). Rat plasma samples containing the drug were extracted by liquid-liquid extraction using a combination of dichloromethane: n-hexane (80:20). A reverse phase chromatographic column C18 hypersil-BDS was used for chromatographic separation with a mobile phase consisting of 50 mM phosphate buffer pH 5.5, acetonitrile and methanol (50:30:20, v/v/v) pumped at a flow rate of 1.2 ml/min. Olanzapine was measured using ultraviolet (UV) detection at 214 nm. The method was validated for precision and accuracy. Results: Separation of compounds of interest was not affected by endogenous interference. Good linearity within the concentration range of 1 - 500 ng/ml in rat plasma was obtained with coefficient of regression (r2) of 0.9986. Liquid-liquid extraction produced comparable recovery to solid phase extraction. Retention time of olanzapine and internal standard (fluoxetine) was 5.0 and 13.4 min, respectively. Lowest limit of quantification (LLOQ) was 1 ng/ml while inter-day and intra-day precision was < 12.5 and 5.1 %, respectively. Accuracy of the method was between 94 and 105 % and the variation of results between two analysts was not significant (p = 0.626). Mean maximum plasma concentration (Cmax) of olanzapine was 412.7 ng/ml, time to attain maximum plasma concentration (tmax) was 1 h and half life (t1/2) was 2.54 h. Conclusion: The proposed method has been successfully validated for precision and accuracy that are within the limits of U.S. Food and Drug Administration (FDA)’s guidance for bioanalyitcal assay validation. The method was successfully applied to preclinical pharmacokinetic analysis of olanzapine in rats Keywords Olanzapine; Antipsychotic; Pharmacokinetics; Rat; Plasma; Bioanalytical assay

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