Development and Validation of Reverse Phase High Performance Chromatography Method for Determination of Fluoxetine in Rat Plasma (Research Article)

Author(s): Swapna. S* and R. Shyam Sunder

Abstract: Purpose: To develop a sensitive and validated reverse phase-high performance liquid chromatographic (RP-HPLC) method for quantification of Fluoxetine in micro-sample of rat plasma using UV detection. Methods: A single oral dose of Fluoxetine (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 of1.2 ml/min. Fluoxetine was measured using ultraviolet (UV) detection at 235 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 Fluoxetine and internal standard (Olanzapine) was5.0and13.4 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 Fluoxetine was 412.7 ng/ml, 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 bio-analytical assay validation. The method was successfully applied to preclinical pharmacokinetic analysis of Fluoxetine in rats.

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