Journal of Bioequivalence & Bioavailability

Abstract

Simultaneous Estimation of Fluoxetine and Norfluoxetine in Plasma by RP-HPLC Employing Pre-Column Derivatization for UV-Sensitivity Enhancement

Smita T Kumbhar, Kundan B Ingale Prafulla B Choudhari and Manish S Bhatia

A rapid high-performance liquid chromatographic method is described for the simultaneous estimation of the widely used antidepressant drug, fluoxetine and its principal metabolite norfluoxetine in human plasma. Pre-column derivatization of norfluoxetine was done using 4-dimethylaminobenzaldehyde (PDAB) to overcome the limitations of sensitivity of norfluoxetine. After liquid-liquid extraction the separation of analytes and internal standard from endogenous matrix interference was achieved using a reversed-phase HIQ sil ODS column (250 mm length x 4.6 mm internal diameter) KYA TECH (Japan) and assayed by ultraviolet absorption at 227 nm. The isocratic mobile phase (1 ml/min.) consisting of acetonitrile: water: triethylamine: 0.01 M Orthophospheric acid (O.P.A.) (70:30:0.5:2) was used to separate fluoxetine, norfluoxetine and the internal standard, nebivolol. The relative retention times were 2.49, 4.24 and 7.29 min for norfluoxetine, fluoxetine and nebivolol, respectively. Chromatographic run time was 10 min and peak area ratios of analytes to IS were used for regression analysis of calibration curve. Linearity was obtained over the concentration range 10-60 μg/ml for both substances. The mean % recovery ± SD was found to be 101.23% ± 1.0 and 100.69 ± 0.67 for fluoxetine and norfluoxetine respectively. The method seems suitable, in terms of accuracy and precision, for the determination of fluoxetine plasma levels of patient; furthermore, it is rapid and sensitive.