A sensitive high performance liquid chromatographic (HPLC) method involving fluorescence detection was developed for the determination of fexofenadine (FEX), known to have low oral bioavailability, in rat plasma. In order to understand the effect of various chromatographic factors on the separation of analytes and to simultaneously optimize the resolution and analysis run time, a response surface method was used. The chromatographic separation was achieved using a Supelco C18-DB (250 mm × 4.6 mm I.D./5 μm particle size) column with mobile phase comprising of ammonium acetate buffer and acetonitrile (63:37, v/v), delivered isocratically at a flow rate of 1.0 mL min-1. Diphenhydramine was used as an internal standard (I.S.). The statistical evaluation of the method was examined and the method was found to be precise and accurate with a linearity range of 1-500 ng mL-1 (r > 0.9980). The intra- and inter-day precision studies showed good reproducibility with coefficients of variation (C.V.) less than 12.26%. The advantages of our method are small sample volume (100 μL), short time of analysis (13 min) and a simple sample extraction and clean-up as compared to the previously published methods. The established method provides a reliable bioanalytical methodology to carry out FEX pharmacokinetics in rat plasma.
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