High-Performance Liquid Chromatographic Method for Determination of Phenytoin in Rabbits Receiving Sildenafil

A validated high-performance liquid chromatographic (HPLC) method for determination of phenytoin (PHN), para-hydroxy metabolite of phenytoin (POH) and sildenafil (SIL) in rabbit plasma is described. The method is based on extraction on Sep-Pak C18 solid support using ethyl acetate and ether as eluents and monitoring at 220 nm. The extracted samples were analyzed by HPLC using Agilent Zorbax Extended C(18) column (150 mm x 4.6 mm internal diameter) and isocratic elution with a mobile phase consist of 29% acetonitrile and 71% sodium acetate solution (0.02 M, pH 4.6). The method was fully validated for linearity and range, selectivity, precision, stability, recovery, and robustness. The linearity of the method was in the range of 0.15 to 39 microg /ml for PHN and 0.15 to 33 microg/ml for both POH and SIL. Limits of detection (LOD) of PHN, POH, and SIL were 0.15 +/- 0.01, 0.15 +/- 0.01, and 0.15 +/- 0.01 microg/ml, respectively. The % recovery of PHN, POH, and SIL from rabbit plasma were, 101.88 +/- 0.12, 99.16 +/- 0.25, and 99.49 +/- 0.33, respectively. The method was applied on plasma collected from rabbits at different time intervals after receiving 30 mg/kg PHN-Na with (and without) 8 mg/kg SIL citrate.