A selective and sensitive analytical method that is reliable and reproducible was developed for the evaluation of phenytoin. Chromatographic separation was performed on Acquity UPLC using sunniest C18 column (2.1X50) mm, 2µm. An isocratic elution of mobile phase having composition of 0.5% formic acid buffer and ACN in 70:30 v/v ratio with flow rate as 0.2ml/min was used with UV detection at 215 nm. Calibration showed that the response of phenytoin was linear in the range of 80-120 µg/mL with r2≥ 0.999. The method was validated and was found to be linear, accurate, robust, specific, precise and rugged.
For rapid quantification of phenytoin in mice plasma a simple, accurate and precise Liquid Chromatographic method with mass detection was developed. Bio analysis was performed using Acquity BEH (50X2.1) mm, 1.7µm column. Mobile phase consisted of 100mM ammonium acetate: ACN: formic acid (50:950:2) and 100mM ammonium acetate: water: formic acid (50:950:2) as eluent at the rate of 0.8mL/min for 2.5 min. The effluence was ionized by Electron Spray Ionization (ESI), negative mode. Niflumic acid was used as the internal standard. The retention time for phenytoin and niflumic acid were found to be 1.30 and 1.71 min respectively. The calibration curve was linear with r2≥0.99 ranging from 100-5000 ng/mL with LLOQ to be 100 ng/mL. Interday and intraday precision was lower than 15% (CV), accuracy ranged from 85-115% and mean extraction recovery was found to be 99.72%. A lipid based formulation of phenytoin, a lipophilic drug was prepared by phase inversion composition (PIC) method. Solubility of phenytoin in different nanoemulsion components viz. oil, surfactant and co-surfactant was determined. Based on the solubility determination and emulsification properties Labrafac oil was selected, Solutol HS15 as the surfactant and Transcutol P as the co-surfactant. Surfactant and co-surfactant were mixed in different volume ratios (1:0, 1:1, 1:2, 2:1, 3:1 and 4:1). Phase diagrams were developed using aqueous titration method as per the titration charts. Ternary phase diagrams were used to evaluate the nanoemulsion existence area. Placebos and then selected formulations NE-B1 and NE-B4 were subjected to thermodynamic stability tests. Formulations were characterized for viscosity, %transmittance, refractive index, droplet size and zeta potential. Droplet size of the optimized formulations was found NE-B1 was 21nm and NE-B4 was 20.44nm respectively and zeta potential was found to be -33.7 and 33.94 mV respectively. Pharmacokinetic studies showed 14.4 folds and 2.7 fold increase in the bioavailability of phenytoin from NE-B1 and NE-B4 respectively.