Box-Behnken Design Assisted Protein Precipitation Optimization for Simultaneous Determination of Metformin Hydrochloride and Alogliptin Benzoate in Plasma along with Pharmacokinetic Application

The study focuses on systematic quality by design oriented approach for the optimization of a sensitive reverse phase liquid chromatographic bioanalytical method for determination of metformin hydrochloride and alogliptin benzoate in plasma along with its validation. Chromatographic separation was carried out on C18 column using gradient mode with mobile phase; 1-octane sulphonic acid (10 mM), acetonitrile and phosphate buffer pH 4, adjusted by orthophosphoric acid, at 1.0 ml/min using 235 nm as detection wavelength. A Box–Behnken design was applied to protein precipitation sample extraction method with centrifugation speed, centrifugation time and volume of plasma as the critical method parameters for maximizing percentage extraction recovery of metformin hydrochloride and alogliptin benzoate as the critical analytical attributes. The optimized condition for centrifugation speed, centrifugation time and volume of plasma considered as the critical method parameters for maximizing extraction recovery were 11 800 rpm, 15 min, 100 μl. This optimized extraction method gave clear samples and resulted in good correlation in the concentration range of 0.022-2.2 and 0.0012-0.12 μg/ml for metformin hydrochloride and alogliptin benzoate, respectively. The mean percentage extraction recoveries at three quality control levels were 90.83-95.87 % for metformin hydrochloride and 94.03-96.73 % for alogliptin benzoate. The plasma concentration time profile showed higher peak plasma concentration for formulation compared to pure drugs indicating that absorption of metformin and alogliptin hydrochloride from formulation was better. The developed liquid chromatographic method presented good quantitative capability, good linearity, higher extraction recovery, simpler operation and short analysis time with low cost.