Abstract

A high performance liquid chromatographic method was developed and validated for the quantitative determination of brinzolamide and brimonidinetartrate. Employing an isocratic RP-HPLC Phenomenex C18 (5 μm, 250×4.6 mm) column resulted in an adequate separation for brinzolamideand brimonidine tartrate with retention time of 5.7±0.345 and 3.8±0.568 min, respectively. Best resolution was achieved with the phosphate buffer (pH 6.6):acetonitrile:methanol (45:15:40) as mobile phase pumped at the flow rate of 1.0 ml/min with the detection wavelength of 254 nm. Regression coefficient for both brinzolamide and brimonidine tartrate was found to be 0.9993 and 0.9965, respectively indicating linearity within the concentration range. Fractional factorial design, 24-1 was applied to assess the robustness of the developed method. Various independent variables selected for robustness testing were wavelength, acetonitrile volume in the mobile phase, flowrate and pH of the mobile phase. It was statistically evinced that retention time of drugs without the loss of resolution between two drugs, is affected by varying the independent variables flow rate and acetonitrile volume in the mobile phase from minimum to maximum. Hence, the limits must be strictly defined for the method conditions; flow rate and acetonitrile volume in mobile phase for optimum separation of drugs with acceptable retention time and resolution. The validation parameters like linearity, precision, accuracy, limit of detection and limit of quantitation were also found to be suitable. The proposed method can hence be successfully applied to quantify brinzolamide and brimonidine tartrate during quality control of formulation.