Method development and validation for simultaneous estimation of brinzolamide and brimonidine in pharmaceutical dosage form by using RP-HPLC

Abstract

A high performance liquid chromatographic method was developed and validated for the simultaneous determination of brinzolamide and brimonidine by employing an isocratic RP-HPLC Kromasil C18 (4.6 x 250mm, 5µm) column resulted in an adequate separation for brinzolamide and brimonidine with retention time of 2.121 min and 2.801min respectively. Best resolution was achieved with the Perchloric acid (0.1%) and acetonitrile with ratio of (58:42) as mobile phase pumped at the flow rate of 1.0 ml/min with the detection wavelength of 251 nm. Regression coefficient for both brinzolamide and brimonidine was found to be 0.998 and 0.998 respectively indicating linearity within the concentration range. 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 during quality control of formulation.