Development of RP-HPLC Method for Simultaneous Determination of Brimonidine Tartrate and Brinzolamide by QbD Approach and Its Validation

A simple, rapid, accurate and precise reversed phase high performance liquid chromatographic method has been developed for the simultaneous determination of brimonidine tartrate (BRT) and brinzolamide (BRZ). A 3(2) factorial design was utilized to aid in method development and optimization. Effective chromatographic separation was achieved using C18 column (250 x 4.6 mm, 5 mu m) as a stationary phase and mobile phase consisted of methanol: 0.01 M ammonium acetate buffer (49.5: 50.5, v/v), pH adjusted to 3.8 with acetic acid at a flow rate of 1.1 mL/min at a detection wavelength of 260 nm. The injection volume was 20 mu L. Quality by design approach was applied to evaluate the effect of two factors i.e. mobile phase composition and flow rate on the various chromatographic responses (area, number of theoretical plates, resolution, retention time and tailing factor). The retention time of BRT and BRZ were found to be 3.96 and 8.34 min; respectively. Calibration curves were found to be linear over the concentration range of 0.2-1.4 mu g/mL for BRT and 1-7 mu g/mL for BRZ. The limit of detection and limit of quantitation for BRT were found to be 0.03 mu g/mL and 0.09 mu g/mL whereas those for BRZ were found to be 0.018 mu g/mL and 0.051 mu g/mL; respectively. The % recovery of the drugs by developed method was found in the range of 99.04 to 101.67 %. The proposed method was found to be precise as well as robust. The method was successfully applied for quantitative determination of BRT and BRZ in in-house dosage form i.e. suspension.