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

Abstract

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 32 factorial design was utilized to
aid in method development and optimization. Effective chromatographic separation was
achieved using C18 column (250 × 4.6 mm, 5μm) 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 µL. 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 µg/mL for BRT and 1-7 µg/mL for BRZ. The limit of detection and limit
of quantitation for BRT were found to be 0.03 µg/mL and 0.09 µg/mL whereas those for
BRZ were found to be 0.018 µg/mL and 0.051 µg/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.