Design of Experiment Utilization to Develop a Simple and Robust RPUPLC Method for Stability Indicating Method of anti glaucoma Ophthalmic Drops

Abstract

Brimonidine Tartrate and Timolol Maleate are used in treatment of glaucoma by decreasing intra ocular
pressure. It is necessary to establish a Validated UPLC method for the assay of Brimonidine Tartrate and
Timolol Maleate as well as Benzalkonium Chloride in eye drops. Using of DOE techniques provides more
information about different factors affects system suitability of new method. DOE led to highly robust methods
through creating design space. The method was performed on Phenomenex Kinetex C18 (50x4.6 mm, 2.6 µm)
and the mobile phase consisted of Methanol and 1- Decane sulphonic acid sodium (pH 3.0, 0.01M) which
pumped at a flow rate 0.6 mL min-1 at 50 °C in gradient manner. 10 µL of drugs sample solutions were
monitored at wavelengths 320 nm. Brimonidine Tartrate, Timolol Maleate and Combigan® ED were stressed
under different conditions in forced degradation studies. Degradation products had a good resolution with main
drugs and Benzalkonium Chloride. The proposed method was validated in terms of linearity, accuracy, precision
and limits of detection and quantitation according to ICH.