A versatile HPLC method with an isocratic single mobile phase system for simultaneous determination of anti-glaucoma formulations containing timolol

Timolol is a non-cardioselective beta blocker and has different combined ophthalmic dosage forms for treatment of glaucoma. This research introduce an HPLC method for the separation of three drugs used in combination with timolol simultaneously by applying isocratic mobile phase system in a single run and the same detection wavelength with short time. The drugs included in the separation procedures are; dorzolamide, brinzolamide, and brimonidine. The HPLC method was carried out through a single mobile phase system, which contains acetonitrile: 0.05 M phosphate buffer at the ratio of 30:70, respectively at pH 3.5 and wavelength of 220 nm. The method, regarding its simplicity allows determination of the studied drugs simultaneously using single run in about 8 minutes. The method was rectilinear in the ranges of concentration: 1.25–25 μg/mL for timolol, 4–80 μg/mL for dorzolamide, 5–50 μg/mL for brinzolamide and 2–20 μg/mL for brimonidine. Different factors affecting the separation are thoroughly studied. The developed method was validated based on the official guidelines and the results were compared statistically with previously published methods and showed non-significant difference.