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: 0.05M phosphate buffer at the ratio of 30:70, respectively at pH 3.5 and wavelength of 220nm. The method, regarding its simplicity allows determination of the studied drugs simultaneously using single run in about 8minutes. The method was rectilinear in the ranges of concentration: 1.25-25µg/mL for timolol, 4-80µg/mL for dorzolamide, 5-50µg/mL for brinzolamide and 2-20µg/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.

Validated HPLC-DAD Method for the Simultaneous Determination of Six Selected Drugs Used in the Treatment of Glaucoma.

This work presents a simple, sensitive, and generic HPLC-diode-array detection method for the simultaneous determination of six drugs prescribed for the treatment of open-angle glaucoma and ocular hypertension. The investigated drugs include brimonidine tartarate (BMN), acetazolamide (AZA), brinzomaide (BZA), dorzolamide HCl (DZA), levobunolol HCl (LVB), and timolol maleate (TIM). Efficient chromatographic separation was achieved using a Thermo Hypersil BDS C18 column (4.6 × 250 mm, 5 µm) with a mobile phase consisting of phosphate buffer pH 5 and acetonitrile in a ratio of 78 + 22. The flow rate was 1 mL/min, and quantification was based on measuring peak areas at 298 nm for TIM and 254 nm for the other drugs. Peaks were perfectly resolved, with retention times at 3.06, 3.87, 4.53, 5.78, 7.31, and 10.78 min for BMN, AZA, DZA, TIM, LVB, and BZA respectively. The developed method was validated according to International Conference on Harmonization guidelines with respect to system suitability, linearity, ranges, accuracy, precision, robustness, and LODs and LOQs. The proposed method showed good linearity in the ranges of 2-80, 2.5-100, 2.5-100, 5-200, 3.75-150, and 1.75-70 µg/mL for BMN, AZA, DZA, TIM, LVB, and BZA, respectively. LODs were 0.20-1.01 µg/mL for the analyzed compounds. Applicability of the proposed method to real-life situations was assessed through the analysis of five different pharmaceutical formulations, and satisfactory results were obtained.

A New UPLC Method with Chemometric Design-Optimization Approach for the Simultaneous Quantitation of Brimonidine Tartrate and Timolol Maleate in an Eye Drop Preparation.

A new ultra-performance liquid chromatography (UPLC) with photodiode array was proposed for the quantitation of Brimonidine Tartrate (BRI) and Timolol Maleate (TIM) in eye drop using experimental design and optimization methodology. A 33 full factorial design was applied to uncover the effects of the selected factors and their interactions on the chromatographic response function for the optimization of experimental conditions in the development of a new UPLC method. As a result, the optimal chromatographic conditions giving a better separation and short analysis time were found to be 49.2°C for column temperature; 0.38 mL/min for flow rate and 56.7 % (v/v) for 0.1 M CH3COOH used in mobile phase. The elution of BRI and TIM was reported as 0.508 and 0.652 min within a short runtime of 1.5 min, respectively. Calibration graphs for BRI and TIM were obtained by the regression of the concentration on the peak area, which was detected at 246 and 298 nm, respectively. The method validation was performed by the analysis of the synthetic mixtures, intra-day and inter-day samples and standard addition samples. This study shows that the optimized and validated UPLC method is very promising and available for the quantification of BRI and TIM in an eye drop formulation.

Determination of phosphate concentration in glaucoma eye drops commercially available in Spain. / Determinación de la concentración de fosfatos en los colirios antiglaucomatosos comercializados en España.

OBJECTIVES: To identify and analyze the phosphate concentration in glaucoma eye drops available in Spain. MATERIAL AND METHODS: Glaucoma medications containing phosphates were identified according to the 2013 Vademecum and the website of the Spanish Agency for Medicines and Medical Devices. Phosphate concentration was determined in these eye drops using ultraviolet molecular absorption spectrophotometry, and pH was determined using scan image analysis algorithms of pH strips. RESULTS: A total of 37 phosphate containing glaucoma eye drops were identified. The mean phosphate concentration was 97.72±75.52mM. The group with higher concentration of active substance was timolol (204.85±42.38mM) followed by brimonidine/timolol (200.9mM). No statistically significant difference was found between brand name (95.65±71.11mM) and generic eye drops (99.14±80mM, P=.892). Although no statistically significant difference was found between products containing preservatives (99.24±76.78mM) and those without preservatives (85.17±72.86mM) (P=.730), a lower phosphate concentration was observed in the preservative-free Timolol and Latanoprost. Single dose samples showed a lower phosphate concentration than multi-dose ones (102.04±75.39 vs. 22.24±2.98mM, P<.001). The mean pH was 7.13±0.63. No statistical correlation was found between phosphate concentration and pH (r: 0.07). CONCLUSION: The phosphate concentration in glaucoma eye drops exceeded the tear film physiological level (1.45mM). No difference was observed between brand names and generic eye drops. Lower phosphate concentration was observed in preservative-free single dose eye drops.

Validated spectrofluorimetric and spectrophotometric methods for the determination of brimonidine tartrate in ophthalmic solutions via derivatization with NBD-Cl. Application to stability study.

Two simple, selective and accurate methods were developed and validated for the determination of brimonidine tartrate (BT) in pure state and pharmaceutical formulations. Both methods are based on the coupling of the drug with 4-chloro-7-nitro-2,1,3-benzoxadiazole in borate buffer (pH 8.5) at 70 °C and measurement of the reaction product spectrophotometrically at 407 nm (method I) or spectrofluorimetrically at 528 nm upon excitation at 460 nm (method II). The calibration graphs were rectilinear over the concentration ranges of 1.0-16.0 and 0.1-4.0 µg/mL with lower detection limits of 0.21 and 0.03, and lower quantification limits of 0.65 and 0.09 µg/mL for methods I and II, respectively. Both methods were successfully applied to the analysis of commercial ophthalmic solution with mean recovery of 99.50 ± 1.00 and 100.13 ± 0.71%, respectively. Statistical analysis of the results obtained by the proposed methods revealed good agreement with those obtained using a comparison method. The proposed spectrofluorimetric method was extended to a stability study of BT under different ICH-outlined conditions such as alkaline, acidic, oxidative and photolytic degradation. Furthermore, the kinetics of oxidative degradation of the drug was investigated and the apparent first-order reaction rate constants, half-life times and Arrhenius equation were estimated. The proposed methods are practical and valuable for routine applications in quality control laboratories for the analysis of BT.