Development and Validation of a Robust Stability-Indicating Reversed Phase High Performance Liquid Chromatography (RP-HPLC) Method for Simultaneous Quantification of Brimonidine Tartrate and Timolol Maleate (Combigan ® ) in Bulk and Pharmaceutical Dosage Forms

Development and Validation of a Robust Stability-Indicating Reversed Phase High Performance Liquid Chromatography (RP-HPLC) Method for Simultaneous Quantification of Brimonidine Tartrate and Timolol Maleate (Combigan ® ) in Bulk and Pharmaceutical Dosage Forms | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Development and Validation of a Robust Stability-Indicating Reversed Phase High Performance Liquid Chromatography (RP-HPLC) Method for Simultaneous Quantification of Brimonidine Tartrate and Timolol Maleate (Combigan ® ) in Bulk and Pharmaceutical Dosage Forms Aktham Mestareehi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7231691/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 13 You are reading this latest preprint version Abstract A robust, rapid, and reproducible reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous quantification of Brimonidine Tartrate and Timolol Maleate in ophthalmic dosage forms. Chromatographic separation was achieved on a Supelco Discovery C18 column (25 cm × 4.6 mm, 5 µm) maintained at ambient temperature, employing isocratic elution with two mobile phases: phase A (buffer at pH 7.0 containing 30 mM triethylamine) and phase B (acetonitrile) in a ratio of 80:20. The flow rate was set at 1.0 mL/min, and detection was performed at 245 nm and 295 nm using a diode array detector (DAD). Method validation, conducted in accordance with ICH Q2(R1), USP, and FDA guidelines, confirmed excellent linearity over the ranges of 0.24–500 ppm for Brimonidine Tartrate and 0.60–1250 ppm for Timolol Maleate. Accuracy results ranged from 99.42% to 99.82% for Brimonidine Tartrate and from 98.71% to 101.10% for Timolol Maleate. Relative standard deviations (RSDs) for precision, specificity, and robustness were all below 2%, demonstrating the method’s consistency and reliability. Additionally, the limits of detection (LOD) were determined to be 0.08 ppm for Brimonidine Tartrate and 0.20 ppm for Timolol Maleate, while the limits of quantification (LOQ) were 0.24 ppm and 0.60 ppm, respectively. Forced degradation studies under various stress conditions, including acid and base hydrolysis and hydrogen peroxide oxidation, demonstrated that the method successfully separated Brimonidine Tartrate and Timolol Maleate from their degradation products, confirming its stability-indicating capability. Notably, both drugs remained stable under thermal and photolytic stress; however, Timolol Maleate was significantly more prone to degradation under strong hydrolytic and oxidative conditions, underscoring the need for stringent control during formulation and storage. Additionally, the three complementary green analytical chemistry (GAC) metrics were evaluated. The method achieved an Eco-Scale score of approximately 75. The GAPI pictogram for this method showed a mixture of green and yellow zones. The AGREE evaluation yielded a score of 0.57 (out of 1.0), indicating moderate greenness. Overall, this method effectively quantified both active pharmaceutical ingredients without interference from excipients or degradation products, supporting its suitability for routine quality control and stability testing of combined ophthalmic formulations. Physical sciences/Chemistry Biological sciences/Drug discovery Health sciences/Medical research Brimonidine Tartrate Timolol Maleate High-Performance Liquid Chromatography (HPLC) Specificity Linearity Accuracy Precision Limit of Detection (LOD) Limit of Quantification (LOQ) Combigan® Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 INTRODUCTION Glaucoma ranks among the leading causes of chronic vision impairment and is recognized as the third most common cause of blindness worldwide. [ 1 ] Elevated intraocular pressure (IOP) remains the primary modifiable risk factor linked to vision loss in glaucoma patients.[ 2 ] Evidence from extensive clinical trials underscores the critical role of promptly lowering IOP to protect against optic nerve damage and preserve visual function. [ 3 ] Importantly, results from the Early Manifest Glaucoma Trial demonstrated that lowering IOP by as little as 1 mmHg is associated with an approximate 10% reduction in the risk of glaucoma progression. [ 4 ] , [ 5 ] Initial treatment for glaucoma generally begins with monotherapy using a single medication. If this approach fails to sufficiently lower IOP, clinicians may either switch to a different single agent or introduce an additional medication. [ 6 ] For patients who require multiple drugs to achieve target IOP, fixed-combination therapies are often favored, as they tend to improve adherence to treatment. [ 7 ] Interestingly, despite guidelines recommending monotherapy as the starting point, real-world practice shows that many patients with primary open-angle glaucoma or ocular hypertension are started directly on combination therapy. [ 8 ] , [ 9 ] Combigan® (Allergan Inc, Irvine, CA, USA)[ 10 ] , [ 11 ] is a fixed-dose ophthalmic formulation combining Timolol Maleate 0.5%, a nonselective beta-blocker, with Brimonidine Tartrate 0.2%, a selective alpha-2 adrenergic agonist. Clinical evidence shows that Combigan provides superior IOP reduction compared to monotherapy with either Timolol or Brimonidine alone, [ 12 ] and it also effectively minimizes IOP fluctuations. [ 13 ] Importantly, the combination does not increase the risk of adverse effects beyond those seen with the individual agents,[ 14 ] and has even been linked to a lower incidence of ocular allergy than brimonidine monotherapy.[ 15 ] Furthermore, multiple studies have found Combigan to outperform other fixed combinations in reducing IOP, [ 16 ] while offering better overall tolerability.[ 17 ] Brimonidine Tartrate (BT) is the tartrate salt of brimonidine (5-bromo-6-(2- imidazolin-2-ylamino) quinoxaline D-tartrate),[ 18 ] an imidazole-based compound that acts as a highly selective alpha-2 adrenergic receptor agonist. By engaging this G-protein-coupled receptor,[ 19 ] brimonidine suppresses intracellular adenyl cyclase activity, leading to decreased production of aqueous humor (AH) (Fig. 1 A). [ 20 ] The drug exhibits a relatively brief systemic elimination half-life (t₁/₂) of about 2–3 hours. Timolol maleate (TM) is chemically described as 2-propanol, 1-(1,1-dimethylethyl)amino-3-[[4-(4-morpholinyl)-1,2,5-thiadiazol-3-yl]-,(S)-,(Z)-2-butenedioate (1:1) (salt)). [ 21 ] TM is classified as a non-selective β-adrenergic blocker lacking both intrinsic sympathomimetic and membrane-stabilizing properties, as illustrated in Fig. 1 B. [ 22 ] Its therapeutic effect in glaucoma arises from blocking β-adrenergic receptors in the ciliary epithelium, thereby lowering aqueous humor production and reducing intraocular pressure. [ 14 ] The United States Pharmacopeia recommends HPLC methods for analyzing this compound in both tablets and ophthalmic solutions, whereas the British Pharmacopoeia outlines a direct spectrophotometric assay for its determination in the same dosage forms. [ 23 ] Several liquid chromatographic methods have been described for quantifying brimonidine tartrate and timolol maleate in pharmaceutical formulations. [ 24 ] , [ 25 ] In a previously reported method, the observed retention times for brimonidine tartrate and timolol maleate were notably brief, measured at approximately 0.51 minutes and 0.65 minutes, respectively; [ 26 ] however, these rapid elution times raise concerns that potential degradants or impurities could be overlooked. Moreover, the method did not include forced degradation stability studies, leaving the influence of possible degradation products on the analytical results unexamined. Another published method relied on an unusually narrow linear concentration range (4–24 µg/mL,10–60.0 µg/mL) for of brimonidine tartrate and timolol maleate respectively, which is not recommended for method development due to its limited applicability and increased risk of analytical errors. [ 27 ] , [ 28 ] This restricted range also hampers the method’s ability to evaluate drug stability and detect degradation products effectively. As a result, the absence of a robust stability-indicating capability emerges as a significant limitation, highlighting the need for a more comprehensive and reliable analytical method. While numerous studies have independently examined the chromatographic analysis of Brimonidine Tartrate and Timolol Maleate, there is still a clear gap in the literature regarding the development of a single RP-HPLC method capable of both simultaneously quantifying these compounds and serving as a stability-indicating assay for combination formulations. To address this gap, the present study aims to develop a simple, rapid, precise, and accurate RP-HPLC stability-indicating method specifically for Combigan®. The method will be validated following ICH and FDA guidelines to ensure compliance with regulatory requirements for quality control and stability testing. Ultimately, this method is intended to offer a reliable analytical tool for monitoring the stability of Combigan®, safeguarding its potency, safety, and therapeutic efficacy throughout its shelf life. MATERIALS & METHODOLOGY Chemicals and Reagents Brimonidine Tartrate (BT, USP) and Timolol Maleate (TM, USP) were used as active pharmaceutical ingredients. Maleic acid from Sigma-Aldrich ReagentPlus®, ≥ 99% purity. Hydrochloric acid (12 N) and sodium hydroxide was purchased from EM Science (USA). Acetonitrile (ACN, HPLC grade) was sourced from Fisher Scientific (USA). Potassium phosphate monobasic was obtained from Merck & Co. (Germany). Phosphoric acid (85%) was sourced from J.T. Baker (USA), and glacial acetic acid was obtained from Mallinckrodt Inc. (USA). Hydrogen peroxide solutions (30% and 3%) were purchased from Fisher Scientific (USA). Deionized water was prepared using a Milli-Q water purification system (Millipore, Bedford, MA, USA). pH buffer standards at pH 4.0, 7.0, and 9.0 were all procured from Fisher Scientific (USA). Chromatography Equipment An Agilent 1100 series HPLC system equipped with a G1311A quaternary pump, G1329A autosampler injector, G1365B DAD detector, G1316A Column Thermostat, and G1314A UV detector was utilized for chromatographic analysis. Data acquisition and processing were performed using ChemStation software. Additional laboratory equipment included a Mettler Toledo analytical balance (model AB265-S, Switzerland), a pH meter (model 3540, UK), and a Bandelin-Sonorex ultrasonic bath (model TK 52, Germany). Precise liquid handling was achieved with an Advantage-Lab variable micropipette (capacity up to 1000 µL). Chromatographic separation of target analytes was carried out using a Supelco Discovery C18 column (25 cm × 4.6 mm, 5 µm). Prior to use, the mobile phase underwent filtration through a 0.45 µm membrane filter (Millipore, Milford, MA) to ensure clarity and remove particulates. Throughout the method, a Sigma refrigerated centrifuge (Germany) and a Stuart vortex mixer (England) were employed to support sample preparation and processing. Additionally, a UV/VIS spectrophotometer (Hitachi U-2910), and a Thermo Nicolet (IR 200) spectrometer were used during the analysis. Pharmaceutical Dosage Combigan® ophthalmic solution, manufactured by Allergan (USA), is formulated to contain Brimonidine Tartrate at a concentration of 0.2% and Timolol Maleate at 0.5%, with NDC 0023-9211-10. Chromatographic conditions Table 1 summarizes the optimized chromatographic conditions established for the analysis of Brimonidine Tartrate and Timolol Maleate. These carefully selected parameters achieve rapid separation and robust detection, ensuring precise quantification of Combigan® components within a streamlined run time. Table 1 Optimized Chromatographic Conditions for Combigan® Method Development Parameters Conditions Column Supelco Discovery C18, 5µm (250 x 4.6 mm) Mobile Phase 20:80 ACN/ monobasic potassium phosphate buffer pH 7.0 with 30 mM TEA Run time 10.0 min Flow Rate 1 ml/min Sample Injector 15µl loop Detection Wavelength 245 nm and 295 nm Column Temperature Ambient Solution Preparation Procedures Comprehensive information on the procedures for sample preparation, the mobile phase formulation, and the analytical techniques employed in this work are described in supplementary material. Stock solution of Brimonidine Tartrate (1,000 ppm) Accurately weigh 50 mg of Brimonidine Tartrate and place it into a 50 mL volumetric flask. Add approximately 40 mL of deionized water and sonicate for about 10 minutes, or until the compound fully dissolves. Carefully fill the flask to the calibration mark with deionized water and mix well to ensure complete homogenization. Stock solution of Timolol Maleate (1,000 ppm) Weigh precisely 50 mg of Timolol Maleate and transfer it into a 50 mL volumetric flask. Add around 40 mL of deionized water and sonicate for roughly 10 minutes or until fully dissolved. Then, top up to the volume mark with deionized water and shake gently to achieve uniform mixing. Working standard solutions of BT (200ppm) and TM (500ppm) Transfer 2 mL of Brimonidine Tartrate (1,000ppm) and 5 mL of Timolol Maleate (1,000ppm) into a 10 mL volumetric flask. Fill the flask to the 10 mL mark with distilled water and mix thoroughly to ensure a uniform solution. Sample Combigan Preparation (600 ppm of BT& 1,500 ppm of TM) Transfer 3 mL of Combigan eye drops and transfer it into a 10 mL volumetric flask. Then, fill the flask up to the 10 mL mark with distilled water, mixing thoroughly to ensure uniform dilution. METHOD DEVELOPMENT & OPTIMIZATION The main aim of this study was to design a simple, efficient, selective, and accurate RP-HPLC method specifically for quantifying Brimonidine Tartrate and Timolol Maleate (Combigan®) in bulk drug form. A critical goal was also to achieve effective separation of these active ingredients from related impurities and potential degradation products, without requiring extra purification steps. Various experimental parameters were systematically examined to identify optimal chromatographic conditions. Emphasis was placed on obtaining a high number of theoretical plates (reflecting superior column efficiency), sharp and symmetrical peak shapes, reduced tailing in raw material analysis, and robust separation of brimonidine tartrate and timolol maleate from impurities and degradants. Method development included a series of targeted trials to refine and validate these conditions, ultimately ensuring consistent, precise, and reproducible analytical performance. [ 28 ] Determination of the Wavelength of Maximum Absorbance To identify the optimal detection wavelength, standard solutions of brimonidine tartrate (80 ppm), timolol maleate (50ppm), and Combigan (40/100 ppm, BT/TM) were scanned using UV spectroscopy over the range of 200–400 nm, with the buffer solution serving as the reference blank. Brimonidine Tartrate exhibited maximum absorbance at 245 nm, Timolol Maleate at 295 nm, and Combigan at both 245 nm and 295 nm. Based on these results, 245 and 295 nm were selected as the analytical wavelength for the quantitative determination of Combigan as seen in Fig. 2 . Infrared (IR) study for Brimonidine Tartrate and Timolol Maleate Approximately 100 mg of potassium bromide (KBr) and 2 mg of each sample Brimonidine Tartrate and Timolol Maleate were weighed separately for analysis. Each finely ground drug sample was thoroughly blended with the powdered KBr and then subjected to high pressure to form a transparent pellet. Under pressure, the potassium bromide fuses, embedding the active compound within a stable matrix to produce a thin disk suitable for infrared (IR) analysis. These prepared KBr pellets were carefully placed in the spectrometer’s sample holder for scanning. The resulting infrared spectra, shown in Figure S1 (Supplementary material), reveal the characteristic absorption bands associated with functional groups present in Brimonidine Tartrate and Timolol Maleate. The identified functional groups are summarized in Table S1 (Supplementary material). Thermo Nicolet IR 200 Spectrometer Column Selection Five different C18 columns were conditioned by sequential flushing with solvent mixtures of 50:50, 75:25 acetonitrile (ACN): water, and finally 100% ACN, each for 30 minutes at a flow rate of 1 mL/min. This step ensured compliance with ICH Q2(R2) guidelines, [ 29 ] targeting tailing factors between 0.9–2.0 and theoretical plate numbers above 2000 for acceptable peak symmetry and column efficiency. Each column was tested twice, with thorough cleaning and equilibration between runs to avoid cross-contamination. Brimonidine Tartrate and Timolol Maleate solutions (200 ppm and 500 ppm) were injected to evaluate column performance, with the columns maintained at ambient temperature to enhance reproducibility (see Fig. 3 and Table S2 (Supplementary material)). All five C18 columns were evaluated under identical chromatographic conditions to allow direct comparison. Columns #2, 4, and 5 failed to meet ICH acceptance criteria due to tailing factors exceeding 2.0, indicating poor peak symmetry. [ 29 ] Column #5 exhibits the longest retention time for Timolol Maleate compared with the other columns due to its stationary phase characteristics, which result in stronger interactions with the analyte. Specifically, the chemistry of column #5 provides higher hydrophobic retention and/or selectivity for Timolol Maleate, causing it to elute later than in the other columns under the same mobile phase and flow conditions. Column #1 met the minimum requirements for tailing factor and theoretical plate count but showed lower efficiency compared to Column #3 and had a retention time too short for degradation studies. Ultimately, Column #3 was selected for further method validation, as it provided the best overall performance with a tailing factor ≤ 2.0 and theoretical plate count ≥ 2000. Additional details are summarized in Fig. 4 and Table 2 . Table 2 Column selection results showing performance data for Peak 1 (Maleic acid), Peak 2 (Brimonidine tartrate), and Peak 3 (Timolol maleate). Column Peak Retention Time (min) Tailing Factor Theoretical Plates Resolution 1 1 1.328 1.603 2571 - 2 2.028 1.152 4928 6.232 3 2.596 1.754 2908 3.638 2 1 2.007 1.636 4469 - 2 3.061 2.275 2921 0.639 3 4.657 3.347 1277 4.190 3 1 3.472 1.349 12420 - 2 4.291 1.337 11171 1.226 3 5.218 1.594 12082 5.368 4 1 2.046 2.603 2449 - 2 3.232 1.900 1471 0.958 3 4.328 2.743 2050 0.175 5 1 2.019 1.453 7313 - 2 3.471 1.916 2502 0.674 3 6.560 3.112 882 5.247 The Combigan® chromatogram displayed three distinct peaks. To identify them, individual standard solutions of 200 ppm Brimonidine Tartrate, 500 ppm Timolol Maleate, and 100 ppm Maleic Acid were injected under optimized conditions to establish their respective retention times as illustrated in Fig. 5 . Selection pH of the Mobile Phase Precise pH control is essential in RP-HPLC to reduce secondary interactions on silica-based columns, which can cause peak tailing and poor separation. [ 30 ] Brimonidine Tartrate and Timolol Maleate solutions (200 ppm and 500 ppm) were tested at pH 2.9, 5.0, and 7.0 using a mobile phase of buffer (phase A) and acetonitrile (phase B) in a 80:20 v/v ratio. As summarized in Table 3 , pH 7.0 produced the best peak shape and the highest theoretical plate count, indicating superior column efficiency. Therefore, a potassium phosphate monobasic buffer at pH 7.0 was selected for further method development and validation. Table 3 Summary of pH Optimization and Selection Combigan® Ophthalmic Solution Brimonidine Tartrate Timolol Maleate Buffer pH Retention Time (min) Tailing Factor Theoretical Plates Resolution Retention Time (min) Tailing Factor Theoretical Plates Resolution 7.0 5.185 1.355 12849 2.164 7.143 1.598 11725 10.057 5.0 4.704 1.181 8061 1.649 6.452 1.496 12550 7.760 2.9 4.662 0.943 4864 1.341 6.176 1.643 13171 3.326 Isocratic Elution Studies Different ratios of buffer (pH 7.0) and acetonitrile (ACN) were evaluated to achieve retention times around 10 minutes, allowing adequate separation of impurities and degradants before the main analyte peaks. Brimonidine Tartrate and Timolol Maleate solutions (200 ppm and 500 ppm) were injected under these conditions. As illustrated in Fig. 6 , initial chromatograms showed peak tailing (TM: Tailing 2.191) due to secondary interactions. To address this, triethylamine (TEA) was added to the buffer, and its concentration was fine-tuned to produce sharper peaks and reduce tailing. Retention times, peak areas, and tailing factors are detailed in Table S3 (Supplementary material). Further optimization of ACN-to-buffer pH 7.0 ratios containing 30 mM TEA determined that a 20:80 v/v composition was optimal, yielding a retention time near 10 minutes and improving the resolution of impurities and degradation products. Nominal Concentration Selection To establish nominal concentrations for Brimonidine Tartrate and Timolol Maleate, calibration curves were generated within the detector’s linear range to ensure proportional response and minimize bias. Stock solutions (600 ppm and 1500 ppm, respectively) were diluted to prepare calibration standards: 0.24–500 ppm for Brimonidine Tartrate and 0.60–1250 ppm for Timolol Maleate. These were analyzed under optimized HPLC conditions, and peak areas (Table 4 ) showed excellent linearity with R² values of 0.9994 for Brimonidine Tartrate and 0.9996 for Timolol Maleate (Fig. 7 ). As a result, 200 ppm of Brimonidine Tartrate and 500 ppm of Timolol Maleate were chosen as nominal concentrations for further method validation. Table 4 Summary of peak areas at different concentrations for determining the nominal concentration Brimonidine Tartrate Timolol Maleate Concentration (ppm) Peak Area Concentration (ppm) Peak Area 0.24 9 0.60 11.5 1 36 2.5 48 10 358 25 476 50 1787 125 2380 100 3576 250 4759 200 6750 500 8986 320 11054 800 14890 400 13327 1000 18043 500 16553 1250 22514 Forced Degradation Studies Forced degradation studies are a fundamental component in evaluating and understanding the intrinsic stability characteristics of pharmaceutical compounds. This approach systematically subjects the drug to a variety of stringent conditions to explore its degradation pathways and identify potential degradation products. Beyond merely observing degradation, such studies also play a pivotal role in partially validating the specificity of analytical methods developed for detecting related substances, including degradation products. Additionally, the chosen stress conditions (concentration, exposure time, and intensity) were selected to ensure sufficient degradation ≤ 10% without causing complete analyte breakdown, in accordance with ICH Q1A (R2) recommendations.[ 31 ] Ultimately, this ensures more accurate impurity detection and contributes to safeguarding the quality, efficacy, and safety of pharmaceutical products used in clinical practice. Acid Degradation Transfer 2 mL of the Brimonidine Tartrate stock solution (1000 ppm) into a screw-cap test tube. Add 2 mL of 3 M HCl, then place the tube on a heating block set to 75°C and heat for 24 hours to induce forced degradation. After heating, allow the solution to return to room temperature, then carefully neutralize by adding 2 mL of 3 M NaOH. Transfer the neutralized solution into a 10 mL volumetric flask and dilute to the mark with deionized water, mixing thoroughly to obtain a final concentration of 200 ppm. In parallel, transfer 2 mL of the Timolol Maleate stock solution (1500 ppm) into a separate screw-cap test tube. Add 2 mL of 1 M HCl, then heat on a heating block at 75°C for 24 hours. After heating, cool to room temperature and neutralize by adding 2 mL of 1 M NaOH. Dilute the neutralized solution appropriately with deionized water, mixing well to achieve a final concentration of 500 ppm. Before HPLC analysis, confirm that each prepared solution is neutral (pH ~ 7 ) using pH indicator strips. Finally, filter each solution through a 0.45 µm membrane filter to remove particulates, then inject into the HPLC system for analysis. Base (Alkali) Degradation Transfer 2 mL of the Brimonidine Tartrate stock solution (1000 ppm) into a screw-cap test tube. Add 2 mL of 3 M NaOH, then place the tube on a heating block maintained at 75°C and heat for 24 hours to induce forced alkaline degradation. After heating, allow the solution to cool to room temperature and carefully neutralize by adding 2 mL of 3 M HCl. Transfer the neutralized solution to a 10 mL volumetric flask and dilute to volume with deionized water, mixing thoroughly to achieve a final concentration of 200 ppm. In parallel, transfer 2 mL of the Timolol Maleate stock solution (1500 ppm) into a separate screw-cap test tube. Add 2 mL of 0.05 M NaOH, then heat on a heating block at 75°C for 24 hours to induce degradation. After heating, cool the solution to room temperature and neutralize by adding 2 mL of 0.05 M HCl. Dilute the neutralized solution as needed with deionized water, mixing well to reach a final concentration of 500 ppm. Prior to HPLC analysis, verify that both prepared solutions are at neutral pH (~ 7) using pH indicator strips. Finally, filter each solution through a 0.45 µm membrane filter to remove any particulates before injecting into the HPLC system for analysis. Hydrogen Peroxide Degradation (Oxidation) Transfer 2 mL of the Brimonidine Tartrate stock solution ( 1000 ppm ) into a screw-cap test tube. Add 2 mL of 3% hydrogen peroxide (H₂O₂), then place the tube on a heating block maintained at 75°C and heat for 24 hours to induce oxidative degradation. After cooling the solution to room temperature, transfer it into a 10 mL volumetric flask, dilute to volume with deionized water, and mix thoroughly to obtain a final concentration of 200 ppm. Prior to HPLC analysis, filter the prepared solution through a 0.45 µm membrane filter to remove any particulates. In parallel, transfer 2 mL of the Timolol Maleate stock solution (1500 ppm) into a separate screw-cap test tube. Add 2 mL of 0.05% hydrogen peroxide (H₂O₂) and heat the mixture on a heating block at 75°C for 24 hours. Once cooled to room temperature, transfer the solution into a 6 mL volumetric flask, dilute to the mark with deionized water, and mix thoroughly to achieve a final concentration of 500 ppm. Filter this solution as well through a 0.45 µm membrane filter before injection into the HPLC system. Thermal Degradation (Heat) Transfer 2 mL of the Brimonidine Tartrate stock solution (1000 ppm) into a screw-cap test tube and place it on a heating block set to 75°C for 24 hours to induce thermal degradation. After heating, allow the solution to cool to room temperature. Transfer the entire contents into a 10 mL volumetric flask, dilute to the mark with deionized water, and mix thoroughly to achieve a final concentration of 200 ppm. Prior to HPLC analysis, filter the prepared solution through a 0.45 µm membrane filter to remove any particulates. Similarly, transfer 2 mL of the Timolol Maleate stock solution (1500 ppm) into a separate screw-cap test tube and heat under the same conditions (75°C for 24 hours). Once cooled to room temperature, transfer the solution into a 6 mL volumetric flask and dilute to volume with deionized water, mixing well to ensure homogeneity before analysis. Photolysis (UV Light) Stress Study Weigh approximately 20 mg of Brimonidine Tartrate and place it in a cuvette. Expose the sample to ultraviolet (UV) light at 245 nm for 24 hours to induce photodegradation. After exposure, accurately transfer 2 mg of the degraded sample into a 10 mL volumetric flask. Add 5 mL of deionized water, then sonicate for 20 minutes or until the drug is completely dissolved. Dilute to volume with deionized water and mix thoroughly to achieve a final concentration of 200 ppm. Prior to HPLC analysis, filter the solution through a 0.45 µm membrane filter to remove particulates. Similarly, weigh approximately 50 mg of Timolol Maleate and place it in a cuvette. Expose the sample to UV light at 295 nm for 24 hours. Following exposure, accurately transfer 5 mg into a 10 mL volumetric flask. Add 5 mL of deionized water and sonicate for 20 minutes or until fully dissolved. Bring to volume with deionized water and mix thoroughly to achieve a final concentration of 500 ppm. Filter this solution through a 0.45 µm membrane filter before injecting into the HPLC system. Brimonidine Tartrate demonstrated notable stability under thermal and photolytic conditions, showing no detectable degradation after 24 hours at 75°C or following exposure to UV light at 245 nm for 24 hours. Under acidic stress (3 M HCl at 75°C for 24 hours), minimal degradation was observed (0.35%). Alkaline conditions (3 M NaOH at 75°C for 24 hours) produced slightly higher degradation (1.45%), indicating greater susceptibility to basic hydrolysis. Oxidative stress using 3% H₂O₂ at 75°C for 24 hours led to moderate degradation (0.798%). Overall, Brimonidine Tartrate was most sensitive to alkaline and oxidative conditions, while remaining highly stable under heat and UV light as illustrated in Table 8 . Timolol Maleate demonstrated excellent stability under thermal (24 h at 75°C) and photolytic (24 h at 295 nm) conditions, showing no detectable degradation. However, it showed significant sensitivity to both acidic and basic hydrolysis, with degradation increasing alongside acid or alkali strength: 22.5% degradation in 3 M HCl and 24.7% in 1 M NaOH. Lower concentrations of acid (1 M HCl) and base (0.05 M NaOH) resulted in 5.8% and 5.6%, respectively. Timolol Maleate was especially susceptible to oxidative stress, with 58.5% degradation after exposure to 3% H₂O₂, indicating pronounced sensitivity to oxidation. Overall, the data highlight that Timolol Maleate is chemically stable under heat and light but degrades notably under strong acidic, basic, and oxidative conditions as seen in Table 5 . Table 5 Overview of Brimonidine Tartrate and Timolol Maleate Degradation Procedures (Thermal, Photolytic, Acidic, Alkaline, and Oxidative); *: Heat it on a heating block at 75°C for the specified duration. Brimonidine Tartrate Stress Condition Time Heated Temperature * Area Degradation Control - - 6052 - Heat 24 hours 75°C 6051 0% Photolysis 24 hours 245 nm 6050 0% 3 M HCl 24 hours 75°C 6030 0.363% 3 M NaOH 24 hours 75°C 5965 1.438% 3% H 2 O 2 24 hours 75°C 6005 0.776% Timolol Maleate Stress Condition Time Heated Temperature* Area Degradation Control - - 8415 - Heat 24 hours 75°C 8414 0% Photolysis 24 hours 295 nm 8415 0% 3 M HCl 24 hours 75°C 6450 23.35% 1 M HCl 24 hours 75°C 7950 5.52% Stress Condition Time Heated Temperature * Area Degradation Control - - 8415 - 1 M NaOH 1 hour 75°C 6402 23.92% 0.1 M NaOH 1 hour 75°C 7200 14.43% 0.05 M NaOH 1 hour 75°C 7949 5.54% Stress Condition Time Heated Temperature * Area Degradation Control - - 8415 - 3% H 2 O 2 1 hour 75°C 3604 57.17% 0.5% H 2 O 2 1 hour 75°C 7610 9.57% Mixed Degradation Study (Acid, Base, and Oxidation) The purpose of the mixed degradation study was to evaluate whether the developed HPLC method could effectively separate Brimonidine Tartrate and Timolol Maleate from their potential degradation products and impurities, thus ensuring reliable quantification and purity assessment [ 29 ]. To establish a baseline, control samples containing 200 ppm of Brimonidine Tartrate, and 500 ppm of Timolol Maleate were prepared and injected into the HPLC system. The resulting chromatogram, presented in Fig. 8 (A), was used to calculate the percentage degradation after subjecting the samples to stress conditions. For the degradation mixture, 1 mL aliquots of Brimonidine Tartrate stress samples that showed less than 10% degradation (treated with 3 M HCl for 24 h, 3 M NaOH for 24 h, and 3% H₂O₂ for 24 h) were combined. Similarly, 1 mL aliquots of Timolol Maleate stress samples (treated with 1 M HCl for 24 h, 0.05 M NaOH for 1 h, and 0.5% H₂O₂ for 1 h) were mixed. The combined solution was filtered using a 0.45 µm membrane filter and analyzed. The chromatogram from this mixed degradation test is shown in Fig. 8 (B). The analysis demonstrated that all degradation peaks were completely separated from the main Brimonidine Tartrate and Timolol Maleate peaks, which appeared at approximately 5.8 min and 8.8 min, respectively. This clear resolution confirms the method’s specificity and its ability to distinguish the active ingredients from their degradation products. Overall, the findings confirm that the developed RP-HPLC method is stability-indicating and fit for accurate quantification of Brimonidine Tartrate and Timolol Maleate in the presence of degradants. A summary of the degradation results for each stress condition is provided in Table 6 . Table 6 Summary of degradation results under different stress conditions for Brimonidine Tartrate and Timolol Maleate; *: Heat it on a heating block at 75°C for the specified duration. Brimonidine Tartrate Stress condition Exposed Time Temperature (°C) * Color Peak Area % Degradation None None None Clear 6052 0% 3M HCl 24 hours 75 Clear 6030 0.363% 3M NaOH 24 hours 75 Clear 5965 1.438% 3.0% H 2 O 2 24 hours 75 Clear 6005 0.776% Mixture solution - - Clear 5960 1.520% Timolol Maleate None None None Clear 8415 ---- 1M HCl 24 hours 75 Clear 7950 5.52% 0.05M NaOH One hour 75 Clear 7949 5.54% 0.5% H 2 O 2 One hour 75 Clear 7610 9.57% Mixture solution - - Clear 7595 9.74% METHOD VALIDATION To comply with GLP and GMP standards, the developed analytical method was validated following ICH Q2A/Q2B, [ 29 ] FDA, [ 32 ] and USP [ 33 ] guidelines. Key validation parameters included: System Suitability : Confirmed consistent HPLC performance before analysis. Specificity : Ensured clear separation of BT and TM from impurities and degradants. Robustness : Tested stability under minor changes in pH and solvent composition. Solution Stability : Verified analyte stability during the test period. Linearity and Range : Confirmed accurate detection across relevant concentrations. Accuracy and Precision : Assessed repeatability, injection consistency, and analyst variability. LOD and LOQ : Defined the lowest detectable and quantifiable levels. This comprehensive validation demonstrated the method’s reliability and suitability for routine analysis. SYSTEM SUITABILITY System suitability testing (SST) is an essential step in HPLC analysis, as outlined in ICH Q2(R1) guidelines, ensuring the entire system including equipment, software, and columns is operating consistently and accurately before running actual samples.[ 29 ] Key acceptance criteria include % RSD of replicate injections not more than 1%, %RSD of retention times not more than 1, number of theoretical plates at least 2000, tailing factor between 0.9 and 2, capacity factor greater than 2, resolution between peaks above 2, and drift within 2%. By meeting these parameters the method confirms it can deliver precise and reproducible results and supports the reliability of subsequent analytical testing. [ 34 ] System suitability evaluation was carried out by injecting two prepared working standard solutions of Brimonidine Tartrate (200 ppm, labeled as WS #1 and WS #2) and Timolol Maleate (500 ppm, also labeled as WS #1 and WS #2). To verify the consistency and reliability of the HPLC system, WS #1 was injected sequentially six times, while WS #2 was injected twice, as detailed in Table 7 . These injections provided data to calculate critical system suitability metrics, such as the %RSD of peak areas and retention times for both WS #1 and WS #2, confirming stable detector response and reproducible retention behavior. Additionally, tailing factors and theoretical plate counts were determined for each analyte peak to assess peak symmetry and column performance, ensuring the method’s robustness and precision. The percentage drift (%Drift) was calculated by using the following equation: $$\:\text{%}\text{D}\text{r}\text{i}\text{f}\text{t}\:=\frac{\text{A}\text{s}-\text{A}\text{c}}{\text{A}\text{s}}\:\text{x}\:100$$ Where, As is the average peak area from six consecutive injections of working standard solution #1, and Ac is the average peak area from two injections of working standard solution #2. Table 7 System Suitability Results for Brimonidine Tartrate and Timolol Maleate Working Standards Brimonidine Tartrate Standard 1 Retention Time Retention Time %RSD Retention Time %Drift Tailing Factor Theoretical Plates Peak Area Peak Area %RSD Peak Area %Drift Injection 1 3.963 0.153% 0.029% 0.999 8201 6155 0.847% 0.550% Injection 2 3.965 0.994 7985 6099 Injection 3 3.953 0.997 8283 6138 Injection 4 3.954 0.983 7940 6051 Injection 5 3.952 0.987 7932 6018 Injection 6 3.951 0.994 8277 6097 Standard 2 Retention Time Retention Time %RSD Tailing Factor Theoretical Plates Peak Area Peak Area %RSD Injection 1 3.959 0.054% 0.990 7961 6097 0.875% Injection 2 3.956 0.988 8198 6022 Timolol Maleate Standard 1 Retention Time Retention Time %RSD Retention Time %Drift Tailing Factor Theoretical Plates Peak Area Peak Area %RSD Peak Area %Drift Injection 1 6.87 0.163% 0.022% 1.253 8689 8432 0.604% 0.137% Injection 2 6.869 1.264 8701 8406 Injection 3 6.845 1.259 8640 8329 Injection 4 6.848 1.245 8648 8415 Injection 5 6.848 1.244 8646 8378 Injection 6 6.853 1.231 8659 8305 Standard 2 Retention Time Retention Time %RSD Tailing Factor Theoretical Plates Peak Area Peak Area %RSD Injection 1 6.859 0.041% 1.265 8352 6097 0.875% Injection 2 6.855 1.268 8340 6022 Specificity Specificity ensures the method can accurately detect and quantify Brimonidine Tartrate and Timolol Maleate without interference from impurities or degradants. Acceptance criteria include a peak purity factor > 990, resolution > 2 for clear separation, and no overlapping peaks. [ 28 ] This confirms the method selectively measures Brimonidine Tartrate and Timolol Maleate in complex sample matrices.[ 29 ] To prepare the mixed degradation samples, 1 mL portions from acid, base, and oxidative stress solutions each showing less than 10% degradation were combined. For Brimonidine Tartrate, this included 1 mL each from samples treated with 3 M HCl for 24 hours, 3 M NaOH for 24 hours, and 3% hydrogen peroxide for 24 hours. In parallel, 1 mL portions were taken from Timolol Maleate stress solutions exposed to 1 M HCl for 24 hours, 0.05 M NaOH for 1 hour, and 0.5% hydrogen peroxide for 1 hour. The mixed solution was thoroughly blended and passed through a 0.45 µm membrane filter to remove particulates. The prepared sample was then analyzed on an Agilent 1100 HPLC system equipped with a diode array detector (DAD). The chromatographic profile obtained from this mixed degradation study is presented in Figure S2 (Supplementary material). Solution Stability Solution stability testing was performed to evaluate the behavior of Brimonidine Tartrate and Timolol Maleate over time under defined environmental conditions, including temperature, light, and humidity. [ 34 ] This step is essential in method validation to ensure accurate and consistent quantification during routine analysis. Stability was assessed by comparing peak areas from the initial injection with those obtained at 24, 48, and 72 hours. Throughout this period, no new peaks or missing peaks were observed in the chromatograms, confirming that the solution remained stable with no significant degradation. The solutions remained stable at room temperature for at least 3 days, as shown in Table 8 . Table 8 Solution Stability of Brimonidine Tartrate and Timolol Maleate Over 3 Days at Room Temperature Combigan® Ophthalmic Solution Brimonidine Tartrate Timolol Maleate Time (hours) Peak Area %Change of Peak Areas Peak Area %Change of Peak Areas 0 7225 ----- 9898 ----- 24 7222 0.042% 9852 0.465% 48 7218 0.097% 9933 0.354% 72 7215 0.138% 9924 0.263% Method Robustness The robustness of an analytical method evaluates its ability to produce consistent and reliable results despite small, deliberate variations in method parameters. As outlined in ICH guidelines, this step is crucial for confirming that the method can withstand routine fluctuations during practical use. [ 29 ] In this study, robustness was tested by slightly altering five key parameters: Buffer pH (7.0 ± 0.2), Flow rate (1.0 ± 0.2 mL/min), Detection wavelength (245 ± 2 nm) and (295 ± 2 nm), Mobile phase B composition (20 ± 2%B), and Injection volume (15 ± 2 µL). The method’s performance under these variations was evaluated using the following acceptance criteria: a tailing factor between 0.9 and 2.0, a theoretical plate count of at least 2000, and resolution (RS) greater than 2.0 between all peaks and the target analyte. Robustness was assessed by deliberately varying critical method parameters: adjusting the mobile phase pH by ± 0.2, changing the percentage of organic solvent (acetonitrile) in the mobile phase by ± 2%, altering the column temperature by ± 2°C, shifting the detection wavelength by ± 2 nm, and modifying the flow rate by ± 0.2 mL/min. These controlled changes showed no significant impact on chromatographic resolution as shown in Table 9 , confirming the method’s robustness. Table 9 Method Robustness Evaluation: Results for Mixed Degradation Samples under Parameter Variations Brimonidine Tartrate Timolol Maleate Parameter Variation Tailing Factor Theoretical Plates Tailing Factor Theoretical Plates Buffer pH 6.8 0.988 8983 1.258 9367 7.0 1.102 9658 1.246 9516 7.2 0.913 10156 0.902 10230 Wavelength (nm) 293 1.057 9341 1.223 9322 295 1.102 9658 1.246 9516 297 1.009 9336 1.197 9321 Wavelength (nm) 243 1.101 7908 0.9982 8123 245 1.090 8109 1.109 8201 247 1.103 8120 1.009 8423 %B composition 18%ACN 1.239 9539 1.265 9851 20%ACN 1.102 9658 1.246 9516 22%ACN 0.921 9620 0.913 9729 Injection Volume ( \(\:\mu\:\) L) 13 1.209 9430 1.065 9457 15 1.212 9610 1.246 9636 17 1.243 9543 1.434 9534 Flow rate mL/min 0.8 1.001 9399 1.065 9457 1 1.120 9680 1.212 9636 1.2 1.321 9483 1.367 9534 Linearity and range for active ingredient The validated linearity of the method was established across the ranges of 0-500 ppm for Brimonidine Tartrate and 250–1250 ppm for Timolol Maleate using pure standards. For analysis of Combigan® dosage form, additional calibration curves were constructed within narrower ranges (100–240 ppm for Brimonidine Tartrate and 250–600 ppm for Timolol Maleate) to reflect actual sample concentrations. The correlation coefficient (R²) for Brimonidine Tartrate and Timolol Maleate must be at least 0.999 to meet acceptance criteria. This ensures a strong linear relationship between concentration and detector response across the tested range, confirming the method’s reliability for accurate quantitative analysis. A stock solution of Brimonidine Tartrate (400 ppm) and Timolol Maleate (1000 ppm) was used to prepare a series of diluted solutions at concentrations of 100 ppm, 160 ppm, 200 ppm, 220 ppm, and 240 ppm for Brimonidine Tartrate, and 250 ppm, 400 ppm, 500 ppm, 550 ppm, and 600 ppm for Timolol Maleate. These solutions were analyzed using HPLC under optimized isocratic elution conditions. The resulting summary of the peak area results provided in Table 10 . The linear regression equations were Y = 46.365 X – 1545.2 (R² = 0.9992) for Brimonidine Tartrate and Y = 25.334 X – 2174.8 ( R² = 0.9996 ) for Timolol Maleate, where Y is the peak area of the standard solution and X is the drug concentration (Fig. 9 ). Table 10 Linearity results for Brimonidine Tartrate and Timolol Maleate Combigan® Ophthalmic Solution Brimonidine Tartrate Timolol Maleate Sample Preparation Concentration (ppm) Peak Area Average Peak Area Concentration (ppm) Peak Area Average Peak Area 1 100 3126 3117 250 4649 4195 2 3167 3949 3 3058 3987 1 160 5875 5852 400 7937 7891 2 5902 7846 3 5780 7890 1 200 7753 7731 500 10597 10539 2 7651 10567 3 7789 10452 1 220 8702 8554 550 11695 11684 2 8589 11605 3 8372 11753 1 240 9835 9676 600 13031 13086 2 9521 13101 3 9672 13126 Accuracy Accuracy of the developed HPLC method focused on evaluating how well the experimentally measured concentrations matched their known theoretical values.[ 28 ] Brimonidine Tartrate and Timolol Maleate solutions were prepared in triplicate at three concentration levels: 80%, 100%, and 120% of the nominal concentrations (200 ppm and 500 ppm, respectively). According to the acceptance criteria, the percent recovery for the active ingredients needed to fall within 95% to 105% of the target value. Meeting these criteria demonstrates that the method consistently and accurately measures the active ingredients and can also reliably detect impurities and degradants over the tested concentration range. These samples were analyzed by injecting them into the HPLC system, and the resulting peak areas were recorded. The percentage recovery of each active ingredient was then calculated using the linear regression equations obtained from the calibration curves: Y = 46.365 X – 1545.2 for Brimonidine Tartrate and Y = 25.334 X – 2174.8 for Timolol Maleate, as shown in Fig. 9 . By applying these equations, the measured concentrations were compared to the theoretical concentrations to calculate percent recovery. The summarized results in Table 11 confirm that the method meets the established accuracy criteria, achieving recoveries within the acceptable range of 95–105%. %Recovery = \(\:\frac{\varvec{C}\varvec{s}\varvec{a}\varvec{m}\varvec{p}\varvec{l}\varvec{e}}{\varvec{C}\varvec{s}\varvec{t}\varvec{a}\varvec{n}\varvec{d}\varvec{a}\varvec{r}\varvec{d}}\varvec{x}100\) Csample is the measured concentration of the sample calculated from the linear regression equation; Cstandard is the known (theoretical) concentration of the prepared sample. Table 11 Accuracy Results for Brimonidine Tartrate and Timolol Maleate Combigan® Ophthalmic Solution Brimonidine Tartrate Timolol Maleate Sample Preparation Concentration (ppm) Peak Area Average Peak Area Recovery Concentration (ppm) Peak Area Average Peak Area Recovery 1 160 5764 5859 99.81% 400 7757 7857 99.00% 2 5843 7843 3 5971 7971 1 200 7598 7674 99.42% 500 10437 10328 98.71% 2 7643 10176 3 7781 10372 1 240 9614 9558 99.78% 600 12981 13190 101.10% 2 9535 13253 3 9526 13336 Method Precision Precision in analytical method validation measures how consistently the method produces similar results under defined conditions. As outlined by ICH guidelines, [ 29 ] precision is evaluated at three key levels: Repeatability – assesses the closeness of results when the same analyst performs multiple injections or measurements on the same day, using identical equipment and procedures. Intermediate Precision – examines method performance within the same laboratory under varied conditions, such as different analysts, days, or instruments, to identify potential sources of variability. Reproducibility – tests the method’s consistency across different laboratories, ensuring broader reliability of results beyond a single lab setting. These levels demonstrate the method’s ability to consistently deliver accurate, reliable, and reproducible data under routine and variable conditions. Repeatability (Method Precision) Repeatability assesses the method’s short-term consistency by examining variability that could result from repeated sample preparation and analysis by the same analyst under identical conditions.[ 35 ] This test highlights the method’s robustness to small operational fluctuations, ensuring reproducible and dependable results in routine analysis. Regulatory guidelines set strict acceptance criteria for repeatability: the %Relative Standard Deviation (%RSD) of peak areas for the target compound must be not more than 1% (NMT 1%). [ 36 ] In this study, repeatability was evaluated by preparing and injecting six independent samples of Brimonidine Tartrate (200 ppm) and Timolol Maleate (500 ppm) into the HPLC system under the validated chromatographic conditions. The %RSD of the peak areas from these six injections was then calculated, demonstrating the method’s precision and compliance with the defined criteria, as summarized in Table 12 . Table 12 Repeatability Precision Results for Brimonidine Tartrate and Timolol Maleate Combigan® Ophthalmic Solution Brimonidine Tartrate Timolol Maleate Sample Preparation Peak Area Average Standard Deviation Peak Area %RSD Peak Area Average Standard Deviation Peak Area %RSD 1 7269 7245 39.80 0.549% 10060 10062 85.28 0.848% 2 7187 9945 3 7232 10040 4 7217 10019 5 7295 10194 6 7268 10116 Injection Precision Injection precision evaluates the method’s ability to consistently produce reliable results by examining instrument-related variability, including potential fluctuations from the injector, detector, column, and data integration process during repeated sample injections.[ 29 ] , [ 37 ] According to the established acceptance criteria, the %Relative Standard Deviation (%RSD) of the peak areas for Brimonidine Tartrate (200 ppm) and Timolol Maleate (500 ppm) must not exceed 1%, ensuring high reproducibility. To assess this, a single preparation of Brimonidine Tartrate (200 ppm) and Timolol Maleate (500 ppm) was injected six consecutive times under the method’s optimized HPLC conditions. The calculated %RSD values confirmed compliance with the acceptance threshold, demonstrating excellent injection precision. The summarized results are provided in Table 13 . Table 13 Injection Precision Results for Brimonidine Tartrate and Timolol Maleate Combigan® Ophthalmic Solution Brimonidine Tartrate Timolol Maleate Injection Peak Area Average Standard Deviation Peak Area %RSD Peak Area Average Standard Deviation Peak Area %RSD 1 7468 7420 63.77 0.859% 10316 10406 84.10 0.808% 2 7306 10456 3 7401 10294 4 7413 10431 5 7452 10512 6 7480 10425 Intermediate precision ( Inter-day Precision /Ruggedness) Intermediate precision of the developed analytical method for Brimonidine Tartrate and Timolol Maleate was evaluated to verify its robustness when subjected to deliberate variations, such as different analysts, instruments, columns, and testing days. [ 29 ] , [ 34 ] This level of validation is essential to demonstrate the method’s reliability beyond controlled laboratory conditions. As specified by acceptance criteria, the %Relative Standard Deviation (%RSD) of peak areas for Brimonidine Tartrate at 200 ppm and Timolol Maleate at 500 ppm must not exceed 1.5%. To assess this, six replicate samples of each analyte were prepared and analyzed under the same optimized chromatographic conditions, but on separate HPLC systems, by different analysts, and across multiple days. The resulting data, summarized in Table 14 , demonstrate the method’s ability to produce consistent and reproducible measurements despite normal variations in testing conditions. Intermediate precision parameters evaluated for this method include: HPLC : 1100 Series HPLC system with MWD (UV/VIS Detector), Agilent Technologies Column : Water XTERRA RP-18 (4.6x250 mm, 5µm) Mobile Phase : Solvent A: 25mM potassium phosphate monobasic buffer, pH 7.0 Solvent B: 100%ACN Solvent Strength : (80:20 v/v) Buffer pH 7.0: ACN Absorbance : 245 nm and 295 nm Flow Rate : 1.0 mL/minute Injection Volume : 15 µL Column Temperature : Ambient Table 14 Intermediate Precision Results Under Varying Analysts, Instruments, and Columns Combigan® Ophthalmic Solution Brimonidine Tartrate Timolol Maleate Sample Preparation Peak Area Average Standard Deviation Peak Area %RSD Peak Area Average Standard Deviation Peak Area %RSD 1 7159 7239 63.96 0.884% 10155 10065 136.24 1.354% 2 7243 9942 3 7215 10170 4 7319 9973 5 7191 10232 6 7308 9916 Limit of Detection (LOD) and Limit of Quantitation (LOQ) The limit of detection (LOD) was evaluated to determine the lowest concentration at which the analyte could be consistently detected, though not necessarily quantified with precision. Brimonidine Tartrate and Timolol Maleate were used as representative compounds to model impurities and degradation products. The LOD was established by assessing the signal-to-noise (S/N) ratio in the chromatographic response, with an acceptance criterion of an S/N ratio ≥ 3, indicating the method's reliable detection capability. To determine the limit of quantitation (LOQ) for Brimonidine Tartrate and Timolol Maleate, a series of dilutions were prepared, and their signal-to-noise ratios were thoroughly evaluated. Preliminary LOQ values were estimated at 0.24 ppm for Brimonidine Tartrate and 0.60 ppm for Timolol Maleate, based on achieving an S/N ratio of ≥ 10 and a %RSD ≤ 10%, as summarized in Table 15 . To confirm these estimates, fresh solutions at 0.24 ppm and 0.60 ppm were injected ten consecutive times into the HPLC system. This allowed for assessment of both precision and detection reliability. As shown in Table 16 , the %RSD for both analytes remained within acceptable limits (≤ 10%), validating these concentrations as the LOQ for the developed method. Table 15 Results of Limit of Detection (LOD) Study for Brimonidine Tartrate and Timolol Maleate solutions Combigan® Ophthalmic Solution Brimonidine Tartrate Timolol Maleate Concentration (ppm) Signal to Noise ratio Concentration (ppm) Signal to Noise ratio 0.04 1.3 0.10 1.2 0.08 3.5 0.20 3.8 0.12 5.9 0.30 5.2 0.16 7.5 0.40 6.9 0.20 8.3 0.50 7.5 0.24 10.5 0.60 10.1 0.28 11.3 0.70 11.0 0.32 14.4 0.80 13.7 0.36 17.0 0.90 15.8 0.40 18.7 1.00 16.9 Table 16 Results of Limit of Quantitation (LOQ) Study for Brimonidine Tartrate and Timolol Maleate solutions Combigan® Ophthalmic Solution No . Brimonidine Tartrate Timolol Maleate Injections Concentration (ppm) Peak Area Peak Area %RSD Concentration (ppm) Peak Area Peak Area %RSD 1 0.24 9.75 4.709% 0.60 12.88 3.660% 2 0.24 9.02 0.60 13.54 3 0.24 10.23 0.60 13.72 4 0.24 9.45 0.60 12.63 5 0.24 10.07 0.60 12.55 6 0.24 9.38 0.60 13.09 Results and Discussion Glaucoma refers to a group of eye diseases marked by the progressive narrowing of the visual field, typically accompanied by optic nerve damage and characteristic optic disc cupping.[ 38 ] Elevated IOP is widely acknowledged as a major risk factor contributing to disease onset and progression. [ 13 ] Clinically, glaucoma most commonly presents as either primary open-angle glaucoma or primary angle-closure glaucoma. Its management involves the use of various pharmacological classes designed to reduce IOP through different physiological mechanisms, thereby helping to slow disease advancement and preserve vision.[ 2 ] Typically, first-line treatment involves typical beta-adrenergic blockers or prostaglandin analogues. When further pressure reduction is necessary, these agents can be combined with or supplemented by other therapies, such as miotics, carbonic anhydrase inhibitors, or sympathomimetic drugs. [ 4 ] COMBIGAN ® (Brimonidine Tartrate and Timolol Maleate ophthalmic solution) is specifically indicated for lowering IOP in patients with chronic open-angle glaucoma or ocular hypertension who do not achieve adequate pressure control with single-agent therapy and when the use of COMBIGAN is considered appropriate.[ 16 ] Beyond reducing IOP, COMBIGAN also helps to minimize long-term fluctuations in eye pressure. By lowering both the average IOP and its variability over time, COMBIGAN aims to slow the progression of visual field loss associated with glaucoma.[ 2 ] Multiple liquid chromatographic methods have been proposed for the quantification of Brimonidine Tartrate (BT) and Timolol Maleate (TM) in pharmaceutical formulations as seen in Table S4 (Supplementary material). However, many of these methods lack full stability-indicating capability or exhibit limitations in sensitivity, linearity range, and robustness. Our proposed RP-HPLC method addresses these shortcomings and demonstrates clear advantages over earlier approaches. One reported method utilized a BDS Hypersil phenyl column with a mobile phase of acetonitrile and phosphate buffer (pH 4.0).[ 23 ] Although the method provided acceptable quantification ranges (BT: 2.0–80.0 µg/mL; TM: 5.0–200.0 µg/mL), it was not stability-indicating and showed only moderate precision. Similarly, another study employed a C18 column with acetonitrile–buffer mixtures, but the linearity range was narrower (BT: 10–50 µg/mL; TM: 4–20 µg/mL), limiting its suitability across a wider concentration span. [ 24 ] UV-spectrophotometric methods offered simplicity but lacked specificity, robustness, and the ability to separate degradation products, making them unsuitable for regulatory-grade stability testing. [ 25 ] Other methods reported extremely short retention times (around 0.5–0.6 minutes), [ 26 ] which risks missing impurities or degradants, especially since forced degradation studies were not performed. Alternative analytical methods have employed relatively limited linear concentration ranges specifically, 4–24 µg/mL for Brimonidine Tartrate and 10–60 µg/mL for Timolol Maleate which restrict their broader applicability and reduce their reliability. While the sensitivity was improved (LOD for BT: 0.05 µg/mL; TM: 0.09 µg/mL), the method still did not evaluate forced degradation studies, limiting its stability-indicating applicability. [ 27 ] Although several researchers have investigated the chromatographic analysis of BT and TM, there remains a gap in developing a unified RP-HPLC method that can simultaneously quantify both drugs while also functioning as a true stability-indicating assay in combination dosage forms. This highlights the need for a comprehensive, robust, and reliable analytical approach to ensure precise quality control and thorough stability evaluation of ophthalmic formulations. In contrast, our proposed method employs a Supelco Discovery C18 column (250 × 4.6 mm, 5 µm) with a mobile phase of acetonitrile and monobasic potassium phosphate buffer (pH 7.0, 30 mM TEA). This setup enables simultaneous detection at 245 nm and 295 nm, with wide linearity ranges (BT: 0.24–500 µg/mL; TM: 0.60–1250 µg/mL). The method demonstrated excellent sensitivity (LOD: BT = 0.08 µg/mL; TM = 0.20 µg/mL), precision (RSD < 1%), and robustness under varied experimental conditions. Importantly, it was validated according to ICH guidelines and included comprehensive forced degradation studies, confirming its stability-indicating nature. Overall, while previous reports achieved partial success in the simultaneous quantification of BT and TM, they either lacked stability indication, had narrower linearity ranges, or demonstrated only moderate robustness. The present method offers a fully validated RP-HPLC approach that is robust, sensitive, stability-indicating, and directly applicable to pharmaceutical quality control and regulatory environments. The system suitability test results show fully resolved peaks with tailing factors near 1, indicating excellent peak symmetry. Over 9,000 theoretical plates were observed, confirming strong column efficiency. For Brimonidine Tartrate, %RSD values for peak area and retention time from six replicates of working standard solution #1 were 0.153% and 0.847%, while for solution #2 (two replicates), they were 0.045% and 0.875%. Drift values were also minimal: %RT Drift at 0.029% and %PA Drift at 0.550%. Similarly, Timolol Maleate exhibited %RSD values of 0.163% and 0.604% (solution #1) and 0.041% and 0.875% (solution #2), with %RT Drift of 0.022% and %PA Drift of 0.137%. These low %RSDs and drift values confirm system repeatability, precision, and stability, satisfying ICH system suitability criteria. [ 29 ] This test was performed before further analyses to validate system readiness. The forced degradation study revealed that Brimonidine Tartrate exhibits high stability under thermal and photolytic stress, with no detectable degradation observed after 24 hours at 75°C or under UV irradiation at 245 nm. Under acidic conditions (3 M HCl), the compound showed minimal degradation (0.363%), while exposure to alkaline conditions (3 M NaOH) led to slightly higher degradation (1.438%), suggesting a greater susceptibility to base-catalyzed hydrolysis. Oxidative stress induced by 3% hydrogen peroxide resulted in moderate degradation (0.776%), indicating some vulnerability to oxidation. Overall, these findings confirm that Brimonidine Tartrate maintains robust stability under most stress conditions, with acidic, alkaline and oxidative environments posing the greatest risk for degradation. Similarly, the forced degradation study demonstrated that Timolol Maleate remains chemically stable under thermal stress at 75°C and photolytic exposure at 295 nm for 24 hours, with no measurable degradation observed. However, the drug displayed pronounced sensitivity to hydrolytic conditions, with degradation increasing in proportion to acid and base concentration; degradation reached 23.35% in 3 M HCl and 23.92% in 1 M NaOH. Timolol Maleate was especially susceptible to oxidative degradation, showing substantial degradation of 57.17% when treated with 3% hydrogen peroxide. Lower concentrations and shorter exposure times led to reduced degradation: 5.52% in 1 M HCl (24 hours), 5.54% in 0.05 M NaOH (1 hour), and 9.57% in 0.5% H₂O₂ (1 hour). These results highlight that while Timolol Maleate is stable under heat and light, it undergoes significant degradation under strong hydrolytic and oxidative stress, underscoring the need for careful control of these conditions during manufacturing, storage, and formulation to ensure product stability and quality. The chromatographic analysis successfully detected and resolved the peaks corresponding to Brimonidine Tartrate, Timolol Maleate, and Maleic Acid. All peaks exhibited excellent resolution, thereby fulfilling the specificity criteria set for analytical separation. Additionally, the peak purity index for each analyte exceeded the acceptable threshold, confirming the absence of co-eluting impurities or interference. The specificity assessment demonstrated that the developed method effectively isolates and quantifies these three components even in the presence of degradation products and potential impurities. Peak purity factor (≥ 999.99), RS ≥ 2.0, and a corresponding three-dimensional chromatogram, which highlight the method’s robustness and demonstrate its adherence to ICH and FDA validation requirements. [ 29 ] Furthermore, the results of the robustness study, all parameters complied with ICH guidelines. The tailing factors fell within the acceptable range (0.9-2) and the number of theoretical plates ≥ 2000, indicating satisfactory column efficiency. Furthermore, the method demonstrated robustness by maintaining consistent performance despite intentional minor variations in solvent composition, buffer pH, flow rate, detection wavelength, and injection volume. To assess solution stability, a freshly prepared ophthalmic solution containing Brimonidine Tartrate (200 ppm) and Timolol Maleate (500 ppm) was immediately analyzed using the RP-HPLC system. The same solution was reinjected after 24, 48, and 72 hours. Throughout this period, the chromatographic profiles showed no appearance of new peaks and no disappearance of existing ones, confirming that the solution remained stable without significant degradation. Specifically, the percent peak area changes for Brimonidine Tartrate at 24, 48, and 72 hours were 0.042%, 0.097%, and 0.138%, respectively, while for Timolol Maleate, the corresponding changes were 0.465%, 0.354%, and 0.263%. These results indicate excellent short-term stability of both active pharmaceutical ingredients (APIs) and further support the robustness of the developed RP-HPLC method, as consistent peak areas reflect the absence of degradation or analyte loss during the study period. Additionally, to evaluate the method’s linearity, five different concentrations of Brimonidine Tartrate and Timolol Maleate standard solutions were prepared, and each was individually injected into the HPLC system. The corresponding peak areas were used to construct calibration curves plotting peak area versus concentration. The resulting linear regression analysis yielded correlation coefficients (R²) of 0.9992 for Brimonidine Tartrate and 0.9996 for Timolol Maleate. These high correlation coefficients satisfy the acceptance criteria for linearity, confirming that the HPLC method provides a consistent and proportional response across the tested concentration range. Overall, these results demonstrate the method’s validity for precise quantitative determination of the active pharmaceutical ingredients. The accuracy of the developed RP-HPLC method for quantifying Brimonidine Tartrate and Timolol Maleate in Combigan® ophthalmic solution was evaluated by recovery studies at three concentration levels corresponding to 80%, 100%, and 120% of the target assay concentrations. Each level was analyzed in triplicate. For Brimonidine Tartrate, the average recovery values obtained were 99.81% at 160 ppm, 99.42% at 200 ppm, and 99.78% at 240 ppm. Similarly, for Timolol Maleate, the recoveries were 99.00% at 400 ppm, 98.71% at 500 ppm, and 101.10% at 600 ppm. All recoveries fell well within the generally accepted range of 95–105%, demonstrating excellent method accuracy across the studied concentration range. These results confirm that the method provides accurate quantification without interference from excipients or other components present in the formulation. The consistency of recoveries across different concentration levels also supports the robustness and reliability of the method for routine quality control and stability testing of Combigan® ophthalmic solution. To further evaluate the accuracy and precision of the proposed method, results were statistically compared with those of reported methods using the Student’s t-test and F-test at the 95% confidence level (Table 17 ). The proposed analytical method demonstrated high accuracy and precision for the determination of Brimonidine Tartrate and Timolol Maleate in comparison with the reference method. [ 27 ] Statistical evaluation using the F-test revealed that the proposed method exhibited significantly lower variance (Brimonidine Tartrate: F = 206,611; Timolol Maleate: F = 7,963), indicating superior precision. The t-test results (Brimonidine Tartrate: t = -2.04; Timolol Maleate: t = -2.43) showed no significant difference between the means of the proposed and reference methods at the 95% confidence level, confirming the reliability and suitability of the proposed method for routine analysis. Overall, these findings demonstrate that the proposed method is statistically comparable to the reference method while offering enhanced precision. Table 17 Statistical comparison of the proposed and reference methods for Brimonidine Tartrate and Timolol Maleate. Data are mean ± SD. F and t values with their 95% confidence critical limits (F_crit, t_crit) are shown. F-test evaluates precision; t-test evaluates accuracy. Drug Name Recovery ± SD Calculated t- values t_critical (α = 0.05) Calculated F- values F_critical (α = 0.05) Proposed methods Reference method Brimonidine Tartrate 99.67 ± 0.0022 100.85 ± 1.00 206,611 2.776 -2.04 19 Timolol Maleate 99.60 ± 0.013 101.41 ± 0.95 7,963 2.776 -2.43 19 To evaluate repeatability, six individually prepared samples of Brimonidine Tartrate (200 ppm) and Timolol Maleate (500 ppm) were analyzed under optimized HPLC conditions. Precision was assessed by calculating the percent relative standard deviation (%RSD) of the peak areas. The %RSD values for Brimonidine Tartrate and Timolol Maleate were 0.549% and 0.848%, respectively. According to ICH guidelines, a %RSD not exceeding 2.0% is considered acceptable, and these results demonstrate that the method consistently delivers precise measurements for both analytes under the same conditions. Injection precision was further evaluated by injecting a single sample of Brimonidine Tartrate and Timolol Maleate six consecutive times into the HPLC system. The resulting %RSD values, 0.859% for Brimonidine Tartrate and 0.808% for Timolol Maleate, also fell within the acceptable range, confirming the method’s reliability for repeated injections. Additionally, intermediate precision was assessed by preparing six separate samples and analyzing them on a different HPLC system. The %RSD values obtained, 0.884% for Brimonidine Tartrate and 1.354% for Timolol Maleate, complied with the specified acceptance criteria. Table S5 (Supplementary material) presents the results from both analysts, allowing proper calculation of inter-day precision and clear presentation of the data. Overall, these results confirm that the method maintains precision and robustness across repeatability, injection precision, and intermediate precision assessments, meeting the requirements set out in the validation protocol. The limit of detection (LOD) for Brimonidine Tartrate and Timolol Maleate was established by preparing a series of dilutions from the stock solution. Each prepared dilution was examined on the HPLC system, and the corresponding signal-to-noise ratios were determined based on the generated chromatograms. The concentrations of 0.08 ppm for Brimonidine Tartrate and 0.20 ppm for Timolol Maleate produced signal-to-noise ratios of 3.5 and 3.8, respectively. These values satisfy the acceptance criterion of a signal-to-noise ratio ≥ 3, and thus were designated as the LODs for the method. To establish the limit of quantitation (LOQ), test solutions at concentrations of 0.24 ppm for Brimonidine Tartrate and 0.60 ppm for Timolol Maleate were each injected into the HPLC system in ten replicate runs. Analysis of the resulting chromatograms showed %RSD values for peak areas of 4.70% and 3.66%, both well below the LOQ acceptance threshold of ≤ 10%. These findings demonstrate that the developed method is sufficiently precise to quantify both analytes reliably at these low concentrations, meeting validation requirements for LOQ performance. To further verify the Limit of Detection (LOD) and Limit of Quantitation (LOQ) values,[ 30 ] the International Council for Harmonisation (ICH) recommends several calculation methods depending on the analytical technique, analyte characteristics, and method suitability.[ 29 ] One widely accepted method involves using the standard deviation of the response (σ) and the slope of the calibration curve (S), calculated with the formulas: \(\:LOD=3.3X\left(\frac{{\sigma\:}}{\text{S}}\right)\:\) and \(\:LOQ=10X\left(\frac{{\sigma\:}}{\text{S}}\right)\) Using data from the linearity study, we determined σ and S for both Brimonidine Tartrate and Timolol Maleate. Applying these formulas, the calculated LOD and LOQ for Brimonidine Tartrate were 0.099 ppm and 0.30 ppm, respectively, which correspond closely to our experimentally obtained signal-to-noise results (LOD = 0.08 ppm, LOQ = 0.24 ppm). Similarly, for Timolol Maleate, the calculated LOD and LOQ were 0.218 ppm and 0.66 ppm, aligning well with the signal-to-noise based findings (LOD = 0.20 ppm, LOQ = 0.60 ppm). These results confirm that our method validation complies with ICH and USP guidelines, supporting the reliability of the developed analytical method. To evaluate the environmental sustainability of the developed method, three complementary green analytical chemistry (GAC) metrics were applied: the Analytical Eco-Scale, the Green Analytical Procedure Index (GAPI), and the AGREE evaluation. [ 39 ] , [ 40 ] The Eco-Scale was calculated by assigning penalty points for hazardous reagents, waste generation, and energy consumption. The main contributor to penalty points was the use of acetonitrile (ACN) as organic solvent in the mobile phase and the direct disposal of waste. Considering dilution as the only sample preparation, room temperature operation (25°C), and a short chromatographic run time (10 min), the method achieved an Eco-Scale score of ~ 75, which classifies it as an acceptable green method (scores ≥ 75). Moreover, the Green Analytical Procedure Index (GAPI) tool was used to obtain a holistic assessment covering the entire analytical workflow from sample collection to final determination. The pictogram for this method showed a mixture of green and yellow zones, reflecting the environmentally favorable aspects such as simple dilution, absence of derivatization, and low energy consumption. However, red zones were observed in solvent usage and waste management, as ACN is toxic, and waste was directly discarded without treatment. Importantly The AGREE evaluation, based on the 12 principles of green analytical chemistry, generated a circular pictogram and a numerical score. The method scored 0.57 (out of 1.0), indicating moderate greenness. High scores were obtained for minimal sample preparation, short analysis time, and absence of derivatization. Lower scores were assigned to principles related to renewable solvents, waste management, and operator safety, due to the use of ACN and its direct disposal. Overall, the three evaluation tools confirmed that the proposed RP-HPLC method possesses several environmentally favorable features such as simplicity, short analysis time, and low energy consumption. However, the main limitations were related to solvent selection and waste handling. Future improvements could include replacing ACN with a greener alternative (e.g., ethanol or water-rich mobile phases), implementing solvent-recycling strategies, and adopting proper hazardous waste management practices. In summary, this validated RP-HPLC method addresses significant analytical limitations reported in previous studies by offering a broader linearity range, robust stability-indicating performance, and compliance with international validation standards. Consequently, this method offers a scientifically robust and practically applicable approach for thorough quality control and stability evaluation of combination ophthalmic formulations, thereby reinforcing ongoing initiatives to preserve therapeutic effectiveness and ensure patient safety in the treatment of glaucoma. CONCLUSION This study successfully developed and validated a robust, accurate, and precise RP-HPLC method for the simultaneous quantification of Brimonidine Tartrate and Timolol Maleate in Combigan® ophthalmic solution. Addressing gaps found in previously published methods, such as insufficient linear ranges, inadequate stability testing, and lack of a unified assay. The new method demonstrated excellent linearity across the tested concentration ranges (R² >0.999), strong system suitability with high theoretical plate counts and low tailing factors, and compliance with ICH acceptance criteria in all validation parameters. The method proved highly repeatable and precise, as confirmed by low %RSD values in repeatability, injection precision, and intermediate precision assessments. Robustness testing further showed consistent performance under deliberate variations in chromatographic conditions. The specificity study verified the method’s ability to resolve the active ingredients from degradants and impurities, supporting its stability-indicating capability. The determined LOD and LOQ values, supported by both signal-to-noise ratios and statistical calculations based on standard deviation and calibration slope, highlight the method’s sensitivity and suitability for detecting and quantifying low levels of Brimonidine Tartrate and Timolol Maleate. The three complementary green analytical chemistry (GAC) metrics were applied and confirmed that the proposed RP-HPLC method possesses several environmentally favorable features such as simplicity, short analysis time, and low energy consumption. Overall, this validated method meets ICH and USP guidelines and offers a comprehensive analytical tool for reliable quality control, routine analysis, and stability testing of Combigan® and similar ophthalmic formulations. It thus represents a significant advancement toward ensuring the safety, efficacy, and quality of combination therapies used in glaucoma management. Abbreviations ACN acetonitrile Avg average FDA Food and Drug Administration HCl hydrochloric acid ICH International Conference on Harmonisation LC liquid chromatography LOD limit of detection LOQ limit of quantitation µL microliter µg/mL microgram per milliliter min minutes N normality Mg milligram ppm parts per million C18 column having octadecyl chain of carbon atom pH negative logarithm of H + concentration RP-HPLC reverse-phase high-performance liquid chromatography RSD relative standard deviation RT retention time R 2 correlation of coefficient UV ultraviolet GAC green analytical chemistry GAPI Green Analytical Procedure Index. Declarations Financial competing interests The authors affirm that they have no financial conflicts of interest related to this work. Declaration of interests The authors state that they have no conflicts of interest to report and no relevant disclosures. Statement of Human and Animal Rights (including Statement of Informed Consent) This manuscript does not involve any research conducted on human participants or animal subjects by the authors. Ethics Statement The datasets analyzed in this research originate exclusively from our own samples, with study approval granted by the institutional ethics committee. Data Availability The datasets supporting the findings of this study are available within the manuscript and from the corresponding author upon reasonable request. AUTHOR INFORMATION Corresponding Author Aktham Mestareehi - Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences , Wayne State University, Detroit, MI, 48201, USA. https://orcid.org/0000-0002-1478-6310; Email: [email protected] Authors Aktham Mestareehi - Department of Applied Pharmaceutical Sciences and Clinical Pharmacy, Faculty of Pharmacy, Isra University, P.O. Box 22, Amman 11622, Jordan Aktham Mestareehi - Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences and Integrated Biosciences, Wayne State University, Detroit, Michigan 48201, United States Author Contributions The manuscript was prepared by the author, who provided approval and consent for the final version. Contributions by A.M. included methodology development, formal analysis, and data curation. Furthermore, A.M. drafted the original manuscript, participated in manuscript review and editing, supervised the project, administered the research, and secured funding. Funding Declaration This research did not receive any specific funding from public, commercial, or non-profit organizations. We express our gratitude to all authors involved and to the publisher for their support. References Resnikoff, S. et al. Global data on visual impairment in the year 2002. Bull World Health Organ 82, 844–851 (2004). Bengtsson, B., Leske, M. 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Supplementary Files SupplementaryMaterials.docx Cite Share Download PDF Status: Published Journal Publication published 05 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 03 Nov, 2025 Reviews received at journal 30 Oct, 2025 Reviewers agreed at journal 30 Oct, 2025 Reviewers agreed at journal 27 Oct, 2025 Reviewers agreed at journal 26 Oct, 2025 Reviews received at journal 08 Oct, 2025 Reviewers agreed at journal 02 Oct, 2025 Reviewers agreed at journal 01 Oct, 2025 Reviews received at journal 30 Sep, 2025 Reviewers agreed at journal 30 Sep, 2025 Reviewers invited by journal 29 Sep, 2025 Submission checks completed at journal 28 Sep, 2025 First submitted to journal 27 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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2","display":"","copyAsset":false,"role":"figure","size":152021,"visible":true,"origin":"","legend":"\u003cp\u003eUV Spectrum of Brimonidine Tartrate, Timolol Maleate, and Combigan using a Hitachi UV/VIS Double Beam Spectrophotometer, Model U-2900\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7231691/v1/ab49c0152ba60ef7030db9c0.png"},{"id":95106714,"identity":"1d770b7a-c414-4f6b-a6da-42ff97ba9aa8","added_by":"auto","created_at":"2025-11-04 11:01:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":71803,"visible":true,"origin":"","legend":"\u003cp\u003eChromatograms obtained using Columns #1 and #2 (C18, 4.6 × 250 mm, 5 µm) following the injection of Brimonidine Tartrate (200 ppm) and Timolol Maleate (500 ppm).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7231691/v1/54cdd4fe4f500df1ef0fa34b.png"},{"id":95106715,"identity":"34a86376-ebb4-4699-b93a-88bc2d81da17","added_by":"auto","created_at":"2025-11-04 11:01:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":63145,"visible":true,"origin":"","legend":"\u003cp\u003eChromatograms obtained using Column #3 Supelco Discovery (C18, 4.6 × 250 mm, 5 µm) following the injection of Brimonidine Tartrate (200 ppm) and Timolol Maleate (500 ppm). Mobile phase (ACN: Buffer; 30:70 v/v)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7231691/v1/dd600a744e74fb1611ba532b.png"},{"id":95106716,"identity":"498837e2-ffdb-4068-8e01-f5969a518b2a","added_by":"auto","created_at":"2025-11-04 11:01:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":112083,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of (A) Combigan® Peaks by Retention Time Using Standard Injections of (B) Maleic Acid (100 ppm), (C) Brimonidine Tartrate (200 ppm), and (D) Timolol Maleate (500 ppm). Mobile phase (ACN: Buffer; 20:80 v/v)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7231691/v1/0af1d716f5f325cf52d777e4.png"},{"id":95106721,"identity":"54c8fc71-e045-433d-a50f-7533fc812e49","added_by":"auto","created_at":"2025-11-04 11:01:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":50403,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of ACN: buffer pH 7.0 ratios and TEA addition on peak shape and retention of Brimonidine Tartrate and Timolol Maleate.\u003c/strong\u003e \u003cem\u003eA) Chromatogram without TEA; B) Chromatogram with 30 mM TEA\u003c/em\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7231691/v1/0ec9206020eed73da9ad613d.png"},{"id":95224062,"identity":"aafc1714-594b-4bac-b55f-a40eab05d3f2","added_by":"auto","created_at":"2025-11-05 16:23:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":179784,"visible":true,"origin":"","legend":"\u003cp\u003eCalibration curves for Brimonidine Tartrate and Timolol Maleate: Peak area versus concentration (ppm) with corresponding regression equations and R\u003csup\u003e2\u003c/sup\u003e values\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7231691/v1/e41552b8391a7552cdb9133a.png"},{"id":95224295,"identity":"14e2ee94-b494-4182-8bf0-1daf98bd0e10","added_by":"auto","created_at":"2025-11-05 16:23:34","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":144413,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Control chromatogram of (A) Brimonidine Tartrate (200 ppm) and (B) Timolol Maleate (500 ppm) showing baseline retention; (C) Zoomed chromatogram after mixed degradation, illustrating complete separation of degradation peaks from main analyte peaks\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7231691/v1/535f51c85e98aa63222d2fea.png"},{"id":95106720,"identity":"5ee5511e-888d-4cc3-9a64-d36af406ca77","added_by":"auto","created_at":"2025-11-04 11:01:44","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":147780,"visible":true,"origin":"","legend":"\u003cp\u003eCalibration curve with error bars showing the relationship between peak area and concentration of Brimonidine Tartrate and Timolol Maleate for the linearity study.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7231691/v1/770d87b793b0837f6596e45f.png"},{"id":100069245,"identity":"584b79c6-8e7f-41f4-8436-817f9550927c","added_by":"auto","created_at":"2026-01-12 16:11:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3682452,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7231691/v1/7fdab3cf-a692-4d70-93c4-3980f17d4a23.pdf"},{"id":95225616,"identity":"e5c5ea29-a5c1-4ace-9cc8-e82863195b65","added_by":"auto","created_at":"2025-11-05 16:25:18","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":751150,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7231691/v1/6099191295b6732d4b4633bb.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development and Validation of a Robust Stability-Indicating Reversed Phase High Performance Liquid Chromatography (RP-HPLC) Method for Simultaneous Quantification of Brimonidine Tartrate and Timolol Maleate (Combigan ® ) in Bulk and Pharmaceutical Dosage Forms","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eGlaucoma ranks among the leading causes of chronic vision impairment and is recognized as the third most common cause of blindness worldwide. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] Elevated intraocular pressure (IOP) remains the primary modifiable risk factor linked to vision loss in glaucoma patients.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] Evidence from extensive clinical trials underscores the critical role of promptly lowering IOP to protect against optic nerve damage and preserve visual function. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] Importantly, results from the Early Manifest Glaucoma Trial demonstrated that lowering IOP by as little as 1 mmHg is associated with an approximate 10% reduction in the risk of glaucoma progression. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] Initial treatment for glaucoma generally begins with monotherapy using a single medication. If this approach fails to sufficiently lower IOP, clinicians may either switch to a different single agent or introduce an additional medication. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] For patients who require multiple drugs to achieve target IOP, fixed-combination therapies are often favored, as they tend to improve adherence to treatment. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] Interestingly, despite guidelines recommending monotherapy as the starting point, real-world practice shows that many patients with primary open-angle glaucoma or ocular hypertension are started directly on combination therapy. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eCombigan\u0026reg; (Allergan Inc, Irvine, CA, USA)[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] is a fixed-dose ophthalmic formulation combining Timolol Maleate 0.5%, a nonselective beta-blocker, with Brimonidine Tartrate 0.2%, a selective alpha-2 adrenergic agonist. Clinical evidence shows that Combigan provides superior IOP reduction compared to monotherapy with either Timolol or Brimonidine alone, [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and it also effectively minimizes IOP fluctuations. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] Importantly, the combination does not increase the risk of adverse effects beyond those seen with the individual agents,[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and has even been linked to a lower incidence of ocular allergy than brimonidine monotherapy.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] Furthermore, multiple studies have found Combigan to outperform other fixed combinations in reducing IOP, [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] while offering better overall tolerability.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eBrimonidine Tartrate (BT) is the tartrate salt of brimonidine (5-bromo-6-(2- imidazolin-2-ylamino) quinoxaline D-tartrate),[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] an imidazole-based compound that acts as a highly selective alpha-2 adrenergic receptor agonist. By engaging this G-protein-coupled receptor,[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] brimonidine suppresses intracellular adenyl cyclase activity, leading to decreased production of aqueous humor (AH) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] The drug exhibits a relatively brief systemic elimination half-life (t₁/₂) of about 2\u0026ndash;3 hours. Timolol maleate (TM) is chemically described as 2-propanol, 1-(1,1-dimethylethyl)amino-3-[[4-(4-morpholinyl)-1,2,5-thiadiazol-3-yl]-,(S)-,(Z)-2-butenedioate (1:1) (salt)). [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] TM is classified as a non-selective β-adrenergic blocker lacking both intrinsic sympathomimetic and membrane-stabilizing properties, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] Its therapeutic effect in glaucoma arises from blocking β-adrenergic receptors in the ciliary epithelium, thereby lowering aqueous humor production and reducing intraocular pressure. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eThe United States Pharmacopeia recommends HPLC methods for analyzing this compound in both tablets and ophthalmic solutions, whereas the British Pharmacopoeia outlines a direct spectrophotometric assay for its determination in the same dosage forms. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] Several liquid chromatographic methods have been described for quantifying brimonidine tartrate and timolol maleate in pharmaceutical formulations. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] In a previously reported method, the observed retention times for brimonidine tartrate and timolol maleate were notably brief, measured at approximately 0.51 minutes and 0.65 minutes, respectively; [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] however, these rapid elution times raise concerns that potential degradants or impurities could be overlooked. Moreover, the method did not include forced degradation stability studies, leaving the influence of possible degradation products on the analytical results unexamined. Another published method relied on an unusually narrow linear concentration range (4\u0026ndash;24 \u0026micro;g/mL,10\u0026ndash;60.0 \u0026micro;g/mL) for of brimonidine tartrate and timolol maleate respectively, which is not recommended for method development due to its limited applicability and increased risk of analytical errors. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] This restricted range also hampers the method\u0026rsquo;s ability to evaluate drug stability and detect degradation products effectively. As a result, the absence of a robust stability-indicating capability emerges as a significant limitation, highlighting the need for a more comprehensive and reliable analytical method. While numerous studies have independently examined the chromatographic analysis of Brimonidine Tartrate and Timolol Maleate, there is still a clear gap in the literature regarding the development of a single RP-HPLC method capable of both simultaneously quantifying these compounds and serving as a stability-indicating assay for combination formulations. To address this gap, the present study aims to develop a simple, rapid, precise, and accurate RP-HPLC stability-indicating method specifically for Combigan\u0026reg;. The method will be validated following ICH and FDA guidelines to ensure compliance with regulatory requirements for quality control and stability testing. Ultimately, this method is intended to offer a reliable analytical tool for monitoring the stability of Combigan\u0026reg;, safeguarding its potency, safety, and therapeutic efficacy throughout its shelf life.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"MATERIALS \u0026 METHODOLOGY","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eChemicals and Reagents\u003c/h2\u003e\u003cp\u003eBrimonidine Tartrate (BT, USP) and Timolol Maleate (TM, USP) were used as active pharmaceutical ingredients. Maleic acid from Sigma-Aldrich ReagentPlus\u0026reg;, \u0026ge;\u0026thinsp;99% purity. Hydrochloric acid (12 N) and sodium hydroxide was purchased from EM Science (USA). Acetonitrile (ACN, HPLC grade) was sourced from Fisher Scientific (USA). Potassium phosphate monobasic was obtained from Merck \u0026amp; Co. (Germany). Phosphoric acid (85%) was sourced from J.T. Baker (USA), and glacial acetic acid was obtained from Mallinckrodt Inc. (USA). Hydrogen peroxide solutions (30% and 3%) were purchased from Fisher Scientific (USA). Deionized water was prepared using a Milli-Q water purification system (Millipore, Bedford, MA, USA). pH buffer standards at pH 4.0, 7.0, and 9.0 were all procured from Fisher Scientific (USA).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eChromatography Equipment\u003c/h3\u003e\n\u003cp\u003eAn Agilent 1100 series HPLC system equipped with a G1311A quaternary pump, G1329A autosampler injector, G1365B DAD detector, G1316A Column Thermostat, and G1314A UV detector was utilized for chromatographic analysis. Data acquisition and processing were performed using ChemStation software. Additional laboratory equipment included a Mettler Toledo analytical balance (model AB265-S, Switzerland), a pH meter (model 3540, UK), and a Bandelin-Sonorex ultrasonic bath (model TK 52, Germany). Precise liquid handling was achieved with an Advantage-Lab variable micropipette (capacity up to 1000 \u0026micro;L). Chromatographic separation of target analytes was carried out using a Supelco Discovery C18 column (25 cm \u0026times; 4.6 mm, 5 \u0026micro;m). Prior to use, the mobile phase underwent filtration through a 0.45 \u0026micro;m membrane filter (Millipore, Milford, MA) to ensure clarity and remove particulates. Throughout the method, a Sigma refrigerated centrifuge (Germany) and a Stuart vortex mixer (England) were employed to support sample preparation and processing. Additionally, a UV/VIS spectrophotometer (Hitachi U-2910), and a Thermo Nicolet (IR 200) spectrometer were used during the analysis.\u003c/p\u003e\n\u003ch3\u003ePharmaceutical Dosage\u003c/h3\u003e\n\u003cp\u003eCombigan\u0026reg; ophthalmic solution, manufactured by Allergan (USA), is formulated to contain Brimonidine Tartrate at a concentration of 0.2% and Timolol Maleate at 0.5%, with NDC 0023-9211-10.\u003c/p\u003e\n\u003ch3\u003eChromatographic conditions\u003c/h3\u003e\n\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the optimized chromatographic conditions established for the analysis of Brimonidine Tartrate and Timolol Maleate. These carefully selected parameters achieve rapid separation and robust detection, ensuring precise quantification of Combigan\u0026reg; components within a streamlined run time.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eOptimized Chromatographic Conditions for Combigan\u0026reg; Method Development\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eConditions\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eColumn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSupelco Discovery C18, 5\u0026micro;m (250 x 4.6 mm)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMobile Phase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20:80 ACN/ monobasic potassium phosphate buffer pH 7.0 with 30 mM TEA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRun time\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.0 min\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFlow Rate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1 ml/min\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample Injector\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15\u0026micro;l loop\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDetection Wavelength\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e245 nm and 295 nm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eColumn Temperature\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAmbient\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eSolution Preparation Procedures\u003c/h3\u003e\n\u003cp\u003eComprehensive information on the procedures for sample preparation, the mobile phase formulation, and the analytical techniques employed in this work are described in supplementary material.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStock solution of Brimonidine Tartrate (1,000 ppm)\u003c/h2\u003e\u003cp\u003eAccurately weigh 50 mg of Brimonidine Tartrate and place it into a 50 mL volumetric flask. Add approximately 40 mL of deionized water and sonicate for about 10 minutes, or until the compound fully dissolves. Carefully fill the flask to the calibration mark with deionized water and mix well to ensure complete homogenization.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eStock solution of Timolol Maleate (1,000 ppm)\u003c/h3\u003e\n\u003cp\u003eWeigh precisely 50 mg of Timolol Maleate and transfer it into a 50 mL volumetric flask. Add around 40 mL of deionized water and sonicate for roughly 10 minutes or until fully dissolved. Then, top up to the volume mark with deionized water and shake gently to achieve uniform mixing.\u003c/p\u003e\n\u003ch3\u003e\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cb\u003eWorking standard solutions of BT (200ppm) and TM (500ppm)\u003c/b\u003e\u003c/div\u003e\u003cp\u003eTransfer 2 mL of Brimonidine Tartrate (1,000ppm) and 5 mL of Timolol Maleate (1,000ppm) into a 10 mL volumetric flask. Fill the flask to the 10 mL mark with distilled water and mix thoroughly to ensure a uniform solution.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSample Combigan Preparation (600 ppm of BT\u0026amp; 1,500 ppm of TM)\u003c/h2\u003e\u003cp\u003eTransfer 3 mL of Combigan eye drops and transfer it into a 10 mL volumetric flask. Then, fill the flask up to the 10 mL mark with distilled water, mixing thoroughly to ensure uniform dilution.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eMETHOD DEVELOPMENT \u0026amp; OPTIMIZATION\u003c/h2\u003e\u003cp\u003eThe main aim of this study was to design a simple, efficient, selective, and accurate RP-HPLC method specifically for quantifying Brimonidine Tartrate and Timolol Maleate (Combigan\u0026reg;) in bulk drug form. A critical goal was also to achieve effective separation of these active ingredients from related impurities and potential degradation products, without requiring extra purification steps. Various experimental parameters were systematically examined to identify optimal chromatographic conditions. Emphasis was placed on obtaining a high number of theoretical plates (reflecting superior column efficiency), sharp and symmetrical peak shapes, reduced tailing in raw material analysis, and robust separation of brimonidine tartrate and timolol maleate from impurities and degradants. Method development included a series of targeted trials to refine and validate these conditions, ultimately ensuring consistent, precise, and reproducible analytical performance. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eDetermination of the Wavelength of Maximum Absorbance\u003c/h2\u003e\u003cp\u003eTo identify the optimal detection wavelength, standard solutions of brimonidine tartrate (80 ppm), timolol maleate (50ppm), and Combigan (40/100 ppm, BT/TM) were scanned using UV spectroscopy over the range of 200\u0026ndash;400 nm, with the buffer solution serving as the reference blank. Brimonidine Tartrate exhibited maximum absorbance at 245 nm, Timolol Maleate at 295 nm, and Combigan at both 245 nm and 295 nm. Based on these results, 245 and 295 nm were selected as the analytical wavelength for the quantitative determination of Combigan as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eInfrared (IR) study for Brimonidine Tartrate and Timolol Maleate\u003c/h2\u003e\u003cp\u003eApproximately 100 mg of potassium bromide (KBr) and 2 mg of each sample Brimonidine Tartrate and Timolol Maleate were weighed separately for analysis. Each finely ground drug sample was thoroughly blended with the powdered KBr and then subjected to high pressure to form a transparent pellet. Under pressure, the potassium bromide fuses, embedding the active compound within a stable matrix to produce a thin disk suitable for infrared (IR) analysis. These prepared KBr pellets were carefully placed in the spectrometer\u0026rsquo;s sample holder for scanning. The resulting infrared spectra, shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e (Supplementary material), reveal the characteristic absorption bands associated with functional groups present in Brimonidine Tartrate and Timolol Maleate. The identified functional groups are summarized in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e (Supplementary material).\u003c/p\u003e\u003cp\u003eThermo Nicolet IR 200 Spectrometer\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eColumn Selection\u003c/h2\u003e\u003cp\u003eFive different C18 columns were conditioned by sequential flushing with solvent mixtures of 50:50, 75:25 acetonitrile (ACN): water, and finally 100% ACN, each for 30 minutes at a flow rate of 1 mL/min. This step ensured compliance with ICH Q2(R2) guidelines, [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] targeting tailing factors between 0.9\u0026ndash;2.0 and theoretical plate numbers above 2000 for acceptable peak symmetry and column efficiency. Each column was tested twice, with thorough cleaning and equilibration between runs to avoid cross-contamination. Brimonidine Tartrate and Timolol Maleate solutions (200 ppm and 500 ppm) were injected to evaluate column performance, with the columns maintained at ambient temperature to enhance reproducibility (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Table S2 (Supplementary material)).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAll five C18 columns were evaluated under identical chromatographic conditions to allow direct comparison. Columns #2, 4, and 5 failed to meet ICH acceptance criteria due to tailing factors exceeding 2.0, indicating poor peak symmetry. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] Column #5 exhibits the longest retention time for Timolol Maleate compared with the other columns due to its stationary phase characteristics, which result in stronger interactions with the analyte. Specifically, the chemistry of column #5 provides higher hydrophobic retention and/or selectivity for Timolol Maleate, causing it to elute later than in the other columns under the same mobile phase and flow conditions. Column #1 met the minimum requirements for tailing factor and theoretical plate count but showed lower efficiency compared to Column #3 and had a retention time too short for degradation studies. Ultimately, Column #3 was selected for further method validation, as it provided the best overall performance with a tailing factor\u0026thinsp;\u0026le;\u0026thinsp;2.0 and theoretical plate count\u0026thinsp;\u0026ge;\u0026thinsp;2000. Additional details are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eColumn selection results showing performance data for Peak 1 (Maleic acid), Peak 2 (Brimonidine tartrate), and Peak 3 (Timolol maleate).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eColumn\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePeak\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRetention Time (min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTailing Factor\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTheoretical Plates\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eResolution\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.328\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.603\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2571\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.028\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.152\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4928\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.232\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.596\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.754\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2908\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.638\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.636\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4469\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.061\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.275\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2921\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.639\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.657\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.347\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1277\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.190\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.472\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.349\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e12420\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.291\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.337\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e11171\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.226\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.218\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.594\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e12082\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.368\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.046\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.603\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2449\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.232\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.900\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1471\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.958\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.328\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.743\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2050\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.175\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.019\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.453\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7313\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.471\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.916\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2502\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.674\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6.560\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.112\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e882\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.247\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe Combigan\u0026reg; chromatogram displayed three distinct peaks. To identify them, individual standard solutions of 200 ppm Brimonidine Tartrate, 500 ppm Timolol Maleate, and 100 ppm Maleic Acid were injected under optimized conditions to establish their respective retention times as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eSelection pH of the Mobile Phase\u003c/h2\u003e\u003cp\u003ePrecise pH control is essential in RP-HPLC to reduce secondary interactions on silica-based columns, which can cause peak tailing and poor separation. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] Brimonidine Tartrate and Timolol Maleate solutions (200 ppm and 500 ppm) were tested at pH 2.9, 5.0, and 7.0 using a mobile phase of buffer (phase A) and acetonitrile (phase B) in a 80:20 v/v ratio. As summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, pH 7.0 produced the best peak shape and the highest theoretical plate count, indicating superior column efficiency. Therefore, a potassium phosphate monobasic buffer at pH 7.0 was selected for further method development and validation.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSummary of pH Optimization and Selection\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e\u003cp\u003eCombigan\u0026reg; Ophthalmic Solution\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eBrimonidine Tartrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e\u003cp\u003eTimolol Maleate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBuffer pH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRetention Time (min)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTailing Factor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTheoretical Plates\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eResolution\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eRetention Time (min)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eTailing Factor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eTheoretical Plates\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eResolution\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.185\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.355\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12849\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.164\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.143\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.598\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e11725\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e10.057\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.704\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.181\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8061\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.649\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.452\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.496\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e12550\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e7.760\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.662\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.943\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4864\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.341\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.176\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.643\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e13171\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e3.326\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eIsocratic Elution Studies\u003c/h2\u003e\u003cp\u003eDifferent ratios of buffer (pH 7.0) and acetonitrile (ACN) were evaluated to achieve retention times around 10 minutes, allowing adequate separation of impurities and degradants before the main analyte peaks. Brimonidine Tartrate and Timolol Maleate solutions (200 ppm and 500 ppm) were injected under these conditions. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, initial chromatograms showed peak tailing (TM: Tailing 2.191) due to secondary interactions. To address this, triethylamine (TEA) was added to the buffer, and its concentration was fine-tuned to produce sharper peaks and reduce tailing. Retention times, peak areas, and tailing factors are detailed in Table S3 (Supplementary material). Further optimization of ACN-to-buffer pH 7.0 ratios containing 30 mM TEA determined that a 20:80 v/v composition was optimal, yielding a retention time near 10 minutes and improving the resolution of impurities and degradation products.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eNominal Concentration Selection\u003c/h2\u003e\u003cp\u003eTo establish nominal concentrations for Brimonidine Tartrate and Timolol Maleate, calibration curves were generated within the detector\u0026rsquo;s linear range to ensure proportional response and minimize bias. Stock solutions (600 ppm and 1500 ppm, respectively) were diluted to prepare calibration standards: 0.24\u0026ndash;500 ppm for Brimonidine Tartrate and 0.60\u0026ndash;1250 ppm for Timolol Maleate. These were analyzed under optimized HPLC conditions, and peak areas (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) showed excellent linearity with R\u0026sup2; values of 0.9994 for Brimonidine Tartrate and 0.9996 for Timolol Maleate (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). As a result, 200 ppm of Brimonidine Tartrate and 500 ppm of Timolol Maleate were chosen as nominal concentrations for further method validation.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSummary of peak areas at different concentrations for determining the nominal concentration\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eBrimonidine Tartrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eTimolol Maleate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eConcentration (ppm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eConcentration (ppm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e48\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e358\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e476\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1787\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2380\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3576\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4759\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6750\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8986\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e320\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11054\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e800\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14890\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13327\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18043\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16553\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e22514\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eForced Degradation Studies\u003c/h2\u003e\u003cp\u003eForced degradation studies are a fundamental component in evaluating and understanding the intrinsic stability characteristics of pharmaceutical compounds. This approach systematically subjects the drug to a variety of stringent conditions to explore its degradation pathways and identify potential degradation products. Beyond merely observing degradation, such studies also play a pivotal role in partially validating the specificity of analytical methods developed for detecting related substances, including degradation products. Additionally, the chosen stress conditions (concentration, exposure time, and intensity) were selected to ensure sufficient degradation\u0026thinsp;\u0026le;\u0026thinsp;10% without causing complete analyte breakdown, in accordance with ICH Q1A (R2) recommendations.[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] Ultimately, this ensures more accurate impurity detection and contributes to safeguarding the quality, efficacy, and safety of pharmaceutical products used in clinical practice.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eAcid Degradation\u003c/h2\u003e\u003cp\u003eTransfer 2 mL of the Brimonidine Tartrate stock solution (1000 ppm) into a screw-cap test tube. Add 2 mL of 3 M HCl, then place the tube on a heating block set to 75\u0026deg;C and heat for 24 hours to induce forced degradation. After heating, allow the solution to return to room temperature, then carefully neutralize by adding 2 mL of 3 M NaOH. Transfer the neutralized solution into a 10 mL volumetric flask and dilute to the mark with deionized water, mixing thoroughly to obtain a final concentration of 200 ppm. In parallel, transfer 2 mL of the Timolol Maleate stock solution (1500 ppm) into a separate screw-cap test tube. Add 2 mL of 1 M HCl, then heat on a heating block at 75\u0026deg;C for 24 hours. After heating, cool to room temperature and neutralize by adding 2 mL of 1 M NaOH. Dilute the neutralized solution appropriately with deionized water, mixing well to achieve a final concentration of 500 ppm. Before HPLC analysis, confirm that each prepared solution is neutral (pH\u0026thinsp;~\u0026thinsp;7\u003cb\u003e)\u003c/b\u003e using pH indicator strips. Finally, filter each solution through a 0.45 \u0026micro;m membrane filter to remove particulates, then inject into the HPLC system for analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eBase (Alkali) Degradation\u003c/h2\u003e\u003cp\u003eTransfer 2 mL of the Brimonidine Tartrate stock solution (1000 ppm) into a screw-cap test tube. Add 2 mL of 3 M NaOH, then place the tube on a heating block maintained at 75\u0026deg;C and heat for 24 hours to induce forced alkaline degradation. After heating, allow the solution to cool to room temperature and carefully neutralize by adding 2 mL of 3 M HCl. Transfer the neutralized solution to a 10 mL volumetric flask and dilute to volume with deionized water, mixing thoroughly to achieve a final concentration of 200 ppm. In parallel, transfer 2 mL of the Timolol Maleate stock solution (1500 ppm) into a separate screw-cap test tube. Add 2 mL of 0.05 M NaOH, then heat on a heating block at 75\u0026deg;C for 24 hours to induce degradation. After heating, cool the solution to room temperature and neutralize by adding 2 mL of 0.05 M HCl. Dilute the neutralized solution as needed with deionized water, mixing well to reach a final concentration of 500 ppm. Prior to HPLC analysis, verify that both prepared solutions are at neutral pH (~\u0026thinsp;7) using pH indicator strips. Finally, filter each solution through a 0.45 \u0026micro;m membrane filter to remove any particulates before injecting into the HPLC system for analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eHydrogen Peroxide Degradation (Oxidation)\u003c/h2\u003e\u003cp\u003eTransfer 2 mL of the Brimonidine Tartrate stock solution \u003cb\u003e(\u003c/b\u003e1000 ppm\u003cb\u003e)\u003c/b\u003e into a screw-cap test tube. Add 2 mL of 3% hydrogen peroxide (H₂O₂), then place the tube on a heating block maintained at 75\u0026deg;C and heat for 24 hours to induce oxidative degradation. After cooling the solution to room temperature, transfer it into a 10 mL volumetric flask, dilute to volume with deionized water, and mix thoroughly to obtain a final concentration of 200 ppm. Prior to HPLC analysis, filter the prepared solution through a 0.45 \u0026micro;m membrane filter to remove any particulates. In parallel, transfer 2 mL of the Timolol Maleate stock solution (1500 ppm) into a separate screw-cap test tube. Add 2 mL of 0.05% hydrogen peroxide (H₂O₂) and heat the mixture on a heating block at 75\u0026deg;C for 24 hours. Once cooled to room temperature, transfer the solution into a 6 mL volumetric flask, dilute to the mark with deionized water, and mix thoroughly to achieve a final concentration of 500 ppm. Filter this solution as well through a 0.45 \u0026micro;m membrane filter before injection into the HPLC system.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eThermal Degradation (Heat)\u003c/h2\u003e\u003cp\u003eTransfer 2 mL of the Brimonidine Tartrate stock solution (1000 ppm) into a screw-cap test tube and place it on a heating block set to 75\u0026deg;C for 24 hours to induce thermal degradation. After heating, allow the solution to cool to room temperature. Transfer the entire contents into a 10 mL volumetric flask, dilute to the mark with deionized water, and mix thoroughly to achieve a final concentration of 200 ppm. Prior to HPLC analysis, filter the prepared solution through a 0.45 \u0026micro;m membrane filter to remove any particulates. Similarly, transfer 2 mL of the Timolol Maleate stock solution (1500 ppm) into a separate screw-cap test tube and heat under the same conditions (75\u0026deg;C for 24 hours). Once cooled to room temperature, transfer the solution into a 6 mL volumetric flask and dilute to volume with deionized water, mixing well to ensure homogeneity before analysis.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003ePhotolysis (UV Light) Stress Study\u003c/h2\u003e\u003cp\u003eWeigh approximately 20 mg of Brimonidine Tartrate and place it in a cuvette. Expose the sample to ultraviolet (UV) light at 245 nm for 24 hours to induce photodegradation. After exposure, accurately transfer 2 mg of the degraded sample into a 10 mL volumetric flask. Add 5 mL of deionized water, then sonicate for 20 minutes or until the drug is completely dissolved. Dilute to volume with deionized water and mix thoroughly to achieve a final concentration of 200 ppm. Prior to HPLC analysis, filter the solution through a 0.45 \u0026micro;m membrane filter to remove particulates. Similarly, weigh approximately 50 mg of Timolol Maleate and place it in a cuvette. Expose the sample to UV light at 295 nm for 24 hours. Following exposure, accurately transfer 5 mg into a 10 mL volumetric flask. Add 5 mL of deionized water and sonicate for 20 minutes or until fully dissolved. Bring to volume with deionized water and mix thoroughly to achieve a final concentration of 500 ppm. Filter this solution through a 0.45 \u0026micro;m membrane filter before injecting into the HPLC system.\u003c/p\u003e\u003cp\u003eBrimonidine Tartrate demonstrated notable stability under thermal and photolytic conditions, showing no detectable degradation after 24 hours at 75\u0026deg;C or following exposure to UV light at 245 nm for 24 hours. Under acidic stress (3 M HCl at 75\u0026deg;C for 24 hours), minimal degradation was observed (0.35%). Alkaline conditions (3 M NaOH at 75\u0026deg;C for 24 hours) produced slightly higher degradation (1.45%), indicating greater susceptibility to basic hydrolysis. Oxidative stress using 3% H₂O₂ at 75\u0026deg;C for 24 hours led to moderate degradation (0.798%). Overall, Brimonidine Tartrate was most sensitive to alkaline and oxidative conditions, while remaining highly stable under heat and UV light as illustrated in Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eTimolol Maleate demonstrated excellent stability under thermal (24 h at 75\u0026deg;C) and photolytic (24 h at 295 nm) conditions, showing no detectable degradation. However, it showed significant sensitivity to both acidic and basic hydrolysis, with degradation increasing alongside acid or alkali strength: 22.5% degradation in 3 M HCl and 24.7% in 1 M NaOH. Lower concentrations of acid (1 M HCl) and base (0.05 M NaOH) resulted in 5.8% and 5.6%, respectively. Timolol Maleate was especially susceptible to oxidative stress, with 58.5% degradation after exposure to 3% H₂O₂, indicating pronounced sensitivity to oxidation. Overall, the data highlight that Timolol Maleate is chemically stable under heat and light but degrades notably under strong acidic, basic, and oxidative conditions as seen in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eOverview of Brimonidine Tartrate and Timolol Maleate Degradation Procedures (Thermal, Photolytic, Acidic, Alkaline, and Oxidative); *: Heat it on a heating block at 75\u0026deg;C for the specified duration.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003eBrimonidine Tartrate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eStress Condition\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eTime Heated\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eTemperature\u003c/b\u003e*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eArea\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eDegradation\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6052\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHeat\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6051\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhotolysis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e245 nm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6050\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3 M HCl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6030\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.363%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3 M NaOH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5965\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.438%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6005\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.776%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTimolol Maleate\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eStress Condition\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eTime Heated\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eTemperature*\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eArea\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eDegradation\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8415\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHeat\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8414\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhotolysis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e295 nm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8415\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3 M HCl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6450\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e23.35%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1 M HCl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7950\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.52%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eStress Condition\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eTime Heated\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eTemperature\u003c/b\u003e*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eArea\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eDegradation\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8415\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1 M NaOH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1 hour\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6402\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e23.92%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.1 M NaOH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1 hour\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e14.43%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.05 M NaOH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1 hour\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7949\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.54%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eStress Condition\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eTime Heated\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eTemperature\u003c/b\u003e*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eArea\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eDegradation\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8415\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1 hour\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3604\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e57.17%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.5% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1 hour\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7610\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.57%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eMixed Degradation Study (Acid, Base, and Oxidation)\u003c/h2\u003e\u003cp\u003eThe purpose of the mixed degradation study was to evaluate whether the developed HPLC method could effectively separate Brimonidine Tartrate and Timolol Maleate from their potential degradation products and impurities, thus ensuring reliable quantification and purity assessment [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. To establish a baseline, control samples containing 200 ppm of Brimonidine Tartrate, and 500 ppm of Timolol Maleate were prepared and injected into the HPLC system. The resulting chromatogram, presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e (A), was used to calculate the percentage degradation after subjecting the samples to stress conditions. For the degradation mixture, 1 mL aliquots of Brimonidine Tartrate stress samples that showed less than 10% degradation (treated with 3 M HCl for 24 h, 3 M NaOH for 24 h, and 3% H₂O₂ for 24 h) were combined. Similarly, 1 mL aliquots of Timolol Maleate stress samples (treated with 1 M HCl for 24 h, 0.05 M NaOH for 1 h, and 0.5% H₂O₂ for 1 h) were mixed. The combined solution was filtered using a 0.45 \u0026micro;m membrane filter and analyzed. The chromatogram from this mixed degradation test is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e (B). The analysis demonstrated that all degradation peaks were completely separated from the main Brimonidine Tartrate and Timolol Maleate peaks, which appeared at approximately 5.8 min and 8.8 min, respectively. This clear resolution confirms the method\u0026rsquo;s specificity and its ability to distinguish the active ingredients from their degradation products. Overall, the findings confirm that the developed RP-HPLC method is stability-indicating and fit for accurate quantification of Brimonidine Tartrate and Timolol Maleate in the presence of degradants. A summary of the degradation results for each stress condition is provided in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSummary of degradation results under different stress conditions for Brimonidine Tartrate and Timolol Maleate; *: Heat it on a heating block at 75\u0026deg;C for the specified duration.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e\u003cp\u003eBrimonidine Tartrate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStress condition\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExposed Time\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003eTemperature (\u0026deg;C) *\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eColor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e% Degradation\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eClear\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6052\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3M HCl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003e75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eClear\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6030\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.363%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3M NaOH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003e75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eClear\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5965\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.438%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3.0% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003e75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eClear\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6005\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.776%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMixture solution\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eClear\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5960\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.520%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTimolol Maleate\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eClear\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8415\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e----\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1M HCl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e24 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eClear\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7950\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5.52%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.05M NaOH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eOne hour\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eClear\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7949\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5.54%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.5% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eOne hour\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eClear\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7610\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e9.57%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMixture solution\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eClear\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7595\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e9.74%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eMETHOD VALIDATION\u003c/h2\u003e\u003cp\u003eTo comply with GLP and GMP standards, the developed analytical method was validated following ICH Q2A/Q2B, [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] FDA, [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] and USP [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] guidelines. Key validation parameters included:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eSystem Suitability\u003c/b\u003e: Confirmed consistent HPLC performance before analysis.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eSpecificity\u003c/b\u003e: Ensured clear separation of BT and TM from impurities and degradants.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eRobustness\u003c/b\u003e: Tested stability under minor changes in pH and solvent composition.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eSolution Stability\u003c/b\u003e: Verified analyte stability during the test period.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eLinearity and Range\u003c/b\u003e: Confirmed accurate detection across relevant concentrations.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eAccuracy and Precision\u003c/b\u003e: Assessed repeatability, injection consistency, and analyst variability.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eLOD and LOQ\u003c/b\u003e: Defined the lowest detectable and quantifiable levels.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eThis comprehensive validation demonstrated the method\u0026rsquo;s reliability and suitability for routine analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eSYSTEM SUITABILITY\u003c/h2\u003e\u003cp\u003eSystem suitability testing (SST) is an essential step in HPLC analysis, as outlined in ICH Q2(R1) guidelines, ensuring the entire system including equipment, software, and columns is operating consistently and accurately before running actual samples.[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] Key acceptance criteria include % RSD of replicate injections not more than 1%, %RSD of retention times not more than 1, number of theoretical plates at least 2000, tailing factor between 0.9 and 2, capacity factor greater than 2, resolution between peaks above 2, and drift within 2%. By meeting these parameters the method confirms it can deliver precise and reproducible results and supports the reliability of subsequent analytical testing. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] System suitability evaluation was carried out by injecting two prepared working standard solutions of Brimonidine Tartrate (200 ppm, labeled as WS #1 and WS #2) and Timolol Maleate (500 ppm, also labeled as WS #1 and WS #2). To verify the consistency and reliability of the HPLC system, WS #1 was injected sequentially six times, while WS #2 was injected twice, as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. These injections provided data to calculate critical system suitability metrics, such as the %RSD of peak areas and retention times for both WS #1 and WS #2, confirming stable detector response and reproducible retention behavior. Additionally, tailing factors and theoretical plate counts were determined for each analyte peak to assess peak symmetry and column performance, ensuring the method\u0026rsquo;s robustness and precision. The percentage drift (%Drift) was calculated by using the following equation:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{%}\\text{D}\\text{r}\\text{i}\\text{f}\\text{t}\\:=\\frac{\\text{A}\\text{s}-\\text{A}\\text{c}}{\\text{A}\\text{s}}\\:\\text{x}\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWhere, As is the average peak area from six consecutive injections of working standard solution #1, and Ac is the average peak area from two injections of working standard solution #2.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSystem Suitability Results for Brimonidine Tartrate and Timolol Maleate Working Standards\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e\u003cp\u003eBrimonidine Tartrate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStandard 1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRetention Time\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRetention Time\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; %RSD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRetention Time\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;%Drift\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTailing Factor\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTheoretical Plates\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003ePeak Area\u0026nbsp;%RSD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003ePeak Area \u0026nbsp; \u0026nbsp; %Drift\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.963\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e0.153%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"8\" rowspan=\"9\"\u003e\u003cp\u003e0.029%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.999\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8201\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6155\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e0.847%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\" morerows=\"8\" rowspan=\"9\"\u003e\u003cp\u003e0.550%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.965\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.994\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7985\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6099\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.953\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.997\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8283\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6138\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.954\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.983\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7940\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6051\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection 5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.952\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.987\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7932\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6018\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection 6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.951\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.994\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8277\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6097\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eStandard 2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eRetention Time\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eRetention Time\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; %RSD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTailing Factor\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eTheoretical Plates\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePeak Area\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003ePeak Area %RSD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.959\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.054%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.990\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7961\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6097\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.875%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.956\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.988\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8198\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6022\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e\u003cp\u003eTimolol Maleate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStandard 1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRetention Time\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRetention Time\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; %RSD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRetention Time\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; %Drift\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTailing Factor\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTheoretical Plates\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003ePeak Area\u0026nbsp;%RSD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003ePeak Area \u0026nbsp; %Drift\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e0.163%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"8\" rowspan=\"9\"\u003e\u003cp\u003e0.022%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.253\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8689\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8432\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e\u003cb\u003e0.604%\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\" morerows=\"8\" rowspan=\"9\"\u003e\u003cp\u003e0.137%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.869\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.264\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8701\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8406\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.845\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.259\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8640\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8329\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.848\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.245\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8648\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8415\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection 5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.848\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.244\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8646\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8378\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection 6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.853\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.231\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8659\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8305\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eStandard 2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eRetention Time\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eRetention Time\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; %RSD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTailing Factor\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eTheoretical Plates\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePeak Area\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003ePeak Area\u0026nbsp;%RSD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.859\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.041%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.265\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8352\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6097\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003e0.875%\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.855\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.268\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8340\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6022\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eSpecificity\u003c/h2\u003e\u003cp\u003eSpecificity ensures the method can accurately detect and quantify Brimonidine Tartrate and Timolol Maleate without interference from impurities or degradants. Acceptance criteria include a peak purity factor\u0026thinsp;\u0026gt;\u0026thinsp;990, resolution\u0026thinsp;\u0026gt;\u0026thinsp;2 for clear separation, and no overlapping peaks. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] This confirms the method selectively measures Brimonidine Tartrate and Timolol Maleate in complex sample matrices.[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] To prepare the mixed degradation samples, 1 mL portions from acid, base, and oxidative stress solutions each showing less than 10% degradation were combined. For Brimonidine Tartrate, this included 1 mL each from samples treated with 3 M HCl for 24 hours, 3 M NaOH for 24 hours, and 3% hydrogen peroxide for 24 hours. In parallel, 1 mL portions were taken from Timolol Maleate stress solutions exposed to 1 M HCl for 24 hours, 0.05 M NaOH for 1 hour, and 0.5% hydrogen peroxide for 1 hour. The mixed solution was thoroughly blended and passed through a 0.45 \u0026micro;m membrane filter to remove particulates. The prepared sample was then analyzed on an Agilent 1100 HPLC system equipped with a diode array detector (DAD). The chromatographic profile obtained from this mixed degradation study is presented in Figure S2 (Supplementary material).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eSolution Stability\u003c/h2\u003e\u003cp\u003eSolution stability testing was performed to evaluate the behavior of Brimonidine Tartrate and Timolol Maleate over time under defined environmental conditions, including temperature, light, and humidity. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] This step is essential in method validation to ensure accurate and consistent quantification during routine analysis. Stability was assessed by comparing peak areas from the initial injection with those obtained at 24, 48, and 72 hours. Throughout this period, no new peaks or missing peaks were observed in the chromatograms, confirming that the solution remained stable with no significant degradation. The solutions remained stable at room temperature for at least 3 days, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSolution Stability of Brimonidine Tartrate and Timolol Maleate Over 3 Days at Room Temperature\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003eCombigan\u0026reg; Ophthalmic Solution\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eBrimonidine Tartrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eTimolol Maleate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTime (hours)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e%Change of Peak Areas\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e%Change of Peak Areas\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7225\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-----\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9898\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-----\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7222\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.042%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9852\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.465%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7218\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.097%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9933\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.354%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7215\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.138%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9924\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.263%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMethod Robustness\u003c/h3\u003e\n\u003cp\u003eThe robustness of an analytical method evaluates its ability to produce consistent and reliable results despite small, deliberate variations in method parameters. As outlined in ICH guidelines, this step is crucial for confirming that the method can withstand routine fluctuations during practical use. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] In this study, robustness was tested by slightly altering five key parameters: Buffer pH (7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2), Flow rate (1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 mL/min), Detection wavelength (245\u0026thinsp;\u0026plusmn;\u0026thinsp;2 nm) and (295\u0026thinsp;\u0026plusmn;\u0026thinsp;2 nm), Mobile phase B composition (20\u0026thinsp;\u0026plusmn;\u0026thinsp;2%B), and Injection volume (15\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026micro;L). The method\u0026rsquo;s performance under these variations was evaluated using the following acceptance criteria: a tailing factor between 0.9 and 2.0, a theoretical plate count of at least 2000, and resolution (RS) greater than 2.0 between all peaks and the target analyte. Robustness was assessed by deliberately varying critical method parameters: adjusting the mobile phase pH by \u0026plusmn;\u0026thinsp;0.2, changing the percentage of organic solvent (acetonitrile) in the mobile phase by \u0026plusmn;\u0026thinsp;2%, altering the column temperature by \u0026plusmn;\u0026thinsp;2\u0026deg;C, shifting the detection wavelength by \u0026plusmn;\u0026thinsp;2 nm, and modifying the flow rate by \u0026plusmn;\u0026thinsp;0.2 mL/min. These controlled changes showed no significant impact on chromatographic resolution as shown in Table\u0026nbsp;\u003cspan refid=\"Tab9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, confirming the method\u0026rsquo;s robustness.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab9\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 9\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMethod Robustness Evaluation: Results for Mixed Degradation Samples under Parameter Variations\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eBrimonidine Tartrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eTimolol Maleate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVariation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTailing Factor\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTheoretical Plates\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTailing Factor\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTheoretical Plates\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eBuffer pH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.988\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8983\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.258\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9367\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.102\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9658\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.246\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9516\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.913\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10156\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.902\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10230\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eWavelength (nm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e293\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.057\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9341\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.223\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9322\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e295\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.102\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9658\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.246\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9516\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e297\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.009\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9336\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.197\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9321\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eWavelength (nm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e243\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.101\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7908\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.9982\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8123\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e245\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.090\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8109\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.109\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8201\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e247\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.103\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8120\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.009\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8423\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e%B composition\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e18%ACN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.239\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9539\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.265\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9851\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20%ACN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.102\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9658\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.246\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9516\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e22%ACN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.921\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9620\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.913\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9729\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eInjection Volume (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003eL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.209\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9430\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.065\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9457\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.212\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9610\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.246\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9636\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.243\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9543\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.434\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9534\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eFlow rate mL/min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9399\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.065\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9457\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.120\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9680\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.212\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9636\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.321\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9483\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.367\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9534\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003eLinearity and range for active ingredient\u003c/h2\u003e\u003cp\u003eThe validated linearity of the method was established across the ranges of 0-500 ppm for Brimonidine Tartrate and 250\u0026ndash;1250 ppm for Timolol Maleate using pure standards. For analysis of Combigan\u0026reg; dosage form, additional calibration curves were constructed within narrower ranges (100\u0026ndash;240 ppm for Brimonidine Tartrate and 250\u0026ndash;600 ppm for Timolol Maleate) to reflect actual sample concentrations. The correlation coefficient (R\u0026sup2;) for Brimonidine Tartrate and Timolol Maleate must be at least 0.999 to meet acceptance criteria. This ensures a strong linear relationship between concentration and detector response across the tested range, confirming the method\u0026rsquo;s reliability for accurate quantitative analysis. A stock solution of Brimonidine Tartrate (400 ppm) and Timolol Maleate (1000 ppm) was used to prepare a series of diluted solutions at concentrations of 100 ppm, 160 ppm, 200 ppm, 220 ppm, and 240 ppm for Brimonidine Tartrate, and 250 ppm, 400 ppm, 500 ppm, 550 ppm, and 600 ppm for Timolol Maleate. These solutions were analyzed using HPLC under optimized isocratic elution conditions. The resulting summary of the peak area results provided in Table\u0026nbsp;\u003cspan refid=\"Tab10\" class=\"InternalRef\"\u003e10\u003c/span\u003e. The linear regression equations were Y\u0026thinsp;=\u0026thinsp;46.365 X \u0026ndash; 1545.2 (R\u0026sup2; = 0.9992) for Brimonidine Tartrate and Y\u0026thinsp;=\u0026thinsp;25.334 X \u0026ndash; 2174.8 \u003cb\u003e(\u003c/b\u003eR\u0026sup2; \u003cb\u003e=\u003c/b\u003e 0.9996\u003cb\u003e)\u003c/b\u003e for Timolol Maleate, where \u003cem\u003eY\u003c/em\u003e is the peak area of the standard solution and \u003cem\u003eX\u003c/em\u003e is the drug concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab10\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 10\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eLinearity results for Brimonidine Tartrate and Timolol Maleate\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003eCombigan\u0026reg; Ophthalmic Solution\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eBrimonidine Tartrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eTimolol Maleate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample Preparation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eConcentration\u003c/p\u003e\u003cp\u003e(ppm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAverage Peak Area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eConcentration\u003c/p\u003e\u003cp\u003e(ppm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAverage Peak Area\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3126\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e3117\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4649\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e4195\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3167\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3949\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3058\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3987\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5875\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e5852\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7937\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e7891\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5902\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7846\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5780\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7890\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7753\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e7731\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10597\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e10539\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7651\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10567\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7789\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10452\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e220\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8702\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e8554\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e550\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e11695\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e11684\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8589\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e11605\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8372\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e11753\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e240\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9835\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e9676\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e600\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13031\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e13086\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9521\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13101\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9672\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13126\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003eAccuracy\u003c/h2\u003e\u003cp\u003eAccuracy of the developed HPLC method focused on evaluating how well the experimentally measured concentrations matched their known theoretical values.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] Brimonidine Tartrate and Timolol Maleate solutions were prepared in triplicate at three concentration levels: 80%, 100%, and 120% of the nominal concentrations (200 ppm and 500 ppm, respectively). According to the acceptance criteria, the percent recovery for the active ingredients needed to fall within 95% to 105% of the target value. Meeting these criteria demonstrates that the method consistently and accurately measures the active ingredients and can also reliably detect impurities and degradants over the tested concentration range. These samples were analyzed by injecting them into the HPLC system, and the resulting peak areas were recorded. The percentage recovery of each active ingredient was then calculated using the linear regression equations obtained from the calibration curves: Y\u0026thinsp;=\u0026thinsp;46.365 X \u0026ndash; 1545.2 for Brimonidine Tartrate and Y\u0026thinsp;=\u0026thinsp;25.334 X \u0026ndash; 2174.8 for Timolol Maleate, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. By applying these equations, the measured concentrations were compared to the theoretical concentrations to calculate percent recovery. The summarized results in Table\u0026nbsp;\u003cspan refid=\"Tab11\" class=\"InternalRef\"\u003e11\u003c/span\u003e confirm that the method meets the established accuracy criteria, achieving recoveries within the acceptable range of 95\u0026ndash;105%.\u003c/p\u003e\u003cdiv id=\"Sec33\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e%Recovery =\u003c/b\u003e \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\varvec{C}\\varvec{s}\\varvec{a}\\varvec{m}\\varvec{p}\\varvec{l}\\varvec{e}}{\\varvec{C}\\varvec{s}\\varvec{t}\\varvec{a}\\varvec{n}\\varvec{d}\\varvec{a}\\varvec{r}\\varvec{d}}\\varvec{x}100\\)\u003c/span\u003e\u003c/span\u003e\u003c/h2\u003e\u003cp\u003e\u003cb\u003eCsample\u003c/b\u003e is the measured concentration of the sample calculated from the linear regression equation; \u003cb\u003eCstandard\u003c/b\u003e is the known (theoretical) concentration of the prepared sample.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab11\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 11\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAccuracy Results for Brimonidine Tartrate and Timolol Maleate\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e\u003cp\u003eCombigan\u0026reg; Ophthalmic Solution\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eBrimonidine Tartrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e\u003cp\u003eTimolol Maleate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample Preparation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eConcentration\u003c/p\u003e\u003cp\u003e(ppm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAverage Peak Area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eRecovery\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eConcentration\u003c/p\u003e\u003cp\u003e(ppm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eAverage Peak Area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRecovery\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5764\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e5859\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e99.81%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7757\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e7857\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e99.00%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5843\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7843\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5971\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7971\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7598\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e7674\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e99.42%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10437\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e10328\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e98.71%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7643\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10176\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7781\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10372\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e240\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9614\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e9558\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e99.78%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e600\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e12981\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e13190\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e101.10%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9535\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e13253\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9526\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e13336\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\u003ch2\u003eMethod Precision\u003c/h2\u003e\u003cp\u003ePrecision in analytical method validation measures how consistently the method produces similar results under defined conditions. As outlined by ICH guidelines, [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] precision is evaluated at three key levels:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eRepeatability\u003c/b\u003e \u0026ndash; assesses the closeness of results when the same analyst performs multiple injections or measurements on the same day, using identical equipment and procedures.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eIntermediate Precision\u003c/b\u003e \u0026ndash; examines method performance within the same laboratory under varied conditions, such as different analysts, days, or instruments, to identify potential sources of variability.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eReproducibility\u003c/b\u003e \u0026ndash; tests the method\u0026rsquo;s consistency across different laboratories, ensuring broader reliability of results beyond a single lab setting.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eThese levels demonstrate the method\u0026rsquo;s ability to consistently deliver accurate, reliable, and reproducible data under routine and variable conditions.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eRepeatability (Method Precision)\u003c/h3\u003e\n\u003cp\u003eRepeatability assesses the method\u0026rsquo;s short-term consistency by examining variability that could result from repeated sample preparation and analysis by the same analyst under identical conditions.[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] This test highlights the method\u0026rsquo;s robustness to small operational fluctuations, ensuring reproducible and dependable results in routine analysis. Regulatory guidelines set strict acceptance criteria for repeatability: the %Relative Standard Deviation (%RSD) of peak areas for the target compound must be not more than 1% (NMT 1%). [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] In this study, repeatability was evaluated by preparing and injecting six independent samples of Brimonidine Tartrate (200 ppm) and Timolol Maleate (500 ppm) into the HPLC system under the validated chromatographic conditions. The %RSD of the peak areas from these six injections was then calculated, demonstrating the method\u0026rsquo;s precision and compliance with the defined criteria, as summarized in Table\u0026nbsp;\u003cspan refid=\"Tab12\" class=\"InternalRef\"\u003e12\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab12\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 12\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRepeatability Precision Results for Brimonidine Tartrate and Timolol Maleate\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e\u003cp\u003eCombigan\u0026reg; Ophthalmic Solution\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eBrimonidine Tartrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e\u003cp\u003eTimolol Maleate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample Preparation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAverage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eStandard Deviation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePeak Area %RSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAverage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eStandard Deviation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ePeak Area %RSD\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7269\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e7245\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e39.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e0.549%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10060\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e10062\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e85.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e0.848%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7187\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9945\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7232\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10040\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7217\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10019\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7295\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10194\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7268\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10116\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eInjection Precision\u003c/h3\u003e\n\u003cp\u003eInjection precision evaluates the method\u0026rsquo;s ability to consistently produce reliable results by examining instrument-related variability, including potential fluctuations from the injector, detector, column, and data integration process during repeated sample injections.[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] According to the established acceptance criteria, the %Relative Standard Deviation (%RSD) of the peak areas for Brimonidine Tartrate (200 ppm) and Timolol Maleate (500 ppm) must not exceed 1%, ensuring high reproducibility. To assess this, a single preparation of Brimonidine Tartrate (200 ppm) and Timolol Maleate (500 ppm) was injected six consecutive times under the method\u0026rsquo;s optimized HPLC conditions. The calculated %RSD values confirmed compliance with the acceptance threshold, demonstrating excellent injection precision. The summarized results are provided in Table\u0026nbsp;\u003cspan refid=\"Tab13\" class=\"InternalRef\"\u003e13\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab13\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 13\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInjection Precision Results for Brimonidine Tartrate and Timolol Maleate\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e\u003cp\u003eCombigan\u0026reg; Ophthalmic Solution\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eBrimonidine Tartrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e\u003cp\u003eTimolol Maleate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAverage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eStandard Deviation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePeak Area %RSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAverage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eStandard Deviation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ePeak Area %RSD\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7468\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e7420\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e63.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e0.859%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10316\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e10406\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e84.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e0.808%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7306\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10456\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7401\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10294\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7413\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10431\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7452\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10512\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7480\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10425\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eIntermediate precision\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eInter-day Precision /Ruggedness)\u003c/span\u003e\u003c/p\u003e\u003cp\u003eIntermediate precision of the developed analytical method for Brimonidine Tartrate and Timolol Maleate was evaluated to verify its robustness when subjected to deliberate variations, such as different analysts, instruments, columns, and testing days. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] This level of validation is essential to demonstrate the method\u0026rsquo;s reliability beyond controlled laboratory conditions. As specified by acceptance criteria, the %Relative Standard Deviation (%RSD) of peak areas for Brimonidine Tartrate at 200 ppm and Timolol Maleate at 500 ppm must not exceed 1.5%. To assess this, six replicate samples of each analyte were prepared and analyzed under the same optimized chromatographic conditions, but on separate HPLC systems, by different analysts, and across multiple days. The resulting data, summarized in Table\u0026nbsp;\u003cspan refid=\"Tab14\" class=\"InternalRef\"\u003e14\u003c/span\u003e, demonstrate the method\u0026rsquo;s ability to produce consistent and reproducible measurements despite normal variations in testing conditions. Intermediate precision parameters evaluated for this method include:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eHPLC\u003c/b\u003e: 1100 Series HPLC system with MWD (UV/VIS Detector), Agilent Technologies\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eColumn\u003c/b\u003e: Water XTERRA RP-18 (4.6x250 mm, 5\u0026micro;m)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eMobile Phase\u003c/b\u003e: Solvent A: 25mM potassium phosphate monobasic buffer, pH 7.0\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSolvent B: 100%ACN\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eSolvent Strength\u003c/b\u003e: (80:20 v/v) Buffer pH 7.0: ACN\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eAbsorbance\u003c/b\u003e: 245 nm and 295 nm\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eFlow Rate\u003c/b\u003e: 1.0 mL/minute\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eInjection Volume\u003c/b\u003e: 15 \u0026micro;L\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eColumn Temperature\u003c/b\u003e: Ambient\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab14\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 14\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eIntermediate Precision Results Under Varying Analysts, Instruments, and Columns\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e\u003cp\u003eCombigan\u0026reg; Ophthalmic Solution\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eBrimonidine Tartrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e\u003cp\u003eTimolol Maleate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample Preparation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAverage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eStandard Deviation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePeak Area %RSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAverage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eStandard Deviation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ePeak Area %RSD\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7159\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e7239\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e63.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e0.884%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10155\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e10065\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e136.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e1.354%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7243\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9942\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7215\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10170\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7319\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9973\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7191\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10232\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7308\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9916\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec37\" class=\"Section2\"\u003e\u003ch2\u003eLimit of Detection (LOD) and Limit of Quantitation (LOQ)\u003c/h2\u003e\u003cp\u003eThe limit of detection (LOD) was evaluated to determine the lowest concentration at which the analyte could be consistently detected, though not necessarily quantified with precision. Brimonidine Tartrate and Timolol Maleate were used as representative compounds to model impurities and degradation products. The LOD was established by assessing the signal-to-noise (S/N) ratio in the chromatographic response, with an acceptance criterion of an S/N ratio\u0026thinsp;\u0026ge;\u0026thinsp;3, indicating the method's reliable detection capability. To determine the limit of quantitation (LOQ) for Brimonidine Tartrate and Timolol Maleate, a series of dilutions were prepared, and their signal-to-noise ratios were thoroughly evaluated. Preliminary LOQ values were estimated at 0.24 ppm for Brimonidine Tartrate and 0.60 ppm for Timolol Maleate, based on achieving an S/N ratio of \u0026ge;\u0026thinsp;10 and a %RSD\u0026thinsp;\u0026le;\u0026thinsp;10%, as summarized in Table\u0026nbsp;\u003cspan refid=\"Tab15\" class=\"InternalRef\"\u003e15\u003c/span\u003e. To confirm these estimates, fresh solutions at 0.24 ppm and 0.60 ppm were injected ten consecutive times into the HPLC system. This allowed for assessment of both precision and detection reliability. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab16\" class=\"InternalRef\"\u003e16\u003c/span\u003e, the %RSD for both analytes remained within acceptable limits (\u0026le;\u0026thinsp;10%), validating these concentrations as the LOQ for the developed method.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab15\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 15\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eResults of Limit of Detection (LOD) Study for Brimonidine Tartrate and Timolol Maleate solutions\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e\u003cp\u003eCombigan\u0026reg; Ophthalmic Solution\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eBrimonidine Tartrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eTimolol Maleate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eConcentration (ppm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSignal to Noise ratio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eConcentration (ppm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSignal to Noise ratio\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e17.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e18.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab16\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 16\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eResults of Limit of Quantitation (LOQ) Study for Brimonidine Tartrate and Timolol Maleate solutions\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003eCombigan\u0026reg; Ophthalmic Solution\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNo\u003c/b\u003e.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003e\u003cb\u003eBrimonidine Tartrate\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTimolol Maleate\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjections\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eConcentration (ppm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePeak Area %RSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eConcentration (ppm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePeak Area %RSD\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e4.709%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e3.660%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13.54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13.72\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12.63\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eGlaucoma refers to a group of eye diseases marked by the progressive narrowing of the visual field, typically accompanied by optic nerve damage and characteristic optic disc cupping.[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] Elevated IOP is widely acknowledged as a major risk factor contributing to disease onset and progression. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] Clinically, glaucoma most commonly presents as either primary open-angle glaucoma or primary angle-closure glaucoma. Its management involves the use of various pharmacological classes designed to reduce IOP through different physiological mechanisms, thereby helping to slow disease advancement and preserve vision.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] Typically, first-line treatment involves typical beta-adrenergic blockers or prostaglandin analogues. When further pressure reduction is necessary, these agents can be combined with or supplemented by other therapies, such as miotics, carbonic anhydrase inhibitors, or sympathomimetic drugs. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] COMBIGAN\u003csup\u003e\u003cb\u003e\u0026reg;\u003c/b\u003e\u003c/sup\u003e (Brimonidine Tartrate and Timolol Maleate ophthalmic solution) is specifically indicated for lowering IOP in patients with chronic open-angle glaucoma or ocular hypertension who do not achieve adequate pressure control with single-agent therapy and when the use of COMBIGAN is considered appropriate.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] Beyond reducing IOP, COMBIGAN also helps to minimize long-term fluctuations in eye pressure. By lowering both the average IOP and its variability over time, COMBIGAN aims to slow the progression of visual field loss associated with glaucoma.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eMultiple liquid chromatographic methods have been proposed for the quantification of Brimonidine Tartrate (BT) and Timolol Maleate (TM) in pharmaceutical formulations as seen in Table S4 (Supplementary material). However, many of these methods lack full stability-indicating capability or exhibit limitations in sensitivity, linearity range, and robustness. Our proposed RP-HPLC method addresses these shortcomings and demonstrates clear advantages over earlier approaches. One reported method utilized a BDS Hypersil phenyl column with a mobile phase of acetonitrile and phosphate buffer (pH 4.0).[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] Although the method provided acceptable quantification ranges (BT: 2.0\u0026ndash;80.0 \u0026micro;g/mL; TM: 5.0\u0026ndash;200.0 \u0026micro;g/mL), it was not stability-indicating and showed only moderate precision. Similarly, another study employed a C18 column with acetonitrile\u0026ndash;buffer mixtures, but the linearity range was narrower (BT: 10\u0026ndash;50 \u0026micro;g/mL; TM: 4\u0026ndash;20 \u0026micro;g/mL), limiting its suitability across a wider concentration span. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] UV-spectrophotometric methods offered simplicity but lacked specificity, robustness, and the ability to separate degradation products, making them unsuitable for regulatory-grade stability testing. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] Other methods reported extremely short retention times (around 0.5\u0026ndash;0.6 minutes), [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] which risks missing impurities or degradants, especially since forced degradation studies were not performed. Alternative analytical methods have employed relatively limited linear concentration ranges specifically, 4\u0026ndash;24 \u0026micro;g/mL for Brimonidine Tartrate and 10\u0026ndash;60 \u0026micro;g/mL for Timolol Maleate which restrict their broader applicability and reduce their reliability. While the sensitivity was improved (LOD for BT: 0.05 \u0026micro;g/mL; TM: 0.09 \u0026micro;g/mL), the method still did not evaluate forced degradation studies, limiting its stability-indicating applicability. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] Although several researchers have investigated the chromatographic analysis of BT and TM, there remains a gap in developing a unified RP-HPLC method that can simultaneously quantify both drugs while also functioning as a true stability-indicating assay in combination dosage forms. This highlights the need for a comprehensive, robust, and reliable analytical approach to ensure precise quality control and thorough stability evaluation of ophthalmic formulations. In contrast, our proposed method employs a Supelco Discovery C18 column (250 \u0026times; 4.6 mm, 5 \u0026micro;m) with a mobile phase of acetonitrile and monobasic potassium phosphate buffer (pH 7.0, 30 mM TEA). This setup enables simultaneous detection at 245 nm and 295 nm, with wide linearity ranges (BT: 0.24\u0026ndash;500 \u0026micro;g/mL; TM: 0.60\u0026ndash;1250 \u0026micro;g/mL). The method demonstrated excellent sensitivity (LOD: BT\u0026thinsp;=\u0026thinsp;0.08 \u0026micro;g/mL; TM\u0026thinsp;=\u0026thinsp;0.20 \u0026micro;g/mL), precision (RSD\u0026thinsp;\u0026lt;\u0026thinsp;1%), and robustness under varied experimental conditions. Importantly, it was validated according to ICH guidelines and included comprehensive forced degradation studies, confirming its stability-indicating nature. Overall, while previous reports achieved partial success in the simultaneous quantification of BT and TM, they either lacked stability indication, had narrower linearity ranges, or demonstrated only moderate robustness. The present method offers a fully validated RP-HPLC approach that is robust, sensitive, stability-indicating, and directly applicable to pharmaceutical quality control and regulatory environments.\u003c/p\u003e\u003cp\u003eThe system suitability test results show fully resolved peaks with tailing factors near 1, indicating excellent peak symmetry. Over 9,000 theoretical plates were observed, confirming strong column efficiency. For Brimonidine Tartrate, %RSD values for peak area and retention time from six replicates of working standard solution #1 were 0.153% and 0.847%, while for solution #2 (two replicates), they were 0.045% and 0.875%. Drift values were also minimal: %RT Drift at 0.029% and %PA Drift at 0.550%. Similarly, Timolol Maleate exhibited %RSD values of 0.163% and 0.604% (solution #1) and 0.041% and 0.875% (solution #2), with %RT Drift of 0.022% and %PA Drift of 0.137%. These low %RSDs and drift values confirm system repeatability, precision, and stability, satisfying ICH system suitability criteria. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] This test was performed before further analyses to validate system readiness.\u003c/p\u003e\u003cp\u003eThe forced degradation study revealed that Brimonidine Tartrate exhibits high stability under thermal and photolytic stress, with no detectable degradation observed after 24 hours at 75\u0026deg;C or under UV irradiation at 245 nm. Under acidic conditions (3 M HCl), the compound showed minimal degradation (0.363%), while exposure to alkaline conditions (3 M NaOH) led to slightly higher degradation (1.438%), suggesting a greater susceptibility to base-catalyzed hydrolysis. Oxidative stress induced by 3% hydrogen peroxide resulted in moderate degradation (0.776%), indicating some vulnerability to oxidation. Overall, these findings confirm that Brimonidine Tartrate maintains robust stability under most stress conditions, with acidic, alkaline and oxidative environments posing the greatest risk for degradation. Similarly, the forced degradation study demonstrated that Timolol Maleate remains chemically stable under thermal stress at 75\u0026deg;C and photolytic exposure at 295 nm for 24 hours, with no measurable degradation observed. However, the drug displayed pronounced sensitivity to hydrolytic conditions, with degradation increasing in proportion to acid and base concentration; degradation reached 23.35% in 3 M HCl and 23.92% in 1 M NaOH. Timolol Maleate was especially susceptible to oxidative degradation, showing substantial degradation of 57.17% when treated with 3% hydrogen peroxide. Lower concentrations and shorter exposure times led to reduced degradation: 5.52% in 1 M HCl (24 hours), 5.54% in 0.05 M NaOH (1 hour), and 9.57% in 0.5% H₂O₂ (1 hour). These results highlight that while Timolol Maleate is stable under heat and light, it undergoes significant degradation under strong hydrolytic and oxidative stress, underscoring the need for careful control of these conditions during manufacturing, storage, and formulation to ensure product stability and quality.\u003c/p\u003e\u003cp\u003eThe chromatographic analysis successfully detected and resolved the peaks corresponding to Brimonidine Tartrate, Timolol Maleate, and Maleic Acid. All peaks exhibited excellent resolution, thereby fulfilling the specificity criteria set for analytical separation. Additionally, the peak purity index for each analyte exceeded the acceptable threshold, confirming the absence of co-eluting impurities or interference. The specificity assessment demonstrated that the developed method effectively isolates and quantifies these three components even in the presence of degradation products and potential impurities. Peak purity factor (\u0026ge;\u0026thinsp;999.99), RS\u0026thinsp;\u0026ge;\u0026thinsp;2.0, and a corresponding three-dimensional chromatogram, which highlight the method\u0026rsquo;s robustness and demonstrate its adherence to ICH and FDA validation requirements. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] Furthermore, the results of the robustness study, all parameters complied with ICH guidelines. The tailing factors fell within the acceptable range (0.9-2) and the number of theoretical plates\u0026thinsp;\u0026ge;\u0026thinsp;2000, indicating satisfactory column efficiency. Furthermore, the method demonstrated robustness by maintaining consistent performance despite intentional minor variations in solvent composition, buffer pH, flow rate, detection wavelength, and injection volume.\u003c/p\u003e\u003cp\u003eTo assess solution stability, a freshly prepared ophthalmic solution containing Brimonidine Tartrate (200 ppm) and Timolol Maleate (500 ppm) was immediately analyzed using the RP-HPLC system. The same solution was reinjected after 24, 48, and 72 hours. Throughout this period, the chromatographic profiles showed no appearance of new peaks and no disappearance of existing ones, confirming that the solution remained stable without significant degradation. Specifically, the percent peak area changes for Brimonidine Tartrate at 24, 48, and 72 hours were 0.042%, 0.097%, and 0.138%, respectively, while for Timolol Maleate, the corresponding changes were 0.465%, 0.354%, and 0.263%. These results indicate excellent short-term stability of both active pharmaceutical ingredients (APIs) and further support the robustness of the developed RP-HPLC method, as consistent peak areas reflect the absence of degradation or analyte loss during the study period. Additionally, to evaluate the method\u0026rsquo;s linearity, five different concentrations of Brimonidine Tartrate and Timolol Maleate standard solutions were prepared, and each was individually injected into the HPLC system. The corresponding peak areas were used to construct calibration curves plotting peak area versus concentration. The resulting linear regression analysis yielded correlation coefficients (R\u0026sup2;) of 0.9992 for Brimonidine Tartrate and 0.9996 for Timolol Maleate. These high correlation coefficients satisfy the acceptance criteria for linearity, confirming that the HPLC method provides a consistent and proportional response across the tested concentration range. Overall, these results demonstrate the method\u0026rsquo;s validity for precise quantitative determination of the active pharmaceutical ingredients.\u003c/p\u003e\u003cp\u003eThe accuracy of the developed RP-HPLC method for quantifying Brimonidine Tartrate and Timolol Maleate in Combigan\u0026reg; ophthalmic solution was evaluated by recovery studies at three concentration levels corresponding to 80%, 100%, and 120% of the target assay concentrations. Each level was analyzed in triplicate. For Brimonidine Tartrate, the average recovery values obtained were 99.81% at 160 ppm, 99.42% at 200 ppm, and 99.78% at 240 ppm. Similarly, for Timolol Maleate, the recoveries were 99.00% at 400 ppm, 98.71% at 500 ppm, and 101.10% at 600 ppm. All recoveries fell well within the generally accepted range of 95\u0026ndash;105%, demonstrating excellent method accuracy across the studied concentration range. These results confirm that the method provides accurate quantification without interference from excipients or other components present in the formulation. The consistency of recoveries across different concentration levels also supports the robustness and reliability of the method for routine quality control and stability testing of Combigan\u0026reg; ophthalmic solution.\u003c/p\u003e\u003cp\u003eTo further evaluate the accuracy and precision of the proposed method, results were statistically compared with those of reported methods using the Student\u0026rsquo;s t-test and F-test at the 95% confidence level (Table\u0026nbsp;\u003cspan refid=\"Tab17\" class=\"InternalRef\"\u003e17\u003c/span\u003e). The proposed analytical method demonstrated high accuracy and precision for the determination of Brimonidine Tartrate and Timolol Maleate in comparison with the reference method. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] Statistical evaluation using the F-test revealed that the proposed method exhibited significantly lower variance (Brimonidine Tartrate: F\u0026thinsp;=\u0026thinsp;206,611; Timolol Maleate: F\u0026thinsp;=\u0026thinsp;7,963), indicating superior precision. The t-test results (Brimonidine Tartrate: t = -2.04; Timolol Maleate: t = -2.43) showed no significant difference between the means of the proposed and reference methods at the 95% confidence level, confirming the reliability and suitability of the proposed method for routine analysis. Overall, these findings demonstrate that the proposed method is statistically comparable to the reference method while offering enhanced precision.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab17\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 17\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eStatistical comparison of the proposed and reference methods for Brimonidine Tartrate and Timolol Maleate. Data are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. F and t values with their 95% confidence critical limits (F_crit, t_crit) are shown. F-test evaluates precision; t-test evaluates accuracy.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eDrug Name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eRecovery\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCalculated\u003c/p\u003e\u003cp\u003et- values\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003et_critical (α\u0026thinsp;=\u0026thinsp;0.05)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCalculated\u003c/p\u003e\u003cp\u003eF- values\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eF_critical (α\u0026thinsp;=\u0026thinsp;0.05)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProposed methods\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eReference method\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBrimonidine Tartrate\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e99.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0022\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e206,611\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.776\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-2.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTimolol Maleate\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e99.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e101.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7,963\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.776\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-2.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTo evaluate repeatability, six individually prepared samples of Brimonidine Tartrate (200 ppm) and Timolol Maleate (500 ppm) were analyzed under optimized HPLC conditions. Precision was assessed by calculating the percent relative standard deviation (%RSD) of the peak areas. The %RSD values for Brimonidine Tartrate and Timolol Maleate were 0.549% and 0.848%, respectively. According to ICH guidelines, a %RSD not exceeding 2.0% is considered acceptable, and these results demonstrate that the method consistently delivers precise measurements for both analytes under the same conditions. Injection precision was further evaluated by injecting a single sample of Brimonidine Tartrate and Timolol Maleate six consecutive times into the HPLC system. The resulting %RSD values, 0.859% for Brimonidine Tartrate and 0.808% for Timolol Maleate, also fell within the acceptable range, confirming the method\u0026rsquo;s reliability for repeated injections. Additionally, intermediate precision was assessed by preparing six separate samples and analyzing them on a different HPLC system. The %RSD values obtained, 0.884% for Brimonidine Tartrate and 1.354% for Timolol Maleate, complied with the specified acceptance criteria. Table S5 (Supplementary material) presents the results from both analysts, allowing proper calculation of inter-day precision and clear presentation of the data. Overall, these results confirm that the method maintains precision and robustness across repeatability, injection precision, and intermediate precision assessments, meeting the requirements set out in the validation protocol.\u003c/p\u003e\u003cp\u003eThe limit of detection (LOD) for Brimonidine Tartrate and Timolol Maleate was established by preparing a series of dilutions from the stock solution. Each prepared dilution was examined on the HPLC system, and the corresponding signal-to-noise ratios were determined based on the generated chromatograms. The concentrations of 0.08 ppm for Brimonidine Tartrate and 0.20 ppm for Timolol Maleate produced signal-to-noise ratios of 3.5 and 3.8, respectively. These values satisfy the acceptance criterion of a signal-to-noise ratio\u0026thinsp;\u0026ge;\u0026thinsp;3, and thus were designated as the LODs for the method. To establish the limit of quantitation (LOQ), test solutions at concentrations of 0.24 ppm for Brimonidine Tartrate and 0.60 ppm for Timolol Maleate were each injected into the HPLC system in ten replicate runs. Analysis of the resulting chromatograms showed %RSD values for peak areas of 4.70% and 3.66%, both well below the LOQ acceptance threshold of \u0026le;\u0026thinsp;10%. These findings demonstrate that the developed method is sufficiently precise to quantify both analytes reliably at these low concentrations, meeting validation requirements for LOQ performance.\u003c/p\u003e\u003cp\u003eTo further verify the Limit of Detection (LOD) and Limit of Quantitation (LOQ) values,[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] the International Council for Harmonisation (ICH) recommends several calculation methods depending on the analytical technique, analyte characteristics, and method suitability.[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] One widely accepted method involves using the standard deviation of the response (σ) and the slope of the calibration curve (S), calculated with the formulas:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:LOD=3.3X\\left(\\frac{{\\sigma\\:}}{\\text{S}}\\right)\\:\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:LOQ=10X\\left(\\frac{{\\sigma\\:}}{\\text{S}}\\right)\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eUsing data from the linearity study, we determined σ and S for both Brimonidine Tartrate and Timolol Maleate. Applying these formulas, the calculated LOD and LOQ for Brimonidine Tartrate were 0.099 ppm and 0.30 ppm, respectively, which correspond closely to our experimentally obtained signal-to-noise results (LOD\u0026thinsp;=\u0026thinsp;0.08 ppm, LOQ\u0026thinsp;=\u0026thinsp;0.24 ppm). Similarly, for Timolol Maleate, the calculated LOD and LOQ were 0.218 ppm and 0.66 ppm, aligning well with the signal-to-noise based findings (LOD\u0026thinsp;=\u0026thinsp;0.20 ppm, LOQ\u0026thinsp;=\u0026thinsp;0.60 ppm). These results confirm that our method validation complies with ICH and USP guidelines, supporting the reliability of the developed analytical method.\u003c/p\u003e\u003cp\u003eTo evaluate the environmental sustainability of the developed method, three complementary green analytical chemistry (GAC) metrics were applied: the Analytical Eco-Scale, the Green Analytical Procedure Index (GAPI), and the AGREE evaluation. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] The Eco-Scale was calculated by assigning penalty points for hazardous reagents, waste generation, and energy consumption. The main contributor to penalty points was the use of acetonitrile (ACN) as organic solvent in the mobile phase and the direct disposal of waste. Considering dilution as the only sample preparation, room temperature operation (25\u0026deg;C), and a short chromatographic run time (10 min), the method achieved an Eco-Scale score of ~\u0026thinsp;75, which classifies it as an acceptable green method (scores\u0026thinsp;\u0026ge;\u0026thinsp;75). Moreover, the Green Analytical Procedure Index (GAPI) tool was used to obtain a holistic assessment covering the entire analytical workflow from sample collection to final determination. The pictogram for this method showed a mixture of green and yellow zones, reflecting the environmentally favorable aspects such as simple dilution, absence of derivatization, and low energy consumption. However, red zones were observed in solvent usage and waste management, as ACN is toxic, and waste was directly discarded without treatment. Importantly The AGREE evaluation, based on the 12 principles of green analytical chemistry, generated a circular pictogram and a numerical score. The method scored 0.57 (out of 1.0), indicating moderate greenness. High scores were obtained for minimal sample preparation, short analysis time, and absence of derivatization. Lower scores were assigned to principles related to renewable solvents, waste management, and operator safety, due to the use of ACN and its direct disposal. Overall, the three evaluation tools confirmed that the proposed RP-HPLC method possesses several environmentally favorable features such as simplicity, short analysis time, and low energy consumption. However, the main limitations were related to solvent selection and waste handling. Future improvements could include replacing ACN with a greener alternative (e.g., ethanol or water-rich mobile phases), implementing solvent-recycling strategies, and adopting proper hazardous waste management practices.\u003c/p\u003e\u003cp\u003eIn summary, this validated RP-HPLC method addresses significant analytical limitations reported in previous studies by offering a broader linearity range, robust stability-indicating performance, and compliance with international validation standards. Consequently, this method offers a scientifically robust and practically applicable approach for thorough quality control and stability evaluation of combination ophthalmic formulations, thereby reinforcing ongoing initiatives to preserve therapeutic effectiveness and ensure patient safety in the treatment of glaucoma.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study successfully developed and validated a robust, accurate, and precise RP-HPLC method for the simultaneous quantification of Brimonidine Tartrate and Timolol Maleate in Combigan\u0026reg; ophthalmic solution. Addressing gaps found in previously published methods, such as insufficient linear ranges, inadequate stability testing, and lack of a unified assay. The new method demonstrated excellent linearity across the tested concentration ranges (R\u0026sup2; \u0026gt;0.999), strong system suitability with high theoretical plate counts and low tailing factors, and compliance with ICH acceptance criteria in all validation parameters. The method proved highly repeatable and precise, as confirmed by low %RSD values in repeatability, injection precision, and intermediate precision assessments. Robustness testing further showed consistent performance under deliberate variations in chromatographic conditions. The specificity study verified the method\u0026rsquo;s ability to resolve the active ingredients from degradants and impurities, supporting its stability-indicating capability. The determined LOD and LOQ values, supported by both signal-to-noise ratios and statistical calculations based on standard deviation and calibration slope, highlight the method\u0026rsquo;s sensitivity and suitability for detecting and quantifying low levels of Brimonidine Tartrate and Timolol Maleate. The three complementary green analytical chemistry (GAC) metrics were applied and confirmed that the proposed RP-HPLC method possesses several environmentally favorable features such as simplicity, short analysis time, and low energy consumption. Overall, this validated method meets ICH and USP guidelines and offers a comprehensive analytical tool for reliable quality control, routine analysis, and stability testing of Combigan\u0026reg; and similar ophthalmic formulations. It thus represents a significant advancement toward ensuring the safety, efficacy, and quality of combination therapies used in glaucoma management.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eACN\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eacetonitrile\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eAvg\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eaverage\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eFDA\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFood and Drug Administration\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eHCl\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehydrochloric acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eICH\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInternational Conference on Harmonisation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eLC\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eliquid chromatography\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eLOD\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003elimit of detection\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eLOQ\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003elimit of quantitation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003e\u0026micro;L\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emicroliter\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003e\u0026micro;g/mL\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emicrogram per milliliter\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003emin\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eminutes\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eN\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003enormality\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eMg\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emilligram\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eppm\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eparts per million\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eC18\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecolumn having octadecyl chain of carbon atom\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003epH\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003enegative logarithm of H\u0026thinsp;+\u0026thinsp;concentration\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eRP-HPLC\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ereverse-phase high-performance liquid chromatography\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eRSD\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003erelative standard deviation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eRT\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eretention time\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eR\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecorrelation of coefficient\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eUV\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eultraviolet\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eGAC\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003egreen analytical chemistry\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eGAPI\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGreen Analytical Procedure Index.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFinancial competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors affirm that they have no financial conflicts of interest related to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors state that they have no conflicts of interest to report and no relevant disclosures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement of Human and Animal Rights (including Statement of Informed Consent)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis manuscript does not involve any research conducted on human participants or animal subjects by the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets analyzed in this research originate exclusively from our own samples, with study approval granted by the institutional ethics committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the findings of this study are available within the manuscript and from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding Author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAktham Mestareehi\u003c/strong\u003e -\u0026nbsp;Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences\u003cem\u003e,\u0026nbsp;\u003c/em\u003eWayne State University, Detroit, MI, 48201, USA.\u003cimg width=\"17\" height=\"12\" src=\"data:image/png;base64,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\" alt=\"image\"\u003ehttps://orcid.org/0000-0002-1478-6310; Email: [email protected] \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthors\u003c/p\u003e\n\u003cp\u003eAktham Mestareehi -\u0026nbsp;Department of Applied Pharmaceutical Sciences and Clinical Pharmacy, Faculty of Pharmacy, Isra University, P.O. Box 22, Amman 11622, Jordan\u003c/p\u003e\n\u003cp\u003eAktham Mestareehi\u0026nbsp;-\u0026nbsp;Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences and Integrated Biosciences, Wayne State University, Detroit, Michigan 48201, United States\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe manuscript was prepared by the author, who provided approval and consent for the final version. Contributions by A.M. included methodology development, formal analysis, and data curation. Furthermore, A.M. drafted the original manuscript, participated in manuscript review and editing, supervised the project, administered the research, and secured funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific funding from public, commercial, or non-profit organizations. We express our gratitude to all authors involved and to the publisher for their support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eResnikoff, S. \u003cem\u003eet al.\u003c/em\u003e Global data on visual impairment in the year 2002. \u003cem\u003eBull World Health Organ\u003c/em\u003e \u003cstrong\u003e82,\u003c/strong\u003e 844\u0026ndash;851 (2004).\u003c/li\u003e\n\u003cli\u003eBengtsson, B., Leske, M. C., Hyman, L., Heijl, A., \u0026amp; Early Manifest Glaucoma Trial Group. Fluctuation of intraocular pressure and glaucoma progression in the early manifest glaucoma trial. \u003cem\u003eOphthalmology\u003c/em\u003e \u003cstrong\u003e114,\u003c/strong\u003e 205\u0026ndash;209 (2007).\u003c/li\u003e\n\u003cli\u003eKass, M. 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Green metrics and green analytical applications: A comprehensive outlook from developing countries to advanced applications. \u003cem\u003eGreen Analytical Chemistry\u003c/em\u003e \u003cstrong\u003e11,\u003c/strong\u003e 100159 (2024).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Brimonidine Tartrate, Timolol Maleate, High-Performance Liquid Chromatography (HPLC), Specificity, Linearity, Accuracy, Precision, Limit of Detection (LOD), Limit of Quantification (LOQ), Combigan®","lastPublishedDoi":"10.21203/rs.3.rs-7231691/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7231691/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA robust, rapid, and reproducible reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous quantification of Brimonidine Tartrate and Timolol Maleate in ophthalmic dosage forms. Chromatographic separation was achieved on a Supelco Discovery C18 column (25 cm \u0026times; 4.6 mm, 5 \u0026micro;m) maintained at ambient temperature, employing isocratic elution with two mobile phases: phase A (buffer at pH 7.0 containing 30 mM triethylamine) and phase B (acetonitrile) in a ratio of 80:20. The flow rate was set at 1.0 mL/min, and detection was performed at 245 nm and 295 nm using a diode array detector (DAD). Method validation, conducted in accordance with ICH Q2(R1), USP, and FDA guidelines, confirmed excellent linearity over the ranges of 0.24\u0026ndash;500 ppm for Brimonidine Tartrate and 0.60\u0026ndash;1250 ppm for Timolol Maleate. Accuracy results ranged from 99.42% to 99.82% for Brimonidine Tartrate and from 98.71% to 101.10% for Timolol Maleate. Relative standard deviations (RSDs) for precision, specificity, and robustness were all below 2%, demonstrating the method\u0026rsquo;s consistency and reliability. Additionally, the limits of detection (LOD) were determined to be 0.08 ppm for Brimonidine Tartrate and 0.20 ppm for Timolol Maleate, while the limits of quantification (LOQ) were 0.24 ppm and 0.60 ppm, respectively. Forced degradation studies under various stress conditions, including acid and base hydrolysis and hydrogen peroxide oxidation, demonstrated that the method successfully separated Brimonidine Tartrate and Timolol Maleate from their degradation products, confirming its stability-indicating capability. Notably, both drugs remained stable under thermal and photolytic stress; however, Timolol Maleate was significantly more prone to degradation under strong hydrolytic and oxidative conditions, underscoring the need for stringent control during formulation and storage. Additionally, the three complementary green analytical chemistry (GAC) metrics were evaluated. The method achieved an Eco-Scale score of approximately 75. The GAPI pictogram for this method showed a mixture of green and yellow zones. The AGREE evaluation yielded a score of 0.57 (out of 1.0), indicating moderate greenness. Overall, this method effectively quantified both active pharmaceutical ingredients without interference from excipients or degradation products, supporting its suitability for routine quality control and stability testing of combined ophthalmic formulations.\u003c/p\u003e","manuscriptTitle":"Development and Validation of a Robust Stability-Indicating Reversed Phase High Performance Liquid Chromatography (RP-HPLC) Method for Simultaneous Quantification of Brimonidine Tartrate and Timolol Maleate (Combigan ® ) in Bulk and Pharmaceutical Dosage Forms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-04 11:01:39","doi":"10.21203/rs.3.rs-7231691/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-03T10:40:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-30T06:42:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"43733089307296363941078657088146866924","date":"2025-10-30T05:21:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"98708776521639266118125132854511866235","date":"2025-10-27T10:23:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"273619345934166241678038159563009202917","date":"2025-10-26T12:22:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-08T06:48:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"17205600944957320408076685002584828423","date":"2025-10-03T00:01:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21512551814252895524992997490469254417","date":"2025-10-01T13:24:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-30T20:02:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28667167163620354791581783364815540306","date":"2025-09-30T19:57:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-29T13:20:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-29T01:15:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-27T19:15:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b3dd1689-9856-4ed3-b15c-d8e960494c79","owner":[],"postedDate":"November 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":57138528,"name":"Physical sciences/Chemistry"},{"id":57138529,"name":"Biological sciences/Drug discovery"},{"id":57138530,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-01-12T16:02:26+00:00","versionOfRecord":{"articleIdentity":"rs-7231691","link":"https://doi.org/10.1038/s41598-025-33716-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-05 15:57:56","publishedOnDateReadable":"January 5th, 2026"},"versionCreatedAt":"2025-11-04 11:01:39","video":"","vorDoi":"10.1038/s41598-025-33716-x","vorDoiUrl":"https://doi.org/10.1038/s41598-025-33716-x","workflowStages":[]},"version":"v1","identity":"rs-7231691","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7231691","identity":"rs-7231691","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}