A Suitable Chromatographic Method for Simultaneous Estimation of Brimonidine and Brinzolamide in Glaucoma Treated Dosage Form: Influence of its Greenness with Use of Green Metric Tools

Introduction

Glaucoma is an eye condition which irreversibly damages the optic nerve invariably leading to blindness. One of the major causes of glaucoma is increased intraocular pressure or IOP^1–4 . The treatment and management of glaucoma generally includes eye medications (drops), laser treatment or surgery. Eye medications focus on controlling / decreasing IOP. Reduction of IOP is the mainstay of treatment of open-angle glaucoma and ocular hypertension. This research work comprises of two drugs namely Brimonidine and Brinzolamide which are used as combination ophthalmic drops to treat ocular hypertension and glaucoma.

Brimonidine tartrate¹ is a nonselective alpha-adrenergic receptor agonist drug which is used as ophthalmic drops for the treatment of ocular hypertension and glaucoma². Its IUPAC name is 5-Bromo-N-(4,5-dihydro-1H-imidazol-2-yl) quinoxalin-6-amine² and its structure is given in figure 1. It is currently present in USP monograph³. When it is used in the form of eye drop medication; it can be used as a short or long term drug lowering ocular pressure in eye treatment. When used in conjunction with some other medications it works more efficiently for minimising intraocular pressure, enhancing uveoscleral outflow in patients having ocular redness⁴, ocular hypertension or open angle glaucoma⁵.

Figure 1: Structure of Brimonidine Tartrate.                           Click here to View Figure

Brinzolamide is a carbonic anhydrase II inhibitor⁶. When used as eye drops, it acts as an ocular hypertension reducing agent. Its IUPAC name is (4R)-4-(ethylamino)-2-(3-methoxypropyl)-1,1-dioxo-3,4-dihydrothieno[3,2-e] thiazine-6-sulfonamide⁷ and its structure is given in figure 2. Ophthalmic form of Brinzolamide is also available in USP monograph⁸. It works in the eye drop dosage form by decreasing the formation of aqueous humor which causes a drop in the intraocular pressure in the interior part of the eyeball^9,10.

Figure 2: Structure of Brinzolamide Click here to View Figure

Monotherapy drug treatment for glaucoma may be insufficient to reach target intraocular pressure (IOP) or tochyphylaxis effect. Single content drug may lose its potency over long periods and hence use of more than one drug exhibits tolerable and satisfactory effect for a long period¹⁰.  Multi drug therapy consisting of Brimonidine and Brinzolamide has been found to be highly effective as anti-glaucoma medication. It has resulted in increased adherence by the patients^11-12 and is also considered to be safe for patients with heart ailments as these drugs do not contain β-blockers. Simultaneous multi drug evaluation techniques are highly favoured as they reduce analysis time and techniques used for simultaneous evaluation of Brimonidine and Brinzolamide have been reported; namely UV-spectroscopic method^13-17, HPTLC¹⁸, RP-HPLC^19-21, UPLC²², spectrofluorimetric²³, LC-UV²⁴, GC/MS²⁵ and Capillary electrophoresis²⁶ methods. The focus of this study is to develop and validate a novel, stability indicating^27-30, simple, specific, accurate, sensitive and environmental friendly RP-HPLC technique; for the concurrent evaluation of Brimonidine and Brinzolamide with a shorter runtime. This study has the potential to drastically reduce time needed to analyse pharmaceutical combination dose formulations of these two drugs, consequently making the analytical process economical and green.

Materials and Methods

Chemicals and reagents

Brimonidine tartrate and Brinzolamide standards were received from Global Calcium Ltd as free samples. Combined product of Brimonidine tartrate and Brinzolamide ophthalmic suspension (Brinzolast), HPLC grade (Milli Q) water, phosphate buffer, 0.1% perchlorate buffer, octane-1-sulfonic acid sodium salt monohydrate (Make- Merck), orthophosphoric acid (Merck), acetonitrile (HPLC grade – Merck) and methanol (HPLC grade – Merck) were obtained from the open market.

Instrument and system

The analytical balance (Sartorius), pH meter (Labindia), ultrasonic bath (Precisonic), chromatographic system: Thermo Scientific HPLC with Chromeleon Software version 7.2 and separation module having PDA or UV-visible Detector (Ultimate 3000) were the equipment used for the method analysis. The HPLC parameters are given in Table 1.

Mobile phase preparation

In order to prepare the mobile phase, 4.68 g octane-1-sulfonic acid sodium salt monohydrate was weighed and transferred into 1L of HPLC grade (Milli Q) water. The resultant solution pH was set to 3.0 with usage of diluted ortho-phosphoric acid. This 0.02M octane-1-sulfonic acid sodium salt monohydrate buffer was added to acetonitrile in the ratio of 65: 35 v/v. Further, filtration through 0.45µ nylon membrane filter and degassing yielded the mobile phase.

Standard preparation

Brimonidine tartrate (10 mg) and Brinzolamide standard (25 mg) were added to 100 mL and 50 mL standard volumetric flasks respectively. These medications on sonication underwent dissolution in the respective volumetric flasks with the addition of 30 mL and 60 mL of the mobile phase. By diluting with the same mobile phase, the appropriate concentrations of clear solutions were obtained. The same mobile phase was used to quantitatively fill 50 mL of the content from 5mL of the resultant solutions into respective volumetric flasks. The final solutions contained 50µg/mL of Brinzolamide (50 ppm) and 10µg/mL of Brimonidine tartrate (10 ppm).

Sample preparation

The test sample drug of Brinzolamide and Brimonidine tartrate in the suspension form (Brinzolast eye drops) manufactured by Mankind Pharma was purchased from Mumbai open market. Density of ophthalmic suspension (weight per mL) was determined by a densitometer and this true value was utilized in the calculations. 0.5 g of this sample solution was taken in a 100 mL standard volumetric flask. Further, after addition of 60 mL of the diluent (mobile phase) this flask was kept for sonication for 5 min with intermediate shaking. Finally the solution was allowed to normalize at controlled room temperature and using the mobile phase further dilution was carried out. The resultant solution was subjected to thorough shaking and shaken well and on filtration through 0.45µ nylon membrane syringe filter, it yielded 10 µg/mL of Brimonidine tartrate and 50 µg/mL of Brinzolamide respectively.

System suitability

The verifying tests for brimonidine and brinzolamide were carried out through the assessment of the performance parameters. Respectively, the performance parameters were having at least countable theoretical plates of 1000, tailing factors not higher than 2.0 and relative standard deviations (RSD) lower than 2.0%.

Method Development

To establish the greatest distinction in Brimonidine and Brinzolamide RT^31-33 several researchers have used various combinations of mobile phase buffers alongside differing stationary phases^34-35. Since both peaks were resolved with decent separation, a flow rate set at 1.0 mL/min was recommended using mobile phase as 0.02 M octane -1-sulfonic acid sodium salt pH 3.0: acetonitrile (65 : 35) v/v. The stationary phase for the column was also selected based on the resolution, clarity of dual peaks and sharpness of both medications of regions of interest. A hypersil BDS C18 (100 mm × 4.6 mm; 5µ) P/N: 28105-104630 was chosen. A wavelength of 254 nm was used as it was optimal for both of the medications and provided great absorbance^36-37. Brimonidine and Brinzolamide retention times were recorded.

Table 1: Optimized HPLC Chromatographic condition.

Developed Method Validation

As per ICH guideline³⁸ Q2(R2) and USP chapter <1225> developed method validation parameters are evaluated. The brief validation parameters outcome is shown along with data and final outcome.

Specificity

As per ICH guidelines, analytical method specificity is defined as the ability to demonstrate the analyte in the presence of matrix component without any obstruction. This was elucidated by the injection of the diluent, inactive matrix, standard and individual content solution finding the obstruction at the Brimonidine and Brinzolamide retention times.

Preparation of the each solution began with use of diluent as such. For inactive matrix solution, the same procedures were were applied as those used for sample preparation. After proper injection in HPLC, it was observed for any obstruction affecting the active content.

Precision

According to the prescribed criterion, in standard preparation both active contents were injected into the HPLC system with six injections each. The relative area percentage deviation of Brimonidine and Brinzolamide area was calculated.

Method Precision

The intraday precision of the developed analytical method was followed by standard precision with injection of six individual sample preparations and the percentage RSD of six assay results was calculated accordingly.

Intermediate Precision

The inter day precision of the developed analytical method was adhered by performing intraday precision on another day by a different individual using same experimental scenario. The individual performed the variation by use of different chemical lots, column and chromatographic system. After the performance of inter day and intraday precision both the sets were calculated for absolute difference.

Linearity and Range

An analytical method is said to be linear if it is demonstrated that the data from the test is exactly proportional to the analyte concentration within the sample’s predetermined range. A line of best fit was plotted, with the concentration of the test substance (in micrograms per mL) graphed along the horizontal axis, against the measured area on the vertical axis. The coefficient of determination, the percentage of Y-intercept, the slope of the regression line, residual sum of squares, and the %RSD of the areas of Brimonidine and Brinzolamide were all determined.

Standard solutions were prepared at concentrations of 10%, 50%, 80%, 100%, 120%, and 150% of target concentrations. For Brimonidine these are 10µg/mL and for Brinzolamide these are 50µg/mL. These six levels were set to include the lower and upper limit of the method range.

Limit of detection and quantification

The lowest concentration of analyte (LOD ) and the lowest concentration of analyte which can be quantified ( LOQ) are parameters which are detected by signal to noise ratio or through a formula derived from standard deviation and slope of linearity curve.

Accuracy

To determine accuracy, three target concentration values were chosen. A normal stock solution was prepared and in triplicate condition spiked with a placebo at distinct concentrations level: 50%, 100% and 150%. In accordance with the methodology, these three levels were injected. All levels showed their recovery within the 98% – 102% range.     

Intermediate precision (Inter-day precision)

The inter day precision parameter established by performing analysis on another day with different instrument using different lot of chemicals. This performance indicates that there is no significant impact on results set. The percentage RSD of 6 sample results set was observed to be 1.56 and 1.22 for Brimonidine and 1.27 and 0.47 for Brinzolamide performed on different days on different instruments using different chemical lots.

Robustness

The robustness is the analytical method parameter which was evaluated by making little but intentional adjustment in the analytical method performance which did not affect the reliance approach of the method. The standard and sample analysis was carried out as per the methodology. The adjustment parameters of the method such as mobile phase buffer pH + 0.2 (pH 2.80 and pH 3.20), column oven temperature + 5°C (35°C and 45°C) and flow change + 0.1 mL of 1.0 mL (0.9 mL/ min and 1.1 mL/min) were carried out.

Solution stability

The solution stability of reference as well as test solution is determined by showing result reliability over the period time. This parameter gives importance after the preparation and injection of both the preparations in the chromatographic system.

Evaluation of Greenness of Proposed Analytical Method

An AES tool, which is based on Van Aken et al’s Eco-Scale for organic synthesis³⁹, is a methodology that analyzes the “greenness” of a process or a method. The penalty point of chemicals based on number of pictogram present in material safety datasheet (MSD) x score for the signal (safe shown as 1 and danger shown as 2). Penalty point is also assigned for the solvent used (<10 mL= 1, between 10-100 mL= 2 and >100 mL= 3). So the total score for acetonitrile is (2 Pictogram x 2(danger) x 2 (10-100 mL )) that is 8. Similarly penalty point for octane- 1- sulphonic acid sodium salt and ortho-phosphoric acid is calculated. In the penalty point instrumental consumption of energy is also shown (0 point for energy less than 0.1 kWh per sample, 1 point for 0.1-1.5 kWh and 2 point for more than 1.5 kWh). In our case HPLC energy consumption is 1 for 0.1-1.5kWh. The waste penalty point is calculated by considering the formula – Run time x flow rate + processing score. In our study, the run time is 5 minutes, flow rate is 1 mL/min and processing score is 3 (no treatment); so the formula became (5 x 1+3) = 8.

The score of the optimized method of our study is 79, an indicator of good greenness. With real green analysis the consumption of reagents, energy and waste is reduced. All the points deducted from the overall penalty score are summated as 100 which signifies an ideal approach⁴⁰.

The second metric tool is NEMI, which incorporates a pictogram with four sections. One of the sections is PBT solvents, which stands for toxic, persistent and bio accumulative. This takes us to the next section which indicates dangers, the next is corrosiveness (usage of solution pH 2-12) and the last is waste. Out of the four parts, the suggested method shows three green sections, which indicates waste, non PBT solvents and non- corrosive conditions. On the other hand, the inclusions of harmful solvents like acetonitrile and ortho-phosphoric acid as stipulated by the Code of Federal Regulations and Environmental Protection 2016⁴¹ represents the blank danger section.

Figure 3: Evaluation of greenness of method using 2 different semi quantitative metric tools Click here to View Figure

AES evaluation: >75 represents excellent green analysis, >50 represents acceptable green analysis, <50 represents inadequate green analysis.

For the green metric assessment⁴², MoGAPI employed five pentagrams which range from one to five. The colours red, yellow and green represents score of three, two and one – indicating high, medium and low environmental impact respectively. Taking into account three red, seven yellow and five green parts of the suggested HPLC method, it could be said that this method has a rather moderate to medium environmental impact. The fourth metric tool AGREE⁴³ employs software that can be downloaded which in turn allows it to use its findings on the 12 principles of green analytical chemistry. The findings were represented as a 12 sectioned colored pictogram on a range between zero to one. A number closer to one and colored dark green indicates a greener approach. It must be noted, however, that AGREE is instant friendly, all rounded and quite simple to use. In relation to their overall greenness, the suggested methods range in colour from pale to dark green. The results obtained from all the green metric tools are detailed of in fig 4.

Figure 4: Evaluation of greenness of method using 2 different quantitative metric tools Click here to View Figure

MoGAPI : excellent green (≥75), acceptable green (50–74), and inadequately green (<50)

AGREE : excellent green (≥0.75), acceptable green (0.5–0.74), and inadequately green (<0.50) 

Results and Interpretention

Specificity

The specificity of method was tested to ensure placebo, other matrix, brimonidine and brinzolamide diluent solution (M. Phase) do not intervene at the retention times for Brimonidine (Rt- 1.7 minutes) and Brinzolamide (Rt- 2.6 minutes) respectively as per Fig. 5

Figure 5: Specificity chromatograph of BRT and BRZ Click here to View Figure

Figure 6: Overlay chromatograph of specificity  Click here to View Figure

Linearity and Range

After the insertion of standard concentrations of Brinzolamide and Brimonidine; the peak area correlation coefficients (R²) of these two drugs were established which indicates the linearity of the chromatographic procedure. BRT and BRZ stock solutions were injected in HPLC system in concentration levels of 10, 50, 80, 100, 120 and 150 percent, while considering 10 ppm BRT and 50 ppm BRZ respectively. For these two drugs the detector response was observed to be linear from 10 to 150 percent of the concentration. Fig 7 represents overlay chromatograph of linearity, Fig 8 and Fig 9 represent the linearity curves of BRT and BRZ along with its residual graph Fig 10 and Fig 11.

Figure 7: Overlay chromatograph of linearity concentration Click here to View Figure

As per the linearity requirements, the R² value (correlation of coefficient) for Brimonidine and Brinzolamide was within the range (not less than 0.995) at 0.9999 and 0.9996 respectively. The graph of linearity is shown in the Fig 8 and Fig 9.

Figure 8: Calibration curve of Brimonidine Click here to View Figure

Figure 9: Calibration curve of Brinzolamide  Click here to View Figure

Figure 10: Residual plot of Brimonidine Click here to View Figure

Figure 11: Residual plot of Brinzolamide Click here to View Figure

Table 2: Linearity summary for BRT and BRZ

 Limit of detection and quantification:

LOD and LOQ were determined by considering slope of the linear calibration curve and standard deviation of the response. The method sensitivity of the established analytical method was evaluated by the LOD and the LOQ values. Data is shown in Table 3.

Table 3: LOD & LOQ values of Brimonidine and Brinzolamide

 Accuracy

The method’s accuracy as measured by percentage recovery fell well within the range of 98 -102% (Table 4 and 5).

Table 4: Accuracy for Brimonidine

 Table 5: Accuracy for Brinzolamide

 Precision

The percentage RSDs of system precision for Brimonidine and Brinzolamide peak values were 0.2 and 0.1 for the standard solution area, which was within the limit of 2.0%. To investigate method precision, six different sample preparations were utilized and the percentage RSD values for the Brimonidine and Brinzolamide content were 1.6 and 1.3 respectively within the limit of 2.0%. In the same manner, six sample preparations were done for intermediate precision and the results indicated that the content measured was 1.2% and 0.5% which fits into the 2% RSD limit range. The measured RSD and the absolute difference between intraday and inter day precision fell within the specified range. Hence, this method was reported as accurate with respect to system precision; inter day precision and intraday precision; refer Table 6 and 7.

Table 6: System precision

Figure 12: System precision Chromatograph  Click here to View Figure

Table 7: Intermediate and method precision

Figure 13: Method precision/ intermediate precision chromatograph of test  Click here to View Figure

Robustness

The robustness parameters such as column oven temperature (+ 5°C), flow rate (+ 0.1mL), mobile phase buffer pH (+ 0.2) were performed and their percentage RSD was calculated (Table 8) which was found within the limit. No significant changes were observed in the results even after changing the above parameters and hence we have concluded that the above method is robust as per ICH validation guideline.

Table 8: Evaluation of Robustness Parameter

Figure 14: Robustness parameter chromatograph of test Click here to View Figure

Figure 15: Overlay graph of test robustness parameter Click here to View Figure

Stability of resulting solution

Solution stability is a key indicator of method validation. To establish the stability of analyte content in solution over a period of time under specific conditions, the sample solution was analyzed from the initial time after preparation at different time intervals. It was observed that standard as well test solutions in our method were stable upto 24hours. This emphasizes the method consistency and results reliability over the time period of method application; refer Table 9.

Table 9: Stability results of test solution

Degradation Study

Hydrolysis degradation study

The hydrolysis stress level of the test sample was checked by refluxing the test sample quantity at 60°C for 6 hours under suitable conditions. The above hydrolytic solution was diluted up to the mark and inserted in the HPLC system in order to verify the stability of the sample. It does not show impact by producing any impurity.

Figure 16: Hydrolytic degradation graph of test solution Click here to View Figure

Thermal degradation study

In order to conduct the thermal degradation study, the test sample was subjected to a temperature of 105°C for 6 hours in the hot oven.  It was allowed to cool to room temperature. Subsequently the test sample was diluted to obtain the desired concentration and injected in the chromatographic device so as to evaluate its thermal stability. This condition does not show impact on assay results by producing any unknown impurity.

Figure 17: Thermal degradation graph of test solution  Click here to View Figure

Photolytic stability study

The stability of the photolytic test sample was studied by irradiating the same to ultraviolet-visible light by placing it in a glass beaker in UV photo chamber for 1.2 million Lux hours or 200 watt hrs/m². The resulting diluted desired solution was then inserted into the HPLC chromatographic system. It shows sensitivity of Brimonidine peak by generating unknown at 1.2 and 2.8 RT.

Figure 18: Photolytic degradation graph of test solution  Click here to View Figure

Acid degradation study

Stress level of the acidic sample was performed by addition of not more than 1 mL of 2N hydrochloric acid into the sample and subjected to boiling at 60°C for 30 minutes duration. The resulting solution on cooling and neutralization with 1 mL of sodium hydroxide solution (2 N) was diluted to obtain the desired concentration. HPLC chromatographic system was used to evaluate its acidic stress level at 60°C which shows not much impact on drug product.

To observe temperature impact on the acidic stress level we further kept the test solution with addition of same acid and volume for 75°C for 3 hours. However no such temperature impact was observed on both drug moieties.

Figure 19: Acidic degradation graph of test solution at 75°C for 3 Hrs.  Click here to View Figure

Base degradation study

Alkali base degradation of the sample was performed by addition of 1 mL of sodium hydroxide (2 N) into the sample and refluxing it for 30 minutes duration at 60°C. The resulting solution on cooling and neutralization with 1 mL of hydrochloric acid solution (2 N) was diluted to obtain the desired concentration. The resulting solution was injected in the HPLC system to evaluate its alkali base stability. The resulting chromatograph shows unknown impurity at 1.1 retention time. This condition shows Brinzolamide peak reduction drastically.

We further degraded the test sample by applying same chemical but keeping sample at 75°C for 3 hours and the resulting desired test solution after dilution was injected in the chromatographic system (Ref Fig.20). The chromatograph shows drastic degradation of both the contents by generating peak at 0.9 and 1.1 RT.

Figure 20: Basic degradation graph of test solution at 75°C for 3 hours  Click here to View Figure

Oxidation degradation study

Oxidative degradation of sample was evaluated by addition of 1 mL of 3% hydrogen peroxide solution into the sample and refluxing for 30 minutes at 60°C. After cooling the resulting solution 1 mL of reducing agent sodium meta-bisulfate solution was added and diluted to obtain the desired concentration. The resulting solution was injected in the HPLC device for peroxide degradation study.

We degraded the sample solution by storing the same reagent in the same quantity at 75°C for an hour in order to observe the effects of the temperature increase on both medications. The resulting solution shows half drastic impact on Brimonidine peak. (Ref Fig.21)

Figure 21: Oxidative degradation graph of test solution at 75°C for 1 Hr. Click here to View Figure

The degradation pathways are shown in Table 10.

Table 10: Evaluation of Forced degradation

The given optimised method shows sensitivity towards stress conditions in the range of 5 to 20 % of degradation. Brimonidine tartrate shows its sensitivity towards photolytic, acid and base conditions. Similarly Brinzolamide exhibits sensitivity to base and oxidative conditions. The above data shows clear stability indication for the given optimised method.

Conclusion

The analytical method that was put forward in this work has been successfully validated for measuring of Brimonidine and Brinzolamide in accordance with the relevant regulations of the USP <1225> and the ICH guideline Q2(R2). This technology has been determined to be robust, linear, accurate, precise and particular. This approach will serve for both qualitative and quantitative analysis of brimonidine and brinzolamide combination throughout the process as well as in the final formulation. The greenness of the method was explained by using various quantitative green metric such as AES, NEMI, MoGAPI and AGREE tools which reiterates  the optimized method is eco- friendly. This indication will be useful as an alternative to many available published methods for the estimation of Brimonidine tartrate and Brinzolamide both single and in combine dosage form for incoming stage testing and stability testing in defined formulation.

Acknowledgement

The resources and facilities required for this research were provided by Chemclues Life Science Pvt. Ltd. in Navi Mumbai, Maharashtra, India, for which the authors are grateful.

Conflict of Interest

The authors disclose that there is no conflict of interest.

Funding Sources

There is no funding Sources

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Abbreviations

ICH: International conference of Harmonization

RP-HPLC: Reverse Phase- High Performance Liquid Chromatography

RSD: Relative Standard Deviation

IOP: Inter Ocular Pressure

QC: Quality Control

NLT: Not Less Than

NMT: Not More Than

BRT : Brimonidine Tartrate

BRZ : Brinzolamide