VALIDATED SPECTROPHOTOMETRIC METHODS FOR SIMULTANEOUS DETERMINATION OF BRINZOLAMIDE AND TIMOLOL MALEATE IN THEIR PURE FORM AND OPHTHALMIC PREPARATION BY

Four sensitive, simple and precise spectrophotometric methods for the simultaneous determination of brinzolamide (BRZ) and timolol maleate (TML) in their pharmaceutical formulation are developed and validated. Method A is the ratio difference spectrophotometric method (RDSM) which can measure the difference in amplitudes between 251 and 265 nm of ratio spectrum for brinzolamide and between 285 and 306 nm of ratio spectrum for timolol. Method B is the mean centering of ratio spectra method (MCR) by measuring the peak amplitude at 252 nm for brinzolamide and 304 nm for timolol. Method C is area under the curve (AUC) in which the area under the curves for brinzolamide and timolol were selected over the ranges of (260265) and (282-288) nm. Method D is the bivariate method (BVM) which depended on the quantitative evaluation of the absorbances at 265 and 285 nm, respectively. Beer’s Lambert’s law for the adopted methods were obeyed over the concentration range of 636 μg/mL for Brinzolamide and 6-42 μg/mL for Timolol. The obtained results were statistically compared with those obtained by the reported methods, showing no significant difference. The suggested methods were validated according to the ICH guidelines and were successfully applied for simultaneous determination of brinzolamide and timolol maleate in bulk drug and in their ophthalmic preparation.


Introduction
Literature survey reports that there are few analytical methods available for simultaneous estimation of BRZ and TML.Three spectrophotometric methods namely simultaneous equation method, Q-absorbance ratio method and ratio first derivative method for the simultaneous determination of BRZ and TML in their mixture form [P.A. Shah et al. 2014] and HPLC method [R.Khatun et al. 2014] were reported.In context to this, the aim of this research paper is to present four recent, sensitive, economical, precise and accurate UV spectrophotometric methods that present wide application for the simultaneous estimation of BRZ and TML in bulk drug and in their ophthalmic preparation without prior separation.Our work can be incorporated in the quality control analysis where time and coast of great value.

Apparatus
JASCO dual beam (Japan) UV-visible spectrophotometer model V-630.The bundle software, spectra manager II was used.The spectral slit width was 2nm and the speed of scan is 1000 nm/min.Sonicator: Bandelin Sonorex RK 510S, Donation of Alexander von Hum-boldt -Foundation, Bonn-Bad, Godesberg, Federal Republic of Germany.

Software
All computations of mean centering method were performed in Matlab® version 7.10 [Matlab 2010].

Materials and reagents
Methanol (Piochem®) spectroscopic grade and distilled water.

Pure standard:
Brinzolamide standard was purchased from Sigma-Aldrich Chemie (GmbH, Germany) and Timolol was kindly supplied by Epico (Cairo, Egypt) ; their purity was reported to be 99.7 % and 99.92 % , respectively according to the reported HPLC method [R.Khatun etal 2014].

Stock standard solution
1-Stock standard solution of BRZ (0.6 mg/mL) An accurately weighed 6 mg of BRZ was dissolved in 10 mL methanol in 10-mL volumetric flask and completed to the volume with the same solvent.

2-Stock standard solution of TML (0.6 mg/mL)
An accurately weighed 6 mg of TML was dissolved in 10 mL methanol in 10-mL volumetric flask and completed to the volume with the same solvent.
Aliquots of the prepared stock solutions were further diluted with methanol to a final volume of 10 mL.The diluted solutions were used as the working solutions for BRZ and TML.

Construction of calibration curves:
Standard solutions of both BRZ and TML in the range of (6-36 µg/mL) and (6-42 µg/mL), respectively, were separately prepared by appropriate dilutions of their respective stock standard solutions in methanol.The absorption spectra of the resulting solutions were measured in the range of 200-400 nm using methanol as blank and stored in the computer.

Ratio difference spectrophotometric method (RDSM)
The stored spectra of BRZ were divided by the spectrum of 12 µg/mL of TML to obtain ratio spectra, as well as the spectra of TML were divided by the spectrum of 12 µg/mL of BRZ.A two calibration curves were constructed relating the difference between the amplitudes of ratio spectra of BRZ and TML at (251 and 265 nm) and (285 and 306 nm), respectively, versus the corresponding concentrations of BRZ and TML, respectively, and the regression equations were computed.

Mean centering of ratio spectra method (MCR)
The stored spectra of BRZ were divided by the spectrum of 12 µg/mL of TML to obtain ratio spectra, as well as the spectra of TML were divided by the spectrum of 12 µg/mL of BRZ, the obtained ratio spectra of both mentioned drugs were mean centered.Two calibration curves were constructed relating the mean center values of BRZ at 252 nm and TML at 304 nm, respectively, versus the corresponding concentrations of BRZ and TML, respectively, and the regression equations were computed.

Area under the curve method (AUC)
The AUC of BRZ was measured between (260 -265) nm for BRZ and between (282 -288) nm for TML.The calibration curve of the mentioned drugs was plotted relating area under the curve versus the corresponding concentrations of BRZ and TML, and then the regression equations and the absorptivity constants were computed.

Bivariate method (BVM)
The absorption spectra of the prepared solutions were measured at 265, 270, 275, 280 and 285 nm.Four calibration curves; 2 calibrations for each component at 2 wavelengths selected using method of Kaiser at 265 and 285 nm, were constructed against the corresponding concentrations of BRZ and TML, and the corresponding regression equations were computed.

Analysis of laboratory prepared mixtures different ratios of BRZ and TML using the proposed methods
Six laboratory prepared mixtures containing different ratios of BRZ and TML were analyzed using the suggested methods.The concentrations of BRZ and TML were calculated from their corresponding regression equations.

Assay of pharmaceutical formulations
AZARGA eye drops bottle each one mL is claimed to contain 10 mg of brinzolamide and 5 mg of timolol maleate equivalent to 5 mg timolol mixed with reported excipients like 0.1% benzalkonium chloride, 0.1 mg mannitol, and 0.1 mg sodium chloride.
One ml of Azarga eye drop was transferred to 10 ml volumetric flask, 8 mL methanol were added and sonicated for 10 minutes then filtered.The volume was completed to the mark with methanol to get 1000µg/mL BRZ and 500µg/mL TML.From the above prepared solution, further dilutions were performed in the obtained linearity ranges using methanol for the adopted spectrophotometric methods.The procedure of each of the suggested methods was applied to determine the concentration of the cited drugs in the prepared dosage form solution.The determination was performed in triplicate for each concentration.Concentrations of BRZ and TML in the prepared samples were calculated from their corresponding regression equations.

Results and discussion
The main target of this paper is to develop four sensitive, simple and precise spectrophotometric methods for the simultaneous determination of (BRZ) and (TML) in bulk drugs and in their pharmaceutical formulation.Since there are few analytical methods available for the simultaneous estimation of Brinzolamide and Timolol in their combined pharmaceutical formulation.
The suggested methods; Ratio difference, Mean centering of ratio spectra, Area under the curve and Bivariate spectrophotometric methods, were applied to resolve the spectral overlap of BRZ and TML in their binary mixture without prior separation steps.

Ratio difference spectrophotometric method (RDSM)
Ratio difference method is simple and accurate method that has the ability for solving severely overlap spectra without prior separation at the same time it does not require any sophisticated apparatus or computer programs.The zero order absorption spectra of the laboratory-prepared mixtures of BRZ and TML are divided by a concentration of 12µg/mL of BRZ and 12µg/mL of TML as a divisor for the determination of BRZ and TML, respectively separately to get the ratio spectra shown in( Fig. 2and 3).Different pairs of wavelengths were selected to choose the optimum wavelengths at which measurements were done, the amplitudes at 251 nm and 265 nm were selected for determination of BRZ and 285 and 306 nm for determination of TML which provide the best selectivity.The concentration of BRZ and TML were obtained using their corresponding regression equations obtained by plotting the difference in the amplitudes at 251-265 nm and 285-306 nm respectively, of the ratio spectra against their corresponding concentrations: RDSM BRZ =0.4020 C-0.4088 r=0.9993RDSM TML =0.2827C-0.0934r=0.9999 Where C is the concentrations of cited drugs µg/mL, RDSM is the difference in peak amplitudes of the ratio spectrum curves and r is the correlation coefficient.

Mean centering method (MCR)
This method depends on the mean centering of ratio spectra.It eliminates the derivative step so the signal-to-noise ratio is enhanced Mean centering method was applied to determine concentration of BRZ in presence of TML and vice versa.For determination of BRZ, the absorption spectra of BRZ were divided by the absorption spectrum of 12µg/mL of TML as a divisor and for determination of TML, the absorption spectra of TML were divided by the absorption of 12µg/mL of BRZ as a divisor.The obtained ratio spectra were mean centered and the concentrations of BRZ and TML were determined by measuring the amplitude at 252 nm and at 304 nm, respectively (Fig. 4and5).The linear regression equation is found to be: Where C is the concentrations of the cited drugs, MCN is the peak amplitude of the mean centered ratio spectrum curve and r is the correlation coefficient.

Area under the curve method (AUC)
AUC [M.M.Abdelrahman et al. 2013] is an accurate, rapid and selective method for the simultaneous determination of components having overlain spectra in binary mixtures by calculating the areas under the spectra in relation to their concentrations.Careful choice of the wavelength ranges at which calculations were done was of great importance, so different pairs of wavelength ranges were selected to choose the optimum wavelength range.
Area under the curves obtained from the scanned spectra over the ranges of wavelengths (260 -265) and (282 -388) nm (Fig. 6 and 7) were recorded and then the corresponding regression equations were obtained for both BRZ and TML.
Where y is the area under curve over the ranges (260-265) and (282-388) respectively, x is the concentration in μg/mL and r is the correlation coefficient Areas under the curve and the absorptivity values at the selected wavelength ranges are used for calculating the concentration of BRZ in presence of TML and vice versa using the following equations: -Where C X ,C Y are the concentrations of the cited drugs.
are the absorptivity of drug X at ( ) nm and at ( )nm , Respectively.
--are the area under the curves at ( ) nm and at ( ) nm, respectively .
The calibration graphs that relate the measured areas under the curve to the concentration of each component in μg/mL were constructed.

Bivariate method (BVM)
This method depends on a simple mathematical algorithm, in which the data ara used from four regression equations, two calibrations for each component at two selected wavelengths using Kaiser method [D.Massart et al. 1988].The absorbance was measured at the selected wavelengths and the corresponding regression equations were calculated for both BRZ and TML.The calculated slope and intercept values were used for obtaining concentration of each compound using the following equation: -A AB1, A AB2 are the absorbances of the drug mixture at the two selected wavelengths (1 and 2), respectively.
-e AB1 , e AB2 are the sum of the intercepts of the linear calibration at two wavelengths (e AB1 = e A1 + e B1 ).
For BRZ and TML mixture,five wavelengths (265,270,275,280,285 nm) were chosen and Kaiser Method is used to select two wavelength at which four regression equations, two calibrations for each component were obtained.The selected wavelengths are 265.0nm and 285.0 nm.At these selected wavelengths, the onecomponent calibration curves were obtained in the range of (6 -36 μg/mL) for BRZ and (6 -42 μg/mL) for TML.The linear regression equations were found to be: Where A is the absorbance value at 265 nm and 285 nm, C is the concentration in μg/mL and r is the correlation coefficient.

Method validation
The applied methods had been validated according to ICH guidelines [ICH 2005] .

Linearity & Range:
The linearity of the methods was evaluated by analyzing six concentrations of BRZ and seven concentrations of TM ranging from 6-36 µg/ml and 6-42 µg/mL, respectively.The linear regression parameters were listed in Table1.

Accuracy
The accuracy of suggested method was checked by analyzing five different concentrations of each drug in triplicate within the linearity range where percentage recoveries suggested satisfactory results of the accuracy of the performed methods as shown in Table 1.The validity of the method was checked by applying the standard addition technique, where satisfactory results are obtained Table 2.

Selectivity
The selectivity of the methods was assessed by the analysis of laboratory prepared mixtures of BRZ and TM in different ratios within the linearity range where good results were obtained as shown in Table 3.

Precision
Precision of the adopted methods was evaluated by performing intra-day and inter-day precision studies.The intra-and inter-day precision were assessed using three different concentrations of each of BRZ and TML in triplicate on the same day and on three successive days using the proposed methods, respectively.The relative standard deviations were obtained Table 1.

Limit of detection (LOD) and Limit of quantification (LOQ):
The LOD and LOQ of the suggested methods were checked and calculated from the standard deviation (σ) of the response and the slope of the calibration curve (S) according to the equations: LOD = 3.3 x σ/S , LOQ = 10 x σ/S and the results were obtained as shown in Table1.

Application of the methods in assay of eye drops
The suggested methods are applicable for the analysis of BRZ and TM in their combined pharmaceutical formulation Azarga eye drops with no interference from the excipients; the results are shown in Table 2.

Statistical analysis
Statistical comparison between the results obtained by the suggested methods and those obtained by the reported HPLC method [R.Khatun etal 2014] showed no significant differences as shown in

Conclusion
From the previous discussion, it could be concluded that the applied methods were simple with minimum manipulation steps, accurate, don't need any sophisticated apparatus and could be easily applied in quality control laboratories.
The developed methods were validated as per ICH guidelines and were found to be precise as revealed from % RSD less than 2, for simultaneous estimation of BRZ and TML in mixture over the applied range.Finally the suggested method could be successfully applied for the routine analysis of BRZ and TML in their pure bulk powders and in their combined ophthalmic preparation without prior separation procedure steps.
Fig. (1): Chemical structure of Brinzolamide (A) and Timolol Maleate (B) It was found that the combination of BRZ and TML gives better effect in treatment of glaucoma than each drug alone, this combination was marketed in the form of eye drops [P.A.Shah et al. 2014].
It can determine the unknown concentration of a component of interest present in a mixture containing both the component of interest and an interfering component [H.M.Lotfy et al. 2014].
A , C B are the concentrations of the analytes.

Table 4 . Table1. Regression parameters and results of determination of pure samples of BRZ and TML by the proposed methods.
RSD% a : the intra-day relative standard deviation of 12 , 24 and 36 μg/mL of BRZ and TML by the proposed methods.RSD% b : & inter-day respectively relative standard deviation of 12 , 24 and 36 μg/mL of BRZ and TML by the proposed methods.

Determination of BRZ and TML in Azarga eye drop by the proposed methods and application of standard addition technique.
* Average of six determinations.**Average of nine determinations over three concentration levels.Table3.