Application of Design of Experiment in Design, Development and Optimization of Stability Indicating RP-HPLC Method for Simultaneous Determination of Montelukast Sodium and Rupatadine Fumarate in Bulk and Formulation

Design of Experiment assisted stability indicating RP-HPLC wasdesigned, developed and optimized using response surface methodology for simultaneous determination of Montelukast sodium and Rupatadine fumarate. Separation was achieved using Acetonitrile: Phosphate buffer (75:25) v/v with pH adjusted to 4.0, flow rate of 1 ml/min with UV detection at 246 nm on RP-C18 column. Stress degradation studies were performed as per scientific guidelines. Method was validated in accordance with regulatory requirements. Results obtained in validation were found to be within specified limit. Montelukast was eluted at 3.99 min and Rupatadine was eluted at 13.25 min respectively. All stress degradation products are very well resolved from drug peak which indicate suitability indicating nature of the developed method. Design of Experiment technique can help in fast and economical optimization of mobile phase which in turn will save time for method development. The developed method is, accurate, sensitive which can be utilized as stability indicating method for identification of degradation products in routine analysis of the drug. Original Research Article Sutar and Magdum; JPRI, 33(50A): 126-144, 2021; Article no.JPRI.77094 127

Present work focusses on use of Design of Experiment technique with response surface methodology as a systematic tool fordesign, development, optimization and validation of stability indicating RP-HPLC method for simultaneous determination of Rupatadine fumarate and Montelukast Sodium.

MATERIALS AND METHODS
Gift samples of Montelukast Sodium and Rupatadine Fumarate were supplied by local pharmaceutical industries. Acetonitrile and Methanol (HPLC Grade were procured from Merk Specialties Pvt. Ltd., Mumbai, and Thomas Baker (chemicals) Pvt. Ltd., Mumbairespectively. Other AR grade chemicals were procured form Research Lab Fine Chem. Industries, Mumbai.

Instrument and Software
Carry-500 Double beam UV-Visible spectrophotometer (Varian) with CARRY software, HPLC Systemhaving 515-pumps with column oven, PDA detectors Auto sampler (Waters) with Empower 2.0 software and pH Meter (Equiptronics) were used for method development. Along with this Design Expert 7.0 (Trial Version)was used for designing and optimizing the method parameters. Microsoft excel was used for statistical analysis.

Experimental Section
As a first step of method development solubility of both drugs was tasted in different solvents to obtain a common solvent which can be used for simultaneous estimation of both drugs in a mixture.

Preparation of Standard Stock Solution
Standard Stock Solutions of Montelukast sodium and Rupatadine fumarate were prepared by transferring accurately weighed 100 mg of drugs to separate 100 ml volumetric flasks, enough methanol was added to the flasks and flasks were swirled for 5 min then final volume was made up to the mark with help of methanol. The concentration of resulting solution was found to be 1 mg/ml. These stock solutions were further diluted to get desired concentration for experimental work.

Preparation of Sample Stock Solution
Formulation sample (Smarti-M, German Remedies) was prepared by accurately weighing twenty tablets containing both drugs in defined ratio (Rupatadine10 mg+ Montelukast sodium 10mg), these tablets were powdered and amount of powder equivalent to 10 mg of Montelukast and 10 mg Rupatadine was transferred to a volumetric flask. The flask was ultra-sonicated for ten minutes after adding enough methanol to the flask. Final volume was made up to the mark and then solution was filtered through Whatman filter paper no 41. This process will separate the excipients from the API (Active pharmaceutical ingredient). Resulting solution was further used for experimental runs after diluting with suitable solvent.

Optimization of Chromatographic
Conditions with Design of Experiment [37][38][39] Earlier reported HPLC method were developed with traditional method development technique that involve changing variable based on the previous knowledge or drug properties. To minimize this trial of mobile phase optimization for method development in HPLC. New technique of systematic design of experiment was implemented for optimization of the mobile phase. This approach is known as design of Experiment approach in which important variable which causes change in retention behavior of the drug candidate were used to develop an optimized mobile phase.

Design of Experiment
First step in DoE experiment was identification of important variable that causes changes in the retention behavior of the drug. From the earlier trial runs and literature review it was found that percentage of organic phase,(%Acetonitrile), pH of the buffer solution, temperature are the important variables that should be considered in the design of experiments. Upper and lower limits of these factors were determined by trial runs on the HPLC system. The next step in the analysis was designing a central composite design (CCD) model which was used for estimating possible combinations of the three factors, a set of 20 experiments with different values for 3 variables was obtained in CCD model this model was used to evaluate the complete set of main effects and interactions. The objective of designing these experiments was to separate the degradation peaks from main drug peak with sufficient resolution. Flow rate, Percentage of organic phase (ACN) and pH of buffer are the three variables likely to have a significant impact on the separation behavior of drug. Resolution Value of Asymmetry factor and retention time are chosen as response variables. In the design of experiment, low-and high-level values of the variables were chosen based on initial experiments.
The Central Composite experimental design is shown in Table 1The data was evaluated using Design-Expert® 7.1 software (Trial Version).
Twenty experiments were carried out using suggested ratio and parameters as per Central Composite model and values of the desired responses were obtained from the software. All the values were added in the software and Response Surface Methodology was used for optimization of the responses which in turn will help in mobile phase optimization with a suitable combination suggested by the systematic experimentation approach.
Following steps were performed in response Surface Methodology

Stress Degradation Studies [40-44]
As the basic objective of the method development was development of a stability indicating assay method both drug samples were subjected to stress degradation conditions there were very few references available at the time of development of this method based on that a systematic way was followed for performing stress degradation of the drugs. Both drugs were subjected to stress studies in Acidic, Alkaline, Neutral, Oxidative, Photo stress degradation along with dry heat stress conditions.

Stress Degradation under Acidic Environment
To check stability of the drugs in acidic environment both the drugs were subjected to acid treatment.Methanolic drug solution was mixed with 0.1 m Hydrochloric acid in equal proportion and the resulting solution was refluxed for 8 hours. After 8 hours solution was neutralized and then diluted to get a concentration of 10 mcg/ml for drug. These diluted solutions were injected in the system and using optimized mobile phase, stability of drugs towards acidic environment was observed.

Stress Degradation under Alkaline Environment
Stability of both drugs in alkaline medium was observed by treating methanolic drug solutions with .1 M sodium hydroxide in equal proportion.These solutions were refluxed for 8 hours and resulting solutions were neutralized and diluted to get desired concentration of 10mcg/ml.Diluted stress samples were injected in the chromatographic system to understand effect of alkaline environment on drug molecules.

Stress Degradation under Neutral Environment
A drug molecule can also undergo degradation at neutral pH value. Methanolic drug solutions were mixed with equal amount of double glass distilled water and the resulting solution was refluxed for 8 hours at 80 0 C.The resulting solutions were diluted and were tested with the optimized mobile phase to study effect of neutral environment on stability of drug molecules.

Stress Degradation under Oxidative Environment
Many drug candidates are very much prone to degradation in oxidative conditions, to observe the effect of oxidative environment methanolic drug solutions were treated with 3% hydrogen peroxide solution and the resulting solution was refluxed for 8 hours at 80 0 C. Resulting solution was diluted to get suitable concentration and was injected in the system and effect of oxidative stress condition was studied in both drug molecules.

Photo Degradation Study
Photo catalytic degradation is observed in many drug candidates owing to their chemical structure. In order to study effect of light on drug sample, methanolic solutions were exposed to sunlight on a bright sunny day and at the end of the day solution was refrigerated and on next day it was brought to room temperature and then again it was exposed to direct sunlight.(Guidelines suggest use of UV lamp with definite power to check this effect) but there are few references where in direct sunlight was used to study photo stability of drug molecule. The resulting solution was diluted to 10 mcg/ml and then solutions were observed for any change in chromatogram.

Stress Degradation under Dry Heat Conditions
In order to check thermal stability of drug moles standard samples of drug were placed in a petri plate and it was placed in a hot air oven for 8 hours at a temperature of 100 0 C.The exposed powder was the transferred to a volumetric flask and it was diluted with methanol to get concentration of 10mcg/ml. SystemSuitability parameters are specified in USP value of these parameters is expressed after analyzing samples in triplicate 2.14 Validation of Developed HPLC Method [45][46] With reference to regulatory requirements of ICH guidelines it is essential to validate any analytical method for its intended purpose. Various validation parameter includes Accuracy, Linearity, Limit of Detection, Limit of Quantitation, Selectivity and Robustness and Ruggedness.

RESULTS
Response surface methodology was used for the optimization of chromatographic parameters. After initial 20 experimental runs as per Central Composite Design values of all response variables were entered in the software and further analysis of significant factors and interdependent terns were identified based on ANOVA test for a quadratic model and significance of models were determined based on lower p value. Model p value and lack of fit values were used for determining the significance of each factor.
The magnitude of the coefficients in the equations for the responses and the lower pvalue (<0.001) indicated that percentage of organic phase, pH of the buffer and flow rate significantly affected the responses.
The optimum conditions were calculated using numerical optimization.
To achieve the composite desirability (di), the response criteria were set as per requirements of the method for the responses the importance factors for all responses were set as 3+.
Derringer's desirability was calculated, and the optimum solution was determined to be a percentage of organic phases ACN 75%, the desirability graph indicated that the maximum desirability was achieved for the pH of 4.0 and at a flow rate of 1.0 ml/min.
To confirm the point prediction values, experiments (n = 3) were conducted to determine the mean responses R1S, R2 S and Rt. The experimental results were found to be R1 S experimental = 2.05, R2 S experimental = 4.21 and Rt experimental = 12.98, representing good agreement with the predicted results.
Four Solutions were found from the design experiments Derringer's desirability was used as evaluating tool results are shown in Table 2.

Solubility of Drugs in Different Solvents
Solubility pattern of both drugs was studied by dissolving them in different solvents. Montelukast was freely soluble in methanol and water, whereas Rupatadine is soluble in methanol.
Initially a mobile phase was optimized for identification of both drugs , but when same mobile phase was used for determination of stability indicating nature of the method, separation of degradation products from pure drug peak resolution was not sufficient and some degradation products were co eluting near retention time of the drugs, in order to solve this issue a new technique of Design of experiments was used and based on previous knowledge central Composite Design was constructed and similar experiments were carried out based on the responses selected an response surface methodology tool was used for optimization of mobile phase according to requirements for separations.
Suggested solution from DoE experiment was used to check applicability of it for explaining stability indicating nature of the method.
It can be observed that the newly designed mobile phase and chromatographic parameters gives desired runtime and sufficient resolution in order to use it as a stability indicating method.

Evaluation of Analytical Method (Method Validation)
Developed analytical proposed methods were validated as per guideline laid by the International Conference on Harmonization (ICH) procedure and United State Pharmacopoeia 24 (USP 24).

Linearity and sensitivity (Limit of Detection and Limit of Quantitation)
Linearity shows the direct relationship of response to the concentration of analyte, it was evaluated by constructing a calibration curve of concentration against peak area for both the drugs separately. Linearity was expressed in terms of correlation coefficient, and it was found to be 0. Limit of detection and limit of quantitation were determined based on the calibration curve data. It was found that minimum concentration detected by the method was 4.06 mcg/ml for Montelukast where as it was found to be 4.10 for Rupatadine fumarate. Minimum quantitation limit was found to be 12.13 mcg/ml and 13.12 mcg/ml for Montelukast and Rupatadine respectively.

Precision:
Precision, reproducibility and accuracy study of the proposed approach were judged by performing replicate investigation of the working standard solutions. Within the linearity calibration curves, the selected concentrations were prepared and analyzed with the developed method to estimate the Intra-day and Inter-days variability. For the Intra-day analysis repeated injection of the selected solution were assessed on the same day. Whereas, Inter-days study were continued five injections were completed for three consecutive days. Precision and reproducibility were expressed in terms of as the % RSD, [ Table 7]. Observation of the resulting values indicates that developed method shows good inter day and Intraday precision.

Accuracy:
To check the degree of accuracy of the method, recovery studies were performed in triplicate by standard addition method at 3 levels of 50%, 100% and 150%. Known amounts of standard Montelukast sodium and Rupatadine were added to pre-analyzed formulation samples separately and were subjected to the HPLC analysis using optimized parameters.
Results of recovery studies are shown in Table 8.
Mean percentage recovery of the analyte was estimated by comparing the concentration obtained from spiked analyte with the actual added concentration. It was observed that mean recovery for both drugs was within 98-102% limit.Also, recovery study outcomes exposed the absence of interference from commonly encountered pharmaceutical excipients present in the selected formulation.      Tables 9 and10.It was observed that even after small alterations in the experimental parameters no significant changes in the results obtained after sample injection.

Analysis of the Marketed Formulation
Optimized mobile phase was used for analysis of pharmaceutical formulation.
Linear regression equation was employed to estimate the amount of Rupatadine and Montelukast tablet. The amount found is calculated which was found to be within the limit of label claim as mentioned in Table 11.

Stress Degradation STUDIES
A stock solution containing 100 mg of drug in 100 ml methanol get final concentration 1mg mL -1 was prepared. This solution was used for force degradation to provide an indication of stability indicting property of the proposed method.

Study of Acid Induced Degradation Product
To check the effect of acid hydrolysis, acid degradation product was injected in the system. Interference of blank Hydrochloric acid was studied by injecting 0.1 M HCl in the system Fig. 8.
Careful observation of blank and degradation chromatogram shows that an additional peakswere present at 6.18 min, 8.79 min and 10.48 min along with drug peak [ Fig. 9] in acid induced hydrolytic conditions for Montelukast Sodium which shows that Montelukast is prone to acid degradation three degradation products were formed in the process of acid hydrolysis. Drug peak showed less than 10% degradation.

Alkali induced Hydrolysis
Initially Blank is injected in the system chromatogram of blank is shown in Fig. 11.
Alkali induced sample of Montelukast sodium shows additional peak at 6.181 min and 10.45 min along with drug peak indicating formation of additional degradation [47-48] products in the alkaline environment and drug is prone to undergo degradation in alkaline conditions. Chromatogram is shown in Fig.12.
Rupatadine fumarate sample which was degraded in alkaline conditions was chromatographed using same optimized mobile phase chromatogram is shown in Fig. 13.
The chromatogram of Rupatadine fumarate does not show additional peak in the alkaline environment suggesting stability of the drug in alkaline conditions.

Degradation under Neutral Conditions
Neutral hydrolysis of both the drugs does not show any additional peaks in the chromatogram.

Preparation of Hydrogen Peroxide induced degradation product:
Peroxide induced degradation sample of Montelukast sodium was analyzed by optimized mobile phase.
It was observed that chromatogram of stress sample shows additional peak at 5.13 and 5.496minalong with drug peak which show that drug is prone to oxidative environment, and it undergoes degradation forming two degradation products. Chromatogram is shown in Fig. 15.
Rupatadine fumarate sample was analyzed and chromatogram was shown in Fig.16. No additional peaks were observed in the chromatogram of Rupatadine in oxidative environment in case of Rupatadine Fumarate.
Photo-degradation product: It was observed that Montelukast chromatogram shows additional peaks at 5.03 min and 10.25 min along with drug peak. When drug was exposed for lesser period of time even then similar peak were obtained after2 hour exposure which shows that Montelukast is undergoing photo catalytic degradation very fast in direct sunlight.
When Rupatadine was exposed to sunlight it does not showed any additional peak in the chromatogram, this shows that drug is very much stable in most of the stress conditions, To study the stability indicating nature of developed method all the degradation samples were mixed together and resulting solution was injected in the HPLC system It was observed that all degradation peaks are well resolved in case of Montelukast sodium. When all degradation samples are mixed and injected in the system no additional peaks were observed in which shows stability of Rupatadine in all stress conditions. These chromatograms of all impurities of Montelukast sodium are resolved properly.
When all stress samples of both drugs were mixed and injected in the HPLC system and optimized chromatographic conditions were used, the resulting chromatogram shows very Comparison of merits over reported stability indicating method and developed method are shown in Table 12.

DISCUSSION AND CONCLUSION
The proposed approach of design of experiment in design, optimization and development of stability indicating HPLC method is accurate, precise, fast and selective for the simultaneous estimation of Montelukast sodium and Rupatadine fumarate in bulk and solid dosage form. The method can be used as a stability indicating method as degradation products are resolved from the drug peaks. The proposed assay outcomes, recovery value for the selected tablets were in good accord with their respective labeled claims Non-interference of the excipients was observed through the analytical run which shows accuracy of the method. All the results of different validation parameters were found to be with in specified limits of regulatory guidelines. Hence this method can be conveniently adapted for the routine quantitative estimation and studying stability indicating nature of drugs.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.