# Novel LC Method Development and Validation for Simultaneous Determination of Montelukast and Doxofylline in Bulk and Pharmaceutical Dosage Forms

Authors: Gadapa Nirupa; A. Siva Kumar; Upendra M. Tripathi
Journal: Journal of Chemistry
 (2013)

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## Abstract

A novel rapid HPLC method was developed for simultaneous determination of montelukast and doxofylline in bulk and pharmaceutical dosage forms. Development of  an analytical method for simultaneous estimation of drugs requires a lot of efforts and of course it is a challenging task. The method was developed by using C18 (150mm×4.6mm, 5 μm) column; mobile phase consisting of methanol and phosphate buffer at pH 4.5; the flow rate of 1.0 mL/min and ultraviolet detection at 280 nm. Both drugs were sufficiently resolved having retention time of 4.7 min and 1.9 min for montelukast and doxofylline, respectively. The method was validated as per ICH Guidelines for various parameters like precision, linearity, accuracy, ruggedness, and robustness. The validated method was applied to the commercially available pharmaceutical dosage form and obtained the desired result.


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## Body

## 1. Introduction

In continuation to our earlier efforts [1, 2]  to support our pharmaceutical fraternity, in this  paper  also  we  are  trying  to   contribute  with  the  analytical  method  of    very important drugs required to cure most common disease,  asthma.Asthma is a chronic inflammatory disease that affects children and adults with symptoms   such   as   a runny   nose,   nasal   congestion,   and   sneezing.   It   causes inflammation of the airways in which the smaller passages of the lungs narrow. This causes wheezing, shortness of breath, mucus production, and coughing.  With the present day’s life style and due to environmental pollution, the population of asthma-infected people is drastically increasing. Hence, it occupies great medical importance. This can be controlled using anti-inflammatory drugs, bronchodilators, or leukotriene modifiers. Due to this reason, we have selected two important drugs, namely, montelukast and doxofylline for the study.  Earlier, one method development for single drug, montelukast, using HPLC has been executed and submitted as a part of the author’s dissertation work [3]. It has encouraged the authors to extend this study with multidrug matrix. In the present study, two drugs montelukast and doxofylline combination has been selected, which are used in the medication and maintenance of asthma.Montelukast  is a leukotriene  receptor antagonist (LTRA) used for the maintenance treatment  of  asthma  and  to  relieve  symptoms  of  seasonal  allergies.  It is usually administered orally. Montelukast is a CysLT1 antagonist and it blocks the action of leukotriene D4 (and secondary ligands LTC4 and LTE4) on the cysteinyl leukotriene receptor CysLT1 in the lungs and bronchial tubes by binding to it. This reduces the bronchoconstriction   otherwise   caused   by   the   leukotriene   and   results in less inflammation. Decreasing   inflammation   reduces   swelling   that   narrows   airways. Montelukast also relaxes bronchial tube walls. Apart from being used to treat asthma, it is also used to treat symptoms of hay fever and allergic rhinitis. Montelukast sodium is   described   chemically   as   [R-(E)]-1-[[[1-[3-[2-(7-chloro-2    quinolinyl)   ethenyl] phenyl]-3-[2-(1-hydroxy-1-methyl ethyl)phenyl]propyl]thio]methyl]cyclopropane acetic acid, monosodium salt.Doxofylline (also known as doxophylline) is a xanthine derivative drug used in the treatment of asthma. It has antitussive and bronchodilator effects and acts as a phosphodiesterase inhibitor. Bronchodilators are drugs used to relieve the symptoms of an asthma attack by clearing mucus from the lungs and reducing swollen airways. Doxofylline is used to treat the symptoms of asthma as well as certain other lung diseases and conditions. It is proven to alleviate symptoms by reducing the urge to cough and helping to increase the airflow to the lungs. Doxofylline is chemically described as 7-(1,3-dioxolan-2-ylmethyl)-1,3-dimethylpurine-2,6-dione. The chemical structures of montelukast and doxofylline are shown in the Figure1.Chemical structure of the drugs.
(a)
Montelukast
(b)
DoxofyllineFor  better  maintenance  therapy  in  patients  suffering  from  asthma  and  chronic obstructive pulmonary disease (COPD), Montelukast and Doxofylline are used in combination.  This drug combination is also available in the market. Hence, these drugs are selected for the analytical studies.For individual estimation of these drugs, several methods are available in the literature [4–15]. Some methods which are available in literature are for the simultaneous estimation of montelukast and levocetirizine [16–19]. Some other methods available in the literature are for the simultaneous estimation of montelukast and bambuterol [20–22]. There is only one method [23] available in the literature for the simultaneous estimation of montelukast and doxofylline and that too has certain limitations. The present method of this paper is faster and user friendly than the method available in the literature for the simultaneous estimation of Montelukast and Doxofylline.


## 2. Experimental Section

### 2.1. Method Development

Selection of UV Maxima. A dilute solution of Montelukast showed the UV maxima at  286  nm  and  the  absorbance  has  been  found  to  be  very  poor  due  to  less chromophoric nature of the compound. On the other hand, Doxofylline has exhibited the UV maxima at 273 nm, and 205 nm and the response was very good. To undertake the combined estimation of both drugs, it has been decided to select the UV maxima of 280 nm, which falls between the maxima of both the drugs and seems to be suitable.Selection of Column. As C18 is the most widely used column, therefore, we decided to develop a method on a common column so that anybody can utilize this method for their application.Selection of the Mobile Phase Composition.  Various mobile phase compositions have been tried to establish the simultaneous estimation of both the drugs. We tried with the simple mixture of methanol and water, which was followed up with the combinations of various Buffers but out of these the combination of methanol and phosphate buffer has been found to be suitable. Buffer pH has also been optimized by performing the estimation at various pH, namely, 3.0, 4.5, and 7.5 and after evaluation of the peak shape and retention time, it has been concluded  to adopt the buffer pH of 4.5.Selection of Column Temperature. This study was quite interesting, and the determination   of   the   combined   drugs   has   been   performed   at   three   different temperatures, namely, 25°C, 40°C and 50°C. It has been found that the retention time of  Doxofylline was unchanged (1.6 min) at all temperatures, whereas, the retention time of Montelukast has been found to be variable at different temperatures as 5.58 min at 25°C,  4.75 min at 40°C, and 4.22 min at 50°C. The representative graph is shown in Figure 2. Actually, 50°C is not a very suitable temperature for a routine analysis.  So it was not selected for carrying out the analysis.  We observed the difference of retention time of Montelukast between analysis at 40°C and 25°C to be almost 1 min. This was quite encouraging. Therefore, we decided to adopt 40°C of analysis temperature to speed up the analysis and elute both the peaks below 5 min, which is really demonstrating for the rapid estimation of this combination drug.Figure 2
Temperature variation chromatograms.Optimised Method of Analysis and Other Chromatographic Conditions Are Below. The Chromatographic column, C18 (150 mm × 4.6 mm, 5μm) was used as a stationary phase. Mobile phase was prepared with Buffer and Methanol (10: 90). Buffer was prepared  by  dissolving  0.01 M  Sodium  Hydrogen  orthophosphate  in  1000  ML  of water. The pH were adjusted to 4.5 with dilute OrthoPhosphoric  acid. The diluents used  for  the  analysis  was  prepared  with  Methanol  and  Water  (80  : 20).  Injection volume was 20μL. The pump flow rate was 1.0 mL/min. The column temperature was maintained at 40°C. The eluent was detected at 280 nm. The run time was 20 min.


### 2.2. Materials, Reagents, and Chemicals

Montelukast and Doxofylline Standards were obtained from Startech Labs. Combination drug tablets used for the experiment, Telekast-D, was manufactured by Intas Pharmaceuticals.  HPLC grade Methanol, Sodium Phosphate, and Orthophosphoric acid were obtained from Merck, Darmstadt, Germany.


### 2.3. Equipments

UV Visible spectrophotometer used was Shimadzu, model UV-2450. The HPLC instrument used was Schimadzu make, model-LC-2010 CHT. Class VP Software was used for data acquisition.


### 2.4. Preparation of Standard Solution

Mixed standard solution   of 0.01 mg/mL of Montelukast   and 0.4 mg/mL of Doxofylline   (treat this as 100% for various experimental purposes) was prepared by taking 25 mg of Montelukast and dissolving in 25 mL diluents. 1 mL of this solution was mixed with 40 mg of  Doxofylline in 100 mL volumetric flask diluted upto the mark with the diluent.


### 2.5. Preparation of Linearity Solutions

For  Linearity  150%,  125%,  100%,  75%,  and  50%  solutions  were  prepared.150% solution was prepared by dissolving 25 mL of Montelukast in 25 mL of diluent. 1.5 mL of this solution was added to 60 mg of Doxofylline and dissolved in 100 mL diluent. 20.83 mL of 150% solution was dissolved in 25 mL diluent for preparing 125% solution. 16.66  mL  of  150%  solution  was  dissolved  in  25  mL  diluent  for preparing 100% solution. 12.5 mL of 150% solution was dissolved in 25 mL diluent for preparing 75% solution. 8.33 mL of 150% solution was dissolved in 25 mL diluent for preparing 50% solution.


### 2.6. Sample Preparation for Accuracy

Three different solutions were prepared for performing the accuracy studies. The first solution was prepared by dissolving 25 mg of Montelukast in 25 mL diluent. 1 mL of this solution was added to 40 mg of Doxofylline in 100 mL volumetric flask and made up the solution with 150% linearity solution. The second solution was prepared by dissolving 25mg of Montelukast in 25 ml diluent. 1mL of this solution was added to 40 mg of Doxofylline in 100 mL volumetric flask and make up the solution with 100% linearity solution. The third solution was prepared by dissolving 25 mg of Montelukast in 25 ml diluent. 1mL of this solution was added to 40mg of Doxofylline in 100 mL volumetric flask and make up the solution with 50% linearity solution.


### 2.7. Preparation of Sample Solution for Batch Analysis

The  method  was  applied  to  the  analysis  of  commercial  sample,  Telekast-D.  The average weight of ten tablets was calculated and found to be 644.93 mg. The tablet was crushed to a homogeneous mixture and 59.43 mg of the tablet was dissolved in 100 mL of the mobile phase. The solution was sonicated for 5 minutes followed by cyclomixing for 5 minutes to extract the drug in solution. The resulting solution was filtered by using Millipore syringe filter (0.42µ).  The resulting clear solution was injected in HPLC in duplicate as per the developed method.


### 2.8. Analytical Method Validation

#### 2.8.1. Specificity of the Method

Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities. This parameter was performed to know the Retention Time of each drug in a  mixture  and  in  the  sample  to  understand  if  any   chromatographic  interference between the analytes or drug-excipient interference is present.


#### 2.8.2. System Suitability

System suitability test is used to verify if the resolution and reproducibility of the chromatographic systems are adequate for the analysis to be done. The tests are based on the fact that the equipment, electronics, samples to be analyzed constitute an integral system that can be evaluated as such. The limits for system suitability were set for theoretical plates, resolution, and asymmetry.


#### 2.8.3. Linearity

Concentrations  of  the  standard  mixture,  50%,  75%,  100%,  125%,  and  150%  were prepared and injected, and chromatogram was recorded. A graph was plotted for  the concentration of the corresponding  drug versus area. The Correlation coefficient (r) for each drug was calculated.


#### 2.8.4. Accuracy

To determine the accuracy in sample preparation, method of standard additions was made for measuring the recovery of the drugs. To the known standard solution concentrations of the drug (50%, 100%, and 150%) were added. Three different solutions were prepared as mentioned in Section2.6. The accuracy was expressed as the percentage of the analyte recovery.


#### 2.8.5. Robustness

To verify the robustness of the method, slight changes to the chromatographic conditions were made to check for any changes in the chromatogram. The analysis was done under variable flow rates, wave lengths, pH, and temperatures. The flow rate as per the developed method was 1 mL/min. This has been purposely changed to 0.8 mL/min and 1.2 mL/min, and the chromatogram was obtained. The wave length as per the developed method was 280nm. This has been purposely changed to 278 nm and 282nm. The pH in the developed method was 4.5. This was changed to 4.3 and 4.7, and the chromatogram was obtained. The column temperature in the developed method was 40°C. This was changed to 39.5°C and 40.5°C, and the chromatogram was obtained.


#### 2.8.6. Ruggedness

To verify the ruggedness of the method, the analysis was done on different days and different  chemists  to check for any changes in the chromatogram.  The percentage RSD for the retention time and area was calculated.


#### 2.8.7. Performance of the Method on Commercial Samples

The method is said to be effective if it can be applied for the analysis of commercial tablets. For this purpose, performance test of the method has been conducted on the market sample Telekast-D, Batch no. 06TTD040 manufactured by Intas Pharmaceuticals.


## 2.1. Method Development

Selection of UV Maxima. A dilute solution of Montelukast showed the UV maxima at  286  nm  and  the  absorbance  has  been  found  to  be  very  poor  due  to  less chromophoric nature of the compound. On the other hand, Doxofylline has exhibited the UV maxima at 273 nm, and 205 nm and the response was very good. To undertake the combined estimation of both drugs, it has been decided to select the UV maxima of 280 nm, which falls between the maxima of both the drugs and seems to be suitable.Selection of Column. As C18 is the most widely used column, therefore, we decided to develop a method on a common column so that anybody can utilize this method for their application.Selection of the Mobile Phase Composition.  Various mobile phase compositions have been tried to establish the simultaneous estimation of both the drugs. We tried with the simple mixture of methanol and water, which was followed up with the combinations of various Buffers but out of these the combination of methanol and phosphate buffer has been found to be suitable. Buffer pH has also been optimized by performing the estimation at various pH, namely, 3.0, 4.5, and 7.5 and after evaluation of the peak shape and retention time, it has been concluded  to adopt the buffer pH of 4.5.Selection of Column Temperature. This study was quite interesting, and the determination   of   the   combined   drugs   has   been   performed   at   three   different temperatures, namely, 25°C, 40°C and 50°C. It has been found that the retention time of  Doxofylline was unchanged (1.6 min) at all temperatures, whereas, the retention time of Montelukast has been found to be variable at different temperatures as 5.58 min at 25°C,  4.75 min at 40°C, and 4.22 min at 50°C. The representative graph is shown in Figure 2. Actually, 50°C is not a very suitable temperature for a routine analysis.  So it was not selected for carrying out the analysis.  We observed the difference of retention time of Montelukast between analysis at 40°C and 25°C to be almost 1 min. This was quite encouraging. Therefore, we decided to adopt 40°C of analysis temperature to speed up the analysis and elute both the peaks below 5 min, which is really demonstrating for the rapid estimation of this combination drug.Figure 2
Temperature variation chromatograms.Optimised Method of Analysis and Other Chromatographic Conditions Are Below. The Chromatographic column, C18 (150 mm × 4.6 mm, 5μm) was used as a stationary phase. Mobile phase was prepared with Buffer and Methanol (10: 90). Buffer was prepared  by  dissolving  0.01 M  Sodium  Hydrogen  orthophosphate  in  1000  ML  of water. The pH were adjusted to 4.5 with dilute OrthoPhosphoric  acid. The diluents used  for  the  analysis  was  prepared  with  Methanol  and  Water  (80  : 20).  Injection volume was 20μL. The pump flow rate was 1.0 mL/min. The column temperature was maintained at 40°C. The eluent was detected at 280 nm. The run time was 20 min.


## 2.2. Materials, Reagents, and Chemicals

Montelukast and Doxofylline Standards were obtained from Startech Labs. Combination drug tablets used for the experiment, Telekast-D, was manufactured by Intas Pharmaceuticals.  HPLC grade Methanol, Sodium Phosphate, and Orthophosphoric acid were obtained from Merck, Darmstadt, Germany.


## 2.3. Equipments

UV Visible spectrophotometer used was Shimadzu, model UV-2450. The HPLC instrument used was Schimadzu make, model-LC-2010 CHT. Class VP Software was used for data acquisition.


## 2.4. Preparation of Standard Solution

Mixed standard solution   of 0.01 mg/mL of Montelukast   and 0.4 mg/mL of Doxofylline   (treat this as 100% for various experimental purposes) was prepared by taking 25 mg of Montelukast and dissolving in 25 mL diluents. 1 mL of this solution was mixed with 40 mg of  Doxofylline in 100 mL volumetric flask diluted upto the mark with the diluent.


## 2.5. Preparation of Linearity Solutions

For  Linearity  150%,  125%,  100%,  75%,  and  50%  solutions  were  prepared.150% solution was prepared by dissolving 25 mL of Montelukast in 25 mL of diluent. 1.5 mL of this solution was added to 60 mg of Doxofylline and dissolved in 100 mL diluent. 20.83 mL of 150% solution was dissolved in 25 mL diluent for preparing 125% solution. 16.66  mL  of  150%  solution  was  dissolved  in  25  mL  diluent  for preparing 100% solution. 12.5 mL of 150% solution was dissolved in 25 mL diluent for preparing 75% solution. 8.33 mL of 150% solution was dissolved in 25 mL diluent for preparing 50% solution.


## 2.6. Sample Preparation for Accuracy

Three different solutions were prepared for performing the accuracy studies. The first solution was prepared by dissolving 25 mg of Montelukast in 25 mL diluent. 1 mL of this solution was added to 40 mg of Doxofylline in 100 mL volumetric flask and made up the solution with 150% linearity solution. The second solution was prepared by dissolving 25mg of Montelukast in 25 ml diluent. 1mL of this solution was added to 40 mg of Doxofylline in 100 mL volumetric flask and make up the solution with 100% linearity solution. The third solution was prepared by dissolving 25 mg of Montelukast in 25 ml diluent. 1mL of this solution was added to 40mg of Doxofylline in 100 mL volumetric flask and make up the solution with 50% linearity solution.


## 2.7. Preparation of Sample Solution for Batch Analysis

The  method  was  applied  to  the  analysis  of  commercial  sample,  Telekast-D.  The average weight of ten tablets was calculated and found to be 644.93 mg. The tablet was crushed to a homogeneous mixture and 59.43 mg of the tablet was dissolved in 100 mL of the mobile phase. The solution was sonicated for 5 minutes followed by cyclomixing for 5 minutes to extract the drug in solution. The resulting solution was filtered by using Millipore syringe filter (0.42µ).  The resulting clear solution was injected in HPLC in duplicate as per the developed method.


## 2.8. Analytical Method Validation

### 2.8.1. Specificity of the Method

Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities. This parameter was performed to know the Retention Time of each drug in a  mixture  and  in  the  sample  to  understand  if  any   chromatographic  interference between the analytes or drug-excipient interference is present.


### 2.8.2. System Suitability

System suitability test is used to verify if the resolution and reproducibility of the chromatographic systems are adequate for the analysis to be done. The tests are based on the fact that the equipment, electronics, samples to be analyzed constitute an integral system that can be evaluated as such. The limits for system suitability were set for theoretical plates, resolution, and asymmetry.


### 2.8.3. Linearity

Concentrations  of  the  standard  mixture,  50%,  75%,  100%,  125%,  and  150%  were prepared and injected, and chromatogram was recorded. A graph was plotted for  the concentration of the corresponding  drug versus area. The Correlation coefficient (r) for each drug was calculated.


### 2.8.4. Accuracy

To determine the accuracy in sample preparation, method of standard additions was made for measuring the recovery of the drugs. To the known standard solution concentrations of the drug (50%, 100%, and 150%) were added. Three different solutions were prepared as mentioned in Section2.6. The accuracy was expressed as the percentage of the analyte recovery.


### 2.8.5. Robustness

To verify the robustness of the method, slight changes to the chromatographic conditions were made to check for any changes in the chromatogram. The analysis was done under variable flow rates, wave lengths, pH, and temperatures. The flow rate as per the developed method was 1 mL/min. This has been purposely changed to 0.8 mL/min and 1.2 mL/min, and the chromatogram was obtained. The wave length as per the developed method was 280nm. This has been purposely changed to 278 nm and 282nm. The pH in the developed method was 4.5. This was changed to 4.3 and 4.7, and the chromatogram was obtained. The column temperature in the developed method was 40°C. This was changed to 39.5°C and 40.5°C, and the chromatogram was obtained.


### 2.8.6. Ruggedness

To verify the ruggedness of the method, the analysis was done on different days and different  chemists  to check for any changes in the chromatogram.  The percentage RSD for the retention time and area was calculated.


### 2.8.7. Performance of the Method on Commercial Samples

The method is said to be effective if it can be applied for the analysis of commercial tablets. For this purpose, performance test of the method has been conducted on the market sample Telekast-D, Batch no. 06TTD040 manufactured by Intas Pharmaceuticals.


## 2.8.1. Specificity of the Method

Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities. This parameter was performed to know the Retention Time of each drug in a  mixture  and  in  the  sample  to  understand  if  any   chromatographic  interference between the analytes or drug-excipient interference is present.


## 2.8.2. System Suitability

System suitability test is used to verify if the resolution and reproducibility of the chromatographic systems are adequate for the analysis to be done. The tests are based on the fact that the equipment, electronics, samples to be analyzed constitute an integral system that can be evaluated as such. The limits for system suitability were set for theoretical plates, resolution, and asymmetry.


## 2.8.3. Linearity

Concentrations  of  the  standard  mixture,  50%,  75%,  100%,  125%,  and  150%  were prepared and injected, and chromatogram was recorded. A graph was plotted for  the concentration of the corresponding  drug versus area. The Correlation coefficient (r) for each drug was calculated.


## 2.8.4. Accuracy

To determine the accuracy in sample preparation, method of standard additions was made for measuring the recovery of the drugs. To the known standard solution concentrations of the drug (50%, 100%, and 150%) were added. Three different solutions were prepared as mentioned in Section2.6. The accuracy was expressed as the percentage of the analyte recovery.


## 2.8.5. Robustness

To verify the robustness of the method, slight changes to the chromatographic conditions were made to check for any changes in the chromatogram. The analysis was done under variable flow rates, wave lengths, pH, and temperatures. The flow rate as per the developed method was 1 mL/min. This has been purposely changed to 0.8 mL/min and 1.2 mL/min, and the chromatogram was obtained. The wave length as per the developed method was 280nm. This has been purposely changed to 278 nm and 282nm. The pH in the developed method was 4.5. This was changed to 4.3 and 4.7, and the chromatogram was obtained. The column temperature in the developed method was 40°C. This was changed to 39.5°C and 40.5°C, and the chromatogram was obtained.


## 2.8.6. Ruggedness

To verify the ruggedness of the method, the analysis was done on different days and different  chemists  to check for any changes in the chromatogram.  The percentage RSD for the retention time and area was calculated.


## 2.8.7. Performance of the Method on Commercial Samples

The method is said to be effective if it can be applied for the analysis of commercial tablets. For this purpose, performance test of the method has been conducted on the market sample Telekast-D, Batch no. 06TTD040 manufactured by Intas Pharmaceuticals.


## 3. Results and Discussion

The present work involves simultaneous estimation of Montelukast and Doxofylline using reverse phase HPLC.  The current trend followed by the industries is developing a methodology which can save sophisticated instruments and chemist’s valuable time by which the product analysis can be done very fast, thereby saving the time. This is the reason why people are more attracted  towards  UFLC (Ultra-Fast Liquid  Chromatography)  [24, 25]  methods,  though  most  of  the  pharmacopeia  still have the HPLC methods only.Keeping all these points in mind, the current method has been developed and it is very fast and encouraging. The developed method was validated with a holistic approach according to ICH guidelines and details of findings are expressed in what follows.


### 3.1. Specificity of the Method

The Retention times of the standard drugs individually were measured and it was found to be 4.78 min and 1.97 min for Montelukast and Doxofylline, respectively. The drugs were mixed and injected for taking the chromatogram. Both drugs were resolved sufficiently in the mixture. Retention time of   both drugs in standard mix was  found  to  be  4.78 min  and  1.97 min  for  Montelukast  and  Doxofylline, respectively. This indicates that there is no chromatographic interference between the analytes. The pharmaceutical dosage form (tablet) was then injected, and the chromatogram was obtained. The retention time of the drugs in the dosage form was found to be 4.78 min and 1.96 min for Montelukast and Doxofylline, respectively. There is no specific change in retention time of both drugs, which indicates that there is no drug-excipient interference. Respective HPLC chromatograms are represented in Figures3, 4 and 5.Figure 3
Individual drugs.Figure 4
Drug mixture.Figure 5
Dosage form.The resolution between Montelukast and Doxofylline was 12.36 which indicates a very good separation. The asymmetry factor for Montelukast and Doxofylline was 0.95 and 0.97, respectively. Therefore, this is a suitable method for the simultaneous estimation of Montelukast and  Doxofylline in drug mixture and dosage forms.


### 3.2. System Suitability

Five injections of the standard mix were injected for this purpose. The retention time, areas, resolution, theoretical plates values, and peak Asymmetry were calculated for standard.  Percentage RSD value was calculated. The Results obtained are given in following Table1.Table 1
System suitability results.
Standard
Average
SD
%RSD
Montelukast
Retention time
4.79
0.002449
0.05
Area
507292.8
4646.642788
0.91
Resolution
12.414
0.074699398
0.6
Theoretical plates
5660.126
88.82016032
1.56
Asymmetry
0.948
0.017888544
1.88
Doxofylline
Retention time
1.97
0.000837
0.04
Area
11536096.2
11488.81385
0.099
Resolution
0
0
0
Theoretical plates
1541.768
11.65102442
0.75
Asymmetry
0.958
0.004472136
0.46


### 3.3. Linearity

The correlation coefficient (r) obtained was calculated and it was found to be   greater than 0.99 for Montelukast and Doxofylline, which is well within the acceptance criteria.  The results are shown in Table2.  The  concentration  was  found  to  be proportional to the area and the response of the detector was determined to be linear over  the  range  of  0.2  to  0.6 mg/mL  for  Montelukast  and  0.5  to  1.5 mg/mL  for Doxofylline as shown in the Figure 6.Table 2
Linearity results.
Linearity range
Correlation coefficient
Montelukast
0.005–0.015 mg/mL
0.9941
Doxofylline
0.2–0.6 mg/mL
0.9935Graphs showing linearity of the drugs.
(a)
(b)


### 3.4. Accuracy

The results indicate that the recoveries are well within the acceptance range of 80–120%;  therefore,  method  is  accurate  and  it  can  be  used  for  the  simultaneous estimation of Montelukast and Doxofylline. The percentage recovery of the results obtained are listed in Table3.Table 3
Results for the accuracy of the method.
Initial conc area
Sol 1 area
50% area
Sol 1-50% area
%Recovery
Sol 1
Montelukast
494506
785653
295518
291177
98.53
Doxofylline
10344817
16526064
6302308
6181247
99.56
Initial conc area
Sol 2 area
100% area
Sol 2-75% area
%Recovery
Sol 2
Montelukast
494506
988418
494506
493912
99.88
Doxofylline
10344817
20787092
10344817
10442275
100.94
Initial conc area
Sol 3 area
150% area
Sol 3-100% area
%Recovery
Sol 3
Montelukast
494506
1201875
712264
707369
99.31
Doxofylline
10344817
25046850
14537835
14702033
101.13


### 3.5. Robustness

Due  to  deliberate  change  in  the chromatographic conditions  of  the  method, like flow rate, pH, wave lengths, and column temperature, excellent  performance  of  the method was observed. This indicates that the method is Robust. The results obtained are shown in Table4.Table 4
Robustness results.
Retention time %RSD
Area %RSD
Different flow rates
0.8 mL/min
Montelukast
0.07
0.48
Doxofylline
0.15
0.16
1.2 mL/min
Montelukast
0.12
0.31
Doxofylline
0.24
0.39
Different pH values
4.3
Montelukast
0.29
0.98
Doxofylline
0.51
0.46
4.7
Montelukast
0.1
0.99
Doxofylline
0.24
0.24
Different wave lengths
278 nm
Montelukast
0.32
0.52
Doxofylline
0.12
0.1
282 nm
Montelukast
0.29
0.76
Doxofylline
0.38
0.49
Different temperatures
39.5°C
Montelukast
0.09
0.34
Doxofylline
0.19
0.15
40.5°C
Montelukast
0.03
0.15
Doxofylline
0.06
0.21


### 3.6. Ruggedness

Data acquired and   %RSD of area and retention time were calculated for various trials and data tabulated in Table5. Based on the data, it is evident that the method is rugged.Table 5
%RSD of the drugs on different days and different analysts.
Retention Time %RSD
Area %RSD
Day 1 & analyst 1
Montelukast
0.29
0.8
Doxofylline
0.12
0.34
Day 2 & analyst 2
Montelukast
0.09
0.49
Doxofylline
0.11
0.41


### 3.7. Performance of the Method on Commercial Formulations

As per the label claim, the commercial tablet, namely, Telekast-D contains 10 mg of Montelukast and 400 mg of Doxofylline.   The quantification of both the drugs was done in the formulation and the results have been found to be in the range of 99.8 to 100.3%. This indicates that the method developed by us can be used for the simultaneous estimation of Montelukast and Doxofylline in any of the pharmaceutical dosage   forms.   The   results   obtained   are      as   shown   below   in   the   Table6.Table 6
Estimation of the drugs in commercial samples.
Telekast-D
Label claim
Acquired data
%Recovery
Montelukast
10 mg/tab
10.03 mg/tab
100.30%
Doxofylline
400 mg/tab
399.48 mg/tab
99.87%


## 3.1. Specificity of the Method

The Retention times of the standard drugs individually were measured and it was found to be 4.78 min and 1.97 min for Montelukast and Doxofylline, respectively. The drugs were mixed and injected for taking the chromatogram. Both drugs were resolved sufficiently in the mixture. Retention time of   both drugs in standard mix was  found  to  be  4.78 min  and  1.97 min  for  Montelukast  and  Doxofylline, respectively. This indicates that there is no chromatographic interference between the analytes. The pharmaceutical dosage form (tablet) was then injected, and the chromatogram was obtained. The retention time of the drugs in the dosage form was found to be 4.78 min and 1.96 min for Montelukast and Doxofylline, respectively. There is no specific change in retention time of both drugs, which indicates that there is no drug-excipient interference. Respective HPLC chromatograms are represented in Figures3, 4 and 5.Figure 3
Individual drugs.Figure 4
Drug mixture.Figure 5
Dosage form.The resolution between Montelukast and Doxofylline was 12.36 which indicates a very good separation. The asymmetry factor for Montelukast and Doxofylline was 0.95 and 0.97, respectively. Therefore, this is a suitable method for the simultaneous estimation of Montelukast and  Doxofylline in drug mixture and dosage forms.


## 3.2. System Suitability

Five injections of the standard mix were injected for this purpose. The retention time, areas, resolution, theoretical plates values, and peak Asymmetry were calculated for standard.  Percentage RSD value was calculated. The Results obtained are given in following Table1.Table 1
System suitability results.
Standard
Average
SD
%RSD
Montelukast
Retention time
4.79
0.002449
0.05
Area
507292.8
4646.642788
0.91
Resolution
12.414
0.074699398
0.6
Theoretical plates
5660.126
88.82016032
1.56
Asymmetry
0.948
0.017888544
1.88
Doxofylline
Retention time
1.97
0.000837
0.04
Area
11536096.2
11488.81385
0.099
Resolution
0
0
0
Theoretical plates
1541.768
11.65102442
0.75
Asymmetry
0.958
0.004472136
0.46


## 3.3. Linearity

The correlation coefficient (r) obtained was calculated and it was found to be   greater than 0.99 for Montelukast and Doxofylline, which is well within the acceptance criteria.  The results are shown in Table2.  The  concentration  was  found  to  be proportional to the area and the response of the detector was determined to be linear over  the  range  of  0.2  to  0.6 mg/mL  for  Montelukast  and  0.5  to  1.5 mg/mL  for Doxofylline as shown in the Figure 6.Table 2
Linearity results.
Linearity range
Correlation coefficient
Montelukast
0.005–0.015 mg/mL
0.9941
Doxofylline
0.2–0.6 mg/mL
0.9935Graphs showing linearity of the drugs.
(a)
(b)


## 3.4. Accuracy

The results indicate that the recoveries are well within the acceptance range of 80–120%;  therefore,  method  is  accurate  and  it  can  be  used  for  the  simultaneous estimation of Montelukast and Doxofylline. The percentage recovery of the results obtained are listed in Table3.Table 3
Results for the accuracy of the method.
Initial conc area
Sol 1 area
50% area
Sol 1-50% area
%Recovery
Sol 1
Montelukast
494506
785653
295518
291177
98.53
Doxofylline
10344817
16526064
6302308
6181247
99.56
Initial conc area
Sol 2 area
100% area
Sol 2-75% area
%Recovery
Sol 2
Montelukast
494506
988418
494506
493912
99.88
Doxofylline
10344817
20787092
10344817
10442275
100.94
Initial conc area
Sol 3 area
150% area
Sol 3-100% area
%Recovery
Sol 3
Montelukast
494506
1201875
712264
707369
99.31
Doxofylline
10344817
25046850
14537835
14702033
101.13


## 3.5. Robustness

Due  to  deliberate  change  in  the chromatographic conditions  of  the  method, like flow rate, pH, wave lengths, and column temperature, excellent  performance  of  the method was observed. This indicates that the method is Robust. The results obtained are shown in Table4.Table 4
Robustness results.
Retention time %RSD
Area %RSD
Different flow rates
0.8 mL/min
Montelukast
0.07
0.48
Doxofylline
0.15
0.16
1.2 mL/min
Montelukast
0.12
0.31
Doxofylline
0.24
0.39
Different pH values
4.3
Montelukast
0.29
0.98
Doxofylline
0.51
0.46
4.7
Montelukast
0.1
0.99
Doxofylline
0.24
0.24
Different wave lengths
278 nm
Montelukast
0.32
0.52
Doxofylline
0.12
0.1
282 nm
Montelukast
0.29
0.76
Doxofylline
0.38
0.49
Different temperatures
39.5°C
Montelukast
0.09
0.34
Doxofylline
0.19
0.15
40.5°C
Montelukast
0.03
0.15
Doxofylline
0.06
0.21


## 3.6. Ruggedness

Data acquired and   %RSD of area and retention time were calculated for various trials and data tabulated in Table5. Based on the data, it is evident that the method is rugged.Table 5
%RSD of the drugs on different days and different analysts.
Retention Time %RSD
Area %RSD
Day 1 & analyst 1
Montelukast
0.29
0.8
Doxofylline
0.12
0.34
Day 2 & analyst 2
Montelukast
0.09
0.49
Doxofylline
0.11
0.41


## 3.7. Performance of the Method on Commercial Formulations

As per the label claim, the commercial tablet, namely, Telekast-D contains 10 mg of Montelukast and 400 mg of Doxofylline.   The quantification of both the drugs was done in the formulation and the results have been found to be in the range of 99.8 to 100.3%. This indicates that the method developed by us can be used for the simultaneous estimation of Montelukast and Doxofylline in any of the pharmaceutical dosage   forms.   The   results   obtained   are      as   shown   below   in   the   Table6.Table 6
Estimation of the drugs in commercial samples.
Telekast-D
Label claim
Acquired data
%Recovery
Montelukast
10 mg/tab
10.03 mg/tab
100.30%
Doxofylline
400 mg/tab
399.48 mg/tab
99.87%


## 4. Conclusion

The novel HPLC method developed is sensitive, unique, precise, user friendly, rapid, and reproducible for simultaneous estimation of Montelukast and Doxofylline in bulk mix and pharmaceutical dosages forms. The method was validated as per the ICH Guidelines.    It  is  concluded  that  this  method  can  be  used  by  the  industries  and academic institutions for their combination drug estimation, which is fast as well as novel.



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*Source: 402723-2012-09-16.xml*