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This article can be downloaded from www.ijpbs.net

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ISSN 0975-6299 Vol 3/Issue 1/Jan Mar 2012

RESEARCH ARTICLE

International Journal of Pharma and Bio Sciences

A VALIDATED STABILITY INDICATING UPLC METHOD FOR MONTELUKAST

IMPURITIES IN MONTELUKAST SODIUM ORAL GRANULES

HANIMI REDDY BAPATUA*

,MARAM RAVI KUMARB, LOVLEEN KUMAR GARG

C, DAMA VENUGOPAL

C

AND A. MALLESWARA REDDYC

aDepartment of Chemistry, J.N.T.University, Kukatpally, Hyderabad-500072, A.P, India.

bDr.Reddy's Laboratories Ltd. CPS, Bachupally, Hyderabad-500072, A.P, India.

cDr.Reddy's Laboratories Ltd. IPDO, Bachupally, Hyderabad-500072, A.P, India.

*Corresponding author

ABSTRACT

Objectives: Montelukast sodium is a selective and orally active leukotriene receptorantagonist. The main objective of this research is to develop a RP-UPLC method forthe determination of impurities in Montelukast sodium Oral Granules.Methods: Chromatographic separation was achieved by using 100 x 2.1 mm, 1.7mAcquity C18 column, 0.03 M phosphate buffer and 1% of Sodium perchlorate, pHadjusted to 4.9 was used as buffer, mobile phase containing a gradient mixture ofsolvent-A: (Buffer and Acetonitrile in 70:30 v/v ratio) and Sol-B: (Buffer andAcetonitrile in 30:70 v/v ratio). Gradient program was 0-4min, sol-B: 40-50; 4-11min-sol-B: 50-50; 11-22min- sol-B: 50-70; 22-25min- sol-B: 70-80; 25-28min- sol-B: 80-90,28-38min- sol-B: 90-40 and 38-45min- sol-B: 40-40. Column temperature was

maintained at 30C, 4L injection volume and run time was 45min. Analytesabsorbance was measured at 225 nm.Results: The developed method was validated as per ICH guidelines with respect tospecificity, limit of detection, limit of quantification, precision, linearity, accuracy,robustness and system suitability. Validation results were found to be satisfactory andthe method applicable for bulk and formulation analysis.

HANIMI REDDY BAPATUDepartment of chemistry, J.N.T.University, Kukatpally, Hyderabad-500072, A.P,

India.

ANALYTICAL CHEMISTRY


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KEYWORDS

Montelukast sodium, Impurities, RP-UPLC, leukotriene receptor antagonist.

INTRODUCTION

Montelukast sodium (1-4) is a selectiveand orally active leukotriene receptor antagonist(LTRAs). It works by blocking the action ofsubstances in the body that cause thesymptoms of asthma and allergic rhinitis. It isused to prevent breathing problem, chesttightness, wheezing and coughing caused byasthma. Montelukast safety results reveal thatintravenous doses of montelukast sodium from

3 to 18 mg and a 10-mg oral dose are weltolerated (5). Montelukast treatment has givenbest results for significant asthma contro(chronic asthma and seasonal aeroallergensensitivity) during the allergy season comparedwith placebo (6). Figure-1 represents thechemical structure of montelukast and itsimpurities.

Figure-1Chemical structure of Montelukast and its related compounds.


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Montelukast have chemical andinstrumental methods.(7-10) But no method wasreported by using ultra peroformance liquidchromatography (UPLC). The present research

is to develop a simple, short runtime andstability indicating UPLC method for thedetermination of montelukast and its six relatedcompounds. The method was developed withlow particle size (1.7 micron) and short 100 mmcolumn. Hence a rapid simple reproduciblestability indicating UPLC method was developedfor the quantitative determination of sixMontelukast impurities in pharmaceutical oraldosage forms. Out of all six impurities two aredegradents MOK-3 Sulphoxide and Styrene

impurity. MOK-3 Sulphoxide is a metaboliteimpurity. MOK-3 Sulphoxide is having anisomer; so it gives two peaks in chromatogram.

MATERIALS AND METHODS

Chemicals and reagentsPure standards were used for this study

(Montelukast and its six impurities namelyMOK-3 Sulphoxide, QUID-8, Saturatedanalogue, MOK-3 Keto, MOK-1 Nitrile and

Styrene). HPLC grade acetonitrile and analyticalgrade NaH2PO4, Sodium perchlorate and orthophosphoric acid were purchased from Merck,Darmstadt, Germany. High purity water wasprepared by using Millipore MilliQ Plus waterpurification system.

EquipmentWaters make UPLC system was used

(equiped with binary solvent manager, a samplemanager and a UV detector, Empower

software). Julabo, Seelbach, Germany makeWater bath equipped with MV controller, Sanyo,Leicestershire, UK photo stability chamber.MACK Pharmatech, Hyderabad, India make dryair oven were used. Waters make Acquity C18100 x 2.1 mm, 1.7m UPLC column was usedfor this study.

Chromatographic ConditionsColumn: Acquity C18 100 x 2.1 mm, 1.7mBuffer: 0.03M phosphate buffer and 1% ofSodium perchlorate and pH was adjusted to 4.9

with ortho phosphoric acidMobile phase: Sol-A: Buffer andacetonitrile in 70:30 v/vSol-B: Buffer and Acetonitrile in 30:70 v/vGradient prog: 0-4min, sol-B: 40-50; 4-11min- sol-B: 50-50; 11-22min- sol-B: 50-7022-25min- sol-B: 70-80; 25-28min- sol-B: 80-9028-38min- sol-B: 90-40 and 38-45min- sol-B40-40.Flow rate: 0.5 mL/minWavelength: 225 nm.

Injection volume: 4 l.Column temp: 30 CDiluent: Milli-Q water : Acetonitrile 40 : 60 v/v

Standard Solutions: A stock solution ofMontelukast (1.0 mg/mL) was prepared bydissolving appropriate amount of drug in thediluent. Working solutions of 50.0 and 2.5g/mL were prepared from the above stocksolution for the related substance determinationA stock solution of impurity (mixture of MOK-3

Keto and styrene impurity) at 0.16 and 0.14mg/mL was also prepared in the diluent. Systemsuitability solution Montelukast (0.5 mg/mL)MOK-3 Keto (3.2 g/mL) and styrene impurity(2.8 g/mL) were prepared by using the aboveimpurity stock.

Sample Solution: Equivalent to 24 mgMontelukast was then transferred to a 50 mLvolumetric flask, 35 mL diluent was addedsonicated for 20 min and diluted to volume to

give a solution containing 480g/mL. Thissolution was centrifuged at 4,000 rpm for10 min.

CALCULATIONS100(1/F) CS / CT) (ri / rS)Here, F is the relative response factor of theimpurity (Table-2); CS is the concentration of


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standard preparation; CT is the concentration ofTest preparation; ri is the peak area for anyimpurity in the Test solution and rS is the peakarea for Standard solution.

RESULTS AND DISCUSSIONS

Development trials were performed withphosphate buffer and acetonitrile as mobilephase, C8 50mm columns were used but themontelukast and keto-impurity were eluted atsame retention time. Finally the separation of allimpurities and montelukast was achieved withoptimized method. System suitability wasevaluated with the replicate injections of the

standard solution. Diluent and diluted standardsolution chromatograms were represented infigure-2 and 3. Interference of the placebo wasstudied and it was found that there is no

interference with all the impurities andmontelukast. Figure-4 represents the placebochromatogram. All known impurities werespiked to test sample and the recovery resultsof the impurities were calculated. Figure-5represents the un-spiked test samplechromatogram and figure-6 represents theimpurities spiked sample. Table-2 representsthe retention time and relative retention time ofmontelukast and impurities.

Table-2Retention time of Montelukast and its impurities.

COMPOUND RT RRT a

MOK-3 Sulphoxide 5.47 & 6.13 0.26 & 0.29

QUID-8 12.01 0.56

Saturated Analogue 16.77 0.79

Montelukast 21.29 1.00

MOK-3 Keto 22.25 1.05

MOK-1 Nitrile 28.27 1.33

Styrene impurity 33.76 1.59aRelative retention times (RRT) were calculated against the retention time (RT) of Montelukast

Figure-2Typical chromatogram of Blank


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Figure-3Typical chromatogram of Montelukast Standard

Figure-4Typical chromatogram of Montelukast System suitability solution

Figure-5Typical chromatogram of placebo

Figure-6Typical chromatogram of test preparation spiked with impurities


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METHOD VALIDATIONSpecificity: Specificity is the ability of themethod to measure the analyte response in thepresence of its potential impurities. Stress

studies were performed for Montelukast sodiumoral granules to provide an indication of thestability indicating property and specificity of theproposed method. Intentional degradation wasattempted to stress condition of UV light (200watt hours / square meter), sun light (1.2 Million

Lux hours ), heat (70C), acid (0.1N HCl), base(0.1N NaOH) and oxidation (0.1% H2O2) toevaluate the ability of the proposed method toseparate Montelukast from its degradation

product. Peak purity test was carried out for theMontelukast peak by using PDA detector instress samples. Degradation has beensummarized in table -3 and stress studychromatograms are represented in figure-7 to11.

Figure-7Chromatogram of Acid stressed Montelukast Sodium Oral Granules 4 mg Test

Figure-8Chromatogram of Base stressed Montelukast Sodium Oral Granules 4 mg Test


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Figure-9Chromatogram of Oxidation stressed Montelukast Sodium Oral Granules 4 mg Test

Figure-10Chromatogram of Sun light stressed Montelukast Sodium Oral Granules 4 mg Test

Figure-11Chromatogram of UV Light stressed Montelukast Sodium Oral Granules 4 mg Test


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Table-3Stress study results.

Stress Condition

Drug product % Impurities

MOK-3Sulphox

ide

QUID-8

Saturated

analogue

MOK-3Keto

MOK-1Nitrile

Styreneimpurity

MaximumUnknownImpurity

RRT

%Impurity

0.1N HCl solon for ~ 1 hour at 70C 0.32 0.02 0.00 0.00 0.0 2.88 0.35 0.54

0.1N NaOH solution for ~ 4 hours at 70C 3.38 0.02 0.00 0.00 0.0 0.06 0.12 0.05

Bench top with 0.1% H2O2 for ~ 15 minutes 8.14 0.02 0.00 0.00 0.0 0.06 0.12 0.04

Water bath with purified water for ~ 10hours/70C.

0.37 0.02 0.00 0.00 0.0 0.06 0.12 0.04

Sunlight about 1.2 Million Lux hours. 1.07 0.03 0.00 0.01 0.02 0.06 1.73 1.23

UV light both at shorter and longerwavelengths for ~ 200 watt hours / square

meter.1.34 0.07 0.00 0.02 0.03 0.05 0.70 1.41

Dry heating done at 70 C for ~ 12 hrs. 1.39 0.03 0.00 0.00 0.0 0.06 0.70 0.16

Humidity at 25C, 90% RH ~7 days 3.54 0.02 0.00 0.00 0.0 0.06 0.12 0.04

Precision: The % R.S.D. for impurity of MOK-3Sulphoxide, QUID-8, Saturated analogue, MOK-3 Keto, MOK-1 Nitrile and Styrene in relatedsubstance method precision study was within2%. The % R.S.D. for impurity MOK-3Sulphoxide, QUID-8, Saturated analogue, MOK-3 Keto, MOK-1 Nitrile and Styrene were wellwithin 2 %, conforming good precision of themethod. %RSD values are presented in table-4.

The precision of the related substancemethod was checked by injecting six individual

preparations of Montelukast (0.48 mg/mL) spikedwith 0.50% of MOK-3 Sulphoxide, QUID-8Saturated analogue, MOK-3 Keto, MOK-1 Nitrileand Styrene with respect to Montelukast analyteconcentration. %R.S.D. of % of impurity for eachMOK-3 Sulphoxide, QUID-8, Saturatedanalogue, MOK-3 Keto, MOK-1 Nitrile andStyrene was calculated. The intermediateprecision of the method was also evaluatedusing different analyst and different instrument inthe same laboratory.

Table-4Regression and Precision Data.

PARAMETERMok-3

SulphoxideQUID-8

Saturatedanalogue

MOK-3 KetoMOK-1Nitrile

Styrene

LOD (g/ml) 0.004 0.009 0.008 0.013 0.009 0.011LOQ (g/ml) 0.016 0.026 0.022 0.036 0.029 0.036

Slope (b) 28715.887430753.403

435476.764

819272.2868 19272.6941

26337.9992

Intercept (a) 349.0255 745.8856 161.3521 540.1722 225.5690 202.568

Correlation coefficient 0.999 0.999 0.999 0.999 0.999 0.999Precision (%RSD) 0.3 0.7 0.5 0.5 0.4 0.4

Inter. precision(%RSD) 0.8 0.8 0.5 0.6 0.4 0.8

Precision at LOQ 6.1 3.0 3.3 2.1 3.2 2.2


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Limits of Detection (LOD) and Quantification(LOQ): The LOD and LOQ for MOK-3Sulphoxide, QUID-8, Saturated analogue, MOK-3 Keto, MOK-1 Nitrile and Styrene were

determined at a signal-to-noise ratio of 3:1 and10:1, respectively, by injecting a series of dilutesolutions with known concentrations. Precisionstudy was also carried out at the LOQ level byinjecting six individual preparations of MOK-3Sulphoxide, QUID-8, Saturated analogue, MOK-3 Keto, MOK-1 Nitrile and Styrene andcalculating the % R.S.D. of the % of impurity.The determination limit of detection, limit ofquantification of all the impurities namelyMontelukast, MOK-3 Sulphoxide, QUID-8,

Saturated analogue, MOK-3 Keto, MOK-1 Nitrileand Styrene are reported in table-4. Theprecision at the LOQ concentrations forMontelukast, MOK-3 Sulphoxide, QUID-8,Saturated analogue, MOK-3 Keto, MOK-1 Nitrileand Styrene were below 7.5 %.Linearity: The result shows that an excellentcorrelation existed between the peak area andconcentration of the analyte. Linear calibrationplot for the related substance method wasobtained over the calibration ranges tested, i.e.

LOQ to 200 % for MOK-3 Sulphoxide, QUID-8,Saturated analogue, MOK-3 Keto, MOK-1 Nitrile

and Styrene. The correlation coefficient obtainedwas 0.999 greater than 0.997 (table 4). Theabove results show that an excellent correlationexisted between the peak area and the

concentration of MOK-3 Sulphoxide, QUID-8Saturated analogue, MOK-3 Keto, MOK-1 Nitrileand Styrene.

Linearity test solutions for the relatedsubstance method were prepared by dilutingstock solutions to the required concentrationsThe solutions were prepared at six concentrationlevels from LOQ to 200% of the specificationlevel. Correlation coefficient value for the slopeand Y-intercept of the calibration curve wascalculated.

Accuracy:The accuracy study of impurities wascarried out in triplicate at LOQ, 50%, 100%150%, and 200% of the target concentrationlevel, 0.5 % of Montelukast analyte concentration(480g/mL). The percentages of recoveries forimpurities were calculated. The percentagerecovery of impurities in Montelukast samplesvaried from 90 to 110 % at LOQ, 50%, 100%150%, and 200% levels of target 0.5 % levelThe LC chromatogram of spiked sample at 0.5%level of all six impurities in Montelukast ora

solution is shown in figure 2. % Recovery valuesfor impurities are presented in table-5.

Table-5Evaluation of Accuracy

% Accuracy

MOK-3

Sulphoxide

QUID-8

Saturated

analogue

Mok-3Keto

Mok-1Nitrile

Styrene

LOQ% Recovery 102.2 107.5 105.7 102.8 103.0 99.8

% RSD 3.50 1.93 5.19 2.72 3.59 4.18

50 %% Recovery 92.0 105.9 100.7 97.8 102.4 101.8

% RSD 4.85 1.80 0.65 1.39 0.34 1.92

100 %% Recovery 100.9 103.5 97.4 95.8 98.0 101.4

% RSD 1.18 0.97 0.93 0.71 1.33 1.25

150 %% Recovery 105.4 103.3 100.7 101.0 98.2 100.7

% RSD 1.90 0.44 0.35 0.23 0.56 0.73

200 %% Recovery 101.9 104.6 96.6 102.3 99.7 101.5

% RSD 0.88 0.78 0.54 0.31 1.14 1.29

% RSD values calculated with three sample recovery at each level.


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Robustness: In all the deliberate variedchromatographic conditions (flow rate, columntemperature and composition of organic

solvent), the resolution between critical pairs,i.e. Montelukast and MOK-3 Keto was greaterthan 1.5, illustrating the robustness of themethod.

The robustness was evaluated,experimental conditions were deliberatelyaltered and the resolution between Montelukast,MOK-3 Sulphoxide, QUID-8, saturatedanalogue, MOK-3 Keto, MOK-1 Nitrile andStyrene was recorded. The flow rate of themobile phase was 0.5 mL/min. To study the

effect of flow rate on the resolution, flow waschanged by 0.1 units from 0.4 to 0.6 mL/min.The effect of the column temperature onresolution was studied at 25 and 35 C insteadof 30 C. The effect of the percent organicstrength on resolution was studied by varyingacetonitrile by 10% +10%; while other mobilephase components was held constant as statedin Section 2.3.

Solution Stability and Mobile Phase

Stability: The solution stability of Montelukastand its impurities in the related substancemethod was carried out by leaving spikedsample solutions in tightly capped ambercoloured volumetric flasks at room temperaturefor 48 hours. Content of MOK-3 Sulphoxide,QUID-8, Saturated analogue, MOK-3 Keto,MOK-1 Nitrile and Styrene were determined forevery 24 hours interval upto the study period.The mobile phase stability was also carried outfor 48 hours by injecting the freshly prepared

sample solutions for every 24 hours interval.Content of MOK-3 Sulphoxide, QUID-8,

Saturated analogue, MOK-3 Keto, MOK-1 Nitrileand Styrene were checked in the test.

No significant changes were observed in

the content of impurities namely MOK-3Sulphoxide, QUID-8, Saturated analogue, MOK3 Keto, MOK-1 Nitrile and Styrene duringsolution stability and mobile phase stabilityexperiments when performed using the relatedsubstance method. The Standard solutionstability is stable for 5 days on bench top andTest solution is stable on bench top for 1 dayMobile phases used during the relatedsubstance determination were stable for 5 days.

CONCLUSIONS

The gradient UPLC method developedfor Montelukast and related substances inpharmaceutical dosage forms is preciseaccurate, linear, robust, rugged and specificSatisfactory results were obtained fromvalidation of the method. The method isstability-indicating and can be used for routineanalysis of production samples and to check thestability of samples of Montelukast ora

granules.

ACKNOWLEDGEMENT

We wish to express our sincere thanks to theManagements of Dr. Reddys LaboratoriesHyderabad, India for their support andencouragement.Cooperation from colleagues and of Research &Development and Analytical Research &Development of Dr.Reddys Laboratories Ltd. is

appreciated.


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