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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 BAPATU A* ,MARAM RAVI KUMAR B , LOVLEEN KUMAR GARG C , DAMA VENUGOPAL C
AND A. MALLESWARA REDDY C
a Department of Chemistry, J.N.T.University, Kukatpally, Hyderabad-500072, A.P, India. b Dr.Reddy's Laboratories Ltd. CPS, Bachupally, Hyderabad-500072, A.P, India. c Dr.Reddy's Laboratories Ltd. IPDO, Bachupally, Hyderabad-500072, A.P, India.
*Corresponding author
ABSTRACT
Objectives: Montelukast sodium is a selective and orally active leukotriene receptor antagonist. The main objective of this research is to develop a RP-UPLC method for the determination of impurities in Montelukast sodium Oral Granules. Methods: Chromatographic separation was achieved by using 100 x 2.1 mm, 1.7m Acquity C18 column, 0.03 M phosphate buffer and 1% of Sodium perchlorate, pH adjusted to 4.9 was used as buffer, mobile phase containing a gradient mixture of solvent-A: (Buffer and Acetonitrile in 70:30 v/v ratio) and Sol-B: (Buffer and Acetonitrile 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. Analytes absorbance was measured at 225 nm. Results: The developed method was validated as per ICH guidelines with respect to specificity, limit of detection, limit of quantification, precision, linearity, accuracy, robustness and system suitability. Validation results were found to be satisfactory and the method applicable for bulk and formulation analysis.
HANIMI REDDY BAPATU Department of chemistry, J.N.T.University, Kukatpally, Hyderabad-500072, A.P, India. ANALYTICAL CHEMISTRY
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ISSN 0975-6299 Vol 3/Issue 1/Jan Mar 2012 KEYWORDS
Montelukast sodium (1-4) is a selective and orally active leukotriene receptor antagonist (LTRAs). It works by blocking the action of substances in the body that cause the symptoms of asthma and allergic rhinitis. It is used to prevent breathing problem, chest tightness, wheezing and coughing caused by asthma. Montelukast safety results reveal that intravenous doses of montelukast sodium from 3 to 18 mg and a 10-mg oral dose are well tolerated (5) . Montelukast treatment has given best results for significant asthma control (chronic asthma and seasonal aeroallergen sensitivity) during the allergy season compared with placebo (6) . Figure-1 represents the chemical structure of montelukast and its impurities.
Figure-1 Chemical structure of Montelukast and its related compounds.
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ISSN 0975-6299 Vol 3/Issue 1/Jan Mar 2012 Montelukast have chemical and instrumental methods. (7-10) But no method was reported by using ultra peroformance liquid chromatography (UPLC). The present research is to develop a simple, short runtime and stability indicating UPLC method for the determination of montelukast and its six related compounds. The method was developed with low particle size (1.7 micron) and short 100 mm column. Hence a rapid simple reproducible stability indicating UPLC method was developed for the quantitative determination of six Montelukast impurities in pharmaceutical oral dosage forms. Out of all six impurities two are degradents MOK-3 Sulphoxide and Styrene impurity. MOK-3 Sulphoxide is a metabolite impurity. MOK-3 Sulphoxide is having an isomer; so it gives two peaks in chromatogram.
MATERIALS AND METHODS
Chemicals and reagents Pure standards were used for this study (Montelukast and its six impurities namely MOK-3 Sulphoxide, QUID-8, Saturated analogue, MOK-3 Keto, MOK-1 Nitrile and Styrene). HPLC grade acetonitrile and analytical grade NaH 2 PO 4 , Sodium perchlorate and ortho phosphoric acid were purchased from Merck, Darmstadt, Germany. High purity water was prepared by using Millipore MilliQ Plus water purification system.
Equipment Waters make UPLC system was used (equiped with binary solvent manager, a sample manager and a UV detector, Empower software). Julabo, Seelbach, Germany make Water bath equipped with MV controller, Sanyo, Leicestershire, UK photo stability chamber. MACK Pharmatech, Hyderabad, India make dry air oven were used. Waters make Acquity C18 100 x 2.1 mm, 1.7m UPLC column was used for this study.
Chromatographic Conditions Column: Acquity C18 100 x 2.1 mm, 1.7m Buffer: 0.03M phosphate buffer and 1% of Sodium perchlorate and pH was adjusted to 4.9 with ortho phosphoric acid Mobile phase: Sol-A: Buffer and acetonitrile in 70:30 v/v Sol-B: Buffer and Acetonitrile in 30:70 v/v Gradient prog: 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. Flow rate: 0.5 mL/min Wavelength: 225 nm. Injection volume: 4 l. Column temp: 30 C Diluent : Milli-Q water : Acetonitrile 40 : 60 v/v
Standard Solutions: A stock solution of Montelukast (1.0 mg/mL) was prepared by dissolving appropriate amount of drug in the diluent. Working solutions of 50.0 and 2.5 g/mL were prepared from the above stock solution for the related substance determination. A stock solution of impurity (mixture of MOK-3 Keto and styrene impurity) at 0.16 and 0.14 mg/mL was also prepared in the diluent. System suitability 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 above impurity stock.
Sample Solution: Equivalent to 24 mg Montelukast was then transferred to a 50 mL volumetric flask, 35 mL diluent was added, sonicated for 20 min and diluted to volume to give a solution containing 480g/mL. This solution was centrifuged at 4,000 rpm for 10 min.
CALCULATIONS 100(1/F) CS / CT) (ri / rS) Here, F is the relative response factor of the impurity (Table-2); CS is the concentration of
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ISSN 0975-6299 Vol 3/Issue 1/Jan Mar 2012 standard preparation; CT is the concentration of Test preparation; ri is the peak area for any impurity in the Test solution and rS is the peak area for Standard solution.
RESULTS AND DISCUSSIONS
Development trials were performed with phosphate buffer and acetonitrile as mobile phase, C8 50mm columns were used but the montelukast and keto-impurity were eluted at same retention time. Finally the separation of all impurities and montelukast was achieved with optimized method. System suitability was evaluated with the replicate injections of the standard solution. Diluent and diluted standard solution chromatograms were represented in figure-2 and 3. Interference of the placebo was studied and it was found that there is no interference with all the impurities and montelukast. Figure-4 represents the placebo chromatogram. All known impurities were spiked to test sample and the recovery results of the impurities were calculated. Figure-5 represents the un-spiked test sample chromatogram and figure-6 represents the impurities spiked sample. Table-2 represents the retention time and relative retention time of montelukast and impurities.
Table-2 Retention time of Montelukast and its impurities.
a Relative retention times (RRT) were calculated against the retention time (RT) of Montelukast
Figure-2 Typical chromatogram of Blank
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ISSN 0975-6299 Vol 3/Issue 1/Jan Mar 2012
Figure-3 Typical chromatogram of Montelukast Standard
Figure-4 Typical chromatogram of Montelukast System suitability solution
Figure-5 Typical chromatogram of placebo
Figure-6 Typical chromatogram of test preparation spiked with impurities
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ISSN 0975-6299 Vol 3/Issue 1/Jan Mar 2012 METHOD VALIDATION Specificity: Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities. Stress studies were performed for Montelukast sodium oral granules to provide an indication of the stability indicating property and specificity of the proposed method. Intentional degradation was attempted to stress condition of UV light (200 watt hours / square meter), sun light (1.2 Million Lux hours ), heat (70C), acid (0.1N HCl), base (0.1N NaOH) and oxidation (0.1% H 2 O 2 ) to evaluate the ability of the proposed method to separate Montelukast from its degradation product. Peak purity test was carried out for the Montelukast peak by using PDA detector in stress samples. Degradation has been summarized in table -3 and stress study chromatograms are represented in figure-7 to 11.
Figure-7 Chromatogram of Acid stressed Montelukast Sodium Oral Granules 4 mg Test
Figure-8 Chromatogram of Base stressed Montelukast Sodium Oral Granules 4 mg Test
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Figure-9 Chromatogram of Oxidation stressed Montelukast Sodium Oral Granules 4 mg Test
Figure-10 Chromatogram of Sun light stressed Montelukast Sodium Oral Granules 4 mg Test
Figure-11 Chromatogram of UV Light stressed Montelukast Sodium Oral Granules 4 mg Test
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ISSN 0975-6299 Vol 3/Issue 1/Jan Mar 2012 Table-3 Stress study results. Stress Condition Drug product % Impurities M O K - 3
S u l p h o x i d e
Q U I D - 8
S a t u r a t e d
a n a l o g u e
M O K - 3
K e t o
M O K - 1
N i t r i l e
S t y r e n e
i m p u r i t y
Maximum Unknown Impurity R R T
%
I m p u r i t y
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% H 2 O 2 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 longer wavelengths 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-3 Sulphoxide, QUID-8, Saturated analogue, MOK- 3 Keto, MOK-1 Nitrile and Styrene in related substance method precision study was within 2%. The % R.S.D. for impurity MOK-3 Sulphoxide, QUID-8, Saturated analogue, MOK- 3 Keto, MOK-1 Nitrile and Styrene were well within 2 %, conforming good precision of the method. %RSD values are presented in table-4. The precision of the related substance method was checked by injecting six individual preparations of Montelukast (0.48 mg/mL) spiked with 0.50% of MOK-3 Sulphoxide, QUID-8, Saturated analogue, MOK-3 Keto, MOK-1 Nitrile and Styrene with respect to Montelukast analyte concentration. %R.S.D. of % of impurity for each MOK-3 Sulphoxide, QUID-8, Saturated analogue, MOK-3 Keto, MOK-1 Nitrile and Styrene was calculated. The intermediate precision of the method was also evaluated using different analyst and different instrument in the same laboratory.
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ISSN 0975-6299 Vol 3/Issue 1/Jan Mar 2012 Limits of Detection (LOD) and Quantification (LOQ): The LOD and LOQ for MOK-3 Sulphoxide, QUID-8, Saturated analogue, MOK- 3 Keto, MOK-1 Nitrile and Styrene were determined at a signal-to-noise ratio of 3:1 and 10:1, respectively, by injecting a series of dilute solutions with known concentrations. Precision study was also carried out at the LOQ level by injecting six individual preparations of MOK-3 Sulphoxide, QUID-8, Saturated analogue, MOK- 3 Keto, MOK-1 Nitrile and Styrene and calculating the % R.S.D. of the % of impurity. The determination limit of detection, limit of quantification of all the impurities namely Montelukast, MOK-3 Sulphoxide, QUID-8, Saturated analogue, MOK-3 Keto, MOK-1 Nitrile and Styrene are reported in table-4. The precision at the LOQ concentrations for Montelukast, MOK-3 Sulphoxide, QUID-8, Saturated analogue, MOK-3 Keto, MOK-1 Nitrile and Styrene were below 7.5 %. Linearity: The result shows that an excellent correlation existed between the peak area and concentration of the analyte. Linear calibration plot for the related substance method was obtained 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 obtained was 0.999 greater than 0.997 (table 4). The above results show that an excellent correlation existed between the peak area and the concentration of MOK-3 Sulphoxide, QUID-8, Saturated analogue, MOK-3 Keto, MOK-1 Nitrile and Styrene. Linearity test solutions for the related substance method were prepared by diluting stock solutions to the required concentrations. The solutions were prepared at six concentration levels from LOQ to 200% of the specification level. Correlation coefficient value for the slope and Y-intercept of the calibration curve was calculated. Accuracy: The accuracy study of impurities was carried out in triplicate at LOQ, 50%, 100%, 150%, and 200% of the target concentration level, 0.5 % of Montelukast analyte concentration (480g/mL). The percentages of recoveries for impurities were calculated. The percentage recovery of impurities in Montelukast samples varied from 90 to 110 % at LOQ, 50%, 100%, 150%, and 200% levels of target 0.5 % level. The LC chromatogram of spiked sample at 0.5% level of all six impurities in Montelukast oral solution is shown in figure 2. % Recovery values for impurities are presented in table-5. Table-5 Evaluation of Accuracy
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Robustness: In all the deliberate varied chromatographic conditions (flow rate, column temperature and composition of organic solvent), the resolution between critical pairs, i.e. Montelukast and MOK-3 Keto was greater than 1.5, illustrating the robustness of the method. The robustness was evaluated, experimental conditions were deliberately altered and the resolution between Montelukast, MOK-3 Sulphoxide, QUID-8, saturated analogue, MOK-3 Keto, MOK-1 Nitrile and Styrene was recorded. The flow rate of the mobile phase was 0.5 mL/min. To study the effect of flow rate on the resolution, flow was changed by 0.1 units from 0.4 to 0.6 mL/min. The effect of the column temperature on resolution was studied at 25 and 35 C instead of 30 C. The effect of the percent organic strength on resolution was studied by varying acetonitrile by 10% +10%; while other mobile phase components was held constant as stated in Section 2.3.
Solution Stability and Mobile Phase Stability: The solution stability of Montelukast and its impurities in the related substance method was carried out by leaving spiked sample solutions in tightly capped amber coloured volumetric flasks at room temperature for 48 hours. Content of MOK-3 Sulphoxide, QUID-8, Saturated analogue, MOK-3 Keto, MOK-1 Nitrile and Styrene were determined for every 24 hours interval upto the study period. The mobile phase stability was also carried out for 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 Nitrile and Styrene were checked in the test. No significant changes were observed in the content of impurities namely MOK-3 Sulphoxide, QUID-8, Saturated analogue, MOK- 3 Keto, MOK-1 Nitrile and Styrene during solution stability and mobile phase stability experiments when performed using the related substance method. The Standard solution stability is stable for 5 days on bench top and Test solution is stable on bench top for 1 day. Mobile phases used during the related substance determination were stable for 5 days.
CONCLUSIONS
The gradient UPLC method developed for Montelukast and related substances in pharmaceutical dosage forms is precise, accurate, linear, robust, rugged and specific. Satisfactory results were obtained from validation of the method. The method is stability-indicating and can be used for routine analysis of production samples and to check the stability of samples of Montelukast oral granules.
ACKNOWLEDGEMENT
We wish to express our sincere thanks to the Managements of Dr. Reddys Laboratories, Hyderabad, India for their support and encouragement. Cooperation from colleagues and of Research & Development and Analytical Research & Development of Dr.Reddys Laboratories Ltd. is appreciated.
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ISSN 0975-6299 Vol 3/Issue 1/Jan Mar 2012 REFERENCES
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