Stability Indicating Assay Method for Montelukast Sodium

software (version # 3.2.1)). Interference from Impurities: All the impurities are Injected indivudialy and spiked into test at 0.3% of test concentrat...

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Mastanaiah Thummisetty et al /J. Pharm. Sci. & Res. Vol.3(8), 2011,1373-1377

Stability Indicating Assay Method for Montelukast Sodium in Pharmaceutical Formulations by RP-HPLC Mastanaiah Thummisetty a,b ,Dr. Jayapal Reddy Samaa,b, . V. Surya Narayana Raob, and. P. Reddannaa a.

Department of Animal Sciences, School of Life Sciences, University of Hyderabad, India. b. Department of Chemistry, Sri Krishnadevaraya Univeristy, Anantapur, India.

Abstract-Montelukast Sodium is used to treat asthma. A simple, precise cost effective and stability indicating RP-HPLC method has been developed and validated for the determination of Montelukast Sodium in pharmaceutical formulations. Separation of Montelukast Sodium from its potentional degradents were achieved with in shorter run time with required resolution, accuracy and precision thus enabling the utility of the method for routine analysis. Chromatographic separation was achieved on a Zorbax SB Phenyl column(50 × 4.6 mm, 1.8μ) using a mobile phase-A consisting of 0.15% trifluro acetic acid in milli-Q grade water and mobile phase-B Consist of 0.15% trifluro acetic acid in acetonitrile at a flow rate of 1.2ml per minute. The detection was made at 238nm. The retention time of Montelukast Sodium was 8.9 minutes. The method was found linear over the range of 5-15 µg per ml. The proposed method was validated as per the ICH and USP guidelines. Key words: Montelukast Sodium, HPLC and validation

INTRODUCTION Montelukast sodium( Fig 1) is chemically (R-(E))-1(((1-(3-(2-(7-chloro-2-quinolinyl) ethenyl)phenyl)3(2-(1-hydroxy-1methylethyl)phenyl)propyl)thio)methyl)cyclopropan eacetic acid, monosodium salt[1–2]. Montelukast is a leukotriene receptor antagonist (LTRA) used for the treatment of asthma and to relieve symptoms of seasonal allergies in children and adults[3–5]. It is a potent selective inhibitor of leukotriene D4 (LTD4) at the cysteinyl leukotriene receptor cysLT1[6–7]. Literature survey reveals that liquid chromatography with fluorescence detector[8], stereoselective high performance liquid chromatography (HPLC) for montelukast and its S-enantiomer[9], column switching HPLC with fluorescence detector[10], semi-automated 96-well protein precipitation[11], HPLC with derivative spectroscopy[12], pressurized liquid extraction followed by HPLC[13] and LC-MS methods[14–16] have been reported for the estimation of montelukast sodium. The present study illustrates development and validation of a Stability indicating simple, accurate and precise method for assay of Montelukast sodium by RP-HPLC in bulk and in tablet dosage form.

MATERIALS AND METHODS I. Chemicals and Reagents Montelukast Sodium working standards were procured from Cipla Labs, and the tested pharmaceutical formulations were procured from commercial pharmacy. Trifluroacetic acid, acetonitrile, methanol were of suitable analytical grade. II. Apparatus and Chromatographic Conditions HPLC analysis was performed on Agilent HPLC system with diode array detector. Separations were carried on a Zorbax SB Phenyl (50 × 4.6 mm, i.d., 1.8 μm particle size) using gradient elution. The flow rate was 1.2 mL min-1. UV detection was performed at 238 nm. HPLC Column temperature was 30°C. Peak identity was confirmed by retention time comparison and the HPLC was operated at room temperature.

Figure 1: Chemical Structure of Montelukast Sodium

III. Preparation of Mobile Phase Mobile Phase-A: 0.15% of trifluroacetic Acid in milli-Q grade water, filtered through a 0.45 μm nylon filter (Millipore, USA) and degassed by sonication prior to use. Mobile Phase-B: 0.15% of trifluroacetic Acid in Acetonitrile, filtered through a 0.45 μm nylon filter (Millipore, USA) and degassed by sonication prior to use. Diluents: Mixed 300ml of milli-Q grade water and 700ml of methanol.

Time (Minute) 0 3 16 17 21

Mobile phase A (%)

Mobile phase B (%)

60 60 49 60 60

40 40 51 40 40

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IV. Preparation of Standard Solution The standard solution of mountaleucast 200ppm was prepared by dissolving the working standard in the diluents. V. Preparation of Sample Solution The sample solution of mountaleucast 200ppm was prepared by transferring 10 tablets of 10mg in to 500ml of volumetric flask, added 350 ml of diluents and sonicated for 30minutes with intermediate shaking and made up to volume with diluents. Centrifuged the portion of solution at 4000rpm for 10minutes. RESULTS AND DISCUSSION Method Development Drug quality control, stability, metabolism, pharmacokinetics, and toxicity studies all necessitate the determination of drugs in pharmaceutical formulations and biological samples. Correspondingly, efficient and validated analytical methods are very critical requirements for all these investigations. Chromatographic parameters were preliminary optimized to develop a stability indicating assay method for mountaleucast with short analyses time (<22 min). Since mountaleucast is highly sensitive Figure 2: Chromatogram of Blank

Figure 4: Chromatogram of Standard

to light and oxidation. It tends to degrade while storage for long time. So these degradents need to separate from main analyte to show the stability indicating assay mehod, to separate the degradents from main analyte chosen the gradient program. The sample retention increases with increased column length so a shorter column (50 x 4.6 mm i.d.1.8µm) was selected to have a shortest possible runtime not compromising on the resolution. In order to identify a suitable organic modifier, various organic solvents like acetonitrile and methanol were tested. Methanol produced high column pressures due to the high viscosity. Acetonitrile was found to display advantageous separations. Various buffers at different pH was verified only trifluro acetic acid is giving the sharp peak compare to other buffers, so trifluro acetic acid at 0.15% was selected. Different gradient programs were tied to separate all the impurities from main analyte with high resolution, optimized the gradient program by choosing initial three minutes isocratic mode followed by linear gradient and initial stabilization mode. Figure 3: Chromatogram of Placebo

Figure 5: Chromatogram of Sample

Figure 6: Chromatogram of Spiked Sample

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Method Validation The above method was validated according to ICH and USP guidelines to establish the performance characteristics of a method (expressed in terms of analytical parameters) to meet the requirements for the intended application of the method. System Suitability In order to determine the adequate resolution and reproducibility of the proposed methodology, suitability parameters including retention time, asymmetry factor, %RSD of retention time and peak areas were investigated. The results are summarized in Table 1. Table: 1 System Suitability Parameter Result Tailing Factor 1.2 %RSD of Peak Area 0.6 %RSD of retention time 0.01

Acceptance Criteria NMT 2.0 NMT 2.0% NMT 2.0%

Specificity The specificity of an analytical method may be defined as the ability to unequivocally determine the analyte in the presence of additional components such as impurities, degradation products and matrix. Specificity was evaluated by preparing the analytical placebo and it was confirmed that the signal measured was caused only by the analytes. A solution of analytical placebo (containing all the tablet excipients except Montelukast was prepared according to the sample preparation procedure and injected. To identify the interference by these excipients, a mixture of inactive ingredients (placebo), standard solutions, and the commercial pharmaceutical preparations were analyzed by the developed method. The representative chromatograms did not show any other peaks, which confirmed the specificity of the method. Peak purity of Montelukast Sodium was also evaluated for confirming the purity of the individual peak of Montelukast.In all the samples Peak purity is more than acceptance limits (Peak purity should be not less than 0.99 on Agilent EZChrom Elite software (version # 3.2.1)). Interference from Impurities: All the impurities are Injected indivudialy and spiked into test at 0.3% of test concentration and injected in to the system. All the impurities are well separated from each other and from main analyte. The Spiked chromatogram was shown in Figure- 6. Forced degradation Studies: Drug product and placebo were subjected to forced degradation at various stressed conditions like acid, base, hydrolysis, peroxide, heat, photo light, U.V

light and Humidity. All the samples were analyzed for peak purity of Montelukast peak. In all the samples Peak purity is more than acceptance limits. (Peak purity should be not less than 0.99 on Agilent EZChrom Elite software (version # 3.2.1)). The results are summarized in Table 2. Table: 2 Forced Degradation Data Sample condition Acid degradation Alkali degradation Peroxide degradation Water degradation UV degradation Photolight Thermal degradation Humidity degradation

% Net degradation

Peak Purity

2.2

1.00

15.2

1.00

13.8

0.99

60°C for 5Hours

8.2

0.99

200 W /m2/hours

1.2

1.00

200 million Lux Hours

5.5

1.00

105°C for 7 Days

5.2

0.99

90% RH at 25°C for 7 days

3.8

0.99

Procedure 1N HCl on bench top for 2Hrs 1N NaOH at 60°C 2Hours 1.0% H2O2 on bench top for 1Hour

Linearity: The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. Linearity of detector response for Montelukast was established by analyzing serial dilutions of a stock solution of the working standard. Five concentrations ranging from 50% to 150% of the test concentration were prepared and analyzed. The final concentration of each solution in µg per mL was plotted against peak area response. Slope, correlation coefficient (R) and intercept were found to be 13794.32, 0.999635and 4057.838. The linear graphs was shown in Figure- 7 Figure 7. Linearity graph for Montelukast

Precision: The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from

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multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility. Six replicate samples were prepared and analyzed as per the sample preparation procedure. Assay of each replicate, the average of 6 replicates, its standard deviation, %RSD and the 95% confidence interval were calculated. . The results are shown in Table 3. Table: 3 Precision Sample No. 1 2 3 4 5 6 Mean ( X ) s %RSD Lower 95% CI Upper 95% CI

% Assay 101.3 100.6 100.6 100.7 100.9 101.0 100.9 0.2739 0.3 100.7 101.1

Accuracy: The accuracy of an analytical procedure expresses the closeness of agreement between the value which

is accepted either as a conventional true value or an accepted reference value and the value found. Recovery study was performed at 50%, 75%, 100%, 125% and 150% of the target concentration by spiking placebo blend with the drug substance. Six replicates each were spiked at 50% & 150% levels, and 3 replicates each at 75%, 100% and 125% levels. Spiked samples were extracted and analyzed. The amount spiked, amount recovered, percent recovery and its mean were calculated. The results are shown in Table 4. Range: The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity. The results are shown in Table 5. Robustness: The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. The variations like flow rate of mobile phase, column temperature, does not have any significant effect on the method performance.

Table: 4 Recovery Sample No. 1 2 3 4 5 6 1 2 3 1 2 3 1 2 3 1 2 3 4 5 6

Spike Level

50%

75% 100% 125%

150%

Table 5: Range Parameter Linearity Precision Accuracy

Amount Spiked, mg

Amount Recovered, mg

Percent Recovery

50.23 50.15 50.46 50.37 50.89 49.48 75.02 75.48 75.36 100.25 100.38 100.85 125.56 124.36 125.56 150.36 150.85 150.36 150.65 149.36 150.12

49.86 50.25 50.05 49.86 50.35 50.15 74.86 74.96 75.85 100.05 100.05 100.15 125.12 125.36 124.86 148.86 150.15 148.96 150.25 148.85 149.12

99.3 100.2 99.2 99.0 98.9 101.4 99.8 99.3 100.7 99.8 99.7 99.3 99.6 100.8 99.4 99.0 99.5 99.1 99.7 99.7 99.3

Acceptance Criteria R ≥ 0.999 %RSD of 6 Replicates NMT 2.0% Recovery 97.0% to 103.0%

Mean Percent Recovery

99.7

99.9 99.6 100.6

99.4

Result 0.999635 0.3% to 0.97% 99.0%-101.4%

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CONCLUSIONS A simple, rapid, cost effective and accurate RPHPLC method was developed for the Stability indicating assay method for Montelukast Sodium in pharmaceutical formulations. The analytical conditions and the solvent system developed provided good resolution between Montelukast Sodium and its potentional degradents within a short run time. The HPLC method was validated and demonstrated good linearity, precision, accuracy, specificity and stability indicating. Thus, the developed HPLC method can be utilized for routine analysis stability studies for Montelukast Tablets. ACKNOWLEDGMENTS The authors are thankful to Cipla labs for providing the working standards of Montelukast Sodium. REFERENCES [1]. Vanheek M, France C F and Compton D S, Pharmacol Exp

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