INTRODUCTION:

Montelukast Sodium (MNKT) chemically, (S,E)-2-(1-((1-(3-(2-(7-chloroquinolin-2-yl)vinyl)phenyl)3-(2-(2-hydroxy propan-2-yl)phenyl)propylthio)methyl)cyclopropyl)acetic acid^1-2 is a Cysteinyl leukotriene receptor antagonist (LTRA) used for the maintenance treatment of asthma and to relieve symptoms of seasonal allergies^3-5. Literature survey reveals that assay of Montelukast Sodium in bulk and tablet dosage form is official in Indian Pharmacopoeia 2010⁶. Ebastine (EBA), chemically, 4-(4-benzhydryloxy-1-piperidyl)-1-(4-tert-butylphenyl) butan-1-one is a non-sedating H₁ antihistamine⁷. Assay of Ebastine in bulk form is official in British Pharmacopoeia⁸. Literature survey reveals that analytical methods,including U.V. spectrophotometer, and HPLCmethods, are available for the determination of Montelukast in pharmaceutical dosage forms. The analytical methods reported for estimation of MNKT and EBA alone and with other drug combination are U.V spectrophotometry^9-17, HPLC^18-24, HPLC/PDA²⁵,LC-MS²⁶and HPTLC^27-29 methods.

The combination of Montelukast sodium and Ebastine has recently been introduced into the market.

However, so far, no method was reported for the simultaneous estimation of Montelukast sodium and Ebastine, in combination. The proposed method is rapid, simple, accurate, and reproducible, and can be successfully employed in the routine analysis of both these drugs simultaneously, in tablet dosage form. The proposed method is optimized and validated as per the ICH guidelines ³⁰. In the present work, a successful attempt has been made to estimate both these drugs simultaneously using First order method by UV Spectrophotometer. This study attempts to useful for routine quality control of Montelukast sodium and Ebastine in pharmaceutical formulation. Structures of both the drugs (MNKT and EBA) are shown in figure 1.

Montelukast Sodium

Ebastine

Fig. 1: Chemical structures of the analytes.

MATERIALS AND METHOD:

Instruments:

Instrument used was an UV-Visible double beam spectrophotometer, make: SHIMADZU (model UV-1800) with a pair of 1 cm matched quartz cells. All weighing was done on analytical balance mettler toledo.

Reagents and chemicals:

A pure drug MNKT was obtained as gift sample from Alembic Pharmaceuticals, Vadodara and EBA was procured as gift sample from Kivi labs Pvt, Baroda. Methanol AR was used as solvent. Calibrated glasswares were used throughout the work.

Marketed formulation:

The marketed formulation studied was Ebast-M tablets manufactured by Micro labs Pvt ltd. Each tablet contains 10 mg Montelukast sodium and 10 mg Ebastine.

Preparation of standard stock solution:

Accurately weighed quantity of MNKT (10 mg) and EBA (10 mg) were transferred to two separate 100 ml volumetric flasks, dissolved in little amount of methanol and diluted to the mark with methanol (stock solutions: 100 μg/ml of MNKT and EBA).

Preparation of working standard solution:

15 mg/ml of MNKT and EBA solution was prepared by diluting 1.5 ml of stock solution to 10 ml with methanol.

First order derivative method:

From the stock solution of 100 μg/ml MNKT and EBA, working standard solutions of the drugs were prepared by appropriate dilution and were scanned in the range of 200-400 nm. Then their first order derivative spectra were scanned using the spectrum mode with medium scan speed using derivative wavelength difference(∆λ) one. From their first order derivative spectra, MNKT was determined at 364 nm (Zero Crossing Point of EBA) and EBA was determined at 256.5 nm (Zero Crossing Point of MNKT) as shown in figure-2. Standard solutions were prepared having concentration 5-25 μg/ml for MNKT and EBA. The absorbances of solutions were measured at 364 nm and 256.5 nm. A calibration curve was prepared by plotting absorbance against respective concentration.

Assay of tablet formulation by First order derivative Method:

Ten tablets were weighed and crushed to obtain a fine powder. An accurately weighed tablet powder equivalent to about 10 mg of MNKT and 10 mg of EBA was transferred to 100 ml volumetric flask and dissolved in 50 ml of methanol. The volume was made up to the mark using methanol as solvent. The resulting solution was filtered through Whatmann filter paper and 1.5 ml of this filtrate was appropriately diluted to get concentration of 15 μg/ml of MNKT and 15 μg/ml of EBA. Absorbance of sample solutions was measured at 364 nm and 256.5 nm and the concentration of two drugs in the sample were determined.

Method validation;

Linearity and range:

Aliquots of standard stock solutions of MNKT and EBA were taken in volumetric flasks and diluted with methanol to get final concentrations in range of 5-25 μg/ml for MNKT and EBA. This calibration range was prepared five times and absorbances were measured at respective wavelengths for each drug separately.

Precision:

Precision of the methods were determined by performing interday variation, intraday variation and method repeatability studies. In interday variation, the absorbance of standard solutions of MNKT and EBA (5-25 μg/ml) were measured on five consecutive days. In intraday variation the absorbances were measured five times in a day. In repeatability study, three concentrations of both the drugs were analysed in triplicate.

Recovery studies:

To study the accuracy of the proposed methods, recovery studies were carried out by standard addition method at three different levels. A known amount of drug was added to preanalyzed tablet powder and percentage recoveries were calculated.

Ruggedness:

The data for ruggedness obtained from two different analysts for MNKT and EBA.

Table 1: Validation Parameters for Simultaneous equation Method

Parameters	MNKT	EBA
Linearity range	5-25 µg/ml	5-25 µg/ml
Correlation coefficient	0.9997	0.9981
Precision (% C.V.)
Repeatability	0.795	1.181
Intraday (n=3)	1.41	1.59
Interday (n=3)	1.53	1.54
Mean % recovery	99.16-101.94%	98.88-101.48%
Ruggudness	0.82-1.80%	1.65-1.90%
Limit of Detection	0.41µg/ml	0.57µg/ml
Limit of Quantification	1.24µg/ml	1.74µg/ml

* MNKT- Montelukast; EBA-Ebastine; RSD-Relative Standard Deviation.

Figure 2: Overlain first order derivative spectra of MNKT (15 µg/ml) and EBA (15 µg/ml) in methanol.

Table 2: Recovery studies

_{Name of Drug}	_{Amount of Drug Added (}µg/ml)	_{First order derivative Method}
_{Name of Drug}	_{Amount of Drug Added (}µg/ml)	_%Recovery*	_SD
MNKT	5	100.55	0.06
EBA	5	100.74	0.064
MNKT	10	101.25	0.041
EBA	10	100	0.11
MNKT	15	100.55	0.19
EBA	15	100.24	0.23

*Mean of Three estimations MNKT- Montelukast; EBA-Ebastine; SD-Standard Deviation.

Table 3: Results of simultaneous estimation of MNKT and EBA in marketed Formulation by First order derivative Method.

Method	mg/tablet		%*of label claim ± S.D.**
Method	MNKT	EBA	MNKT	EBA
_{First order derivative Method}	10	10	100.4±0.08	99.81±0.08

RESULTS AND DISCUSSION:

The proposed methods were validated as per ICH guideline. The plot of absorbances versus respective concentrations of MNKT and EBA were found to be linear in the concentration range of 5-25 μg/ml for both drugs with correlation coefficient 0.9997 at 364 nm and 0.9981 at 256.5 nm for First order derivative method. Precision was calculated as interday and intraday variations and % RSD was found to be less than 2 for First order derivative method Table 1. The accuracy of method was determined at 80, 100 and 120 % level. The % recovery ranges from 98.88 to 101.66 for these methods as shown in Table 2. The % RSD of ruggedness for MNKT ranges from 0.82 to 1.8%, while for EBA it was found to be 1.65 to 1.9% for this method for MNKT and EBA respectively. Results for all validation parameters are presented in table 1, 2 and 3. The two _methods can be successfully _used for simultaneous estimation of MNKT and EBA_{in their combined tablet dosage form.}Marketed tablets were analyzed and results obtained were in the range of 98-102 %( Table 3).

CONCLUSION:

The proposed methods give accurate and precise results for determination of MNKT and EBA in marketed formulation (tablet) without prior separation and are easily applied for routine analysis. The most striking feature of both the methods is its simplicity and rapidity. Method validation has been demonstrated by variety of tests for linearity, accuracy, precision and ruggedness. The developed methods have several advantages, as they are simple, accurate and precise. The proposed methods were successfully applied to determination of these drugs in commercial tablets.

ACKNOWLEDGEMENT:

The authors are thankful to Alembic Pharmaceuticals, Vadodara and Kivi labs, Baroda for providing pure gift samples of Montelukast sodium and Ebastine respectively. The authors are also thankful to the Principal, Parul Institute of Pharmacy College for providing necessary facilities.

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Received on 12.02.2013 Modified on 01.03.2013

Asian J. Research Chem. 6(3): March 2013; Page 232-235