INTRODUCTION:

Montelukast sodium (MON) [(R-(E))-1-(((1-(3-(2-(7-chloro-2-quinolinyl) ethenyl) phenyl) -3-(2-(1-hydroxy-1-methylethyl) phenyl) propyl)thio) methyl) cyclopropaneacetic acid, monosodium salt] is leukotrine receptor blocker, with molecular formula C₃₅H₃₅ClNNaO₃S administered orally as tablet in the dose of 5-10 mg per day ^[1] (Fig. 1). Fexofenadine HCL, is Fexofenadine HCl (FEX), chemically designated as (±)-4-[1-hydroxy-4-(4- hydroxydiphenylmethyl)-1- piperidinyl]-butyl]-∝,∝-dimethyl benzene acetic acid hydrochloride is a histamine H1 receptor antagonist used in patients with allergic rhinitis.The molecular weight is 538.13 and the empirical formula is C₃₂H₃₉NO₄•HCl^[2](Fig.2). Few analytical techniques such as spectrophotometry, HPLC ^[3-8] have been reported for the individual and simultaneous determination of MON and other antiretroviral analogues. As far as Fexofenadine HCL is concerned, few reports are available for its estimation in bulk and formulation such as spectrophotometry, HPLC and bioanalytical methods^[9-17]. The method was devloped and validated in accordance with International Conference on Harmonization (ICH) guidelines ^[18].

Fig.1structure of montelukast

Fig.2 structure of Fexofenadine HCl

MATERIALS AND METHODS:

Instrumentation

An UV-Visible double beam spectrophotometer (Varian Cary 100) with 10 mm matched quartz cells was used. Electronic balance (Model Shimadzu AUW-220D) was used for weighing.

Reagents and chemicals

Pure drug sample of both MON (% purity 99.86) and FEX (% purity 99.92) was kindly supplied as a gift sample by Cipla Ltd. These samples were used without further purification. Spectroscopy grade methanol was used throughout the study. Tablets each containing 10 mg of MON and 120 mg of FEX used for analysis were MONTAIR-FX manufactured by Cipla Ltd.

Preparation of Standard Stock Solutions and calibration Curve

Standard stock solutions of pure drug containing 1000μg/mL of MON and FEX were prepared separately in methanol. Standard stock solutions were further diluted with methanol to get working standard solutions of analytes in the concentration range of 2-10 μg/mL and 24-120 μg/mL of Montelukast sodium (MON) and Fexofenadine HCL (FEX), respectively and scanned in the range of 200-400nm. For ratio derivative amplitudes (at interval 1.2 and filter size 9) of ratio spectra were measured at 288.17nm and 289.12nm for MON and FEX, respectively. First derivative amplitudes of ratio spectra and concentrations were used to construct calibration curve.

Preparation of Sample Solution and Formulation analysis

Twenty tablets were weighed accurately and a quantity of tablet powder equivalent to 10mg of MON and FEX (120mg) was weighed and dissolved in the 30 mL of methanol with the aid of ultrasonication for 7 min and solution was filtered through Whatman paper No. 41 into a 100 mL volumetric flask. Filter paper was washed with same solvent, adding washings to the volumetric flask and volume was made up to the mark with methanol. The solution was suitably diluted further with methanol to get required final concentration of MON (6μg/mL) and FEX(72µg/mL).(fig 3)

Fig 3: Overlay spectra of FEX, MON and Formulation

Theoretical aspects of Ratio Derivative

The method involves dividing the spectrum of mixture by the standardized spectra of each of the analyte to get ratio spectra and first derivative of ratio spectrum was obtained which was independent of concentration of divisor .Using appropriate dilutions of standard stock solution, the two solutions were scanned separately. The ratio spectra of different MON standards at increasing concentrations were obtained by dividing each with the stored spectrum of the standard solution of FEX (72 μg/mL) as shown in (Fig 4A).Wavelength 288.17nm was selected for the quantification of MON in MON +FEX mixture. The ratio and ratio derivative spectra of the solutions of FEX at different concentrations were obtained by dividing each with the stored standard spectrum of the MON (6μg/mL) as shown in (Fig 4B). Wavelength 289.12nm was selected for the quantification of FEX in MON + FEX mixture. Measured analytical signals at these wavelengths were proportional to the concentrations of the drugs over the selected concentration range. Calibration curves were prepared from the measured signals at the selected wavelength and concentration of the standard solutions. The concentrations of MON (C_MON) and FEX (C_FEX) in solution of tablets was calculated by using equations(1) and (2), respectively.

At288.17nm:C_MON .... (1)

At289.12 nm: C_FEX = … (2)

Fig.4A: First Derivative of ratio spectra of 2,4,6,8,10µg/ml of MON when 72µg/ml of FEX is used as divisor

Fig.4B: First Derivative of ratio spectra of 24,48,72,96,120µg/ml of FEX when6µg/ml of MON is used as divisor

Validation of Analytical method

Validation is the process of establishing documented evidence that provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes. The method was validated for linearity, accuracy, precision, sensitivity, robustness, etc. in accordance with International Conference on Harmonization (ICH) guidelines

1) Accuracy

Accuracy of the method was determined in terms of % recovery of standard. Recovery studies were carried out by addition of standard drug solution at the level of 80%, 100% and 120% to the pre analyzed sample. Results of the recovery study were found to be within the acceptance criteria 100±10 %, indicating a good degree of sensitivity of the method towards detection of analytes in sample. Accuracy results for MON and FEX are shown in Table no 1.1 and 1.2 respectively.

2) Solution Stability

Method stability was checked by analyzing solution kept in fridge and at room temperature by both methods. Solution at room temperature was stable for 12 hours and solution in fridge was stable for 30 days (% RSD < 2).

3) Precision of the Method

Method repeatability was determined by six times repetitions of assay procedure. For intra-day precision method was repeated 5 times in a day and the average % RSD was determined. Similarly the method was repeated on five different days for inter-day precision and average % RSD was determined (Table 2).

4) Linearity

The linearity of the proposed method was evaluated for each drug by analyzing a series of different concentrations of each of FEX,MON ; within the range stated in Table .3 The assay was performed according to the experimental conditions previously established. The absorbance values for FEX and MON were measured, at the specified wavelengths (Table 3), and plotted against its concentration. A straight line was obtained in each case. The statistical analysis of these graphs using least squares method was made for the slope, intercept and correlation coefficients. The results obtained show that the linearity of calibration graphs and the compliance with Beer`s law for the both drugs i.e FEX and MON Fig no(5A and 5B). The correlation coefficients of calibration plots for MON and FEX (Table 3)

5) Limit of Quantification (LOQ) and Limit of Detection (LOD)

LOD and LOQ were calculated statistically from formula shown in equation No 3 and4 respectively:

….. (3)

….. (4)

Where, SD: Standard Deviation of y- intercepts of regression lines,

The LOD and LOQ of MON and FEX by statistical and visualization methods were mentioned in Table (3).

6) Sensitivity

Absorbance of standard solutions of MON and FEX was taken at 288.17 and 289.12 nm. Sandell’s sensitivity (П) for drugs was calculated from the following formula, at both wavelengths^.(19)

П (μg/cm^-3 AU) =×0.001

The Sandell’s sensitivity for Montelukast was calculated at 288.17 nm. and for Fexofenadine at 289.12 nm..(Table 3

7) Robustness

Robustness of the method was determined by repeating the proposed method by other analyst in the same laboratory and calculating % RSD. The results are indicated in Table 4.

Table 1.1 Accuracy result for Montelukast

Ingredient	Recovery	Drug	Amount Spiked	Amount recovered	% Mean Recovery,
Ingredient	Level %	Amount (µg/ml)	(µg/ml)	(µg/ml)	% RSD (n=3)
MON	80%	6	4.8	10.7	99.074,0.7407
	100%	6	6	11.89	99.0833,0.333
	120%	6	1.2	7.1	98.61,0.111111

Table 1.1 Accuracy result for Fexofenadine.

Ingredient	Recovery	Drug	Amount Spiked	Amount recovered	% Mean Recovery,
Ingredient	Level %	Amount (µg/ml)	(µg/ml)	(µg/ml)	% RSD (n=3)
FEX	80%	72	57.6	129.4	99.845,0.67901
	100%	72	72	143.69	99.784,0.7222
	120%	72	86.4	158	99.747,0.4747

Table 2 Determination of precision

Sr no	Reproducibility (%)	(Intra-day precision) (n=4)(%)	(Inter-day precision) (n=3)(%)
1	99.02716	99.02716	99.2057
2	99.55	100.4089	99.3763
3	100.0667	100.2095	99.02716
4	100.45	99.97441	98.02734
5	100.15	100.266	99.31679
6	100.58	100.0756	99.31679
Mean	99.97064333	99.993595	99.045013
Std. deviation	0.584738518	0.496867626	0.513668
RSD	0.005849102	0.004968995	0.0051862
%RSD	0.584910228	0.496899453	0.5186207

Table 3: Optical characteristics of the proposed methods and result of linearity,sensitity and formulation analysis

SR NO	PARAMETERS		MONTELUKAST (MON)	FEXOFENADINE (FEX)
1	Wavelength	nm	288.17	289.12
2	Beer’s law limit	(μg/mL)	2-10	24-120
3	Linearity	(μg/mL)	2-10	24-120
		Regression Equation*	Y=0.084X+0.055	Y=0.00045X+0.003
		R²	0.999	0.999
4	Sensitivity	μg/cm-3 AU	0.00035	0.02018
5	Formulation Analysis (%Assay, %RSD), n=6	MONTAIR-FX	98.00%	100.66%
6	LOD	(μg/mL)	0.1043	0.144
7	LOQ	(μg/mL)	0.31	0.371

RESULT AND DISCUSSION:

Using appropriate dilutions of standard stock solution the two solutions were scanned separately. Beer’s law is obeyed in the concentration range of 2-10 µg/mL and 24-120 µg/mL for MON and FEX, respectively. Under experimental conditions described, calibration curve, assay of tablets and recovery studies were performed.Correlation coefficient was greater than 0.999 for both the drugs .A critical evaluation of proposed method was performed by statistical analysis of data where slope, intercept, correlation coefficients are shown in Table 3. The proposed methods were also evaluated by the assay of commercially available tablets containing MON and FEX. The results of formulation analysis are presented in Table 3. Recovery was found in the range of 99.07–99.61% for MON (Table no 1.1) and 99.74 – 99.84% for FEX (Table 1.2) by ratio derivative method . The accuracy is evident from the data as results are close to 100 % and standard deviation is low. The method was found to be validated in accordance with ICH guidelines.

Fig no 5: Calibration cure of A) Fexofenadine and B) Montelukast

Table 4 Results of Robustness

Sr. no	Analyst I	Analyst II
1	99.02716	99.2057
2	100.51	99.3763
3	100.0667	99.02716
4	100.1053	98.02734
5	100.1172	99.31679
6	100.397	99.31679
Mean	100.037227	99.0450133
Std. deviation	0.52632396	0.51366797
RSD	0.00526128	0.00518621
%RSD	0.5261281	0.51862073

CONCLUSION:

The validated spectrophotometric method employed here proved to be simple, economical, precise and accurate. Thus the method is useful for performing IPQC test and for routine simultaneous determination of MON and FEX in tablet dosage form.

ACKNOWLEDGEMENT:

The authors wish to express their gratitude to Cipla Ltd. India for gift samples of pure Montelukast sodium and Fexofenadine HCL, respectively. The authors are also thankful to the Principal and Management of MAEER’s Maharashtra Institute of Pharmacy for providing necessary facilities.

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Received on 15.02.2012 Modified on 01.03.2012

Asian J. Research Chem. 5(5): May 2012; Page 637-641