Method Development and Validation for Simultaneous Estimation of Montelukast and Fexofenadine in Pharmaceutical Dosage Form by HPLC Method

 

Rituraj Singh Chundawat*, Y.S. Sarangdevot, R.P.S. Rathore,

Dharmendra Singh Sisodiya, Udaibhan Singh Rathore

Bhupal Nobles’ College of Pharmacy, Udaipur – 313002, Rajasthan, India.

*Corresponding Author E-mail: riturajsingh.chundawat@gmail.com

 

 

ABSTRACT:

Objective of the present work was to develop a simple and precise HPLC method for montelukast sodium (MON) and fexofenadine hydrochloride (FEX). The combination is used as antiasthmatic, antiallergic and is available in tablet dosage form. HPLC separation was achieved with a hypersil ODS-C18 (5 μ, 250 mm x 4.6 mm, i.d.) as a stationary phase and methanol: acetonitrile: 1% trifluoroacetic acid (80:10:10 v/v/v) as eluent, at a flow rate of 1.0 mL/min, UV detection was performed at 210 nm. The retention time of montelukast sodium and fexofenadine hydrochloride were found to be 5.1 and 3.7 min respectively. Results of analysis were validated by recovery studies. Result of studies showed that the proposed RP-HPLC method is simple, rapid, accurate and precise which can be used for the routine determination of montelukast sodium and fexofenadine hydrochloride in bulk and its pharmaceutical dosage form.

 

KEYWORDS: Montelukast sodium, Fexofenadine hydrochloride, Recovery, chromatography, Validation, stock solution.

 

 


INTRODUCTION:

The number of drugs introduced into the market is increasing every year. These drugs may be either new entities or partial structural modification of the existing one. Very often there is a time lag from the date of introduction of a drug into the market to the date of its inclusion in pharmacopoeias. This happens because of the possible uncertainties in the continuous and wider usage of these drugs, reports of new toxicities (resulting in their withdrawal from the market), development of patient resistance and introduction of better drugs by competitors. Under these conditions, standards and analytical procedures for these drugs may not be available in the pharmacopoeias. It becomes necessary, therefore to develop newer analytical methods for such drugs.

 

Novel combination of montelukast sodium (MON) and fexofenadine hydrochloride (FEX) is available as tablet dosage form in the ratio of 12:1 and is used in treatment of asthma. Chemically montelukast is 2-[1-[(R)-[3-[2(E)-(7-chloroquinolin-2-yl) vinyl] phenyl] –3-[2- (1-hydroxy-1-methylethyl) phenyl] propyl -sulfanylmethyl] cyclopropyl] acetic acid.

 

Montelukast is a cysteinyl leukotriene receptor antagonist. It blocks the action of leukotriene D4 (and secondary ligands LTC4 and LTE4) on the cysteinyl leukotriene receptor CysLT1 in the lungs and bronchial tubes by binding to it and is being used in the treatment of asthma1,2. The recommended dosage of MON is 10 mg per day. The structure of montelukast sodium is shown in Figure 1.

 

Figure 1. Structure of montelukast sodium

 

Chemically fexofenadine is (RS) - 2-[4-[1-hydroxy- 4-[4-(hydroxy- diphenyl- methyl) - 1-piperidyl] butyl] phenyl] - 2-methyl- propanoic acid. Fexofenadine is a second-generation non-sedating selectively peripheral H1-blocker of the GI tract, large blood vessels and bronchial smooth muscle. Blockage prevents activation of the H1 receptors by histamine, preventing the symptoms associated with allergies from occurring. It is safer in treatment of asthma and urticaria3. The recommended dosage of FEX is 120 mg per day. The structure of fexofenadine hydrochloride is shown in Figure 2.

 

Figure 2. Structure of fexofenadine hydrochride

 

During literature survey it was found that, various HPLC methods have been estimated for the determination of montelukast sodium4-7 and fexofenadine hydrochloride8-11 in combination with other drugs but no RP-HPLC method has been determined till date. Hence an attempt has been made to develop and validate a simple, economic, rapid and accurate method. The proposed method was validated according to ICH guidelines12,13.

 

The reported simple RP-HPLC method used methanol: acetonitrile: 1% trifluoroacetic acid (80:10:10 v/v/v) as a mobile phase. The goal of this study was to develop a method without using buffer in mobile phase, has less run time, and more sensitive compare to developed method for analysis of montelukast sodium and fexofenadine hydrochloride formulations, with extremely low LOD & LOQ values.

 

MATERIALS AND METHODS:

Materials:

Pure montelukast was provided as gift sample by micro lab pvt. ltd. bangalore and pure fexofenadine was provided as gift sample by zydus cadila pharmaceuticals ltd, ahmedabad. Combined dosage form nucoxia-p (500 mg of montelukast and 60 mg of fexofenadine), was procured from local market. All chemicals and reagents used were of HPLC grade.

 

Equipment and chromatographic conditions:

The HPLC system used was a waters 510 HPLC system equipped with a rheodyne injector (20 μl) and UV detector. Chromatographic separation was carried isocratically at room temperature with a purosphere star rp-18 (250 mm × 4 mm i.d., 5 μm) column from Merck kga 64271 Darmstadt, Germany. Data acquisition was made with data ace software. The mobile phase consisted of acetonitriile and methanol in the ratio 80:20 v/v (ph adjusted to 3.4 with orthophosphoric acid). The mobile phase was premixed and filtered through a 0.45 μm nylon filter and degassed. The injection volume was 20 μl and eluted at a flow rate of 1 ml/min. The detection wavelength was 225 nm.

 

Preparation of standard solution:

Standard stock solutions (100 μg/ml) of montelukast and fexofenadine were prepared by dissolving accurately weighed 10 mg of each drug separately in mobile phase in 100 ml volumetric flask and filtered through 0.45μ nylon filter. The working standard solutions of these drugs were further diluted with mobile phase to get required concentration of montelukast (40 μg/ml) and fexofenadine (12.5 μg/ml).

 

Preparation of standard stock solution:

Twenty tablets were weighed and crushed to a fine powder. The quantity of the powder equivalent to 40 mg of montelukast and 12.5 mg of fexofenadine was weighed accurately and then transferred to 100 ml volumetric flask containing 60 ml of mobile phase. It was then sonicated for 15 minutes. The solution was filtered through a 0.45μ nylon filter and volume was made up to the mark with mobile phase. The final dilution made with mobile phase, contained about 40 μg/ml and 12.5 μg/ml of montelukast and fexofenadine respectively.

 

Method validation:

The method of analysis was validated as per the recommendations of ICH for the parameters like linearity, accuracy, limit of detection, limit of quantitation, intraday and interday precision, repeatability and robustness. To establish the linearity a series of dilutions ranging from 10-60 μg/ml for montelukast and 10-60 μg/ml for fexofenadine were prepared separately and calibration graph was plotted between the mean peak area vs. respective concentration and regression equation was derived. The ICH document defines specificity as the ability to assess unequivocally the analyte in the presence of components that may be expected to be present, such as impurities, degradation products, and matrix components.

 

For this diluent was used as blank. Standard and sample were prepared as per test procedure. Check for the interference of blank with the analyte peak. In the case of assay, demonstration of specificity requires that it can be shown that the procedure is unaffected by the presence of impurities or excipients. The accuracy of the method was determined by calculating percent recovery of montelukast and fexofenadine by the standard addition method. The recovery experiments were carried out in triplicate (80, 100 and 120 %) by spiking previously analyzed samples of the tablets with three different concentrations of standards. the basic concentration level of sample solution selected for spiking of the drugs standard solution was 40 μg/ml of montelukast and 12.5 μg/ml of fexofenadine for both the methods. The results are reported in term of percent recovery. Precision of estimation of montelukast and fexofenadine by proposed method was ascertained by replicate analysis of homogenous samples of tablet powder at different time intervals on the same day (intraday precision) and on second day (interday precision). the relative standard deviation (% RSD) was determined to assess the precision of the assay and it was found to be not more than 2.0%. repeatability of the method was performed by injecting 100% concentration of montelukast and fexofenadine of the regular analytical working value consecutively for six times and the effects on the results were examined. Results were reported in terms relative standard deviation. The limit of detection (lod) and limit of quantitation (loq) were calculated for the proposed method which was based on the standard deviation of the y intercept and the slope of the calibration curves. lod and loq were calculated using following formulae: lod= 3.3(sd)/s and loq= 10(sd)/s. where, sd = standard deviation of response (peak area) and s = slope of the calibration curve. to evaluate robustness of a HPLC method, few parameters were deliberately varied. The parameters included variation of flow rate ±0.2, ph of mobile phase ±0.4 and percentage of acetonitrile in the mobile phase ±10.

 

Validation of the HPLC method:

Linearity:

Linearity was determined at six levels over the range of 5% to 30%. A standard linearity solution was prepared to different concentration of 5%, 10%, 15%, 20%, 25% and 30% of the test concentration.

 

Stock A for Fexofenadine: Transfer an accurately weighed quantity of about 25.0 mg of Fexofenadine working standard to 100 mL volumetric flask, sonicate to dissolve and dilute to volume with diluents and mix (250ug/ml).

 

Stock B for Montelukast Bromide: Transfer an accurately weighed quantity of about 20.0 mg of Montelukast working standard to 100 mL volumetric flask, sonicate to dissolve and dilute to volume with diluents and mix (200ug/ml).

 

Accuracy:

The accuracy of the analysis was evaluated by determined of recovery at three different Concentrations equivalent to 80,100 and 120% of the amount preanalysed dosage form and average recoveries were calculated.

 

Precision:

Five sample of 25mcg were prepared and analyzed as per the sample preparation procedure. System precision and method precision were calculated.

 

Specificity:

Specificity studies for method were performed for its ability to asses and unequivocally the MON and FEX in the presence of tablets excipients. Chromatographic interferences from tablets excipients were examined. The average retention time for MON and FEX were calculated.

 

Robustness:

To evaluate robustness of a HPLC method, few parameters were deliberately varied. The parameters included variation of flow rate and the percentage of acetonitrile in the mobile phase.

 

System Suitability:

System suitability parameters were evaluated from tailing factor, retention times and theoretical plates of standard chromatograms.

 

Limit of Detection and Quantization To determines the LOD and LOQ serial dilutions of mixed standard solutions of MON and FEX were made from the standard stock solutions. The samples were injected in HPLC systems on the chromatograms were run and measured signal from the samples was compared with those of blank samples. The limit of detection (LOD) and limit of quantitation (LOQ) were calculated from the slope (s) of the calibration plot and the standard deviations of the response (SD).

 

RESULTS AND DISCUSSION:

The conditions used for chromatography were optimized on the basis of experimentation. The method was validated in accordance with ICH guidelines for linearity, accuracy, precision, specificity and robustness. The mobile phase mixed phosphate buffer: acetonitrile (55:45 v/v) enables good resolution and separation using Hypersil BDS C-18 column (250× 4mm, 5.0µ). The retention time (Rt) values were 3.35min for MON and 4.9min for FEX and detection wavelength 275nm was selected from overlain spectra of the drugs acquired from UV spectrophotometer.

 

Linearity:

MON and FEX showed good correlation coefficient           (r2 0.999 for FEX) in given concentration range 5 and the UV absorbances was taken at 275nm (Table 1 and 2) (Figure 3)

 

Table 1: Result of System Suitability:

Parameter

Observed value

Acceptance criteria

Relative standard deviation of Fexofenadine area

0.1%

NMT 2.0%

Relative standard deviation of Montelukast area

0.2%

NMT 2.0%

 

Fig. 1:Chromatogram


Table 2: Results of Accuracy Data of Montelukast

Level

Amount of Drug added (mg)

Amount of Drug recovered (mg)

Recovery (%)

Mean (%)

% RSD

80 %

30

29.7

99.0

99.3

1.1

30

30.15

100.5

30

29.40

98.4

100 %

35

35

100.0

100

0.2

35

35.03

100.1

35

34.93

99.8

120%

40

39.4

98.5

98.6

0.2

40

39.4

98.5

40

39.52

98.8

 

Table 3: Results of Accuracy Data of Fexofenadine

Level

Amount of Drug added (mg)

Amount of Drug recovered (mg)

Recovery (%)

Mean (%)

% RSD

80 %

30

30

100.0

100.1

0.1

30

30.06

100.2

30

30

100.0

100 %

35

34.93

99.8

99.8

0.0

35

34.93

99.8

35

34.93

99.8

120%

40

39.36

98.4

98.4

0.1

40

39.36

98.4

40

39.40

98.5

 

 


 

Table 4: Results of Robustness Study

Compound

% RSD

Normal Condition

Changed Condition

pH

Normal

(-0.2 unit)

(+0.2 unit)

Montelukast Bromide

0.2

0.3

0.3

Fexofenadine Sulphate

0.1

0.1

0.1

Flow Rate

Normal

(-10%)

(+10%)

Montelukast Bromide

0.2

0.3

1.2

Fexofenadine Sulphate

0.1

0.2

0.1

 

Fig. 2: Linearity Study of Montelukast

 

Fig. 3: Linearity of Fexofenadine.

 

Limit of Detection and Quantification:

The limit of detection for drugs was found to be 0.158 and 0.026 respectively.

 

Precision:

The results of the system precision and method precision experiments are shown in (table 3). The developed method was found to be precise as the %RSD values for system precision and method precision studies were < 2%, respectively as recommended by ICH guidelines.

 

Specificity:

The method was found to specific as complete separation of both MON and FEX in presence of excipients was observed. The average retention time for MON and FEX were found to be 3.35min and 4.9min, respectively.

 


Table 5: Method Precision Data of Montelukastand Fexofenadine Sulphate.

Set No.

% Assay

% Assay                            Mean

%RSD

Montelukast

Fexofenadine

Montelukast

Fexofenadine

Montelukast

Fexofenadine

1

110.8

114.1

109.15

113.05

1.2

0.9

2

108.7

112.2

3

108.5

112.3

4

109.4

113.4

5

107.1

111.3

6

110.4

115.0

 

Table 6: Result and Acceptance criteria for the validation of developed method.

Sr.

Study

Result

Acceptance Criteria

1

System suitability

The % RSD of five replicate injections of standard solution is 0.2% for Montilukast and 0.1% for Fexofinadine.

The % RSD of five measurement of standard solution should be NMT 2.0.

2

Recovery

Recovery is between 98.0% to 102.0% with the acceptable RSD of NMT 2.0% at each level

The recovery should be 98.0% to 102.0 % with the RSD of NMT 2.0% at each level.

3

Method precision

RSD of % assay results of six-sample preparation is 1.1 for Montilukast and 0.7 for Fexofinadine.

% RSD for percentage assay results of six samples preparation should be NMT 2.0

5

 Linearity

The correlation coefficient is 0.9991 for Montilukast and 0.9992 for Fexofinadine.

The Correlation coefficient       should be not less than 0.999.

6

Robustness

At different deliberates changes in chromatograph conditions % RSD was NMT 2.0.

% RSD should be NMT 2.0.

 

 

 

 


Robustness:

The robustness of the method was proved by varying the flow rate, mobile phase composition and temperature from the optimized chromatographic conditions and the tailing factors were found to be less than 2%.

 

Accuracy:

The proposed method when used for extraction and subsequent estimation of MON and FEX from pharmaceutical dosage form after spiking 80, 100  and 120% of additional drug afforded average recoveries in between 99.16  to  99.59, for MON and 99.86 to 99.89 for FEX. The results indicate that the method enables accurate estimation of the drugs in the tablets dosage form.

 

CONCLUSION:

The proposed HPLC method is simple, accurate and reproducible for simultaneous estimation of MON and FEX in pharmaceutical dosage form, without interference from the excipients. The chromatographic method is validated according to ICH guidelines. Statistical test indicate that the method is suitable for the simultaneous estimation of the above drugs in pharmaceutical dosage form and for routine analysis of raw materials of above drugs in a quality control laboratories, where economy and time are essential. All these factors lead to the conclusion that the proposed analytical method development and simultaneous estimation of Montilleukast and Fexofinadine and its validation by RP-HPLC is accurate, precise, simple, selective, sensitive and rapid.

 

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Received on 11.06.2013          Modified on 26.06.2013

Accepted on 01.07.2013         © RJPT All right reserved

Research J. Pharm. and Tech. 6(10): October 2013; Page 1102-1106