Objective: A successful attempt has been made to develop simple, accurate, economic and rapid methods for the estimation of Montelukast and Fexofenadine in tablet formulation by RP-HPLC and to validate the methods, as per the guidelines given by ICH requirements to assure that the method consistently meets the predetermined specifications and quality attributes.

Method: Separation of Montelukast and Fexofenadine successfully achieved on RP-HPLC with Thermo 2080 system, P4000 Quaternary pump, UV 6000 PDA detector, ARP-C18 (250 mm X 4.6 mm), 5? column or equivalent utilizing mobile phase composed of Acetonitrile: Phosphate buffer (pH2.8)in the ratio of 70:30 v/v as mobile phase at a flow rate of 1mL/min and the eluates was monitored at 245 nm.

Result: Chromatogram showed a peak at a retention time of 3.624 min and 5.178 min for Montelukast and Fexofenadine respectively. The method for simultaneous estimation was validated for system suitability, linearity, precision, accuracy; specificity, ruggedness, robustness; LOD and LOQ. Recovery of Montelukast and Fexofenadine were found to be in the range of 99.2% and 99.13% and showing linearity in the range of 10-50 ?g/ml. The S/N for LOD was found to be 2.96 and 2.78 and LOQ were found 9.96 and 9.78for estimation of Montelukast and Fexofenadine respectively.

Conclusion: The developed method was found to be accurate, precise, linear, specific and reproducible for the simultaneous determination of Fexofenadine and Montelukast in formulation. Developed method can be successfully applied for the quantitative determination of Montelukast and Fexofenadine in Bulk drug and Pharmaceutical dosage form.

Montelukast; Fexofenadine Potassium; RP-HPLC

Montelukast is chemically known as 2-[1-[[(1R)-1-[3-[(E)-2-(7-chloroquinolin-2 yl) ethenyl] phenyl]-3-[2-(2-hydroxypropan-2yl) phenyl] propyl] sulfanylmethyl] cyclopropyl] acetic acid. Chemically Montelukast belongs to the class of organic compounds known as 1,3-diarylpropanoids (linear).Being Leukotriene receptor antagonist, Montelukast is used as an alternative to anti-inflammatory medications in the management and chronic treatment of asthma and exercise-induced bronchospasm (EIB) [1-3]. The chemical structure of MON is shown in (Figure 1) 

Figure 1: Structure of Montelukast

Fexofenadine is 2-[4-[1-hydroxy-4-[4-[hydroxy (diphenyl) methyl] piperidin-1-yl] butyl] phenyl]-2-methylpropanoic acid, is Fexofenadine is a second generation antihistamine that is used for the treatment of allergic rhinitis, angioedema and chronic urticarial [1,4]. The chemical structure of FEXO is shown in (Figure 2) 

Figure 2: Structure of Fexofenadine 

Fexofenadine is a selective, second-generation H1 receptor antagonist and non-sedative in nature, which have an additional impact on the inflammatory mediators. Montelukast is a highly selective type I receptor antagonist of leukotriene D4. The leukotrienes modifiers have both anti-inflammatory and bronchodilator properties [5-7]. The literature search ascertained that addition of an antihistamine to Montelukast has added benefit. The combination therapy of Montelukast with antihistamine provides enhancing and complimentary effects thereby reducing the symptoms effectively. Fexofenadine along with Montelukast is more effective than antihistaminic alone in control of allergic rhinitis symptoms [8-9]. 

Numerous methods were developed for the analysis of both Fexofenadine and Montelukast in combination such as UV-Vis spectroscopies, HPLC and LCMS/ MS methods. Instantaneous estimation of these compounds by RP-HPLC methods were showing more time of analysis and complicated procedures; hence the present study was focused on chromatographic analysis of Fexofenadine and Montelukast in a less time consuming simultaneous analysis of these compounds inactive ingredient (API) and Pharmaceutical dosage form which found in the pharmaceutical market [10-11].

Chemical and Reagents

Montelukast and Fexofenadine obtained as gift sample from Cipla Ltd. Mumbai. Vysov-M tablets containing 500 mg Montelukast and 50 mg Fexofenadine were purchased from market. Water (HPLC grade), Methanol (HPLC grade), Acetonitrile (HPLC grade), Potassium dihydrogen phosphate, Sodium dihydrogen phosphate, Sodium hydroxide, Hydrochloric acid, Triethylamine, Ortho phosphoric acid were purchased from Thermocil Fine Chem Ltd. And remaining reagents were of Analytical grade.

Instrumentation

The analysis was carried out on a HPLC system (Thermo 2080 system) equipped with UV 6000 PDA  detector, pressure controlled by P4000 Quaternary pump. C18 column (particle size 5??m, 250?mm × 4.6?mm in dimension) was used for separation. UV spectrophotometer used was UV 3092, (Lab India, Mumbai) and pH meter used was Thermo electron corporation Orion 2 star.

Solution preparation

Preparation of mobile phase: Buffer 300 ml (30%) and 700 ml of Acetonitrile HPLC (70%) were mixed and degassed in ultrasonic water bath for 5minutes and filtered through 0.45 ? filter under vacuum filtration.

Diluent Preparation: Mobile phase was used as Diluent.

Preparation of standard solution (Mixed standard) : 10 mg of Montelukast and 10mg of Fexofenadine working standards were accurately weighed and transferred into a 100 ml clean dry volumetric flask add about 70ml of diluent was added and sonicated to dissolve it completely and the volume was made up to the mark with the same solvent. (Stock solution) Further 1.2ml of Montelukast & 3ml of Fexofenadine was pippeted from the above stock solution into a 10ml volumetric flask and diluted up to the mark with diluent.

Preparation of sample solution

MONTAIR-FX Tablets were weighed and powdered in glass mortar. The powder equivalent to the amount of active ingredient present in 10 tablets was transferred into a 100 ml clean dry volumetric flask, 70 ml of diluent was added to it and was shaken by mechanical stirrer and sonicated for about 30minutes by shaking at intervals of five minutes each and was diluted up to the mark with diluent and let it stand until the residue settles before taking an aliquot for further dilution (stock solution). 0.6ml of upper clear solution was transferred to a 10 ml volumetric flask and diluted with diluent up to the mark and the solution was filtered through 0.45 µm filter before injecting into HPLC system.

Chromatographic conditions

Table 1: Optimized chromatographic conditions

A. System Suitability

Sample solution of Montelukast and Fexofenadinewere injected three times into HPLC system as per test procedure. The system suitability parameters were evaluated from standard chromatograms obtained, by calculating the % RSD of retention times, tailing factor, theoretical plates and peak areas from three replicate injections.

B. Accuracy

Assay was performed in triplicate for various concentrations of Montelukast and Fexofenadine equivalent to 50, 100, and 150 % of the standard amount was injected into the HPLC system per the test procedure. The percentage recovery and percentage relative standard deviation[% RSD] were taken into consideration for testing accuracy.

C. Precision

Repeatability (intra-day precision): 10 mg of Montelukast and 10mg of Fexofenadine, working standards were accurately weighed and transferred into a 100ml clean dry volumetric flask after adding about 70ml of diluent, it is  sonicated to dissolve it completely and volume was made up to the mark with the same solvent. Further 1.2ml of Montelukast and 3ml of Fexofenadine, of the solution A was pipetted into a 10ml volumetric flask and diluted up to the mark with diluent. The standard solution was injected for five times and the areas for all five injections were measured in HPLCIntermediate precision: The standard solution was injected for five times and the areas for all five injections were measured in HPLC. The % RSD for the area of six replicate injections was calculated for system precision and 20 ?L of sample solution was injected for six times and the peakarea of the resulting chromatogram was used for the calculation of standard deviation and relative standard deviation for method precision

D. Linearity

Pipette out specific volume of from stock solution to 100 ml with mobile phase. The concentration of the solution becomes 4-20?g of Montelukast and 10-50?g of Fexofenadine.

E. Specificity

For the simultaneous determination of Montelukast and Fexofenadine potassium, the specificity requires that the method should not be affected by the presence of other components. Solutions of mobile phase, sample solution, standard solution were injected into liquid chromatography. Retention times of samples and standard were compared.

F. LOD and LOQ

The detection and quantification limits for the Montelukast and Fexofenadine were performed and calculated using S/N ratio method.

G. Robustness

Table 2: Robustness conditions for Montelukast and Fexofenadine potassium

As per the USP-XXVI system suitability tests were carried out on freshly prepared standard stock solution of Montelukast and Fexofenadine. These parameters signify good sensitivity, more ruggedness and robustness of the method.

Selection of wavelength

The standard solution of and Montelukast and Fexofenadine were separately scanned at different concentration in the range of 200-400 nm and the ?max was determined. The overlain spectrum of both the drugs was also run as shown in (Figure 3). Hence the complete method was preceding with the wavelength 245 nm.

Figure 3: Overlay UV spectra of Montelukast and Fexofenadine

Analytical Method Development

Several trials were made to get good peak resolution, acceptable plate count and tailing factor. Method was optimized for the simultaneous estimation of Montelukast and Fexofenadine in bulk and Pharmaceutical dosage form.

Figure 4: Standard chromatogram for Montelukast and Fexofenadine

Figure 5: Sample Chromatogram for Montelukast and Fexofenadine

The retention times for Montelukast and Fexofenadine were found to be 3.624 and 5.178 respectively. Resolution between two analytes is good. No peak asymmetry was observed. No other impurity interference was seen. All the results were found to be within the acceptance criteria. Hence the method was considered to be optimized.

Method validation

A. System suitability studies 

Standard solution of Montelukast and Fexofenadine was determined under proposed condition chromatogram indicating satisfactory % RSD of peak responses, theoretical plates, and asymmetry and retention time. From the system suitability studies it was observed that% RSD of retention time was found to be 0.2, % RSD of peak area was found to be0.2. Theoretical plates were found to be more than 3500. USP tailing factor was found to be 1.48 for Montelukast and 1.52 for Fexofenadine.  All the parameters were within the limit. Results are summarized in (Table 3, 4)

Table 4: Chromatogram values for system suitability of Fexofenadine

B. Accuracy

The percentage recoveries of pure drug from the analyzed solution of formulation are calculated in the recovery range from 50% to 150%. The summary of accuracy results are tabulated in (Table 5, 6)

Sample No.	Spike Level	Amount(?g/ml) added	Amount(?g/ml) Found	% Recovery	Mean % Recovery
1	50%	5	4.96	99.2%	100.2%
		5	4.99	99.8%
		5	5.1	103%
2	100%	10	9.92	99.1%	99.3%
		10	9.93	99.3%
		10	9.99	99.6%
3	150%	15.3	15.2	98.5%	99.2%
		15.3	15.3	99.2%
		15.3	15.3	100%

Table 5: Percentage recovery results for Montelukast

Sample no	Spike Level	Amount(?g/ml) added	Amount(?g/ml) found	% Recovery	Mean % Recovery
1	50%	5	4.8	98%	100%
		5	5.1	101%
		5	5	100%
2	100%	10	9.88	98.7%	99.12%
		10	9.90	99%
		10	9.93	99.4%
3	150%	14.7	14.71	99.3%	99.68%
		14.7	14.77	99.8%
		14.7	14.78	99.78%

Table 6: Percentage Recovery results for Fexofenadine

The % recovery for 50%, 100% and 150% accuracy level of Montelukast and Fexofenadine was found to be within the range of 99.2-100.2% and 99.12-100% respectively (98.0 to 102.0%).

C. Precision 

1. Repeatability

The RSD of % Recovery for Montelukast and Fexofenadine chromatograms of repeatability precision and intermediate precision is calculated. The results of precision are summarized in (Table 7, 8)

Injection No	Peak area	% Recovery
1	3480635	99.3%
2	3463599	100%
3	3498778	99.0%
4	3498779	99.7%
5	3490275	99.1%
Mean	3486413	99.47%
SD	0.41	0.416
%RSD	0.43	0.42

Table 7: Sample chromatogram values for repeatability of Montelukast

Injection No	Peak Area	% Recovery
1	323862	99.1%
2	325248	99.7%
3	322052	99.1%
4	328132	99.35%
5	328654	100%
Mean	325589.6	99.51
SD	2802.2	0.32
%RSD	0.85	0.36

Table 8: Sample chromatogram values for repeatability of Fexofenadine

The % RSD for area of five standard injections of repeatability of Montelukast and Fexofenadine was found to be 0.42 and 0.36 respectively (NMT 2).

2. Intermediate precision 

Comparison of both the results obtained for two different analysts shows that the assay method was rugged for analyst-analyst variability.The results of intermediate precision (Ruggedness) were found to be within the limits and are tabulated in (Table 9-12)

Parameter	Peak Area(mV.?)	% Assay
Avg*	3486376	99.13%
SD	0.41	0.416
% RSD*	0.98	0.42

*Average of five determinations

Table 9: Intermediate precision results for Montelukast (Day-1, Analyst-1)

Parameter	Peak Area(mV.?)	% Assay
Avg*	326553.6	99.24%
SD	2802.2	0.32
%RSD	0.85	0.36

*Average of five determinations

Table 10: Intermediate precision results for Fexofenadine Day-1, Analyst-1)

Parameter	Peak Area(mV.?)	% Assay
Avg*	3488808	99.14%
SD	0.41	0.416
%RSD	0.98	0.43

*Average of five determinations

Table 11: Intermediate precision results for Montelukast (Day-2, Analyst-2)

Parameter	Peak area(mV.?)	%Assay
Avg*	326647	99.07%
SD	2802.2	0.32
%RSD	0.85	0.26

Table 12: Intermediate precision results for Fexofenadine (Day-2, Analyst-2)

The % RSD for the area of five standard injections for intermediate precision of Montelukast and Fexofenadine was found to be 0.42 and 0.36 for day-1, analyst-1and 0.43 and 0.26 for day-2, analyst-2 respectively (NMT 2).

3. Linearity

Linearity study was performed in the concentration range of 4-20 ?g / ml for Montelukast and 10-50 ?g/ml. For Fexofenadine, the Calibration curve for the linearity was shown in (Figure 6, 7).  The data of linearity is tabulated in (Table 13, 14)

Montelukast:

Figure 6: Calibration curve of Montelukast

Sr. no	Concentration(µg/ml)	Area
1	4	2011573
2	8	2681556
3	12	3390742
4	16	4161135
5	20	4964754
Correlation Coefficient		0.9986

Table 13: Linearity result for Montelukast

Fexofenadine:

Figure 7: Calibration curve of Fexofenadine

Sr. No	Concentration(µg/ml)	Area
1	10	189398
2	20	258338
3	30	321849
4	40	394695
5	50	459758
Correlation Coefficient		0.9997

Table 14: Linearity result for Fexofenadine

Correlation coefficient of Montelukast and Fexofenadine calibration curve was found to be 0.9986 and0.9997 respectively (NMT 0.999), which shows that Montelukast and Fexofenadine shows linearity forgiven range.

4. Specificity

There is no interference observed due to placebo and sample at the retention time of analyte which shows that the method was specific. The chromatograms for specificity studies (standard, sample, placebo and blank) are represented as (Figure 8-11).

Figure 8: Standard chromatogram for Montelukast and Fexofenadine 

Figure 9: Sample chromatogram for Montelukast and Fexofenadine

5. Limit Of Detection (LOD) & Limit Of Quantification (LOQ)

Limit of Detection (LOD)

Limit of Detection was found to be 0.003?g/ml for Montelukast and 0.09?g/ml for Fexofenadine. The chromatograms are shown in (Figure10,11).

For Montelukast

Figure 10: LOD chromatogram of Montelukast

Limit of detection was found to be 2.96 for Montelukast

For Fexofenadine

Figure 11: LOD chromatogram of Fexofenadine

Limit of detection was found to be 2.78 for Fexofenadine (NMT 3).

Limit of Quantification (LOQ)

The limit of quantification was calculated from the linearity curve method using slope, and standard deviation of intercepts of calibration curve. The chromatograms are shown in (Figure 12,13)

For Montelukast

Figure 12: LOQ Chromatogram of Montelukast

For Fexofenadine

Figure 13: LOQ Chromatogram of Fexofenadine

Limit of quantification was found to be 9.96 for Montelukast and 9.78 for fexofenadine (NMT 10).

6. Robustness

a) Effect of variation in flow rate

As the % RSD of retention time and asymmetry were within limits for variation in flow rate (± 0.1 ml). For this reason the allowable flow rate should be within 0.9 ml to 1.1 ml. The results of robustness for effect of variation in flow rate are tabulated in Table 15

Flow rate	RT of Montelukast	RT of Fexofenadine potassium
0.9 mL/ min	3.623	5.175
1.1 mL/ min	3.653	5.204

Table 15: Robustness value for change in flow rate

b) Effect of variation in mobile phase composition 

The mobile phase composition was varied as ± 10% organic phase and the results of robustness for effect of variation in mobile phase composition are tabulated in Table 16.

Sr. No	Change in organic composition in the mobile phase	RT of Montelukast	RT of Fexofenadine potassium
1	10% less	3.802	5.408
2	10% more	3.618	5.130

Table 16: Robustness values for change in mobile phase composition

A simple RP-HPLC method was developed and validated successfully for simultaneous estimation of Fexofenadine and Montelukast in tablet dosage form. The present study was validated as per the ICH guidelines and the method was found to be accurate, precise, linear, specific and reproducible for the simultaneous determination of Fexofenadine and Montelukast in formulation. From the comprehensive validation conducted, it was concluded that the method is stable andcould be used throughout shelf life of the drug. Hence this study can be extended by studying the degradation kinetics of Fexofenadine and Montelukast determination by RP-HPLC method and also its estimation in plasma and biological fluids.

Conflict of Interest

The author(s) confirms that this article content has no conflicts of interest.

Acknowledgement

The authors are highly thankful to the Principal Dr. V.K. Deshmukh for providing the necessary research facilities to carry out the research work.

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*Corresponding Author: Popat Mohite, Department of Pharmaceutical Quality Assurance and PG studies, MES’s College of Pharmacy, India. E-mail: mohitepb@rediffmail.com

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