INTRODUCTION:

Allergy-induced nasal passage irritation and infection is referred to as allergic rhinitis. It happens when an allergen triggers an excessive immunological response in the body, which include dust, insects, and chemical particles. body's reaction, causing symptoms such as watery, itchy eyes, sneezing, rhinorrhea, and congestion in the nose and sinuses. Numerous drugs and pharmacological combinations are used to treat allergy infections.¹

Fexofenadine hydrochloride (FEX) (figure1) (±)-4-[1-hydroxy-4-(4hydroxydiphenylmethyl) -1-piperidinyl]-butyl]-∝, ∝-dimethyl benzene acetic acid hydrochloride relieves the symptoms of seasonal allergic rhinitis, often known as hay fever, which include runny nose, sneezing, red, watery, itchy eyes, as well as adult-onset itching of the nose, throat and upper side of the mouth. Terfenadine is a carboxylic acid metabolite of Fexofenadine also nonsedating selective histamine H1 receptor antagonist. This drug, which is supplied therapeutically or used as a P-glycoprotein probe in the form of a combination of R- and S-enantiomers, has an asymmetric carbon in its chemical structure.² It is almost insoluble in hexane and easily soluble in methanol and ethanol. It is also slightly soluble in water and chloroform. The empirical formula is C₃₂H₃₉NO₄.HCl and the molecular weight is 538.13. Recent research has shown that simultaneously administering an anti-leukotriene and an antihistamine to treat allergic rhinitis results in much improved symptom relief as compared to a slight improvement in symptoms of rhinitis with only the individual treatments.³

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. By binding to the cysteinyl leukotriene receptor CysLT1, it treats asthma. Leukotriene D4 (together with secondary ligands LTC4 and LTE4) prevents CysLT1 from acting in the lungs and bronchial tubes.^4-5 The Montelukast chemical structure is depicted in figure 2.

There are many methods which identifies and quantifies the fexofenadine and montelukast in single or in combined dosage form. There are literatures available to determine the amount of montelukast and fexofenadine hydrochloride in individual and in combined form by RP-HPLC^6-8, UV spectrophotometry^9-10, HPTLC^11-12, in biological fluid by RP-HPLC^13-14, LC/MS¹⁵, LC/MS/MS¹⁶, stability indicating method^17-18and UPLC^19.

Figure 1. Structure of Fexofenadine

Figure 2. Structure of Montelukast

MATERIAL AND METHODS:

A gift sample of Pure fexofenadine and montelukast was received from Swapnroop Drugs and Pharmaceuticals, Chha. Sambhajinagar, India. Methanol and acetic acid of HPLC grade were obtained from Omkar Traders Private Limited, Mumbai. The combined dose formulation MONTIFLEX (MITS Life Sciences Pvt Ltd) was acquired from the market. All HPLC grade chemicals and reagents are used during study.

Instrumentation:

Thechromatographic analysis was performed using an HPLC binary gradient system (AGILENT 1100 series) with a reciprocating pump (1100). In the chromatographic study, the stationary phase was Agilent RP C-18 (5μm; 4.6 x 250 mm ID) column and a combination of A methanol-0.05% formic acid was used as the mobile phase. The data was processed using CHEMSTATION software. A DAD detector was used for the detection of wavelength, which occurred at 248nm. A digital balance for measuring and a Labman ultrasonicator for dissolving any remaining drug particles were used.

Working wavelength determination:

By using the isobestic point, the wavelength for the further study was determined. Initially, the absorbance of both drugs was measured separately. Then, by overlaying the graph, the absorbance point where the absorbance of both drugs was the same was identified. From the isobestic point, a wavelength of 248 nm was selected for further study. Graph with overlaps showed in Figure 3.

Figure 3. Overlapping Spectrum of FEX and MON

Preparation of Buffer Solution:

Combine 0.1ml of formic acid and 100ml of HPLC-grade water to make a 0.1% formic acid buffer. A 3.5 PH was adjusted.

Standard Stock Solution Preparation:

Weighing of Fexofenadine 120mg and Montelukast 10 mg was done precisely. Measured amount was dissolved in 50ml methanol to get 200µg/ml Montelukast and 2400µg/ml Fexofenadine. Drug particles that remained after three to five minutes of sonication were removed by filtration. This solution was used to study the validation parameters.

Sample Solution Preparation:

Twenty tablets were weighed precisely to determine their average weight. Crush the tablet into powder form, then weigh and transfer the amount equal to 120mg of FEX and 10mg of MON into a 50ml methanol-filled volumetric flask. After sonicating the sample for fifteen minutes, let it cool to ambient temperature.

Chromatographic Condition:

The suitable chromatographic condition is shown in Table 1. An Agilent RP C-18 (5μm; 4.6 x 250mm ID) column equipped with DAD detector were used. Mobile phase consists of Methanol: 0.1% Formic Acid (pH 3.5) in a ratio 40:60% v/v at a flow rate of 0.8ml/min. We used a wavelength of 248nm for detection.

Table 1. Optimized chromatographic conditions

Parameters	Chromatographic Condition
Mode of elution	Isocratic
Mobile Phase	Methanol: 0.1% Formic Acid (pH 3.5) 40:60 % v/v.
Column	Agilent RP C-18 (5μm; 4.6 x 250mm ID
Flow Rate	0.8ml/min
Runtime	10min
Injection Volume	20µL
Detection Wavelength	248nm
Temperature	28⁰C
Retention Time	4.523 for FEX and 6.380 for MON

RESULT AND DISCUSSION:

Linearity:

The method is said to be linear if the test results from tests are exactly proportionate to the analyte concentration in the sample. An injection of six distinct concentrations was injected. and a graph was plotted to assess the method's linearity as shown in figures 4 and figure 5. Linearity was found in the range of 24-120 µg/ml for FEX and 2-10µg/ml for MON with a correlation coefficient of 0.999.

Table 2: Linearity values of Fexofenadine and Montelukast

Parameters	Fexofenadine	Montelukast
Linearity range (µg/mL)	24-120	2-10
Regression coefficient ± SD	0.999± 5.11	0.999±0.83
Slope ± SD	78.717 ±5.11	166.93±0.83
Intercept ± SD	282.61± 5.11	19.305±0.83

Figure 4. Calibration curve for Fexofenadine.

Figure 5. Calibration curve for Montelukast.

Intraday Precision and Interday Precision:

The variation in findings from several studies of the same samples conducted in a single day is measured by intraday precision. Three duplicates of the sample solutions peak areas were measured the same day of injection. The three duplicate injection results peak regions as a percentage of RSD shouldn't be greater than 2. In terms of interday precision, percentage RSD and results variability measurements from many analyses of the same samples on several days were computed. Table 3 presents the evaluated results.

Robustness:

Robustness was examined by implementing slight modifications to the chromatographic conditions. Minor adjustments were made to mobile phase ratio (39:61 and 41:59), flow rate (0.8ml/min ± 0.1ml/min) and detection wavelength (248nm±1nm). The %RSD was calculated.Variation in a flow rate, pH, wavelength and mobile phase were specifically evaluated, calculated and summarized in table 4.

Table 4: Robustness values of Fexofenadine and Montelukast.

Sr. No	Chromatographic Condition	Changes	% RSD of FEX	% RSD of MON
1.	Flow (-0.1ml/min)	0.7	0.08	0.16
2.	Flow (+0.1ml/min)	0.9	0.11	0.36
3.	M.P (-0.1ml/min)	39+61	0.11	0.10
4.	M.P (+0.1ml/min)	41+59	0.08	0.24
5.	Wavelength (-1)	247	0.09	0.24
6.	Wavelength (+1)	249	0.04	0.16

LOD and LOQ:

The minimum quantity which is detectable in a sample from background noise but not quantitated is the LOD (limit of detection). The smallestamount of substance in a sample that can be precisely and accurately quantified is the LOQ (limit of quantitation). Three times the noise level was used to determine LOD, and ten times the noise level was used to calculate LOQ.

The table no 5 depicted the results of LOD and LOQ.

LOD = 3.3 σ / S.

LOQ = 10 σ / S.

where σ = Standard Deviation; S = Slope.

Table 5. LOD and LOQ Values of FEX and MON

Sample	LOD	LOQ
Fexofenadine	0.2142231	0.6491609
Montelukast	0.0164081	0.0497214

Accuracy:

Accuracy is defined as the closeness of agreement between a measured value and a true or accepted value. It assessed by comparing the measured values obtained using the method to reference or true values. The method’s accuracy was assessed through recovery studies conducted at three different levels of concentration (80%, 100%, and 120%). To perform the recovery studies, a specific concentration of the standard drug (80%, 100%, and 120%) was added to previously analysed sample solutions. Recovery of Fexofenadine evaluated in the range of 99.48 to 102.63 and Montelukast in the range of 100.13% to 100.7%. Results are showed in table 6 from which process is said to be accurate.

Table 6: Recovery values of Fexofenadine and Montelukast.

Drug	Level	Analyte amount (mg)	Recovery amount (mg)	Mean % recovery	RSD %
Fexofenadine	80%	19.2	19.13	99.65	0.24
	100%	24	23.91	99.64	0.10
	120%	28	28.76	102.70	0.10
Montelukast	80%	1.6	1.60	100.13	0.47
	100%	2	2.02	100.75	0.37
	120%	2.4	2.42	100.70	0.42

System Suitability Studies:

The test for system suitability was conducted by injecting a standard solution of FEX and MON six times into the system. Different parameters, including resolution, tailing factor and theoretical plates were assessed to ensure the reproducibility of the chromatographic system. All the results were found to be within specifications proving the suitability of the method. Table 7 shows results for system suitability.

Table 7: Summary of system suitability parameters.

Parameters	Fexofenadine	Montelukast	Acceptance criteria
Tailing factor	0.79	0.74	≤2
Retention time	4.523	6.380	≥2
Theoretical plates	4699	6459	≥2000

Ruggedness:

By conducting the experiment with various instruments, operators, and comparable column types, the robustness of the method was ascertained. Table 8 shows the Ruggednessvalues of Fexofenadine and Montelukast.

Table 8. Ruggedness values of Fexofenadine and Montelukast

Drug

Analyst-1

(Peak area)

Analyst-2 (Peak area)

RSD %

Fexofenadine

7937.348

7932.568

3.380

0.043

Montelukast

1375.863

1378.635

1.960

0.142

SUMMARY AND CONCLUSION:

According to ICH guidelines an easy-to-use, precise, and accurate RP-HPLC technique was developed and validated for FEX and MON in both pure and pharmaceutical dose formulations. The %RSD for validation parameters was found to be less than 2, suggests that method is appropriate for identifying and quantifyingthe amount of FEX and MON. This method considered more cost effective as compared to other reported method. It is suitable for the tablet analysis, making it a viable option for routine analysis in quality control of Fexofenadine and Montelukast in the pharmaceutical marketed formulations.

ABBREVIATIONS:

FEX- Fexofenadine; MON- Montelukast; RP-HPLC- Reverse Phase High Performance Liquid Chromatography; %RSD- Relative Standard Deviation; SD – Standard Deviation.

ACKNOWLEDGEMENT:

The author thanks to Swapnroop Drugs and Pharmaceuticals, Aurangabad, Maharashtra, India for providing gift samples of Fexofenadine and Montelukast respectively.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

REFERANCES:

1. Sreedevi Adikay, Mounika Bhavanasi. A Stability Indicating RP-HPLC Method for Simultaneous Estimation of Acebrophylline, Montelukast, and Fexofenadine in Bulk and Pharmaceutical Dosage Forms, Int. J. Pharm. Investigation. 2023; 13(2): 306-312. DOI: 10.5530/ijpi.13.2.040.

2. Hitesh Vekaria, Vipul Limbasiya, Piyush Patel, Development and validation of RP-HPLC method for simultaneous estimation of Montelukast Sodium and Fexofenadine hydrochloride in combined dosage form, Journal of Pharmacy Research. 2013; 6(1): 134-139. ISSN 0974-6943, https://doi.org/10.1016/j.jopr.2012.11.028.

3. Sevgi Karakuş, İlkayKüçükgüzel, Ş.GünizKüçükgüzel, Development and validation of a rapid RP-HPLC method for the determination of cetirizine or fexofenadine with pseudoephedrine in binary pharmaceutical dosage forms. Journal of Pharmaceutical and Biomedical Analysis. 2008; 46(20: 295-302, https://doi.org/10.1016/j.jpba.2007.10.018.

4. Injamamul Haque, Ravikumar, Narayanaswamy VB, Mohibul Hoque. Formulation and Evaluation of Montelukast Sodium Fast Dissolving Tablets. Asian J. Pharm. Res. 2016; 6(3): 159-169.

5. Nishant Sarode, G. S. Chhabra, Shailesh Luhar, Anil Jadhav. Development and Validation of RP-HPLC Method for the Estimation of Montelukast Sodium in Bulk and In Tablet Dosage Form. Research J. Science and Tech. 2011; 3(5): 257-260.

6. Rituraj Singh Chundawat, Y.S. Sarangdevot, R.P.S. Rathore, Dharmendra Singh Sisodiya, Udaibhan Singh Rathore. Method Development and Validation for Simultaneous Estimation of Montelukast and Fexofenadine in Pharmaceutical Dosage Form by HPLC Method. Research J. Pharm. and Tech. 2013; 6(10): 1102-1106.

7. Baokar Shrikrishna, Ranpise Nisharani. Analytical Method Development and Validation for Simultaneous Estimation of Montelukast and Ebastine by HPLC. Research J. Pharm. and Tech. 2015; 8(1): 1- 5.

8. Ramesh J, Jayalakshmi B, Vijayamirtharaj R, Arul Prakasam K. C. Simultaneous Estimation of Montelukast Sodium and Levocetrizine Hydrochloride by RP-HPLC Method. Asian J. Research Chem. 2010; 3(4): 1069-1072.

9. Anuruddha R Chabukswar, Vishnu P. Choudhari, Shailesh N. Sharma, Nilesh A. Bari, Dhaval Ghuge. Ratio derivative spectrophotometry method for simultaneous estimation of Montelukast and Fexofenadine HCl in their combined dosage form. Asian J. Research Chem. 2012; 5(5): 637-641.

10. Md. Jakaria, Md. Hazrat Ali, Md. Areeful Haque, Mohammed Abu Sayeed, Shoayeb Ahmed. In vitro Comparative Forced Degradation Study of Different Brands and Active form of Montelukast sodium using UV Spectrophotometer. Asian J. Pharm. Ana. 2015; 5(1): 26-30.

11. Tamilselvi N, Sruthi K, Arivukkarasu R, Vanathi P, Deepthi Visakh. Development of Validated HPTLC Method for Simultaneous Estimation of Fexofenadine Hydrochloride and Montelukast Sodium in Tablet Dosage form. Research J. Pharm. and Tech. 2016; 9(4): 469-473. doi: 10.5958/0974-360X.2016.00087.1

12. S. N. Meyyanathan, P. A. Shirsode, and B. Suresh. Analysis of fexofenadine in pharmaceutical preparations by high performance thin layer chromatography, Indian Drugs. 2005; 42(4): 248–250.

13. M. Miura, T. Uno, T. Tateishi, and T. Suzuki. Determination of fexofenadine enantiomers in human plasma with high performance liquid chromatography. Journal of Pharmaceutical and Biomedical Analysis. 2007; 43(2): 741–745.

14. T. Uno, N. Yasui-Furukori, T. Takahata, K. Sugawara, and T. Tateishi. Liquid chromatographic determination of fexofenadine in human plasma with fluorescence detection. Journal of Pharmaceutical and Biomedical Analysis. 2004; 35(4): 937–942.

15. B. Sudhakar, Karipe Akshaya, Ramya Sri. S. Analytical Method Development and Validation for the Simultaneous Estimation of Bilastine and Montelukast by RP-HPLC. Asian Journal of Research in Pharmaceutical Sciences. 2023; 13(2): 79-4.

16. M. Gergov, J. N. Robson, I. Ojanpera, O. P. Heinonen, and E. ¨ Vuori. Simultaneous screening and quantitation of 18 antihistamine drugs in blood by liquid chromatography ionspray tandem mass spectrometry. Forensic Science International. 2001; 121(1-2): 108–115.

17. I. Fu, E. J. Woolf, and B. K. Matuszewski. Determination of fexofenadine in human plasma using 96-well solid phase extraction and HPLC with tandem mass spectrometric detection. Journal of Pharmaceutical and Biomedical Analysis. 2004; 35(4): 837–846.

18. Kakad SB, Kolhe MH, Dukre TP. A Review on Pharmaceutical Validation. International Journal of Pharmaceutical Quality Assurance. 2020; 11(3): 338-342.

19. Mohamed Mustafa, S. Amuthalakshmi, C.N. Nalini. Simultaneous UPLC Estimation of Fexofenadine HCl and Montelukast Sodium Tablets. Research J. Pharm. and Tech. 2017; 10(2): 557-561.

Received on 01.04.2024 Revised on 14.11.2024

Accepted on 05.05.2025 Published on 02.08.2025

Available online from August 08, 2025

Research J. Pharmacy and Technology. 2025;18(8):3468-3472.

DOI: 10.52711/0974-360X.2025.00499

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Concentration (µg/ml)	Fexofenadine		Concentration (µg/ml)	Montelukast
Concentration (µg/ml)	% RSD		Concentration (µg/ml)	% RSD
	Intraday	Interday		Intraday	Interday
48	0.03	0.03	4	0.03	0.22
72	0.06	0.07	6	0.10	0.17
96	0.00	0.02	8	0.01	0.06