Analytical Method Development and Validation for the Simultaneous Estimation of Bilastine and Montelukast by RP-HPLC

 

B. Sudhakar1*, Karipe Akshaya1, Ramya Sri. S2

1Department of Pharmaceutical Analysis, Samskruti College of Pharmacy,

Affiliated to JNTUH University, Hyderabad 501301, Telangana, India.

2Department of Pharmacy, University College of Technology,

Osmania University, Hyderabad, Telangana, 500007, India.

*Corresponding Author E-mail: sudhakarspkg@gmail.com

 

ABSTRACT:

A new, simple, rapid and precise reverse phase high performance liquid chromatographic method has been developed for the validation of Bilastine and Montelukast in its pure form as well as in combined marketed formulation. Chromatography was carried out on a Phenomenex Luna C18 (4.6mm×250mm) 5µm particle size column using a mixture of Methanol: Phosphate Buffer (pH-4.2) (37:63% v/v) as the mobile phase at a flow rate of 1.0ml/min, thedetection was carried out at 260nm. The retention time of the Bilastine and Montelukast was found to be was 2.133, 3.692±0.02 min respectively. The method was validated according to ICH guidelines for linearity, sensitivity, accuracy, precision, specificity and robustness. The method produce linear responses in the concentration range of 20-60mg/ml of Bilastine and 10-30mg/ml of Montelukast.The inter-day and intra-day precisions were found to be within limits. The method precision for the determination of assay was below 2.0% RSD. The method is useful in the quality control of bulk and pharmaceutical formulations.

 

KEYWORDS: Bilastine and Montelukast, RP-HPLC, Validation, Accuracy, Precision.

 

 

INTRODUCTION:

High Performance Liquid Chromatography (HPLC) is the fastest growing analytical technique for the analysis of drugs. Chromatographic separation in HPLC is the result of specific interaction between sample molecules with both the stationary and liquid mobile phases. HPLC has been rapidly developed with the introduction of new pumping methods, more reliable columns and wide range of detectors.1 In the era of developed and modified chromatographic techniques, the HPLC is still the simplest, most reliable, easy handling and worldwide used technique in the various stages of drug development2.

 

Bilastine or 2-[4-[2-[4-[1-(2-ethoxyethyl) benzimidazol-2-yl]piperidin-1-yl]ethyl]phenyl]-2-methylpropionic acid, is selective Histamine H1 receptor antagonist, leading to decreased nasal congestionand urticaria.3 The absorption of Bilastine is fast, linear and dose proportional; it appears to be safe and well tolerated at all doses levels in healthy population4. 

 

 

Fig. 1: Chemical structure of bilastine5

 

Bilastine is an antiallergenic agent, which helps alleviate allergic symptoms such as nasal inflammation and urticarial by combining and preventing H1 receptor activation. Bilastine has decreased the severity of allergic effects due to histamine release from mast cells 6.

Montelukast Sodium (1-[[[(1R)-1-[3-[(1E)-2-(7-chloro-2-quinolinyl) ethenyl] phenyl]-3-[2-(1-hydroxy-1-methylethyl) phenyl]-propyl] thio] methyl] cyclopropaneacetic acid, monosodium salt is a white colored powder and it is freely soluble in ethanol, methanol, and water7. Montelukast is a potent, selective and orally active leukotrines receptor antagonist that inhibits the cysteinyl leukotrines CysLT1 receptor used in the treatment of asthma8. The recommended dosage of MON is 10mg per day9.

 

 

Fig. 2: Chemical structure of bilastine10

 

MATERIALS AND METHODS:

Bilastine from Sura labs, Montelukast from Sura labs, Water and Methanol for HPLC from LICHROSOLV (MERCK), Acetonitrile for HPLC from Merck, Potassium Dihydrogen Phosphate from Merck.

Validation methods procedures followed as per ICH guidelines.

 

RESULTS AND DISCUSSION:

Optimized Chromatogram (Standard):

Mobile phase ratio: Methanol: Phosphate Buffer (pH-4.2) (37:63 v/v)

Column: Phenomenex Luna C18 (4.6mm×250mm) 5µm particle size

Column temperature: 35ŗC

Wavelength: 260nm

Flow rate: 1ml/min

Injection volume: 10µl

Run time: 6minutes

 

 

Figure 3-: Optimized Chromatogram (Standard)

 

Table 1-: Optimized Chromatogram (Standard)

S. No.

Name

RT

Area

Height

USP Tailing

USP Plate Count

Resolution

1

Bilastine

2.133

526389

86756

1.56

5679

 

2

Montelukast

3.692

1687285

367532

1.79

8685

9.8

 

Observation:

From the above chromatogram it was observed that the Bilastine and Montelukast peaks are well separated and they shows proper retention time, resolution, peak tail and plate count. So it’s optimized trial.

 

 

Optimized Chromatogram:

 

Figure 4-: Optimized Chromatogram (Sample)

Table 2-: Optimized Chromatogram (Sample)

S. No.

Name

Rt

Area

Height

USP Tailing

USP Plate Count

Resolution

1

Bilastine

2.166

536587

77464

1.57

5789

 

2

Montelukast

3.629

1695846

378564

1.80

8795

10.01

 

System Suitability:

Table 3-: Results of system suitability for Bilastine

S. No.

Peak Name

RT

Area (µV*sec)

Height (µV)

USP Plate Count

USP Tailing

1

Bilastine

2.152

526358

86598

5695

1.56

2

Bilastine

2.157

526548

86254

5652

1.57

3

Bilastine

2.141

526854

86598

5627

1.56

4

Bilastine

2.133

526598

86245

5692

1.57

5

Bilastine

2.166

524874

86521

5641

1.56

Mean

 

 

526246.4

 

 

 

Std. Dev.

 

 

787.353

 

 

 

% RSD

 

 

0.149617

 

 

 

Table 4-: Results of system suitability for Montelukast

S. No.

Peak Name

RT

Area (µV*sec)

Height (µV)

USP Plate Count

USP Tailing

Resolution

1

Montelukast

3.674

1682821

1686958

8659

1.56

9.8

2

Montelukast

3.631

1682726

1685745

8675

1.57

9.9

3

Montelukast

3.625

1687361

1685421

8692

1.56

9.8

4

Montelukast

3.692

1682811

1685242

8642

1.57

9.8

5

Montelukast

3.629

1683816

1685364

8635

1.58

9.8

Mean

 

 

1683907

 

 

 

 

Std. Dev.

 

 

1982.03

 

 

 

 

% RSD

 

 

0.117704

 

 

 

 

 

Assay (Standard):

Table 5-: Peak results for assay standard of Bilastine

S. No

Name

RT

Area

Height

USP Tailing

USP Plate Count

Injection

1

Bilastine

2.152

526358

86598

1.56

5698

1

2

Bilastine

2.198

526584

86784

1.57

5687

2

3

Bilastine

2.179

529658

86253

1.56

5639

3

 

Table 6-: Peak results for assay standard of Montelukast

S. No.

Name

RT

Area

Height

USP Tailing

USP Plate Count

Injection

1

Montelukast

3.646

1687589

365879

1.80

8659

1

2

Montelukast

3.604

1685987

365854

1.79

8697

2

3

Montelukast

3.610

1685974

369854

1.80

8675

3

 

Assay (Sample):

Table-7: Peak results for Assay sample of Bilastine

S. No

Name

RT

Area

Height

USP Tailing

USP Plate Count

Injection

1

Bilastine

2.152

536859

87584

1.58

5789

1

2

Bilastine

2.150

532654

87965

1.59

5784

2

3

Bilastine

2.187

532685

87465

1.58

5769

3

 

Table-8: Peak results for Assay sample of Montelukast

S. No

Name

RT

Area

Height

USP Tailing

USP Plate Count

Injection

1

Montelukast

3.646

1698568

378562

1.81

8759

1

2

Montelukast

3.651

1698574

375847

1.80

8795

2

3

Montelukast

3.601

1698547

376584

1.81

8745

3

 

% ASSAY =

  Sample area        Weight of standard     Dilution of sample     Purity      Weight of tablet

 ___________ ×   ________________ × _______________×_______×______________×100

  Standard area      Dilution of standard    Weight of sample       100          Label claim

 

= 99.89%

The % purity of Bilastine and Montelukast in pharmaceutical dosage form was found to be 99.89%

 

Linearity:

Chromatographic Data for Linearity Study of Bilastine:

 

Table 9: Chromatographic Data for Linearity Study of Bilastine

Concentration mg/ml

Average Peak Area

20

272897

30

402986

40

526389

50

649785

60

769287

 

 

Fig 5-: Calibration Curve of Bilastine

 

Chromatographic Data for Linearity Study of Montelukast:

 

Table 10-: Chromatographic Data for Linearity Study of Montelukast

Concentration mg/ml

Average Peak Area

10

1000237

15

1448768

20

1887285

25

2365897

30

2826845

 

 

Fig-6: Calibration Curve of Montelukast

 

Repeatability:

Table 11: Results of repeatability for Bilastine:

S. No.

Peak Name

Retention time

Area

(µV*sec)

Height

(µV)

USP Plate Count

USP  Tailing

 

1

Bilastine

2.157

526358

86598

5689

1.56

2

Bilastine

2.159

524856

86542

5687

1.57

3

Bilastine

2.186

526985

86578

5684

1.56

4

Bilastine

2.160

528654

86354

5689

1.56

5

Bilastine

2.170

528457

86958

5639

1.56

Mean

 

 

527062

 

 

 

Std.dev

 

 

1569.114

 

 

 

%RSD

 

 

0.297709

 

 

 

 

Table 12: Results of Repeatability for Montelukast:

S. No.

Peak Name

Retention time

Area (µV*sec)

Height (µV)

USP Plate Count

USP  Tailing

1

Montelukast

3.603

1687589

367859

8659

1.79

2

Montelukast

3.608

1685987

368547

8679

1.80

3

Montelukast

3.600

1685987

367985

8645

1.80

4

Montelukast

3.696

1685754

365874

8695

1.79

5

Montelukast

3.629

1685985

364589

8625

1.79

Mean

 

 

1686260

 

 

 

Std.Dev

 

 

749.493

 

 

 

%RSD

 

 

0.044447

 

 

 

 

Accuracy:

Table-13: The accuracy results for Bilastine

%Concentration

(at specification Level)

Area

Amount Added

(ppm)

Amount Found

(ppm)

% Recovery

Mean Recovery

50%

267011.3

20

20.063

100.315%

100.28%

100%

523752.3

40

40.118

100.295%

150%

778457.3

60

60.133

100.221%

                                                                               

Table-14: The accuracy results for Montelukast

%Concentration

(at specification Level)

Area

Amount Added

(ppm)

Amount Found

(ppm)

% Recovery

Mean Recovery

50%

972876.3

10

10.094

100.94%

100.48%

100%

1900122

20

19.998

99.99%

150%

2851152

30

30.156

100.52%

 

Limit of Detection:

The    detection  limit  of  an  individual  analytical  procedure  is  the  lowest  amount  of analyte in a sample which can be detected but not necessarily quantitated as an exact value.

 

LOD= 3.3 × σ / s

 

Where 

σ = Standard deviation of the response   

S = Slope of the calibration curve

 

Bilastine:

Result:

= 1.04µg/ml

 

Montelukast:

Result:

= 3.12µg/ml

Quantitation Limit:

The  quantitation  limit  of  an  individual  analytical  procedure  is  the  lowest  amount  of analyte  in  a  sample  which  can  be  quantitatively  determined. 

 

LOQ=10×σ/S

Where 

σ = Standard deviation of the response   

S = Slope of the calibration curve

 

Bilastine:

Result:

=2.1µg/ml

 

Montelukast:

Result:

=6.3µg/ml

Robustness

 

Table 15-: Results for Robustness

Bilastine

Parameter used for sample analysis

Peak Area

Retention Time

Theoretical plates

Tailing factor

Actual Flow rate of 1.0 mL/min

526389

2.133

5679

1.56

Less Flow rate of 0.9 mL/min

542685

2.210

5264

1.54

More Flow rate of 1.1 mL/min

526483

2.184

5426

1.52

Less organic phase

516854

2.200

5163

1.57

More Organic phase

506898

2.172

5098

1.51

 

Table 16-: Results for Robustness

MONTELUKAST

Parameter used for sample analysis

Peak Area

Retention Time

Theoretical plates

Tailing factor

Actual Flow rate of 1.0 mL/min

1687285

3.692

8685

1.79

Less Flow rate of 0.9 mL/min

1725468

4.498

8265

1.68

More Flow rate of 1.1 mL/min

1652847

3.505

8415

1.59

Less organic phase

1687485

4.504

8326

1.62

More organic phase

1674524

3.512

8415

1.63

 

CONCLUSION:

In the present investigation, a simple, sensitive, precise and accurate RP-HPLC method was developed for the quantitative estimation of Bilastine and Montelukast in bulk drug and pharmaceutical dosage forms.

 

This method was simple, since diluted samples are directly used without any preliminary chemical derivatisation or purification steps.

 

Bilastine was found to be freely soluble in chloroform, soluble in water and in glacial acetic acid, slightly soluble in ethanol and in acetonitrile and practically insoluble in ethyl acetate and in n-hexane. Montelukast was found to be soluble in organic solvents such as ethanol, DMSO, and dimethyl formamide, soluble in water.

 

Methanol: Phosphate Buffer (pH-4.2) (37:63 v/v) was chosen as the mobile phase. The solvent system used in this method was economical.

 

The % RSD values were within 2 and the method was found to be precise.

 

The results expressed in Tables for RP-HPLC method was promising. The RP-HPLC method is more sensitive, accurate and precise compared to the Spectrophotometric methods.

 

This method can be used for the routine determination of Bilastine and Montelukast in bulk drug and in Pharmaceutical dosage forms.

 

ACKNOWLEDGEMENT:

Thе Authors arе thankful to the Management and Principal, Department of Pharmacy, Samskruti College of Pharmacy, Hyderabad, for extending support to carry out the research work. Finally, the authors express their gratitude to the Sura Pharma Labs, Dilsukhnagar, Hyderabad, for providing research equipment and facilities.

 

REFERENCES:

1.      Govinda Rao Kamala, Sowjanya Vadrevu, Malipeddi Haripriya. Method Development and Validation for Simultaneous Estimation of Omeprazole and Domperidone by RP-HPLC. Asian J. Pharm. Ana. 5(4): 2015; 195-205. doi: 10.5958/2231-5675.2015.00031.9

2.      Hamid Khan, Mushir Ali, Alka Ahuja, Javed Ali. Validated HPLC-UV Method for Simultaneous Determination of Some Anti-Inflammatory and Analgesic Drugs. Asian J. Pharm. Ana. 2016; 6(3): 183-187.

3.      Ankita Shinde, G.B. Gajeli, Sneha Ubale, Vinod Matole. Simultaneous HPLC Method Development and Validation of Bilastine and Montelukast in Bulk and Formulation. Research Journal of Pharmacy and Technology. 2021; 14(11):6061-5. doi: 10.52711/0974-360X.2021.01053.

4.      Pardeshi P. P., Gaware V. M., Dhamak K. B. Development and Validation of RP-HPLC Method for the Estimation of Bilastine from bulk and Formulation. Asian J. Pharm. Ana. 2020; 10(2):109-111. doi: 10.5958/2231-5675.2020.00019.8

5.      https://en.wikipedia.org/wiki/Bilastine.

6.      A. M. Beltagi, I. A. Lashin, W. A. Essa, A. A. Hathoot, M. Abdel Azzem. Evolution and effectiveness of HPLC Technique for rapid estimation of an Antiallergenic agent Bilastine. Asian Journal of Pharmaceutical Analysis. 2021; 11(2):57-2. doi: 10.52711/2231-5675.2021.00011.

7.      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. 5(1): Jan.- March 2015; Page 26-30. doi: 10.5958/2231-5675.2015.00005.

8.      Dipti G. Phadtare, Amol R. Pawar, Rajashri R. Kulkarni, Govind K. Patil. Method development and validation of Montelukast sodium in bulk and tablet formulation by HPLC. Asian J. Research Chem. 2016; 9(7): 339-342. doi: 10.5958/0974-4150.2016.00051.1

9.      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. 6(10): October 2013; Page 1102-1106.

10.   https://en.wikipedia.org/wiki/ Montelukast

11.   Sįnchez MLF. Chromatographic techniques, European RTN Project, GLADNET, retrieved on 05-09-2013.

12.   Snyder LR, Kirkland JJ, Glach JL. Practical HPLC Method Development, John Wiley and Sons, New York, 1997; 158-192.

13.   McpolinOona.an Introduction to HPLC for Pharmaceutical Analysis. Mourne Training Service. 11-12.

14.   Charde MS, Welankiwar AS and Kumar J. Method development by liquid chromatography with validation. International Journal of Pharmaceutical Chemistry.2014; 4(2):57-61.

15.   Ranjit Singh. HPLC method development and validation. J Pharm Educ Res2013; 4(1): 26-33.

16.   Snyder LR, Kirkland JJ, Dolan JW. Introduction to modern liquid chromatography. John Wiley & Sons. New York. 2011.

17.   Xiang Y, Liu Y, Lee ML. Ultrahigh pressure liquid chromatography using elevated temperature. Journal of Chromatography. 2006; 1104(1): 198-202.

18.   International journal of novel trends in pharmaceutical sciences 2013; 3(1): 15-23.

19.   Lindholm J. Development and Validation of HPLC method for Analytical and Preparative Purpose. Acta Universities Upsaliensis Uppsala. 2004; 13-14.

20.   Snyder LR, Kirkland JJ, Glach JL. Practical HPLC Method Development, 2nd edition. New York. John Wiley &Sons. 1997; 233-291.

21.   Sethi PD. Introduction – High Performance Liquid Chromatography, 1st edn, CBS Publishers, New Delhi. 2001; 1-28.

22.   FDA Guidance for Industry (2000)-Analytical Procedures and Method Validation, Chemistry, Manufacturing, and Controls Documentation, Center for Drug Evaluation and Research (CDER) and Center for Biologics Evaluation and Research (CBER).

23.   Kayode J, Adebayo. Effective HPLC method development. Journal of Health, Medicine and Nursing.2015; 12: 123-133.

24.   Gad S. Pharmaceutical manufacturing handbook of regulations and quality. John wiley and sons; 2006.

25.   Webster P. Analytical procedures and method validation. Environmental protection agency; 2001.

26.   Weston A, Brown PR. HPLC and CE Principles and Practice. Academic press California; 1997.

27.   Shah RS, Pawar RB, Gayakar PP. An analytical method development of HPLC. International Journal of Institutional Pharmacy and Life Sciences. 2015; 5(5): 506-513.

28.   ICH Q2 (R1) Validation of Analytical Procedures: Text and Methodology. International Conference on Harmonization, IFPMA, Geneva; 2005.

29.   ICH Q2A. Text on Validation of Analytical Procedures, International Conference on Harmonization. Geneva; 1994.

30.   ICH Q2A. Text on Validation of Analytical Procedures, International Conference on Harmonization. Geneva; 1995.

31.   Sura, R. S., CVS, S., & rachamalla, S. S. (2022). Bioanalytical RP-HPLC Method Development And Validation Of Clopidogrel Bisulfate In Wistar Rat Plasma And Its Application To Pharmacokinetic Study.  International Journal of Applied Pharmaceutics, 14(1), 106–111. https://doi.org/10.22159/ijap.2022v14i1.43328

 

 

 

 

 

 

Received on 15.10.2022           Modified on 12.11.2022

Accepted on 01.12.2022   ©Asian Pharma Press All Right Reserved

Asian J. Res. Pharm. Sci. 2023; 13(2):79-84.

DOI: 10.52711/2231-5659.2023.00015