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RESEARCH ARTICLE

 

Analytical Method Development and Validation for Simultaneous Estimation of Montelukast and Ebastine by HPLC

 

Baokar Shrikrishna1,2*, Ranpise Nisharani3

1Shivnagar Vidya Prasarak Mandal’s College of Pharmacy, Malegaon (Bk.), Tal- Baramati, Dist- Pune, Maharashtra, India- 413115.

2Department of Pharmaceutical Sciences, Pacific Academy of Higher Education and Research (PAHER) University, Udaipur, Rajasthan, India- 313003

3Department of Pharmaceutical Sciences, Sinhgad College of Pharmacy, Vadgaon (Bk.), Pune, Off Sinhgad Road, Pune, Maharashtra, India- 411041.

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

 

ABSTRACT:

A RP-HPLC method was developed and validated for simultaneous estimation of montelukast and ebastine in bulk as well as in tablet formulation according to ICH guidelines.  The chromatographic separations of drugs were achieved on Younglin (S.K) isocratic system with Lichrocart C18 column (4.6 mm×250 mm, 5µm).  The mobile phase consisted by methanol and water (80: 20) at pH 3 adjusted by ortho phosphoric acid.  The flow rate was adjusted to 1 ml/min and UV detection was carried out at 268 nm. The retention time for montelukast and ebastine were found to be 6.56 and 2.95 min respectively.  The detector was showed linear responses over the concentration range 5 – 25 µg/mL for both drugs by showing a good correlation coefficient of 0.999.   This proposed method is highly sensitive, precise and accurate which reduces cost of analysis; hence recommended for routine quality analysis in laboratories.

 

KEYWORDS: Montelukast; ebastine; validation; simultaneous estimation.

 

 


INTRODUCTION

Montelukast (MTS) is 1-[[[(1R)-1-[3-[(1E)-2-(7-Chloro-2-quinolinyl) ethenyl] phenyl]-3-[2-(1-hyroxy-1-methyl-ethyl) phenyl] propyl] thio] methyl] cyclopropaneacetic acid. 

 

Fig 1 Montelukast Sodium1

 

MTS is freely soluble in ethanol, methanol, water and practically insoluble in acetonitrile.  MTL is a selective, potent and orally active antagonist of the cysteinyl, CysTL1, leukotriene receptor used for the treatment of asthma in children and adults2-4. 

 

 

 

 

Received on 13.09.2014       Modified on 21.10.2014

Accepted on 09.12.2014      © RJPT All right reserved

Research J. Pharm. and Tech. 8(1): Jan. 2015; Page 01-05

DOI: 10.5958/0974-360X.2015.00001.3

Montelukast blocks the action of leukotriene D4 on the cysteinyl leukotriene receptor CysLT1 in the lungs and bronchial tubes by binding to it.  This reduces the bronchoconstriction otherwise caused by the leukotriene and results in less inflammation.  Because of its method of operation, it is not useful for the treatment of acute asthma attacks. Again because of its very specific locus of operation, it does not interact with other allergy medications such as theophylline. Literature survey reveals several methods for MTS such as analytical method development and validation by RP-HPLC5-6,  stability indicating HPLC, HPLC coupled with ESI-MS/MS7, capillary electrophoresis8, derivative spectroscopy9, HPLC simultaneous estimation with other drugs10-11, TLC-Densitometry12, HPLC and HPTLC13-14, liquid-liquid extraction method by using HPLC with fluorescence detector15, derivative  spectrophotometry16. Ebastine (EBS) is 1-[4-(1,1-Dimethylethyl) phenyl]-4-[4-(diphenylmethoxy) piperidin-1-yl] butan-1-one.

Fig. 2 Ebastine

EBS is very soluble in methylene chloride and sparingly soluble in methanol. It is used in antihistaminic treatment17. Ebastine, a piperidine derivative, is a long-acting, non-sedating, second-generation histamine receptor antagonist that binds preferentially to peripheral H1 receptors. It has antihistaminic, antiallergic activity and prevents histamine-induced broncho-constriction.  It does not have significant sedative or antimuscarinic actions. Literature survey for EBS revealed several methods such as, individual determination of EBS by HPLC18-19, stability indicating  LC Method20, HPTLC21, spectrofluorimetry22, HPLC simultaneous estimation with other drugs23, simultaneous spectrophotometric estimation24, spectrophotometric absorption ratio method25, HPLC-DAD26. Literature survey reveals several methods for simultaneous determination of MTS and EBS by HPLC27-30  but this present work describes new and kinetic validated RP- HPLC method with different retention time.

 

EXPERIMENTATION:

Instrumentation:

The analysis was performed by using Younglin (S.K) isocratic system consisting UV detection at 344 nm for MTS and 252 nm for EBS. The separation was achieved on a C18 Lichrocart column (4.6mm × 250mm, 5µm).

 

MATERIALS:

Reference standards for MTS and EBS were gifted by Micro Labs Pvt. Limited, Pondicherry.  Pharmaceutical preparation for combination of MTS and EBS (Ebast-M) was obtained from local market.  Methanol of analytical reagent grade and HPLC grade, ortho phosphoric acid, water of HPLC grade were purchased from Merck Ltd., India.

 

Preparation of mobile phase:

The mobile phase consisting methanol: water (80:20; v/v) and pH adjusted to 3 by ortho phosphoric acid. The flow rate was adjusted to 1 ml/min.

 

Preparation of standard stock solutions:

Weighed accurately 10 mg of MTS and EBS and transferred to separate volumetric flask of 10 ml. Sufficient amount of mobile phase was added and drugs were dissolved to give a stock solution of 1000 µg/mL each.

 

Preparation of test solution:

Weighed accurately powdered tablet equivalent to 10 mg of MTS and EBS, transferred to volumetric flask, mixed it well with 100 ml of mobile phase to prepare 100µg/mL.  From above solution 1.5 ml was taken and diluted to 10 ml with mobile phase to get a solution containing 15µg/mL of MTS and EBS each.  The amounts of MTS and EBS per tablet were calculated by extrapolating the value of area from the calibration curve by using UV detection at isoabsorptive point of 268 nm (fig. 3). Procedure was repeated six times with the same tablet formulation. And obtained results are tabulated in Table 1.

 

Fig.3 Overlaying spectra of MTS and EBS

 

Table 1 Assay of MTS and EBS

Sr. No.

Amount present (mg)

Amount found in mg

% Label claim

MTS

EBS

MTS

EBS

MTS

EBS

1

10

10

9.906

9.902

99.10

99.03

SD

 

1.235

0.894

% RSD

 

1.246

0.903

*All the results were average of 6 readings, (n=6)

 

Calibration:

MTS and EBS were showed linear responses in the range  5-25 µg/mL at their respective maxima, which were validated by least square method. For simultaneous estimation of MTS and EBS, a series of standard solution were prepared by diluting appropriate volume. The scanning of MTS and EBS were carried out in the range of 200 – 400 nm against selected mobile phase. MTS and EBS showed maximum absorbance at 344 nm and 252 nm respectively.

 

System Suitability:

According to United States Pharmacopoeia, system suitability tests were integral part of LC method in the course of optimizing the conditions of proposed method.  The system suitability test solutions were injected and chromatographic parameters for MTS and EBS were evaluated for proving the system suitability. (Table 2)

 

Table 2 System Suitability study

System Suitability Parameters

MTS

EBS

Retention Time (Min)

6.58

2.98

Theoretical Plates

5302.5

3545.4

Area Under Curve

659.13

690.34

Tailing Factor

0.96

1.16

 

 

Resolution of MTS and EBS:

Standard solution of MTS and EBS were injected to get a chromatogram.  The retention time of MTS and EBS were found to be 6.56 and 2.95 min respectively.  Chromatogram of MTS and EBS is shown in Fig 4.

 

Fig 4 HPLC chromatogram of MTS and EBS

 

RESULTS:

VALIDATION31-37:

Validation of analytical method is a process to establish performance characteristics of developed method which meets the requirement with intended analytical applications. This method was validated according to ICH guideline for linearity, precision, accuracy, robustness, LOD and LOQ.

 

Linearity:

The linearity of an analytical method is the ability to obtain test results which are directly proportional to concentration in sample.This was studied by analyzing five different concentrations in selected range of 5 – 25 µg/mL for MTS and EBS. The analytical data for Linearity is tabulated in Table 3.

 

Precision:

The precision of an analytical method expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.   Precision was determined by repeatability, inter-day and intra-day experiments.  Standard solution containing MTS and EBS were injected six times. The mean amount and standard deviation (SD) value of each constitute were calculated and mentioned in Table 3.

 

Accuracy:

The accuracy of an analytical method is the closeness of the test result obtained by that method to true value. The accuracy was calculated from the test results as the percentage of analyte recovered by the assay. These studies were performed by standard addition method at 80 %, 100 % and 120 % levels as stated in ICH Guideline. Results are tabulated in Table 4.

 

Robustness:

The robustness is the capacity of method to remain unaffected by small but deliberate changes in chromatographic conditions. Robustness was studied by testing the influence of small changes in column temperature (±5°C), change in flow rate (±10 %) and changes in mobile phase composition (75:25). The obtained results are mentioned in Table 5.

 

LOD and LOQ:

LOD and LOQ for the optimized method were performed as per ICH guidelines. LOD was expressed by establishing the minimum level at which the analyte can be reliably detected. LOQ was considered as the lowest concentration of analytes in standards that can be reproducibly measured with acceptable accuracy and precision. LOD and LOQ) were separately determined at a signal to noise ratio (S: N) of 3: 10 and which was based on calibration curve. The standard deviation of y intercept and slope of the regression line were used. The LOD and LOQ were calculated by using following formulas and results are mentioned in Table 6.

 

LOD = 3.3 × D / S

LOQ = 10 × D / S

 

Where,

S = Slope of regression line, D = Standard deviation of y- intercept on the regression line.

 

Table 3 Validated analytical parameters

Parameters

MTS

EBS

Linearity Range

5 – 25 µg/mL

5 – 25 µg/mL

Intercept

-0.30

-1.53

Slope

0.025

0.024

Corre. Coef.

0.999

0.999

Inter day Preicision

(%RSD, n=3)

0.250

0.403

Intraday Preicision

(%RSD, n=3)

0.522

0.479

 

Table 4 Recovery Studies of MTS and EBS

Level of Recovery

Drug

% Recovery

Standard

Deviation*

%   RSD

80 %

MTS

99.25

0.395

0.397

EBS

100.33

0.497

0.495

100 %

MTS

99.9

0.507

0.508

EBS

99.76

0.475

0.476

120 %

MTS

99.91

0.409

0.409

EBS

100.14

0.395

0.394

*Denotes average of three determinations.

 

Table 5 Robustness Studies of MTS and EBS

Factor

Retention Time

Factor

Retention Time

Flow Rate

(mL/min)

MTS

EBS

Mobile Phase (v/v)

MTS

EBS

0.9

6.63

2.96

79:21

6.66

2.96

1.0

6.70

2.95

80:20

6.60

2.95

1.1

6.63

2.96

81:29

6.56

2.91

Mean

6.65

2.95

Mean

6.60

2.94

S.D.

0.0404

0.0057

S.D.

0.0503

0.0264

%RSD

0.6074

0.1952

%RSD

0.7618

0.8999

 

 

 

Table No. 6 Results of LOD and LOQ

Sample

LOD (µg/mL)

LOQ (µg/mL)

MTS

1.05

3.20

EBS

1.13

3.44

 

 

DISCUSSION:

In literature various methods are discussed for MTS and EBS with each other or with another drug combination separately. Sony LK. et.al. (2012)26 reported simultaneous estimation of ebastin with phenylephrine hydrochloride by UV absorption method whereas RIM and Tarek (2012)27 in his study reported HPLC and UV method for MTS and EBS but this showed more retention time. Anand J et.al (2013)28, Jangid RK et.al. (2013)29, Savsani JJ et.al (2012)30 and Ambadkar SG et.al. (2013)31 were reported separately HPLC methods for simultaneous estimation of MTS and EBS showing linearity range at 5-35 µg/mL, 1.2 ml/min flow rate and etc. this indicating scope to minimize total cost of analysis.

 

CONCLUSION:

Proposed study describes HPLC method for the simultaneous estimation of MTS and EBS in bulk as well as in tablet formulation. This method showed good separation of two compounds with less retention time than any method listed in reference. This method is useful to minimize total cost of analysis. The method is validated and found to be simple, sensitive, accurate, precise and robust.  Hence proposed method is suggested for routine quality analysis of MTS and EBS in laboratories.

 

ACKNOWLEDGEMENT:

The authors are wish to thank Principal and Management of Shivnagar Vidya Prasarak Mandal’s College of Pharmacy, Malegaon (Bk), Tal- Baramati, Dist-Pune, Department of Pharmaceutical Sciences, Pacific Academy of Higher Education and Research (PAHER) University, Udaipur, Rajasthan and Scan Research Bioanalytical Laboratories, Bhopal, for providing us required lab facilities with enthusiastic environment.

 

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