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ejbps, 2015, Volume 2, Issue 4,295-305.

SJIF Impact Factor 2.062

2349-8870 European Journal Biomedical European Journal of of Biomedical and Pharmaceutical ISSN Sciences Volume: 2 AND Issue: 3 295-305 Pharmaceutical sciences

Alagar et al.

Year: 2015

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RP-HPLC ESTIMATION OF MONTELUKAST SODIUM AND OLOPATADINE HYDRO CHLORIDE IN PHARMACEUTICAL TABLET DOSAGE FORM Alagar Raja.M1*, Saikumar.CH1.Rao.KNV1, DavidBanji1, SelvaKumar.D2 1

Department of Pharmaceutical Analysis & Quality Assurance, Nalanda College of Pharmacy, Nalgonda, Telangana State-508001, India. 2

School of Pharmacy, Taylors University, Subangjaya, Malaysia.

Article Received on 10/05/2015

Article Revised on 07/06/2015

Article Accepted on 01/07/2015

ABSTRACT

*Correspondence for Author

A simple, economic, accurate and precise HPLC method was

Dr. Alagar Raja. M

successfully

Department of

development, the estimation was carried out by using the Hypersil C18

Pharmaceutical Analysis &

column (4.6 X 150 mm) with 5µm particle size. Injection volume of

Quality Assurance, Nalanda

20μl is injected and eluted with the mobile phase Acetonitrile: 0.02M

College of Pharmacy,

developed.

In

simultaneous

RP-HPLC

method

Nalgonda, Telangana State-

potassium dihydrogen phosphate 0.1% triethylamine) in the ratio of 60:

508001, India.

40 and the buffer pH is maintained at 5.0 and adjusted with 0.01M Phosphoric acid, which is pumped at a flow rate of 1.0 ml/min.

Detection was carried out at 270 nm. The peaks obtained were sharp with retention time of 3.133 min Olopatadine Hydro Chloride for 5.137min for Montelukast Sodium. The peaks were well resolved with a resolution factor of 5.03. The method was precisely applied to the tablet formulation and the results obtained were accurate and reproducible. System suitability studies were also carried out which includes theoretical plates, resolution factor and tailing factor etc. And validation study compared as per ICH guideline. KEYWORDS: HPLC, Montelukast sodium, Olopatadine Hydro Chloride. INTRODUCTION Montelukastsodium[1] ischemically1- [[[(1R)-1- [3-(1E)-2- (7-chloro-2quinolinyl)ethenyl] phenyl] -3-[2-(1-hydroxy-1.-methylethyl)phenyl]propyl]thio]-methyl] cyclopropaneacetiacid, monosodium www.ejbps.com

salt Selectively

antagonist

leukotriene

D4(LTD4),at

the cysteinyl 295

Alagar et al.

European Journal of Biomedical and Pharmaceutical Sciences

leukotriene receptor, cysLT1 ,in the human airway montelukast inhibit action of LTD4at the cyst1 receptor, preventing airway edema, smooth muscle contraction and enhance secretion of thick viscous mucous. Olopatadine

hydrochloride[2]

is

chemically2-[(2Z)-2-[3-(dimethylamino)propylidene]-9-

oxatricyclo[9.4.0.0{3,8}]pentadeca-1(11),3(8),4,6,12,14-hexaen-5-yl]acetic acid. It is a selective histamine H1antagonist that binds to the histamine H1receptor. This block the action of endogenous histamine, which subsequently leads to temporary relief of the negative symptoms brought on by histamine. Olopatadine is devoid of effects on alpha-adrenergic, dopamine and muscarnic type 1 and 2 receptors.[3-4] literature survey[5-12]regarding quantitative analysis of these drugs revealed that there were several analytical methods for montelukast sodium using extractive spectrophotometry, HPLC and HPTLC, RP-HPLC. Extractive spectrophotometry, HPTLC methods have been reported for estimation of olopatadine hydrochloride. There is only first order derivative spectroscopic method is reported for the estimation of montelukast sodium. So in present study simple, sensitive, specific, accurate and precise new RP-HPLC method is described for the estimation of these two drugs in combined dosage forms. MATERIAL AND METHOD Material Apparatus Instrument used was an UV-Visible double beam spectrophotometer, shimadzu (model UV1800) with a pair of 1 cm matched quartz cells. Reagents and Chemicals: Montelukast sodium was kindly supplied as a gift sample from Cipla Ltd. Solan (India). Olopatadine hydrochloride was kindly supplied as a gift samples from Sun PharmaLtd, Sikkim (India). Method Method Development and Optimization of Chromatographic Conditions. Solubility From the literature review, Montelukast and Olopatadine are freely soluble in acetonitrile and methanol and ethanol slightly soluble in water.

Selection of chromatographic method

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Alagar et al.


Proper selection of the method depends upon the nature of the sample (ionic/ionisable/ neutral molecule), its molecular weight and solubility. The drugs selected in the present study are polar in nature and hence reversed phase or ion-pair or ion exchange chromatography method may be used. The reversed phase HPLC was selected for the separation because of its simplicity and suitability. Selection of wavelength (λmax) The sensitivity of the HPLC method which uses UV detection depends upon the proper selection of wavelength. An ideal wavelength is one that gives good response for all the drugs to be detected. A UV spectrum of Montelukast and Olopatadine was recorded between 200400nm and was obtained by measuring the absorption of 10µg/ml solution of Montelukast and Olopatadine in methanol prepared from stock solution was shown in Fig:1. The spectrum was obtained by using 1cm quartz cell using methanol as reference solution, λmax of Montelukast and was 274 nm, λ max of Olopatadine was 269 nm and isosbestic or isoabsorptive point to be found was 270 nm.

Figure: 1 selection of wavelength for Montelukast and Olopatadine Selection of mobile phase Initially the mobile phase tried was methanol and water, methanol and acetonitrile, acetonitrile and buffer with various combinations of pH as well as varying proportions. Finally, the mobile phase was optimized to acetonitrile: buffer (0.02M potassium dihydrogen phosphate, pH = 5 adjusted with Phosphoric acid, 0.1ml of triethylamine) in proportion 60:40 v/v respectively. www.ejbps.com

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Optimization of column The method was performed with various C18 columns like, thermo BDS column, Inertsil and Hypersil column. Symmetry C18 (4.6 x 150mm, 5µm) was found to be ideal as it gave good peak shape and resolution at 1.0ml/min flow-rate.Based on the above study, the following chromatographic conditions were selected for the simultaneous estimation of drugs in multicomponent dosage forms. Table: 1 Optimized Chromatographic Conditions System Stationary phase Mobile phase Ratio pH Flow-rate Injection volume Detection wavelength Temperature Run-time Diluent Elution Needle wash

WATERS e2695 system with auto sampler Hypersil C18 column (150×4.6mm, 5µm) Acetonitrile: 0.02M potassium dihydrogen phosphate 0.1% triethylamine) 60:40v/v 5.0 adjusted with Phosphoric acid 1.0 ml/min 20µL 270 nm Ambient temperature (300C) 10min Mobile phase Isocratic elution Water: Acetonitrile (90:10v/v)

Estimation of drugs in tablet formulation by developed RP-HPLC method Mobile phase preparation Buffer preparation: Prepare 0.02M potassium dihydrogen phosphate in 0.1 % of Triethyl amine and adjust pH of resulting solution to 5.0 with ortho phosphoric acid. Mix buffer and Acetonitrile at 60: 40 ratio sonicate the resulting solution and degass it using vacuum filtration through 0.4 micron membrane filter. Standard stock solution preparation Weigh and transfer10 mg of Montelukast working standard and 5 mg of Olopatadine working standard into 25 mL volumetric flask, add 10 mL of diluent and sonicate to dissolve and dilute to volume with diluent.

Standard preparation

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Transfer10 mL of standard stock solution into 50 mL volumetric flask and dilute to volume with diluent. Sample Preparation Finely grind pre weighed 20 tablets. Transfer grinded sample quantitatively equivalent to 10 mg of Montelukast and 5 mg of Olopatadine in to 25 mL volumetric flask add 10 mL of diluent, sonicate to dissolve for 10 minutes and dilute to volume with diluent. Further filter the solution through filter paper. Dilute 10 ml of filtrate to 50 ml with mobile phase. Procedure Inject 20 µL of blank solution, placebo solution, Standard solution, Disregard peaks due to blank and placebo if any. Assay procedure Inject 20µL of the standard and sample solutions into the HPLC system and the chromatograms were recorded and measured the areas for the Montelukast and Olopatadine peaks and calculate the %Assay by using following formula.

Where, At = average area counts of sample preparation. AS = average area counts of standard preparation. WS= Weight of working standard taken in mg. Wt = Weight of sample taken in mg. Dt = sample dilution DS= standard dilution Estimation of Drugs in Marketed Formulation by Developed RP-HPLC Method. In RPHPLC method, HPLC conditions were optimized to obtain, an adequate separation of eluted compounds. Mobile phase and flow rate selection was based on peak parameters (Height, Theoretical plates, Tailing or Symmetry factor), run time and resolution.

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Fig: 2 chromatogram of standard drugs of

Fig: 3 chromatogram of tablets of

Montelukast & Olopatadine

Montelukast & Olopatadine

Validation of Developed RP-HPLC Method Validation of an analytical method is the process to establish by laboratory studies that the performance characteristic of the method meets the requirements for the intended analytical application. Performance characteristics were expressed in terms of analytical parameters. Linearity Appropriate aliquots of Olopatadine and Montelukast standard stock solution (0.2 -1.2ml) were taken in different 100ml volumetric flasks and resultant solution was diluted up to the mark with HPLC grade mobile phase to obtain final concentration 20-120 µg/ml for Olopatadine and 10-60 µg/ml for Montelukast. These solutions were injected into chromatographic system. The chromatograms were obtained and peak area was determined for each concentration of drug solution and calibration curves for the two drugs were constructed by plotting peak area against applied concentrations. The slope, intercept and correlation coefficient (r2) were also determined. An excellent correlation exists between peak area and concentration of drugs within the concentration range. Calibration curves for Olopatadine and Montelukast were shown in the Fig.4&5. The Linear detector response for Olopatadine and Montelukast is demonstrated by concentration versus Area. Over the range of 25 to 150% with respect to the target concentration (Dosage).

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Fig: 4 Calibration graph of Montelukast

Fig: 5 Calibration graph of Olopatadine

Hydrochloride Accuracy Accuracy of the method was determined by recovery experiments (standard addition method). To the formulation, the reference standards of the respective drugs were added at the level of 100 %. These were further diluted by procedure as followed in estimation of formulation. The resulting sample solutions were analyzed by HPLC. For assay methods samples are prepared in three concentrations of 50%, 100%, and 150% respectively. Table: 2 Recovery studies for Montelukast and Olopatadine. S .No Injection-1 Injection-2 Injection-3 Average Amount Recovered %Recovery

Accuracy-- 80% Olp Mon Area Area 849365 2992547 851575 3092213 846451 2989899 849130 3024886



79.77

79.63

99.61

99.04

119.60

119.68

99.72

99.53

99.61

99.04

99.66

99.73

Precision The precision of the method was demonstrated by repeatability and intermediate precision (inter-day precision studies). System Precision Preparation of solution Dilute 10 ml of standard stock solution with 50 ml of diluent. Inject the above solution six times.

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Method Precision Preparation of solution Dilute 10 ml of standard stock solution, with 50 ml of diluent.Prepare six solutions and inject each solution. Repeatability (system and method precision) studies were performed by injecting six (6) repeated injections of 100% concentration from tablet solution within a day. Peak area and % RSD were calculated and reported. The Inter-day precision studies, was done by injecting six (6) repeated injections of 100% concentration from tablet solution for six consecutive days. Peak area and %RSD were calculated and reported in Table3, 4 &5. Table: 3 system precision results for Montelukast and Olopatadine . S No

Name

1 S-Precision-1 2 S-Precision-2 3 S-Precision-3 4 S-Precision-4 5 S-Precision-5 6 S-Precision-6 Average Standard Deviation RSD

Olp RT Area 3.119 1023151 3.120 1022514 3.119 1027334 3.119 1028062 3.119 1032945 3.120 1032041 3.119 1027675 0.0005 4340.6 0.0166 0.422

Mon RT Area 5.465 3704945 5.466 3710105 5.466 3721253 5.467 3721383 5.467 3735906 5.464 3736309 5.466 3721650 0.001 12889.47 0.02 0.35

Table: 4 Method precision results for Montelukast and Olopatadine . S No

Name

1 M-Precision-1 2 M-Precision-2 3 M-Precision-3 4 M-Precision-4 5 M-Precision-5 6 M-Precision-6 Average Standard Deviation RSD

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Olp RT Area 3.121 1031254 3.125 1040125 3.124 1029569 3.121 1030201 3.125 1033220 3.123 1041153 3.123 1034254 0.0018 5109.0 0.0587 0.494

Mon RT 5.468 5.461 5.462 5.467 5.469 5.463 5.465 0.003 0.06

Area 3712121 3721545 3736356 3745485 3729954 3740254 3730953 12417.99 0.33

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Table: 5 Intermediate precision results for Montelukast and Olopatadine. S No

Name

1 S-Precision-1 2 S-Precision-2 3 S-Precision-3 4 S-Precision-4 5 S-Precision-5 6 S-Precision-6 7 M-Precision-1 8 M-Precision-2 9 M-Precision-3 10 M-Precision-4 11 M-Precision-5 12 M-Precision-6 Average Standard Deviation

RT 3.119 3.120 3.119 3.119 3.119 3.120 3.121 3.125 3.124 3.121 3.125 3.123 3.121 0.002

Olp Area 1023151 1022514 1027334 1028062 1032945 1032041 1031254 1040125 1029569 1030201 1033220 1041153 1030964 5677.465

RT 5.465 5.466 5.466 5.467 5.467 5.464 5.468 5.461 5.462 5.467 5.469 5.463 5.465 0.002

Mon Area 3704945 3710105 3721253 3721383 3735906 3736309 3712121 3721545 3736356 3745485 3729954 3740254 3726301 13008.129

Table; 6 Result data of Validation summary S. no 1 2 3 4 5 6 7

Parameter Accuracy Precision Correlation coefficient LOD LOQ Ruggedness Method precision More flow Less flow Robustness More wave length Less wave length

Montelukast 99.7% 0.01 0.999 0.6243 1.81 0.05 1.008 1.39 1.555 1.093

Olopatadine 100% 0.35 0.999 1.8729 1.58 0.33 0.938 1.321 1.19 0.895

ICH acceptance limit 98-102% %RSD < 2 Not less than 0.999 S/N =3 S/N =10 %RSD < 2 %RSD < 2

CONCLUSION A simple, economic, accurate and precise HPLC method was successfully developed. In simultaneous RP-HPLC method development, the estimation was carried out by using the Hypersil C18 column (125 X 4.6 mm) with 5µm particle size. Injection volume of 20μl is injected and eluted with the mobile phase Acetonitrile: 0.02M potassium dihydrogen phosphate 0.1% triethylamine) in the ratio of 60: 40 and the buffer pH is maintained at 5.0 and adjusted with 0.01M Phosphoric acid, which is pumped at a flow rate of 1.0 ml/min. Detection was carried out at 270 nm. The peaks obtained were sharp with retention time of 3.133 min Olopatadine Hydro Chloride for 5.137min for Montelukast Sodium. The peaks were well resolved with a resolution factor of 5.03. The method was precisely applied to the

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tablet formulation and the results obtained were accurate and reproducible. System suitability studies were also carried out which includes theoretical plates, resolution factor and tailing factor etc. Hence, the chromatographic method developed for Olopatadine Hydro Chloride and Montelukast Sodium are rapid, simple, sensitive, precise, and accurate. The RP-HPLC was useful in the analysis of respective drugs in pharmaceutical formulation. ACKNOWLEDGMENTS I express my deep thanks and gratitude to my respectable, beloved guide, our principal, Viceprincipal, miltan lab Pondicherry & my Parents & friends for providing all the encouragement & facilities to complete my research work successively. REFERENCES 1. Kalyankar tukaramaM, wale rishi R a and kakde rajendra B, Development and Validation of RP-HPLC Method for Estimation of Montelukast Sodium and Fexofenadine Hydrochloride in Pharmaceutical Preparations

Chemical Science Transactions, 2013;

2(3): 889-899. 2. Dyade G.K., Sharma A.K., Indian drugs, 2001; 38(2): 75-78. 3. Jadhav G.P., More H.N., Mahadik K.R., Indian drugs, 1998; 35(8): 475-480. 4. Gangwal S., Trivedi P., Indian drugs, 1998; 35(5): 291-295. 5. Connors K.A., A textbook of pharmaceutical Analysis, 3 rd edition, John wiley and sons, 1999; 221-224. 6. Panda S.K., Sharma A.K., and Sahu L.K., Indian Journal of Pharmaceutical sciences, 2002; 64(6): 540-544. 7. Sethi P.D., Qualitative Analysis of drugs in Pharmaceutical Formulations, 3rd edition, 1997; 182-184. 8. Solutions Mahajan Anand1, Gandhi Purvi S1., Pandita Nancy1, Gandhi Santosh V.2, Deshpande Padmanabh, Validated High Performance Thin Layer Chromatographic Method for Estimation of Olopatadine Hydrochloride as Bulk drug and in Ophthalmic, International Journal of ChemTech Research, 2010; 2(3): 1372-1375. 9. Varun Pawar,Sanjay Pai and Roa G.K., Development and Validation of UV Spectrophotometric Method for Simultaneous Estimation of Montelukast Sodium and Bambuterol Hydrochloride in Bulk and Tablet Dosage Formulation ,Jordan Journal of Pharmaceutical Sciences, 2008; 1(2): 676 – 684. www.ejbps.com

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10. Suddhasattya dey, vikram reddy, swetha.b, sandeep kumar. d., p.n. murthy, sudhir kumar sahoo, dhiraj kumar, s. subhasis patro and subhasis mohapatra,

Method development and

validation for the estimation of olopatadine in bulk and pharmaceutical dosage formsand i ts stress degradation studies

using Uv-Vis spectrophotometric

method,

International

Journal of Pharmacy and Pharmaceutical Sciences, 2010; 2(4): 0975-1491. 11. Patel Nilam K. and Pancholi S. S, Spectrophotometric Determination of Montelukast Sodium and Levocetirizine Dihydrochloride in Tablet Dosage Form by AUC Curve Method, Scholars Research Library,Der Pharma Chemica, 2010; 3(5): 135-140. 12. ICH Harmonized Tripartite Guideline. Validation of Analytical Procedures: Text and Methodology Q2 (R2), International Conference on Harmonization, Geneva, Switzerland Nov, 2005.

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