INTERNATIONAL RESEARCH JOURNAL OF PHARMACY ISSN Research Article
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1 INTERNATIONAL RESEARCH JOURNAL OF PHARMACY ISSN Research Article RP- HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF MONTELUKAST SODIUMAND DESLORATIDINE FROM BULK AND TABLETS FORMULATION Kalyankar Tukaram M 1*, Kokate Ranjeet H 1 and Kakade Rajendra B 2 1 Department of Pharmaceutical Chemistry, S R T M University Nanded (MS) India 2 University Dept. of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Campus, Nagpur (MS), India Article Received on: 11/05/12 Revised on: 09/06/12 Approved for publication: 02/07/12 * dr.kalyankartm@gmail.com ABSTRACT A simple, rapid, accurate, precise and validated High performance Liquid Chromatographic method for the simultaneous estimation of Desloratidine(DES) and Montelukast sodium(mon) in bulk and Multicomponant formulation. The Reversed-phase liquid chromatographic analysis was performed on a Hypersil C18 column (250mm, 4.6mm i.d., 5 μm particle size) column with mobile phase Acetonitrile: 0.05M Potassium Dihydrogen ortho-phosphates with 1.5ml Triethylamine of ph 3 (65: 35 v/v) and column temperature at 40 C. The flow rate of the mobile phase was adjusted to 1.3 ml/min and the injection volume was 10 μl. Detection was performed at 210 nm. The retention time for DES and MON were 2.12 min and min respectively. The method was validated and shown to be linear for DES and MON in µg/ml (r 2 =0.998) and µg/ml (r 2 =0.998) respectively. The relative standard deviation of DES and MON for intra-day was and respectively, inter-day was found to be, and respectively. The developed RP- HPLC method is suitable for estimation of Montelukast sodium and Desloratidine in tablet formulation. Hence this method can be used in quality control for routine analysis of the finish drug product. KEY WORDS: Montelukast sodium, Desloratidine,RP-HPLC, Simultaneous estimation. INTRODUCTION Chemically desloratidine (DES) is 8- Chloro-6; 1- dihydro-11 (-4piperidinylidine) -5H- benzo [5, 6] Cycloheptal [1, 2-b] pyridine, descarboethoxyloratidine. Its molecular formula is C 19 H 19 ClN 2 and molecular weight g/mole. It is not yet official in any Pharmacopoeia. It is non sedating peripheral histamine H1 receptor antagonist, the active metabolite of loratidine. A survey of literature reveals that DES is estimated by HPLC, HPTLC, spectrophotometry, special data analysis. There is no single method for this combination. The present HPLC method was validated as per ICH guidelines. Chemically montelukast sodium is-[r-(e)]-1-[1-[3-[2-7- chloro2quinolinyl) ethenyl] phenyl] -3-[2-(1-hydroxy1 (methylethyl) phenyl] propyl] Thio] methyl] cyclopropane acetic acid, monosodium salt. Its molecular formula is C 35 H 35 ClNNaO 3 S having molecular weight g/mol. It is a leukotriene receptor antagonist, used in the treatment of asthma. Montelukast selectively antagonizes leukotriene D 4 (LTD 4 ) at the cysteinyl leukotriene receptor, CysLT 1, in the human airway. Montelukast inhibits the actions of LTD 4 at the CysLT 1 receptor, preventing airway edema, smooth muscle contraction, and enhanced secretion of thick, viscous mucus. MATERIALS AND METHODS Reagents and Chemicals Pharmaceutical grade Montelukast and Desloratidine, were pursued as a gift sample from the Vasudha Pharma Chem Limited, Hyderabad, (AP) India. Acetonitrile (HPLC grade) and Methanol (HPLC grade) was purchased from Rankem Pvt. Ltd. Pharmaceutical formulation Marketed formulation Mondeslor tablet containing DES 5 mg and MON 10 mg was used as sample; purchased from local pharmacy. Calibrated glassware was used throughout the work. Method Development Different mobile phases containing methanol, water, Acetonitrile, and different buffers in different proportion were tried and finally of these Acetonitrile: 0.05 M Potassium Dihydrogen ortho-phosphates with 1.5 ml Triethylamine of ph 3 (65: 35 v/v) was selected as mobile phase which gave good resolution and acceptable peak parameters for both Desloratidine and Montelukast sodium Apparatus and chromatographic Conditions Chromatographic separation was performed on a PerkinElmer series 200, with PDA detector, autosampler HiQ SiLC18 (250mm x 4.6mm i.d., 5 μm particle size) was used for the separation. The mobile phase containing mixture of Acetonitrile: 0.05 M Potassium Dihydrogen ortho-phosphates with 1.5 ml Triethylamine of ph 3 (65: 35 v/v) was delivered at a flow rate of 1.3 ml/min with detection at 210 nm. The mobile phase was filtered through a 0.2 and degassed. The injection volume was 10 µl; analysis was performed at temperature 40 ºC. Preparation of Standard Solutions 10 mg of DES & 20 mg of MON was weighed accurately and transferred to 50 ml volumetric flask and dissolved in 25 ml of mobile phase and then the volume was made up to the mark with mobile phase to get 100 µg/ml and 200 µg/ml of stock solution of DES and MON respectively. Preparation of Sample Solutions Twenty tablets were weighed accurately and triturate to produce a fine powder. A quantity equivalent to 5 mg of DES and 10 mg of MON were weighed and transferred to 100 ml volumetric flask. Add 50 ml mobile phases and sonicate for 3 min. Made up the volume up to 100 ml with mobile phase again sonicate for 10 min and filtered through 0.45 μm membrane filter paper. By appropriate dilution of this solution with mobile phase obtain a sample solution within the concentration range for two drugs. A 10 μl volume of each sample solution was injected into the HPLC system for Page 343
2 six times under the chromatographic condition as stated above. The peak area of each peak was measured at 210 nm. With the optimized chromatographic conditions, a steady baseline was recorded, the mixed standard solution was injected and the chromatogram was recorded. The retention time of Desloratidine and Montelukast sodium, were found to be 2.12 and min respectively. This procedure was repeated for the sample solution obtained from the formulation. The proposed method was found to be specific and no interference from common tablet excipients like lactose, starch etc. was observed. VALIDATION The proposed method was validated by studying several parameters such as Linearity, Precision, Accuracy, Limit of Detection (LOD), Limit of quantization, and Robustness. Linearity and Range The linearity of the system was investigated by serially diluting the stock solutions to give concentrations in the range of 10 µg/ml to 60 µg/ml for Desloratidine and 20 to 120 µg/ml for Montelukast sodium. Calibration curves were obtained by plotting the response factor vs. concentration. The calibration curves are as shown in Fig.6 and Fig.7 for Desloratidine and Montelukast sodium respectively. The equations of the regression are given in table 1. Precision of method The precision of the method was demonstrated by interday and intraday variation studies. In the intraday studies, 3 repeated injections of sample solutions were made in a day and the response factor of drug peaks and percentage RSD were calculated.in the inter day variation studies, 3 repeated injections of sample solutions were made in 3 consecutive days. The Repeatability of sample applications and measurement of peak area were expressed in term of % R. S. D. and found to be less than 2%. The results of precision studies are given in tables 2, 3 4, and 5. Recovery studies The recovery studies were performed at 80, 100 and 120 % of test concentration as per ICH guideline. Recovery was within the range of 100 ± 2% which indicates the accuracy of the method. The result of recovery studies along with its statistical validation are given in Tables 6 and 7. Robustness The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variation in method parameters and provides an indication of its reliability during normal usage. The result of robustness studies along with its different parameters are given in Table 8,9,10 and 11. RESULT AND DISCUSSION The proposed method was found to be simple and linear in the concentration range of 10 to 60 mg/ml for Desloratidine and 20 to 120 mg/ml Montelukast sodium respectively. The method was found to be accurate and precise as indicated by recovery studies and the% RSD not more than 2.0. Moreover LOD and LOQ for Desloratidine were found to be µg/ml and µg/ml, respectively and for Montelukast sodium were µg/ml and µg/mL, respectively. Thus the method is specific and sensitive. ACKNOWLEDGEMENT Authors are thankful to Vasudha Pharma Chem Limited, Hyderabad. (A.P.) India, for providing us the gift sample of the pure drug and to the director School of Pharmacy, S R T M University, Nanded, for providing necessary research facilities. REFERENCES 1. Godge RK, Damle MC, Pattan SR, Kendre PN. RP- HPLC method for simultaneous estimation of Pseudoephidrine Sulphate and Desloratidine from bulk and tablets. Asian J. Research Chem 2009; 2(2): Eldin AB, Tohamy ME. Development and validation of an HPLC method for the determination of Montelukast and its degradation products in pharmaceutical formulation using an experimental design, Acta Pharmaceutica Science 2011;1: Rathore AS, Mahadik KR. Development of validated HPLC and HPTLC methods for simultaneous determination of Levocetirizine Dihydrochloride and Montelukast Sodium in bulk drug and pharmaceutical dosage form, Pharm Anal Acta 2010;1: Devi NK, Madhavi BR, Mrudula BS. New RP- HPLC method for the analysis of Montelukast Sodium in pharmaceutical dosage forms. International Journal of ChemTech Research 2010; 2 (1): Rawat IS. Simultaneous RP-HPLC Estimation of Levocetirizine Hydrochloride and Montelukast Sodium in tablet dosage form. International Journal of Pharm Tech Research 2011; 3: Rote AR, Niphade VS. Determination of Montelukast Sodium and Levocetirizine Dihydrochloride in combined pharmaceutical dosage form by RP-HPLC. Lat. Am. J. Pharm 2010;29 (6): Azure IA. Development of a stability-indicating HPLC method for the determination of Montelukast in tablets and human plasma and its applications to pharmacokinetic and stability studies, Saudi Pharmaceutical Journal 2012; 12: Sharma MC. Simultaneous estimation and validation of Pseudoephidrine Sulfate and Desloratidine from bulk and tablets as hydrotropic solubilizing agent. Journal of Current Pharmaceutical Research. 2010; 01: Patel DJ, Patel SA, Patel DJ. Simultaneous determination of Montelukast Sodium and Bambuterol Hydrochloride in tablet dosage form by ultraviolet spectrophotometry. International Journal on Pharmaceutical and Biomedical Research 2010; 1(3): ICH, Q2A Validation of Analytical Procedures: Consensus Guidelines; ICH Harmonized Tripartite Guidelines, ICH, Q2B Validation of Analytical Procedures: Methodology, Consensus, Consensus Guidelines; ICH Harmonized Tripartite Guidelines, Table 1. Linear regression data for calibration curve of DES and MON Drug Linearity Range (µg/ml) Slope Intercept Regression Coefficient DES MON Table 2. Repeatability Data Sr. No. Concentration (µg/ml) Peak Area % of found DES MON DES MON DES MON Table 3. Statistical validation Drug Mean AUC S. D. R.S.D. DES MON Table 4. Intraday Precision data Drug Mean* S. D. R.S.D. DES MON Table 5. Interday Precision data Drug Mean* S. D. R.S.D. DES MON Page 344
3 Level of recovery 80% 100% 120% Table 6. Result of Recovery Study Amount present (mg) Added conc. (mg) Total amount recovered % Recovery (mg) Level of % recovery Flow Rate (ml/min) Table 7-Statistical validation % Mean recovery S.D. % R.S.D. DES MON DES MON DES MON Table 8. Flow Rate (ml/min) Retention time Tailing factor Peak area % content found *Mean ±S. D. ph 2.3 ± ± ± ± ± Table 9. ph of Buffer Retention time Tailing factor Peak area % content found *Mean Temp. ( 0 c) 2.70 ± ± ± ± ± ±0.589 Table 10: Column Temperature Retention time Tailing factor Peak Area % content found Mean 2.30 ± ± ± ± ± ± ±0.131 Table 11: Ratio of Mobile Phase Ratio Retention time Tailing Factor Peak Area % content found 63: : : Mean 2.45 ± ± ± ± ± ±0.230 Table 12-Study of System Suitability Parameter Sr. No. Parameters DES MON 1 Retention time (min) Resolution Tailing Factor Theoretical plate Peak area Page 345
4 Cl N N + H Fig 1. Structure of Desloratidine Cl N S COONa H 3 C CH 3 CH 3 Fig 2. Structure of Montelukast sodium Fig 3. Chromatogram of Desloratidine. Fig 4. Chromatogram of Montelukast sodium Page 346
5 Fig 5. Chromatogram of mixture desloratidine and montelukast sodium respectively. Fig.6: Calibration curve for DES Fig.7: Calibration curve for MON Source of support: Nil, Conflict of interest: None Declared Page 347
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