Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385                                                                   ...
Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385                                                      INTRODUCTION ...
Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385        Levocetirizine is a third generation non-sedative antihista...
Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385         Sol-A: weighed accurately 1g of potassium hydrogen phospha...
Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385        System suitability parameters were established by injecting...
Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385                                      Table-1: System suitability (...
Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385                              Figure-4: Standard solution system su...
Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385                                         Figure-5: Overlaid Lineari...
Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385     Alsarra I. Development of a stability-indicating HPLC method f...
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A simple rp hplc method for simultaneous analysis of pseudoephedrine, bambuterol, levocetirizine and montelukast in pharmaceutical dosage forms

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A simple rp hplc method for simultaneous analysis of pseudoephedrine, bambuterol, levocetirizine and montelukast in pharmaceutical dosage forms

  1. 1. Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385 ISSN:2249-5347 IJSID International Journal of Science Innovations and Discoveries An International peer Review Journal for Science Research Article Available online through www.ijsidonline.info A SIMPLE RP-HPLC METHOD FOR SIMULTANEOUS ANALYSIS OF PSEUDOEPHEDRINE, BAMBUTEROL, LEVOCETIRIZINE AND MONTELUKAST IN PHARMACEUTICAL DOSAGE FORMS 1Department of Chemistry, JNT University, Kukatpally, Hyderabad, AP, India; 2AR&D, Custom Pharmaceutical Services, Dr. Hanimi Reddy Bapatu*1, Maram Ravi Kumar2, Useni Reddy Mallu3, R.S. Murthy1 and Harikishan Reddy Ganthi3 Reddys Laboratories Ltd, Bachupally, Hyd-72, India; 3Department of Chemistry, Sri Krishnadevaraya University, Anantapur, AP, India A simple and economic reverse phase high performance liquid chromatography ABSTRACT (RP-HPLC) method has been developed for the estimation of Pseudoephedrine, Bambuterol, Levocetirizine and Montelukast in bulk and tablet dosage forms. Separation was achieved on C18 column (150 x 4.6mm i.d., 5μm) using gradient mobile phase Sol-A:Received: 13.02.2012Accepted: 19.06.2012 buffer (weighed accurately 1gm of potassium di-hydrogen phosphate in to 1000mL of HPLC grade water) and Sol-B: acetonitrile gradient program (0-4min, sol-A:98-98; 4- 8min- sol-A: 98-75; 8-12min- sol-A: 75-70; 12-15min- sol-A: 70-45; 15-20min- sol-A: 45-*Corresponding Author 20; 20-25min- sol-A: 20-98 and 25-30min- sol-A: 98-98), pumped in to the column at flow rate of 1.0 ml/min and the detection of eluent from the column was carried out using variable wavelength detector at 210nm. The total run time was 30 min and the column oven temperature was maintained at 35°C. The retention times of Pseudoephedrine, Bambuterol, Levocetirizine and Montelukast were 4.1 min, 10.9 min, 15.4 and 21.1 min, respectively. The standard curves were linear over the concentration range of 10-60 μg/ml. The method was validated as per ICH guidelines. Validation studies demonstrated that the proposed RP-HPLC method is simple, specific, rapid,Address: reliable and reproducible. The suitability of the proposed method for the routine quality INTRODUCTIONName: control analysis in bulk and tablet dosage forms.Hanimi Reddy BapatuPlace:JNT University,Hyderabad, IndiaE-mail:hanimi.b@gmail.com International Journal of Science Innovations and Discoveries, Volume 2, Issue 3, May-June 2012 377
  2. 2. Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385 INTRODUCTION Montelukast sodium is a leukotriene receptor antagonist and used in the treatment of asthma. it is a hygroscopic,optically active, white to off-white powder and freely soluble in ethanol, methanol, and water and practically insoluble inacetonitrile. Side effects of montelukast are skin rash, bruising, severe tingling, numbness, pain, muscle weakness, mood orbehavior changes, anxiety, depression, tremors or shaking, severe sinus pain, swelling or irritation or worsening asthma.General dose of montelukast is for adults and adolescents 15 years of age and older: one 10-mg tablet, for pediatric patients 6to 14 years of age: one 5-mg chewable tablet and for pediatric patients 2 to 5 years of age: one 4-mg chewable tablet or onepacket of 4-mg oral granules. For pediatric patients 12 to 23 months of age: one packet of 4-mg oral granules. Safety andeffectiveness in pediatric patients less than 12 months of age with asthma have not been established. Studies are in progresson clinical trials in patients with asthma to evaluate the relative efficacy of morning versus evening dosing. Thepharmacokinetics of montelukast are similar whether dosed in the morning or evening. Efficacy has been demonstrated forasthma when montelukast was administered in the evening without regard to time of food ingestion. Bambuterol hydrochloride is a direct acting sympathomimetic with predominantly -adrenergic activity (β2-agonist)and an ester prod rug of β2 adrenergic agonist and it is known to a producing of terbutaline but has better therapeutic totoxicity ratio and acts as an endogenous reservoir. Bambuterol is a pro-drug of terbutaline that is slowly converted into thebody to the form. Pseudoephedrine is a decongestant that shrinks blood vessels in the nasal passages. It is used in the treatment of nasaland sinus congestion, congestion of the tubes that drain fluid from you inner ears. Do not give pseudoephedrine to a childyounger than 4 years old it can give adverse effects of death. Pseudoephedrine Bambuterol Figure-1: Chemical structures of active ingredients Levocetirizine Montelukast International Journal of Science Innovations and Discoveries, Volume 2, Issue 3, May-June 2012 378
  3. 3. Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385 Levocetirizine is a third generation non-sedative antihistamine cetirizine. Chemically it is the active enantiomer ofcetirizine. It works by blocking histamine receptors and it may not prevent the actual release of histamine from mast cells, butprevents in binding to its receptors. This in turn prevents the release of other allergy chemicals and increased blood supply tothe area and provides relief from the typical symptoms of hay fever.Dosage forms list Tablets Montelukast 4 mg chewable tablets Dosage form Composition Montelukast 5 mg chewable tablets Montelukast 10 mg tablets Montelukast oral granule packets -- containing 4 mg per packet Psedoepidrine HCl-15mg/60mg pseudoephedrine hydrochloride USP 120mg/tablet pseudoephedrine hydrochloride USP 240mg/tablet Bambuterol Tablets 10 mg Bambuterol Tablets 20 mg Levocetirizine dihydrochloride 5mg tablet. Syrup Bambuterol hydrochloride 5mg/5ml syrup pseudoephedrine hydrochloride 30mg/5mL Literature survey reveals that numerous methods (1-27) have been reported for the analysis of Montelukast Sodium,bambuterol, Pseudoephedrine and Bambuterol for individual and combination drug products but not method available for thedetermination of four drugs in single time. The present research work is to develop a single and simple method for thedetermination of these four drugs by HPLC method. MATERIALS AND METHODS A waters HPLC system consisting of alliance 2695, agilent 1200 series HPLC instrument with UV-Visible detector, twoInstruments:systems were operated by Empower software. Waters make X-Terra C18 1504.6mm, 5µm column, Mettler Toledo madeanalytical balance were used for this study Pure (not less than 98.5%) standards of all active ingredients, HPLC grade acetonitrile and water; AR grade ofMaterials:Potassium Dr-Hydrogen orthophosphate were used.Mobile phase: International Journal of Science Innovations and Discoveries, Volume 2, Issue 3, May-June 2012 379
  4. 4. Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385 Sol-A: weighed accurately 1g of potassium hydrogen phosphate, transferred in to 1000ml of HPLC water and mixed.Filtered the final solution through a 0.4μm membrane filter; Sol-B: HPLC grade acetonitrile. Mixed the HPLC water and acetonitrile in the ratio of 1:1 (v/v).Diluent: Transferred the 40mg of Levocetirizine standard in to 100mL volumetric flask and added 10ml of methanol and shakeStandard solution:well then added 40mg of each Montelukast sodium, Bambuterol and Pseudoephedrine standards in to the same volumetricflask, 50ml of diluent added and sonicated to dissolve and made up to volume with diluent. Further diluted the 5ml of resultingsolution into 50ml with diluent (each standard 40ppm). Prepared the all dosage forms to get each active ingredient equal to 40microgram per mL with diluent and analyzed.Test solution:Chromatograph : Waters/ Agilent HPLC system with Empower software.Chromatographic conditionsMobile phase : Solution-A and solution-B with gradient elution.Gradient program : (0-4min, sol-A: 98-98; 4-8min-sol-A: 98-75; 8-12min-sol-A: 75-70; 12-15min-sol-A: 70-45; 15- 20min-sol-A: 45-20; 20-25min-sol-A: 20-98 and 25-30min-sol-A: 98-98;)Column : Waters make X-Terra C18 1504.6mm, 5µm.Flow rate : 1.0 mL per minDetection : 210nmInjection volume : 10 μLRetention time : Pseudoephedrine-4.1, Bambuterol-10.9, Levocetirizine-15.4 and Montelukast-21.1.Run time : 30 min.Column oven temp : 35°C : All active ingredients were quantified with the following calculation. Sample area x standard concentration x Potency of standardCalculation -------------------------------------------------------------------------- X 100 Standard area x sample concentration x 100 RESULTS AND DISCUSSION The mobile phase was chosen after several trials with methanol, acetonitrile, water and buffer solutions in variousMethod Developmentproportions and at deferent pH values. Flow rates between 0.5 and 1.5/min were studied. A flow rate of 1.0 ml/min gave anoptimal signal to noise ratio with a reasonable separation time. Using a reversed-phase C18 column, the retention times ofPseudoephedrine, Bambuterol, Levocetirizine and Montelukast were 4.1 min, 10.9 min, 15.4 and 21.1 min respectively. Totaltime of analysis was less than 30 min. The maximum absorption of Pseudoephedrine, Bambuterol, Levocetirizine andMontelukast together as detected at 210nm and this wavelength was chosen for the analysis. The chromatogram at 210nmshowed a complete resolution of all peaks (Figure-3). International Journal of Science Innovations and Discoveries, Volume 2, Issue 3, May-June 2012 380
  5. 5. Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385 System suitability parameters were established by injecting the freshly prepared standard solution (each activeSystem suitability40microgram per mL/five replicate injections) in to the chromatographic system. The percent relative standard deviation forpeak area and retention time results found to be satisfactory. System suitability chromatograms were represented in figure-4and tabulated the results in table-1 and 2. The precision of the method was demonstrated by inter day and intraday variation studies. In the intraday studies, sixPrecision:repeated injections of standard and sample solutions were made and the area of drug peaks and percentage RSD werecalculated. In the inter day variation studies, six repeated injections of standard and sample solutions were made for threeconsecutive days and area of drug peaks and percentage RSD were calculated. From the data obtained, the developed HPLCmethod was found to be precise. Linearity is determined by calculating the regression line using a mathematical treatment of the linearity results vsLinearity:analyte concentration (10microgram per mL to 60microgram per mL for each ingredient) of the standard solution. Linearitygraph was plotted against peak area and concentration of solution. The correlation coefficient value found to be within thelimit 0.999. The linearity chromatograms shown in figure-5 and linearity results tabulated in table-4 and linearity plots wererepresented in graph-1. Each factor selected (except columns from different manufacturers) was changed at three levels (−0.1, 0 and 0.1). One Robustness and Ruggedness:factor at the time was changed to estimate the effect. Thus, replicate injections (n = 6) of mixed standard solution at threeconcentration levels were performed under small changes of three chromatographic parameters (factors). Insignificantdifferences in peak areas and less variability in retention time were observed Figure-2: Diluent chromatogram International Journal of Science Innovations and Discoveries, Volume 2, Issue 3, May-June 2012 381
  6. 6. Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385 Table-1: System suitability (Area %RSD) Active Ingredient Standard solution Area Pseudoephedrine Name Inj-1 Inj-2 Inj-3 Inj-4 Inj-5 Average %RSD Bambuterol 16524569 16522905 16477184 16701716 16634850 16572245 0.56 Levocetirizine 1868913 1789289 1849078 1841880 1852652 1840362 1.64 Montelukast 2043705 2038930 2033310 2027799 2036356 2036020 0.29 2900859 2902302 2901514 2899794 2916066 2904107 0.23 Figure-3: Standard chromatogram Table-2: System suitability (Retention time %RSD) Standard solution Retention time (min) PseudoephedrineActive Ingredient Name Inj-1 Inj-2 Inj-3 Inj-4 Inj-5 Average %RSD Bambuterol 4.11 4.12 4.12 4.12 4.12 4.118 0.11 Levocetirizine 10.89 10.9 10.9 10.91 10.91 10.902 0.08 Montelukast 15.38 15.39 15.39 15.39 15.4 15.39 0.05 21.09 21.1 21.11 21.1 21.11 21.102 0.04 Table-3: System suitability (USP Tailing factor) Standard solution Tailing factor Pseudoephedrine Active Ingredient Name Inj-1 Inj-2 Inj-3 Inj-4 Inj-5 Bambuterol 1.0 1.0 1.0 1.1 1.1 Levocetirizine 1.2 1.1 1.1 1.1 1.1 Montelukast 0.9 0.9 0.9 0.9 0.9 0.8 0.8 0.8 0.8 0.8 Table-4: System suitability (USP Resolution) Standard solution Resolution Resolution between Pseudoephedrine and Bambuterol Active Ingredient Name Inj-1 Inj-2 Inj-3 Inj-4 Inj-5 Resolution between Bambuterol and Levocetirizine 11.18 11.54 11.69 11.91 12.11 Resolution between Levocetirizine and Montelukast 21.69 2186 21.6 21.48 21.74 26.37 26.66 26.4 26.69 26.37 International Journal of Science Innovations and Discoveries, Volume 2, Issue 3, May-June 2012 382
  7. 7. Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385 Figure-4: Standard solution system suitability chromatograms Table-5: Linearity Results. Linearity solutions areaActive Ingredient Linearity Linearity Linearity Linearity Linearity Linearity level- Co-relation Name level-2 level-3 level-4 level-5 level-6 1 (25%) Coefficient (50%) (75%) (100%) (125%) (150%)Pseudoephedrine Bambuterol 3929981 8152866 12291125 16516368 20666550 24676668 0.999970 Levocetirizine 410793 870869 1322585 1795780 2232422 2769734 0.999639 Montelukast 481666 1001290 1513533 2028715 2535195 3048322 0.999994 680955 1425061 2167601 2915401 3657614 4401669 1.000000 International Journal of Science Innovations and Discoveries, Volume 2, Issue 3, May-June 2012 383
  8. 8. Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385 Figure-5: Overlaid Linearity chromatograms A gradient RP-HPLC method developed and validated for the simultaneous determination of Pseudoephedrine, CONCLUSIONBambuterol, Levocetirizine and Montelukast in both bulk and tablet dosage form. The validation results reveals that, methodhave good precision and accuracy, which proves the reliability of the proposed method. The retention time ofPseudoephedrine is 4.2min, Bambuterol is 10.9min, Levocetirizine is 15.3min and Montelukast is 21.1min. linearity resultswere found to be linear for Pseudoephedrine is 0.9999, Bambuterol is 0.999, Levocetirizine is 0.9999 and Montelukast is1.000. The short runtime and low solvent consumption are advantageous for applying in quality control analysis. Sweetman SC, editor. Martindale: The Complete Drug Reference. 33rd ed. London: Pharmaceutical Press; 2002. p. 768. REFERENCES Budavari S, editor. The Merck Index. 12th ed. Whitehouse Station, NJ: Merck & Co Inc; 1996. p. 1070.1. Morrow JD, Roberts LJ. In: Goodman and Gilmans The Pharmacological Basis of Therapeutics. 10th ed. Hardman JG,2. Limbird LE, Gilman AG, editors. New York: McGraw-Hill; 2001. p. 669.3. Al-Rawithi S, Al-Gazlan S, Al-Ahmadi W, Alshowaier I, Yusuf A, Raines D. Expedient liquid chromatographic method with fluorescence detection for montelukast sodium in microsamples of plasma. J Chromatogr B Biomed Sci Appl.4. 2001;754:527–31 Ochiai H, Uchiyama N, Takano T, Hara K, Kamei T. Determination of montelukast sodium in human plasma by column- switching high performance liquid chromatography with fluorescence detection. J Chromatogr B Biomed Appl.5. 1998;713:409–14. Chauhan B, Shubha Rani, Nivsarkar M, Padh H. New liquid liquid extraction method for determination for montelukast in small volume human plasma samples using HPLC with fluorescence detector. Indian J Pharm Sci. 2006;68:517–20.6. Liu L, Cheng H, Zhao JJ, Rogers JD. Determination of montelukast (MK-0476) and S-enatiomer in human plasma by stereoselective high performance liquid chromatography with column switching. J Pharm Biomed Anal. 1997;15:631–8.7. Radhakrishna T, Narasaraju A, Ramakrishna M, Satyanarayana A. Simultaneous determination of montelukast and loratadine by HPLC and derivative Spectrophotometric methods. J Pharm Biomed Anal. 2003;31:359–688. International Journal of Science Innovations and Discoveries, Volume 2, Issue 3, May-June 2012 384
  9. 9. Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385 Alsarra I. 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 Pharm J. 2004;12:136–43.9.10. Sweetman SC, editor. Martindale: The Complete Drug Reference. 33rd ed. London: Pharmaceutical Press; 2002. p. 761.11. Budavari S, editor. The Merck Index. 12th ed. Whitehouse Station, NJ: Merck & Co Inc; 1996. p. 163.12. The British Pharmacopoeia. 3rd ed. Vol. 1. London: Stationary Office Books; 2001. pp. 174–5.13. Zhang Y. Determination bambuterol hydrochloride tablets by. Jiangsu Pharm Clin Res. 2001;9:13–4.14. Bartolincic A, Druskovic V, Sporec A, Vinkovic V. Development and validation of HPLC methods for the enantioselective analysis of bambuterol and albuterol. J Pharm Biomed Anal. 2005;36:1003–1015. Wannerberg O, Persson B. Liquid chromatographic methods for the determination of bambuterol hydrochloride and related compounds. J Chromatogr A. 1988;435:199–203.16. Harris R, Hodgkinson P, Larsson T, Muruganatham A. Quantification of bambuterol hydrochloride in a formulated product using solid-state NMR. J Pharm Biomed Anal. 2005;38:858–64.17. Chauhn B, Rani Shubha, Nivasarkar M, Padh H, Indian Journal of Pharmaceutical Sciences, 2006, 68 (4), 517-520.18. Alsarra I, Khalil NY, Sultan M, Al-Ashaban R, Pharmazie, 2005, 60 (11), 823-826.19. Smith GA, Rawls CM, Kunka RL, Pharm. Res, 2004, 21 (9), 1539-1544.20. Arayne MS et al., Simultaneous quantitation of cefpirome and cetirizine or levocetirizine in pharmaceutical formulations and human plasma by RP-HPLC, Journal of Ana. Chem, 2008, 63, 881-887.21. D. W. Empey, G. A. Young, E. Letley, G. C. John, P.Smith, K. A. McDonnell, L. R. Bagg, D. T.Hughes, Br.J. Clin. Pharmacol., 9, 351 (1980).22. H. Richardson, B.A. Bidlingmeyer, J. Pharm. Sci. 73 (1984) 1480.23. A. Yacobi, Z.M. Look, C.M. Lai, J. Pharm. Sci. 67 (1978)166824. M. K. Chao, I. J. Holcomb, S. A. Fusari, J. Pharm. Sci.,68, 1463 (1979)25. B. R. Thomas, X. Fang, P. Shen, S. Ghodbane, J. Pharm.Biomed. Anal., 12, 85 (1994)26. M. D. Paciolla, S. A. Jansen, S. A. Martellucci, A. A.Osei, J. Pharm. Biomed. Anal., 26, 143 (2001).27. The United States Pharmacopeia 24, United States Pharmacopeial Convention, Inc, Rockville, 2000, p. 32. International Journal of Science Innovations and Discoveries, Volume 2, Issue 3, May-June 2012 385

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