Analytical Method Development And Validation For The Determination Of .
ISSN: 2230-7346R. Vani et al. / JGTPS / 5(4)-(2014) 2248 – 2252(Research Article)Journal of Global Trends in Pharmaceutical SciencesJournal home page: www.jgtps.comANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE DETERMINATION OFLORATADINE, AMBROXOL HYDROCHLORIDE AND GUAIPHENESIN USING REVERSE PHASEHPLC METHOD IN BULK AND LIQUID DOSAGE FORMR. Vani1*B. Vijaya kumar2G. Krishna Mohan31ABSTRACTResearch Scholar, JNTU-K,Kakinada2Jangaon Institute ofPharmaceutical Sciences, Warangal.3Center for PharmaceuticalSciences, JNTU-H Hyderabad.A new simple, accurate, precise and reproducible RP-HPLC method has beendeveloped for the simultaneous estimation of loratadine, ambroxol hydrochloride andguaiphenesin in bulk drug & liquid dosage form using C18 column (Waters, 250 x 4.6mm, 5 μm) in isocratic mode. The mobile phase consisted of 0.1 M di potassiumPhosphate buffer (pH 5.5) and methanol in the ratio of 50:50 v/v. The detection wascarried out at 245 nm. The method was linear over the concentration range for Loratadine6-14μg/ml, ambroxol hydrochloride36-84μg/ml and guaiphenesin 60-140μg/ml. therecoveries of loratadine, ambroxol hydrochloride and guaiphenesin were found to be100.68-98.8%, 100.34-98.52%, 100.71-98.54% respectively. The validation of methodwas carried out utilizing ICH-guidelines. The described HPLC method was successfullyemployed for the analysis of pharmaceutical formulations containing combined dosageform.Keywords: Loratadine, Ambroxol Hydrochloride and Guaiphenesin, Reverse PhaseHPLC, Validation.INTRODUCTION:DescriptionLoratadineTable 1: Drug profileAmbroxol HclGuaiphensineStructureIUPAC nameMol. formulaPkaMol.wtCategoryethyl 4-(8-chloro-5,6-dihydro-11Hbenzo[5,6] cyclohepta[1,2-b]pyridin11-ylidene) Allergic Agents, Antipruritics,Histamine H1 Antagonists, NonSedatingLoratadine is a derivative of azatadine and asecond generation histamine H1 receptor antagonist usedin the treatment of allergic rhinitis and urticaria. UnlikeAddress for correspondenceR. Vani*Deccan School of Pharmacy, HyderabadPhone: most classical Antihistamines (histamine H1 antagonists)it lacks central nervous system depressing effects such asdrowsiness. Ambroxol is a secretolytic agent used in thetreatment of respiratory diseases associated with viscid orexcessive mucus. It is the active ingredient ofMucosolvan, Lasolvan or Mucoangin. The substance is amucoactive drug with several properties includingsecretolytic and secretomotoric actions that restore thephysiological clearance mechanisms of the respiratorytract which play an important role in the body’s naturalR. Vani et al, JGTPS, 2014, Vol. 5(4): 2248 - 22522248
defence mechanisms. It stimulates synthesis and releaseof surfactant by type II pneumocytes. Surfactants acts asan antiglue factor by reducing the adhesion of mucus tothe bronchial wall, in improving its transport and inproviding protection against infection and irritatingagents. Guaifenesin An expectorant that also has somemuscle relaxing action. It is used in many coughpreparations. Guaifenesin (glyceryl guaiacolate) has thechemical name 3-(2-methoxyphenoxy)-1,2-propanediol.Its molecular formula is C10H14O4with a molecularweight of 198.21. It is a white or slightly gray crystallinesubstance with a slightly bitter aromatic taste. One gramdissolves in 20 mL water at 25 C; it is freely soluble inethanol. It is Guaifenesin is readily absorbed from the GItract and is rapidly metabolized and ometric methods have been reported for thedetermination of LOR, AMB, GUA in single and ion of LOR and AMB, GUA in bulk and liquiddosage form were reported by using spectrophotometric,.However very few HPLC methods were reported for thesimultaneous estimation of LOR and AMB,GUA in liquiddosage form. The aim of present work was to develop andvalidate as per ICH guidelines [20], a sensitive HPLCmethod that can be applied for simultaneous estimation ofLOR and AMB, GUA.MaterialsLOR and AMB, GUA were received gratis fromHetero drugs, Hyderabad and were used as received.HPLC grade acetonitrile was purchased from SD FineChem Pvt. Ltd. (Mumbai, Maharashtra). Ultra-pure waterwas obtained from ELGA (Bucks, UK) water purificationunit. .All other chemicals were of analytical reagentgrade.The volume of injection was 10 µl, prior toinjection of the analyte, the column was equilibrated for30–40 min with the mobile phase. The eluents weredetected at 245 nm. The developed method was validatedin terms of specificity, linearity, accuracy, limit ofdetection (LOD), limit of quantification(LOQ), intra-dayand inter-day precision and robustness for the assay ofLOR and AMB,GUA as per ICH guidelines.Preparation of standard solutions:Standard stock solutions of Guaifensine,Ambroxol Hcl & Loratadine (microgram/ml) wereprepared by dissolving 50mg Guaifensine, 30mgAmbroxol Hcl & 5mg Loratadine dissolved in sufficientmobile phase. After that filtered the solution using 0.45micron syringe filter and Sonicated for 5min and dilute to100 ml with mobile phase. Pipette out 5ml & furtherdilute to 50ml.Preparation of sample solution:Pipette out 5ml of the syrup solution anddissolved in 50 ml of mobile phase sonicate the solutionfor about 30mints and filter through 0.45 micron filterfrom this pipette out 5ml and make up to 50mlRESULTS AND DISCUSSION:Method Development:Number of mobile phase and their differentproportions were tried and finally was selected as 0.1 MDipotassium Phosphate buffer (pH 7) and acetonitrile inthe ratio of 70:30 v/vappropriate mobile phase whichgave good resolution and acceptable system suitabilityparameters. The results of system suitability parameterswere shown in table 3. The chromatogram of workingstandard solution is shown in Fig 1. The summaries ofChromatographic conditions were given in table 2.EXPERIMENTAL WORK:Table 2: Summary of Chromatographic conditionsChromatographic conditionsThe HPLC system (Agilent 1220 series)consisted of quaternary gradient system (600 Controller),in-line degasser (Agilent), UV detector and Manualsampler. Data was processed using EZchrome software(Agilent). Isocratic elution of the mobile phase 0.1 MDipotassium Phosphate buffer (pH 7) and acetonitrile inthe ratio of 70:30 v/v with the flow rate of 1 ml/min.Separation was performed on a C18 (250 x 4.6 mm i.d, 5μ particle size) analytical column and a pre-column toprotect the analytical column from strongly bondedmaterial. Integration of the detector output was performedusing the EZ chrome software to determine the peak area.The contents of the mobile phase were filtered through a0.45 µm membrane filter and degassed by sonicationbefore use. Mobile phase was used as diluents. The flowrate of the mobile phase was optimized to 1 ml/min whichyields a column back pressure of 110–112 kg/cm. The runtime was set at 10 min and a column temperature wasmaintained at 35 ater’s C18 (250X4.6X5)2Mobile Phase35Flow rateDetectionWavelengthDetector7Rt’s48910110.1 M Dipotassium Phosphatebuffer (pH 7) and acetonitrile in theratio of 70:30 v/v1 ml/min245nmUV detectorLOR – 7.0503 Min AMB- 5.280MinGUA – 3.107MinInjectionvolumeColumnTemperatureRun timeDiluentR. Vani et al, JGTPS, 2014, Vol. 5(4): 2248 - 225210 μl35 C10 minsMobile Phase2249
Fig. 1: Typical Chromatogram of Loratadine, Ambroxol hydrochloride and GuaiphenesinTable 3: System suitability parametersS. No12345ResultsLoratidine Ambroxol HCL GuaiphenesinRetention Time (min) 7.05035.2803.107Tailing1.3131.5181.926Theoretical Plates (n) 562873807337613Resolution factor (R)6.188Similarity Factor1.0124 (Limit: 0.98 – 1.2)ParameterMethod Validation:AccuracyRecovery assessment was obtained by usingstandard addition technique which was by adding knownquantities of pure standards at three different levels in50%, 100%and 150% to the pre analyzed sampleformulation. From the amount of drug found, amount ofdrug recovered and percentage recovery were calculatedwhich sense to conformation that the proposed methodwas accurate. The results were tabulated in Table 4.Table 4: Results of AccuracyS.No123GUAAMBAmountAmount AmountAmountMean %Mean .5214*Mean % Recovery of 6 replicates; **Mean % Recovery of 3 replicates% conc isionThe intraday and inter day precision of theproposed method was determined by analyzing mixedstandard solution of GUA and AMB,LOR atLORAmountfound(µg\ml)9.8912.0813.93Mean %recovery98.9100.6899.48concentration 50µg/mL and 30µg/mL, 5µg/mL 3 timeson the same day and on 3 different days. The resultsshown in table 4 were reported in terms of relativestandard deviation.Table 5: Results of Precision (%Assay)LORATIDINESample Area - 1498.8003.103499.8203.113499.3163.1157STD0.0177% RSD0.57Sample No.123456AvgAMBROXOLSample Area - 09% RSD0.17GUAIPHENESINSample Area - 0459.6777.067461.4257.060460.6897.065STD0.008% RSD0.11R. Vani et al, JGTPS, 2014, Vol. 5(4): 2248 - 22522250
Linearity:Calibration graphs were constructed by plottingpeak area vs concentration of GUA and AMB and LORthe regression equations were calculated. The calibrationgraphs were plotted over 5different linear concentrationsin the range of 60-140µg/ml for GUA and 36-84µg/ml forAMB,6-14µg/ml. Aliquots (10 ml) of each solution wereinjected under the operating chromatographic conditiondescribed above [Number of replicates (n 5)]. Thelinearity graphs were shown in fig 2& 3, 4.Fig 4Limit of detection (LOD) and limit of quantitation(LOQ):The limit of detection (LOD) and limit ofquantitation (LOQ) of GUA and AMB,LOR weredetermined by calculating the signal-to-noise(S/N) ratioof 3:1 and 10:1, respectively according to InternationalConference on Harmonization guidelines. LOD values forGUA and AMB, LOR were found to be 16.63µg/mL &1.55µg/mL, 0.20µg/mL respectively. LOQ values forGUA and AMB, LOR were found to be50.44µg/mL &4.71µg/mL, 0.62 µg/mL respectively.Fig 2Assay of the liquid dosage form:The proposed validated method was successfullyapplied to determine GUA and AMB,LOR in liquiddosage form. The results obtained for GUA and AMB,LOR were comparable with corresponding labeledamounts. The results were tabulated in table 4.Fig 3Table 6: Assay of the dosage formGUALabeledSample No.amount(mg/ tablet)12345650AverageSTD PURITYCAL% 956510.658502.850AMBSample 81199.249.69 mg99.39Labeledamount(mcg/tablet)Standard Area Sample .511Average1968.245STD PURITYCAL% Assay:FORCED DEGRADATION:a) Hydrolytic degradation under acidic condition: tosample 0.1N HCl is added and refluxed for 30minutes at 60oC.b) Hydrolytic degradation under alkaline condition:0.1N NaOH refluxed for 30 minutes at 64450.249Average 458.4458STD PURITYCAL% 7466.757465.190899.35.04mg100.76c) Oxidative degradation: to sample 1% of H2 O2 isadded and refluxed for 30minutes at 60od) Thermal degradation: Sample is exposed to 105oCfor about 1 hours.e) Photolytic degradation: Sample was exposed tosunlight for 24 hoursR. Vani et al, JGTPS, 2014, Vol. 5(4): 2248 - 22522251
Table 7: Forced degradation studyParameter Area of GUA Area of AMBAcidBaseSun lightHeat493.836502.845510.34527.851Formula to calculate assayAssay1949.451993.3051990.472052.678Area of%%% Degradation of AMBDegradation of LORLOR Degradation of .2500.1246.88.09.72. Net degradation Assay of sample – Assay ofdegradation sample.Acceptance criteria for forced degradation: the netdegradation should be between 1%-50%Result: Forced degradation is done to establish that themethod is suitable even at a high temperature. In table 7 itis seen that Even after the degradation there is no shift inretention time. This shows that even after degradationthere is no impact on method. By this it can be said thatthis method indicates “Stability”.CONCLUSIONS:The proposed method has advantage ofsimplicity and convenience for the separation andquantization of GUA and AMB, LOR in the combinationwhich can be used for the assay of their dosage form.Also, the low solvent consumption and short matographic procedure. The method is accurate,precise, rapid and selective for simultaneous estimation ofloratadine, ambroxol hydrochloride and guaiphenesin inits dosage form. Hence it can be conveniently adopted forroutine analysis.ACKNOWLEDGMENTS:The authors are grateful to Principal,Management of Deccan School of Pharmacy, Hyderabad,India for providing necessary facilities to carry out thisresearch project.REFERENCES:1. Gagandeep, Navdeep kaur Gill, Karan, G.S Sarma, ArtiThakkar * et al., developed Simultaneous determinationof Ambroxol, Guaifensine, levosalbutamol inpharmaceutical formulation with the use of fourrapidderivative spectrophotometric method, J. Chil.Chem. Soc. vol.57 no.4 Concepción 20123.4.5.6.7.8.M. Abdelkawy F. Metwaly, N. El Raghy M.Hegazy and N.Fayek Simultaneous determination ofAmbroxol Hydrochloride and Guaifenesin by HPLC,TLC-Spectrodensitometric and multivariate calibrationmethods in pure form and in Cough Cold Formulations,Journal of Chromatography and sepration technique,Published August 06, 2011Tripathi K.D. Essential of Medical Pharmacology, 5thEdn, Jaypee Brothers Medical publisher New Delhi.International Conference on Harmonization (ICH) ical for Human Use: Harmonized Triplicateguideline onValidation of Analytical procedures:Methodology, Recommended for Adoption at Step 4 ofthe ICH Process on November 1996 by The ICHSteering Committee, IFPMA, Switzerland.PatelPriyankaV*,ChaudhriBharatG spectrophotometricmethodforsimultaneousestimation of Ambroxol hydrochloride, loratadine,guaifensine, In combined syrup formulation, InventiRapid: Pharm Analysis & Quality Assurance,publication date: 2013/2/8 .Nirav C. Patel, Dipti B. Patel, Pruthviraj K. drochloride, Guaifenesin and LevosalbutamolSulphatein Syrup, Asian Journal Of Research InChemistry. Volume 6, Issue 4, Apr 2013.Krishna Veni,Nagappan MeyyanathanSN,Rajinikanth B Raja, Suresh Reddy, Jeyaprakash MR, ARP-HPLC Method for Simultaneous Estimation al Formulation. Research J. Pharm. andTech. 1(4): Oct.-Dec. 2008,Nirav C. Patel 1, Dipti B. Patel 1, Pruthviraj K.EstimationofChaudhari 1. nandLevosalbutamol Sulphatein SyrupHow to cite this article:R. Vani*, B. Vijaya kumar, G. Krishna Mohan: Analytical method development and validation for the determination ofLoratadine, Ambroxol Hydrochloride and Guaiphenesin using Reverse Phase HPLC method in bulk and Liquid DosageForm 5(4): 2248-2252. (2014)All 2010 are reserved by Journal of Global Trends in Pharmaceutical Sciences.R. Vani et al, JGTPS, 2014, Vol. 5(4): 2248 - 22522252
The validation of method was carried out utilizing ICH-guidelines. The described HPLC method was successfully employed for the analysis of pharmaceutical formulations containing combined dosage form. Keywords: Loratadine, Ambroxol Hydrochloride and Guaiphenesin, Reverse Phase HPLC, Validation. R. Vani1* ABSTRACT B. Vijaya kumar2 G. Krishna Mohan3
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