Development and Validation of RP-HPLC Method for - International ...

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701______________________________________________________________________Research PaperDevelopment and Validation of RP-HPLC Method for theestimation of ZafirlukastA. Lakshmana Rao 1* and V. Naga Jahnavi 21V. V. Institute of Pharmaceutical Sciences, Gudlavalleru, A. P., India2Aditya Institute of Pharmaceutical Sciences and Research, Surampalem, A. P., India__________________________________________________________________________________ABSTRACTA rapid and sensitive high performance liquid chromatographic method was developed for theestimation of zafirlukast in bulk drug. Zafirlukast was chromatographed on a reverse phase C 18 column in amobile phase consisting of acetone, methanol and 0.05M KH 2 PO 4 buffer (pH 3.0 adjusted withorthophosphoric acid) in the ratio 10:20:70 v/v. The mobile phase was pumped at a flow rate of 1.0 mL/minand eluents were monitored at 242 nm. The calibration curve was linear in the range of 10-70 µg/mL. Theintra- and inter-day variation was found to be less than 1% showing high precision. The mean recovery of thedrug from the solution containing 70µg/mL was 70.221 µg/mL, indicating high accuracy of the proposedmethod. Due to its simplicity, rapidness, high precision and accuracy of the proposed HPLC method may beused for estimating zafirlukast in bulk drug.Keywords: Zafirlukast , HPLC, Estimation.INTRODUCTIONZafirlukast 1 [Fig 1] is chemically, 4-(5-cyclopentyloxy-carbonylamino-1-methyl-indol-3-ylmethyl)-3-methoxy-N-o-tolylsulfonylbenzamidean anti-asthmatic drug. It is a selective andcompetitive receptor antagonist of leukotriene D4and E4, components of slow reacting substance ofanaphylaxis. Literature survey reveals that variousUV 2 , HPLC 3-6 and capillary electrophoresis 7methods have been reported for the determinationof zafirlukast in pure form. The aim of this study isto develop a simple, rapid, precise and accurateRP-HPLC method for the determination ofzafirlukast in bulk samples.Fig 1: Chemical structure of zafirlukast____________________________*Address for correspondence:E-mail: dralrao@gmail.comEXPERIMENTALChemicals and reagentsMethanol (HPLC grade), acetone (HPLC grade)was supplied by Standard Reagents, Hyderabad.Potassium dihydrogen phosphate andorthophosphoric acid of AR grade were obtainedfrom Qualigens Fine Chemicals Ltd., Mumbai.Zafirlukast was a gift sample by Sumages PharmaPvt. Ltd., Bhimavaram.InstrumentationThe separation was carried out on gradientHPLC system (Shimadzu) with Shimadzu binaryHPLC LC 20AT pump, Shimadzu APD 20A UV-Visible absorbance detector, Spinchrom softwareand RP-C 18 column (250mmx4.6mm I.D.; particlesize 5µm).Chromatographic conditionsThe mobile phase consisting of acetone,methanol and phosphate buffer (pH 3.0 adjustedwith orthophosphoric acid) were filtered through0.45 µ membrane, degassed and were pumped fromthe solvent reservoir in the ratio of 10:20:70 v/vand was pumped into the column at a flow rate of1.0 mL/min. The detection was monitored at242nm and the run time was 8 min. The volume ofinjection loop was 20 µL prior to injection of thedrug solution the column was equilibrated for atleast 30 min. with the mobile phase flowingVol. 1 (2) Oct – Dec 2010 www.ijrpbsonline.com 102

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701through the system. The column and the HPLCsystem were kept in ambient temperature.ProcedureStock solution of zafirlukast was prepared bydissolving 100 mg of zafirlukast in100 mL standardvolumetric flask containing 10 mL acetone. To this,20 mL of methanol was added, sonicated for 10min and finally the volume was made up with70mL of buffer, so as to give 1 mg/mL solution.Subsequent dilutions of this solution were madewith mobile phase to get concentrations of 10µg/mL - 70 µg/mL. The standard solutionsprepared as above were injected into the 20µL loopand the chromatogram was recorded in Fig 2. Theretention time of zafirlukast was found to be1.910min.Fig 2: Chromatogram of zafirlukastThe calibration curve was constructed byplotting concentration vs peak area ratio. Theamount of zafirlukast present in sample wascalculated through the standard calibration curve.The linearity experiment was carried out five timesto ascertain accuracy and precision of the method.The peak area ratios of the drug vs concentrationwere found to be linear and the results arefurnished in Table 1.Validation of proposed methodSelectivity of the method was assessed on thebasis of elution of zafirlukast using the abovementioned chromatographic conditions. To studythe specificity, linearity, precision, accuracy, limitof detection, limit of quantification, robustness andsystem suitability parameters has been validated forthe determination of zafirlukast. The results arefurnished in Table 2.Table 1: Calibration of HPLC methodConc. of zafirlukast(µg/ml)Peak Area*% C.V10 128.4814 0.935420 259.3664 0.712430 393.5928 0.982440 510.9160 0.932850 650.4222 0.254160 780.4138 0.925170 911.9268 0.7710*Mean of five determinationsTable 2: Validation summarySystem suitability ResultsTheoritical plates (N) 2910Linearity range (µg/mL) 10-70Retention time (min.) 1.910Asymmetric factor 2.000Correlation coefficient 0.9992LOD (µg/mL) 0.0104LOQ (µg/mL) 0.0314LinearityThe standard curve was obtained in theconcentration range of 10-70 µg/mL. The linearitywas evaluated by linear regression analysis usingthe least square method. It was found thatcorrelation coefficient and regression analysis arewithin the limits.PrecisionThe precision was determined in terms of intradayand inter-day precision. The intra-day andinter-day variation in the peak area of drug solutionwas calculated in terms of coefficient of variation(C.V.) obtained by multiplying the ratio of standarddeviation to mean with 100. The results arefurnished in Table 3.Limit of detection (LOD) and limit ofquantification (LOD)The LOD and LOQ for zafirlukast werepredicted basing on the parameters of standarderror of estimate and slope, calculated fromlinearity of the response data of zafirlukast.AccuracyThe accuracy of the HPLC assay methodwas assessed by adding known amount of drugsolution to a drug solution of known concentrationand subjecting the samples to the proposed HPLCmethod. The recovery studies were replicated 3times. The accuracy was expressed in terms ofrecovery and calculated by multiplying the ratio ofmeasured drug concentration to the expected drugconcentration with 100 so as to give the percentagerecovery. The results are furnished in Table 4.RESULTS AND DISCUSSIONBy applying the proposed method, the run timeof the method was set at 8 min and zafirlukastappeared on the typical chromatogram at 1.910min., which indicates a good base line. When thesame drug solution was injected 5 times, theretention time of the drug was same. Linearityrange was observed in concentration range of 10-70µg/mL. The regression equation of zafirlukastconcentration over its peak area ratio was found tobe X=0.063039Y-7.263371 (r=0.9992) where X isthe concentration of zafirlukast (µg/mL) and Y isthe respective peak area. The proposed HPLCmethod was also validated for intra-day and interdayvariation. The coefficient of variation in theVol. 1 (2) Oct – Dec 2010 www.ijrpbsonline.com 103

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701peak area of the drug for 5 replicate injections wasfound to be less than 1%. The asymmetry factorwas found to be 2.000, which indicated asymmetricnature of peak. The number of theoretical plateswas found to be 2910, which indicates efficientperformance of the column. The limit of detectionand limit of quantitation was found to be 0.0104µg/mL and 0.0314 µg/mL, indicates the sensitivityof the method. To optimize the chromatographicconditions, various combinations of acetone,methanol and phosphate buffer were tested. Theuse of acetone, methanol and phosphate buffer inthe ratio of 10:20:70 v/v resulted in peak with goodshape and resolution. The high percentage ofrecovery of zafirlukast, 100.706 indicates that theproposed method is highly accurate.Table 3: Inter- and intra-day precisionConcentrationof zafirlukastMeasured concentration of zafirlukast(g/ml)(µg/ml)Intra-day Inter-dayMean % C.V Mean % C.V(n=3)(n=3)20 23.718 0.6512 23.463 0.752040 39.631 0.3968 39.462 0.167560 56.924 0.6484 56.383 0.97205. Ficarra R, Ficarra P, Tommasini S, Melardi S,Calabro ML, Furlanetto S and Semreen M.Validation of a LC method for the analysis ofzafirlukast in a pharmaceutical formulation. JPharm Biomed Anal 2000;23(1):169-174.6. Bui KH, Coleen MK, Connie TA, Bruce KB.Determination of zafirlukast, a selectiveleukotriene antagonist, human plasma bynormal-phase high-performance liquidchromatography with fluorescence detection. JChromatogr B 1997;696 (1):131-136.7. Suslu I, Demircan S, Altinoz S and Kir S.Optimisation, validation and application of acapillary electrophoretic method for thedetermination of zafirlukast in pharmaceuticalformulations. J Pharm Biomed Anal2007;44:16-22.Amountof drugadded(µg/mL)CONCLUSIONTable 4: Recovery studiesMean (±SD)amount found(µg) (n=3)Mean % ofrecovery(n=3)30 30.541(±0.976) 101.80350 50.000(±0.071) 100.00070 70.221(±0.723) 100.316The proposed HPLC method was found to besimple, rapid, precise, accurate and sensitive for theestimation of zafirlukast in bulk drug. Hence, thismethod can easily and conveniently adopt forroutine analysis of zafirlukast in pure form.REFERENCES1. The Merck Index, 13 th Edition, Merck & Co.,Inc., White House Station, NJ, (2001) 1806.2. Suslu I and Altinoz S. UV spectrophotometricdetermination of zafirlukast in pharmaceuticalformulations. Hacettepe University Journal ofthe Faculty of Pharmacy 2007;27(1):33-46.3. Suslu I and Altinoz S. A reversed-phase highperformanceliquid chromatographic method forthe determination of zafirlukast inpharmaceutical formulations and humanplasma. J AOAC Int 2006;89(6):1557- 1572.4. Radhakrishna T, Satyanarayana J andSatyanarayana A. Determination of zafirlukastby stability indicating LC and derivativespectrophotometry. J Pharm Biomed Anal2002;30(3):695-703.Vol. 1 (2) Oct – Dec 2010 www.ijrpbsonline.com 104