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 DEVELOPMENT AND VALIDATION OF RP-UPLC ANALYTICAL METHOD FOR SIMULTANEOUS ESTIMATION OF EMTRICITABINE, RILPIVIRINE, TENOFOVIR DISOPROXIL FUMARATE AND ITS PHARMACEUTICAL DOSAGE FORMS

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(1)Kavitha K.Y et al. IRJP 2013, 4 (1) INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com. ISSN 2230 – 8407 Research Article. DEVELOPMENT AND VALIDATION OF RP-UPLC ANALYTICAL METHOD FOR SIMULTANEOUS ESTIMATION OF EMTRICITABINE, RILPIVIRINE, TENOFOVIR DISOPROXIL FUMARATE AND ITS PHARMACEUTICAL DOSAGE FORMS Kavitha K.Y1*, Geetha.G1, Hariprasad.R1, Venkatnarayanan.R2, Kaviarasu. M.1 1PSG College of Pharmacy, Peelamedu, Coimbatore, Tamilnadu, India 2RVS College of Pharmacy, Sulur, Coimbatore, Tamilnadu, India Article Received on: 14/11/12 Revised on: 10/12/12 Approved for publication: 29/12/12. *E mail: kaviky1978@rediffmail.com ABSTRACT Developing a single analytical method for estimation of individual drug from a multidrug composition is a very challenging task. A simple, rapid, precise, and reliable simultaneous RP -UPLC method was developed for the separation and estimation of three drugs emtricitabine, rilpivirine and tenofovir in bulk drug mix and pharmaceutical dosage forms. Chromatography was carried on an aquity BEH C18 column using acetonitrile and phosphate buffer pH 3 in ratio of 55:45 as mobile phase at a flow rate of 0.35 ml/min with detection at 261 nm. The retention times of the emtricitabine, tenofovir disoproxil fumerate and rilpivirine were about 0.6, 1.05 and 2.95 minutes respectively. The detector response is linear from 12-48μg/ml and 3.2-12.80μg/ml concentration for emtricitabine, tenofovir and rilpivirine respectively. The respective linear regression equation being Y=32727.95 + 6072.57 for emtricitabine, Y=17956.31x – 1066.28 for tenofovir disoproxil fumerate and Y=28648.79x + 1739.92 for rilpivirine. The limit of detection and Limit of quantification was 0.03, 0.06 and 0.07 μg/ml and 0.08, 0.15 and 0.18μg/ml for emtricitabine, tenofovir and rilpivirine respectively. The percentage assay of emtricitabine, tenofovir disoproxil fumerate and rilpivirine were about 99.50, 100.2 and 99.46 % respectively and percentage recovery for average of five different concentrations were 99.65%, 99.62% and 100.34% respectively. The method was validated as a final verification of method development with respect to precision, linearity, accuracy, ruggedness, and robustness. The validated method was successfully applied to the commercially available pharmaceutical dosage form, yielding very good and reproducible result. KEYWORDS: Emtricitabine, Tenofovir disoproxil fumerate , Rilpivirine, UPLC, Validation.. INTRODUCTION The multi drug combinations of nucleoside reverse transcriptase inhibitors (NRTI’s) and non nucleoside reverse transcriptase inhibitors (NNRTI’s) are effective in the therapy of Human Immunodeficiency Virus (HIV) infection.It is used as a part of highly active Anti-Retroviral Therapy (HAART), for the treatment of HIV.1,2 Three drug FDC comprising of Emtricitabine,Tenofovir disoproxil fumarate, Rilpivirine form one of the first line regimensin HIV-therapy.3 Emtricitabine The chemical name of emtricitabine is 5fluoro-1-[(2R,5S)-2(hydroxymethyl)-1,3-oxathiolan-5yl]cytosine. Emtricitabine is the (-) enantiomer of a thio analog of cytidine, which differs from other cytidine analogs in that it has a fluorine in the 5-position.4,5. Rilpivirine It is available as the hydrochloride salt. The chemical name for rilpivirine hydrochloride is 4-[[4-[[4-[(E)2-cyanoethenyl]-2,6-dimethylphenyl]amino]2pyrimidinyl]amino]benzonitrile monohydrochloride. 4,5. Tenofovir disoproxil fumarate Tenofovir DF is a fumaric acid salt of the bis isopropoxy carbonyl oxymethyl ester derivative of tenofovir. The chemical name of tenofovir DF is [[bis[[(isopropoxycarbonyl)oxy]methoxy] 9-[(R)-2 phosphinyl] methoxy] propyl]adenine fumarate (1:1). 4,5 On the literature survey it was found that various above mentioned antiretroviral drugs were estimated by several analytical methods either alone or in combination by HPLC417 ,HPTLC18,19, Spectrophotometric methods20-30etc. There is. no reported method for estimation of emtiricitabine, tenofovir disoproxil fumarate and rilpivirine in their tablet dosage form by RP-UPLC. The present work describes a simple, precise and accurate reversed phase UPLC method for the simultaneous estimation emtiricitabine, tenofovir disoproxil fumarate and rilpivirine in combined dosage form. The method was validated according to procedures and acceptance criteria based on FDA guidelines31 and recommendations of ICH.32 MATERIALS AND METHODS Materials Emtricitabine, Tenofovir disoproxil fumerate and Rilviprine were obtained as a gift sample from Aurobindo Pharma Ltd, Hyderabad. HPLC grade Methanol, Acetonitrile (Merck) and AR grade sodium dihydrogen orthophosphate (Merck) was used. Milli-Q water was used in mobile phase preparation. Commercially available Emtricitabine,Tenofovir disoproxil fumerate and Rilviprine dosage forms (Complera/Eviplera , Gilead Sciences) were purchased from market. Equipments UV-Visible spectrophotometer used was Shimadzu, Model1650. The UPLC instrument used was waters Acquity system. Empower Software was used for data acquisition. A Sartorius analytical micro balance, an ultra sonic cleancer, pH meter L1610ELICO, micropipettes and micro-pore filtration assembly etc were also used. Analytical Method Chromatographic Condition The analysis was carried out on Acquity BEH C 18 column (50 x 2.1 mmi.d., 1.7µm). The mobile phase consisted of a mixed and degassed solution of acetionitrile and phosphate buffer in the ratio of 55:45(v/v).the buffer solution was prepared and adjusted to pH 3 with orthophosphoric acid filtered through a 0.22 µm membrane filter . Maintained column oven temperature at 25°C and detection with PDA Page 150.

(2) Kavitha K.Y et al. IRJP 2013, 4 (1) detector at 261nm. The injection volume was 5 µl. the flow rate of mobile phase was maintained at 0.35ml/min. Standard and Sample Preparation Standard stock solutions were prepared by dissolving 40mg of emtricitabine, 60mg of tenofovir disoproxil fumarate and 5mg of rilpivirine in 10ml of mobile phase and sonicated for 15min and the volume was made upto 100ml with mobile phase. From the standard stock preparation 10ml solution was taken into 100ml flask, further diluted with mobile phase to get the concentration of 40µg/ml, 60µg/ml and 5µg/ml emtiricitabine, tenofovir and rilpivirine respectively. Twenty tablets were accurately weighed and crushed into a. fine powder. The powder equivalent to one tablet was taken in 250 ml volumetric flask. About 150 ml diluent was added, shaken for 5min on rotary shaker and then sonicated for 15mins with intermediate shaking. Then the volume was finally made upto the mark (250ml). Sample solution was centrifuged at 5000 rpm for 5 min to get a clear solution. Then supernatant solution was diluted to twenty times to get a final sample solution of a concentration of 40, 60 and 5 µg/ml for the emtricitabine, tenofovir disoproxil fumerate and rilpivirine. These solutions were filtered through membrane filter.. Table 1 System Suitability Parameters Compound Parameter Emtricitabine Tenofovir Rilpivirine Retention time(Rt) in min ±%RSD 0.60±0.09 1.09±0.05 2.95±0.04 Tailing factor(Tf) 0.91 1.02 1.09 Injection repeatability(%RSD)* 0.095 0.129 0.262 Resolution(Rs)** 9.34 12.94 Capacity factor(K’) 1.33 4.54 7.63 Theoretical plate(N) 2427.94±0.60 4079.71±0.48 6453.73±0.25 RSD = Relative standard deviation, *RSD of peak area of five consecutive injections at a concentration of 10µg/ml of each drug., ** Resolution between (Emtricitabine & Tenofovir) &( Tenofovir & Rilpivirine). Compound emtricitabine tenofovir rilpivirine.. Table 2 Linearity Parameters for Calibration Curve of the Three Drugs(n=6) Slope Intercept Concentration range (µg/ml) Mean ±SD CV% Mean ±SD 12-48 32727.95 ± 272.57 2.50 16556±529.1 12-48 17956.31± 106.28 3.01 8765±241.5 3.2-12.80 28648.79± 173.92 1.92 16937±483.1. R2 CV% 3.19 2.91 2.85. 0.9995 0.9998 0.9996. Table 3 Intraday and Interday Precision Results of emtricitabine, tenofovir and rilpivirine. Compound %RSD(Intraday) %RSD(Interday) emtricitabine 0.51 0.56 tenofovir 0.48 0.54 rilpivirine 0.59 0.65 Drug emtricitabine. tenofovir. rilpivirine. Level% 50 100 150 50 100 150 50 100 150. Table 4 Results of Accuracy Study Amount added(µg/ml) Amount found(µg/ml) 15.00 14.97 30.00 29.98 45.00 44.78 15.00 14.97 30.00 29.95 45.00 44.99 4.00 3.96 8.00 7.79 12.00 11.94. % recovery 99.85 99.89 99.74 99.94 99.85 99.68 98.58 98.99 99.09. Mean recovery% 99.83. SD 0.21. % RSD 0.21. 99.44. 0.59. 0.59. 99.11. 0.71. 0.72. Robust condition Flow rate0.34 ml/min Flow rate0.36 ml/min Buffer :CAN(53:47) Buffer :CAN(57:43) Column temperature 23°C Column temperature 27°C Wavelength 259nm Wavelength 263nm. Table 4 Evaluation Data of Robustness Study of emtricitabine System Suitability Parameters % assay asymmetry Theoretical plates 100.08 2440 1.24 99.77 2456 1.22 99.63 2453 1.25 100.01 2456 1.24 99.88 2434 1.23 100.03 2432 1.24 99.99 2458 1.24 100.11 2478 1.25. % RSD 0.23 0.20 0.24 0.47 0.27 0.19 0.28 0.27. Robust condition Flow rate0.34 ml/min Flow rate0.36 ml/min Buffer :CAN(53:47) Buffer :CAN(57:43) Column temperature 23°C Column temperature 27°C Wavelength 259nm Wavelength 263nm. Table 5 Evaluation Data of Robustness Study of tenofovir System Suitability Parameters % assay asymmetry Theoretical plates 101.08 4141 1.15 99.77 4160 1.02 99.93 4059 1.17 101.01 4156 1.10 99.85 4034 1.13 101.03 4138 1.14 98.99 4057 1.19 99.81 4170 1.16. % RSD 0.28 0.27 0.26 0.37 0.27 0.16 0.38 0.25. Page 151.

(3) Kavitha K.Y et al. IRJP 2013, 4 (1) Robust condition Flow rate0.34 ml/min Flow rate0.36 ml/min Buffer :CAN(53:47) Buffer :CAN(57:43) Column temperature 23°C Column temperature 27°C Wavelength 259nm Wavelength 263nm Day 1. 2. Complera. Table 6 Evaluation Data of Robustness Study of rilpivirine System Suitability Parameters % Assay Theoretical plates Asymmetry 99.98 6351 1.10 99.97 6460 1.12 99.95 6409 1.07 101.09 6415 1.11 99.97 6403 1.16 99.83 6399 1.04 100.99 6407 1.09 99.91 6417 1.10. Table 7 RSD of the Drugs on Different Days and Different Analysts Analyst Component Rt %RSD Stand. area % RSD 1 Emtricitabine 0.03 0.01 Tenofovir 0.08 0.02 0.03 0.01 Rilpivirine 2 Emtricitabine 0.04 0.04 Tenofovir 0.06 0.02 Rilpivirine 0.07 0.01 Table 8: Estimation of the Drugs in Commercial Samples. Component Label claim Acquired data 199.01mg/tab Emtricitabine 200 mg/tab Tenofovir 300.06mg/tab 300 mg/tab Rilpivirine 25 mgltab 24.85mg/tab. % RSD 0.18 0.17 0.26 0.23 0.27 0.19 0.29 0.27. Sample area %RSD 0.04 0.02 0.02 0.05 0.06 0.07. Assay% 99.50% 100.2% 99.46%. Fig 1 Typical Chromatogram of Standard Mixture of emtricitabine(0.6min),tenofovir(1.05min) and rilpivirine (2.95 min). Fig 2 Linearity Graph for emtricitabine. Page 152.

(4) Kavitha K.Y et al. IRJP 2013, 4 (1). Fig 3 Linearity Graph for Tenofovir disoproxil fumarate. Fig 4 Linearity Graph for rilpivirine. RESULT AND DISCUSSION For successful method validation, preliminary tests were performed with the objective to select adequate and optimum conditions. Parameters, such as choice of analytical column, pH of buffer, mobile phase composition and proportion, detection wavelength and other factors were exhaustively studied. Various reversed columns were used and isocratic mobile phase system was tried. Development Trial 1 When chromatography was carried out at 25° C on a 250 × 4.6 mm i.d., 5μm Phenomenex Gemini C18 column with the isocratic mobile phase of 0.02 M aqueous phosphate buffer and methanol (30:70 v/v, pH 4.0) at a flow rate of 1.0 ml/min, late elution of analyte with peak tailing and high column pressure was observed. Hence, the second experiment was carried out with acetonitrile as an organic modifier. Development Trial 2 When chromatography was carried out at 25° C on a 250 × 4.6 mm i.d., 5μm Phenomenex Gemini C18 column with the isocratic mobile phase of 0.1 % orthophosphoric acid and acetonitrile (50:50 v/v) at a flow rate of 1.0 ml/min, a satisfactory separation of the three drugs was achieved with good resolution and minimal tailing. Method Transfer on UPLC With an aim to develop a rapid, sensitive, simple and reliable UPLC method which can estimate the three components in less than five minutes. Hence, the above satisfactory separation of the three drugs was achieved on a Acquity BEH. C18 (50 x 2.1 mm,1.7 µm)column with a mobile phase of acetonitrile and phosphate buffer pH 3 (55:45, v/v) at a flow rate of 0.35 ml/min. Quantification was achieved with PDA detection at 261 nm based on the peak area. Better resolution of the peaks with clear base line separation was found. The emtricitabine, tenofovir disoproxil fumarate and rilpivirine were eluted at 0.6 min,1.09 min and 2.95 min respectively. Fig 1 Method Validation The developed LC method was validated to confirm that the method was suitable for its intended purpose as described in ICH guidelines .the described method extensively validated in terms of specificity, system suitability, linearity, accuracy, precision, limit of detection and limit of quantification and robustness. System sutability The system suitability tests represent an integral part of the method and are used to ensure adequate performance of the chromatographic system. The parameters, retention time theoretical plates, tailing factor, peak asymmetry and repeatability were evaluated using five replicate injections of the drugs at a concentration of emtricitabine(30 µg/ml), tenofovir disoproxil fumarate(30 µg/ml) and rilpivirine 8 µg/ml). acceptance criteria for system sutability, asymmetry should not be more than 2.0, theoretical plate should not be less than 2000 and %RSD of peak area should not be more than 2.0, were full fill during all variation parameters. The Page 153.

(5) Kavitha K.Y et al. IRJP 2013, 4 (1) result of system suitability study of the developed assay method was shown in the table1 Specificity and Selectivity The selectivity of the method is depicted by the three sharp well resolved peaks for emtricitabine, tenofovir disoproxil fumarate and rilpivirine obtaine at their retention times, 0.6 min,1.09 min and 2.95 min respectively. The specificity of the method was checked by comparison of chromatograms obtained from standard, sample and the corresponding placebo. The retention time of the drug standard and the drugs from sample solutions were identical, conforming specificity of the method. The method was also selective because there was no interference observed from any of the excipients in the tablets formulation tested. Linearity The linearity of this method was determined at seven concentration levels ranging from 12-48μg/ml and 3.212.80μg/ml concentration for emtricitabine, tenofovir and rilpivirine respectively. The calibration curves were constructed by plotting peak area versus concentration of emtricitabine, tenofovir and rilpivirine . The respective linear regression equation being Y=32727.95x + 6072.57 for emtricitabine fig 2, Y=17956.31x – 1066.28 for tenofovir disoproxil fumerate fig 3 and Y=28648.79x + 1739.92 for rilpivirine fig 4. Where x is the concentration in µg/ml and y is the peak area in absorbance unit. Limit of Detection(LOD) and Limit of Quantification(LOQ) The limit of detection and limit of quantification were evaluated by serial dilutions of for emtricitabine, tenofovir and rilpivirine stock solution in order to noise ratio of 3:1 for LOD and 10:1 for LOQ as per ICH guideline. The LODs for for emtricitabine, tenofovir and rilpivirine were found to be 0.03, 0.o6 and 0.07 µg/ml, while LOQs were 0.08,0.15 and 0.18 µg/ml. Precision Study The method precision (intraday) study showed that the results of percentage assay in six different samples preparation of same sample were within limits(%RSD<2) as shown in Tables 3. The intermediate precision study was performed within laboratory variation by different analysis, on different days, different instruments and different column by using standard and sample solution of the same sample in method precision and the results were compared with method precision. Accuracy The accuracy of the method was evaluated in triplicate at the concentration levels 50, 100, and 150% of the target test concentration (30µg/ml of emtricitabine, tenofovir and 8 µg/ml rilpivirine). The percentages of the recoveries were calculated. The percentages of the recoveries obtained were 99.8± 0.21, 99.44±0.72 and 99.11±0.59 for emtricitabine, tenofovir and rilpivirine respectively(Table 4). The recovery of the method was good. Robustness The robustness was studied by evaluating the effect of small but deliberate variation in the chromatographic conditions. Te conditions studied were flow rate (±0.01), composition of mobile phase (53:47 and 57:43,v/v),column temperature (±2°) and wavelength of detection(±5nm). The result of robustness study of the developed assay method was established in table 5 ,6 &7. Ruggedness To test the ruggedness of the method, the analysis was done on different days and different chemists to check for any. changes in the chromatograph. The percentage RSD for the retention time and area was calculated.Data acquired and compared, % RSD of area and Rt has been calculated and tabulated in Table 7. Based on the data, it is evident that the method is Rugged. Performance of the Drug/Batch Analysis One market sample have been analysed to see the performance of the method. Tablet taken was complera which contains 200 mg of emtricitabine, 300 mg of tenofovirdisoproxil fumarate and 25 mg of rilpivirine. Results obtained have been summarized in the Table 8. Stability of Sample Solution The sample solution was stable up to 48 hr at 5°C temperature and did not show any appreciable change in sample area. CONCLUSION Thus, to summarize, the proposed UPLC method of analysis was found to be precise and accurate, as depicted by the statistical data of analyis. The developed method is nontedious, with a very simple mobile phase composition, extremly small flow rate (0.35ml/min), and relativelyshort runtime, i.e., 5.0 min utilizing an isocratic elution mode with wavelenth monotoring. All these factors enable rapid quantitation and simultaneous analysis of the three antireteroviral drugs in bulk and from commercial formulation with out any excipient interference. It can, therefore, be concluded that the reported method could find practical application as an economical and rapid quality –control tool for simultaneous analysis of the thre drugs from their combined dosage forms in both research and indutrial quality –control laboratories. REFERENCES 1. Rouzes A, Berthoin K, Xuereb F. Simultaneous Analysis of Antiretroviral drugs, Journal of chromatography B 2004; 813, 209-216. 2. Madhusudan reddy Induri, Bhagavan Raju M, Rajendra Prasad Y, Pavan kumar reddy K and Sarva Raidu CH. Simultaneous quantification of Efavirenz and Lamivudine in tablet dosage form by liquid chromatography. Indian Journal of Pharmaceutical Education and Research 2011; 45Suppl 4: 305-309. Raffanti S, Haas D, Anti Microbial agents: Anti-retroviral agents, Mc 3. Graw-Hill, New York, 1990. 4. C.Vanitha,P.Vishnuvardan,R.Ramalingam,M.Alagarraja.Simultaneous determination of efavirenz,Lamivudine and tenofovir disoproxil fumarate in tablet dosage form by using RP-HPLC. Journal of Pharmacy Research, 2012;5 suppl 6:3185-3188. 5. Sentenac S, Fernadez C, Thrillers A lechat P, Aymard G.Sensitive determination of tenofovir in human plasma samples using reversedphase liquid chromatography, J chromatogr, B 2003; 793 Suppl 2: 317324. 6. Kandagal PB, Manjunatha DH, Seetharamappa JS, Kalanur S. RPHPLC Method for the Determination of Tenofovir in Pharmaceutical Formulations and Spiked Human Plasma. Analytical Letters 2008 41 Suppl 4: 561-570. 7. Appala raju N, Begum S, Simultaneous. RP-HPLC Method for the estimation of the Emtricitabine, Tenofovir Disoproxil fumarate and Efavirenz in tablet dosage forms. Res J Pharm Techn 2008; 1 Suppl 4: 522-525. 8. Rajesh S, Pooja G. A Validated RP-HPLC Method for Simultaneous Estimation of Emtricitabine and Tenofovir Disoproxil Fumarate in a Tablet Dosage Form. Eurasian J Anal Chem 2009; 4 Suppl 3:, 276284. Konda Ravi Kumar, A. Suneetha, N.Srilakshmi. A validated LC 9. method for the estimation of rilpivirine in API and pharmaceutical dosage form.2012;5 Suppl 8:4434-4436. 10. TripatiSharma, neelam Mishra,Swapan k.Moitra Sudam C.S.I, Dannana G. Sankar. A validated RP-HPLC method for estimation of Tenofovir disoproxil fumarate in bulk and pharmaceutical ormulation.2012;5 Suppl 3:108-110. 11. Droste, J. A. H.; Aarnoutse, R. E. Burger, D. M.Determination of Emtricitabine in Human Plasma using HPLC with Fluorometric. Page 154.

(6) Kavitha K.Y et al. IRJP 2013, 4 (1) 12. 13.. 14.. 15.. 16. 17.. 18. 19. 20.. 21.. Detection. Journal of Liquid Chromatography & Related Technologies, 2007; 30 Suppl 10: 2769-2778. Mangaonkar K, Desai A. Simultaneous estimation of Emtricitabine, Tenofovir disoproxil fumerate & Efavirenz from tablets by RP-HPLC method. Indian Drugs 2008; 45: 188-192. Sentenac S, Fernandez C, Thuillier A, Lechat P, Aymard G. Sensitive determination of Tenofovir in human plasma samples using reversedphase liquid chromatography. Journal of chromatography B Analytical Technology Biomed Life Sciences 2001; 793: 317-324. Mangaonkar K, Desai A. Simultaneous estimation of Lamivudine and Tenofovir disoproxil fumerate in tablets by isocratic reverse phase high performance liquid chromatographic method. Indian Drugs 2008; 45; 119-122. Sentenac S, Fernandez C, Thuillier A, Lechat P, Aymard G. Sensitive determination of tenofovir in human plasma samples using reversedphase liquid chromatography. J Chromatogr B Analyt Technol Biomed Life Sci. 2001;793:317–24. Kandagal PB, Manjunatha DH, Seetharamappa J, Kalanur SS. RPHPLC method for the determination of tenofovir in pharmaceutical formulations and spiked human plasma. Anal Lett.2008;41:561–70. D.Pranitha,Vanitha.C. Prince Francies, M.Alagar Raja, Vishnuvardhan, Surender M.Daniel Balaji.Simulataneous estimation of emtiricitabine, tenofovir disoproxil fumarate and rilpivirine in bulk form by RPHPLC method. Journal of Pharmacy, 2012;5 Suppl 8:4600-4602 Joshi M, Nikalje AP, Shahed M, Dehghan M. HPTLC method for the simultaneous estimation of emtricitabine and tenofovir in tablet dosage form. Indian J Pharm Sci 2009;71 Suppl 1:95-97. Pradeep K, Dwivedi SC, Ashok K. Validated HPTLC Method for the Determination of Tenofovir as Bulk Drug and in Pharmaceutical Dosage Form. Res J Chem Sci 2011;1 Suppl 7, 2011, 33-37. Sapnil A Ghorpade, Monali S. Sali, Atul H. Kategaonkar, Dhaval M. Patel, Vishnu P, Choudhari Bhanudas, S Kuchekar. Simultaneous determination of emtricitabine and tenofovir by area under curve and dual wavelength spectrophotometric method. Journal of the Chilean Chemical Society,2010;55 Suppl 1: 115-117. Pratap Reddy, I.E. Chakravarthy. New spectrophotometric determination of Tenofovir disoproxil fumarate in bulk and pharmaceutical dosage form. Journal of applied chemistry 2012;1(2):29-33.. 22. T Shela Rani,K.Sujatha,K.Chitra, Don Mathew Jacob, Ramya yandapalli, D.Manoj and B.Sushma.Spectrophotometric method for estimation of tenofovir disoproxil fumarate in tablet.Research and review journal of pharmaceutical 2012;1Suppl 1:9-12. 23. Onah JO, Ajima U.Spectrophotometric Determination of Tenofovir Disoproxil Fumarate After Complexation with Ammonium Molybdate and Picric Acid. Int J Drug Dev & Res,2011 Jan-March; 3 Suppl 1:199204. 24. Sudha T, Saminthan J, Hemlatha PV, Ravikumar VR. Estimation of Tenofovir and Emtricitabine in Bulk and in tablet Dosage form by Spetrophotometric method. Int J Biopharmaceutics 2010; 1; 26-30. 25. Anindita B, Aurobinda P, Amit KM, Dannana GS, Swapna KM, Sudam CSI. Development and Validation of Spectrophotometric method for determination of Emtricitabine and Tenofovir Disoproxil Fumarate in Bulk and Tablet dosage Form. Int J Pharm Tech Res 2011; 3 Suppl 3: 1874-1882. 26. Arjun Goje, Yogeswaran.P,David Banji.Spectrophotometric Method for the Simultaneous Estimation of Lamivudine and Tenofovir disoproxil fumarate in Pure and in Tablet Formulation, Der Pharmacialettre2010; 2 Suppl 5: 221-228. RP, 27. Ramesh J, Arul Prakasam KC, Sowjanya C, Chintham Morampudi. Simultaneous Estimation of Lamivudine and Tenofovir Disoproxil Fumarate in Pure and in Tablet Formulation by First Derivative Spectrophotometric Method. IJPI’S J Anal Chem 20011; 1Suppl 6: 16-22. 28. Bojja sowmiya, Thimmaraj N, Manisha kumar,Nerella Raghunathan. Simultaneous determination of Tenofovir disoproxil fumarate and lamivudine by uv spectrophotometric method. International journal of pharmacy and pharmaceutical science research 2012;2Suppl 1: 9-15. 29. Atul AA, Charushila HB, Sanjay JS. Application of UVSpectrophotometric methods for estimation of tenofovir Disoproxil fumarate in tablets. Pak J Pharm Sci 2009; 22Suppl 1: 27-29. 30. Patel S, Baghel US, Rajesh P, Prabhakar D, Engla G, Nagar PN. Spectrophotometric method developm fumarate and Emtricitabine in bulk drug and tablet dosage form. Int J Pharm Clinic Res2009; 1 Suppl 1: 28-30. 31. Bioanalytical Method Validation Guidance for Industry, CDER, FDA, US Department of Health and Human Service, Rockville, MD (2001). 32. ICH Hermonized Tripartite Guidance for Validation of Analytical Procedure: Methodology (1996).. Source of support: Nil, Conflict of interest: None Declared. IRJP is an official publication of Moksha Publishing House. Website: www.mokshaph.com. All rights reserved.. Page 155.

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Figure

Table 2 Linearity Parameters for Calibration Curve of the Three Drugs(n=6)
Table 6 Evaluation Data of Robustness Study of rilpivirine
Fig 4  Linearity Graph for rilpivirine

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