Page561 Indo American Journal of Pharmaceutical Research, 2013 ISSN NO: 2231-6876 Journal home page: http:///index.php/en/ INDO AMERICAN JOURNAL OF PHARMACEUTICAL RESEARCH ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF SUNITINIB MALATE IN BULK AND ITS PHARMACEUTICAL DOSAGE FORM USING RP-HPLC Iffath Rizwana 1, Dr. K. Vanitha Prakash 2 *, Dr. G. Krishna Mohan 3 1 Research Scholar, School of Pharmaceutical Sciences, JNTU-K, Kakinada & DeccanSchool of Pharmacy, Hyderabad, A.P. India 2 Department of Pharmaceutical Analysis, SSJ College of Pharmacy, Gandipet,Hyderabad, A.P. India. 3 Centre for Pharmaceutical Sciences, IST, JNTU Hyderabad, A.P. India. ARTICLE INFO Article history Received27/12/13 Available online 31/01/14 Keywords Validation, RP-HPLC, Sunitinib malate ABSTRACT A simple, specific and accurate RP-HPLC method was developed for the determination of Sunitinib malate in bulk and its pharmaceutical dosage form. Hypersil ODS C18 (4.6 x 150mm, 5 m,) column was used in isocratic mode with mobile phase containing Acetonitrile : phosphate buffer ph 3.0 (ph adjusted with orthophosphoric acid) in the ratio of 50:50 v/v. The flow rate was 1.0 ml/ min and eluents was monitored at 248nm. The retention time Sunitinib malate was found to be 2.567 min, respectively. The linearity for Sunitinib malate was in the range of 10-50 µg/ml respectively. The mean recovery of Sunitinib malate was found to be 99.5%, respectively. The proposed method was validated according to ICH guidelines and successfully applied to the estimation of Sunitinib malate in capsule dosage form. The developed method was very simple, economic, accurate and precise and can be used for routine quality control examination of capsules of Sunitinib malate. Corresponding author Dr. K. Vanitha Prakash M. Pharm, Ph.D Professor and Principal, SSJ College of Pharmacy, Gandipet, Hyderabad, A.P. India +91-9885355562, iffathriz@gmail.com prakash.karanam@gmail.com Please cite this article in press as Dr. K. Vanitha Prakash et al. Analytical Method Development And Validation for the Estimation of Sunitinib Malate in Bulk and its Pharmaceutical Dosage Form Using RP-HPLC. Indo American Journal of Pharm Research.2014:4(01). Copy right 2013 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Page562 INTRODUCTION Figure 1: Structure of Sunitinib Malate Sunitinib is an orally available inhibitor of multiple tyrosine kinases. Sunitinib has proven efficacy as single agent in several solid tumor types and is approved for use in advanced renal cell cancer (RCC) and imatinib-resistant or intolerant gastrointestinal stromal tumors (GISTs) (1,2). N-(2-diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-2,4-dimethyl-1Hpyrrole- 3-carboxamide (Chemical structure was shown in Fig.1)Sunitinib inhibits cellular signaling by targeting multiple receptor tyrosine kinases (RTKs).These include all receptors for platelet-derived growth factor (PDGF-Rs) and vascular endothelial growth factor receptors (VEGFRs), which play a role in both tumor angiogenesis and tumor cell proliferation. The simultaneous inhibition of these targets therefore leads to both reduced tumor vascularization and cancer cell death, and ultimately tumor shrinkage. Sunitinib also inhibits KIT (CD117), the RTK that (when improperly activated by mutation) drives the majority of gastro intestinal stromal cell tumors. It has been recommended as a second-line therapy for patients whose tumors develop mutations in KIT that make them resistant to imatinib, or who become intolerant to the drug [3,4]. In addition, sunitinib inhibits other RTKs. These include: like RCC, GIST does not generally respond to standard chemotherapy or radiation. Imatinib was the first cancer agent proven effective formetastatic GIST and represented a major development in the treatment of this rare but challenging disease. However, approximately 20% of patients do not respond to imatinib (early or primary resistance), and among those who do respond initially, 50% develop secondary imatinib resistance and disease progression within 2 years. Prior to sunitinib, patients had no therapeutic option once they became resistant to imatinib [5,6]. Literature survey revealed that there are very few methods reported on determination of sunitinib in human plasma using LC/MS/MS [7, 8] and in Sweat of a Patient using LC/MS/MS [9]. An Rp-HPLC method [3] was reported on the biological samples. As far the knowledge of the authors were concerned there is no method reported on the estimation of sunitinib in bulk and pharmaceutical dosage forms using Rp-HPLC, so a simple, accurate, precise, reproducible method was developed and validated according to ICH guidelines [10] for the estimation of sunitinib in bulk and pharmaceutical dosage form. MATERIALS AND METHODS A Waters HPLC system consisting of a Water 2695 binary gradient pump, an inbuilt auto sampler, a column oven and Water 2487 dual wavelength absorbance detector (DAD) was employed throughout the analysis. The data was acquired using Empower 2 software. The column used was Hypersil ODS C18 (4.6 x 150mm, 5 m,) sonerex sonicator was used for enhancing dissolution of the compounds. An Adwa digital ph meter was used for ph adjustment. Analytically pure Sunitinib malate was obtained as gift sample from Natco Pharma Ltd., (Hyderabad, India). The capsules of sunitinib was purchased from local pharmacy. Acetonitrile, methanol, water (E. Merck, Mumbai, India) were of HPLC grade, while ortho-phosphoric acid and potassium dihydrogen phosphate (S. D. Fine Chemicals, Mumbai, India) were of Analytical grade used for the preparation of mobile phase. Preparation of mobile phase and stock solutions: Potassium dihydrogen phosphate was weighed (6.8 g) and dissolved in 1000 ml of water. Finally the ph was adjusted to 3.0 with ortho phosphoric acid. The solution was sonicated for 10 minutes and filtered using Whatman filter paper (No.1) and used. For the estimation of Sunitinib malate from the capsules, twenty capsules were taken and their contents were mixed thoroughly. Average weight was calculated. capsule content or the powder equivalent to 100mg was weighed accurately and transferred into a 100ml volumetric flask, dissolved and dilute up to mark with diluent (1000ppm). Mix well and filter through 0.45µm filter. Chromatographic conditions: A reverse phase C18 column equilibrated with mobile phase phosphate buffer-methanol adjusted to ph 3.0 was used. Mobile phase flow rate was maintained at 1.0 ml/min and eluents was monitored at 248 nm. The sample was injected using a 20 µl fixed loop, and the total run time was 6.0 min. Appropriate aliquot of Sunitinib malate stock solutions was taken in different 10 ml volumetric flasks and diluted up to the mark with mobile phase to obtain final concentrations of 10, 15,20,25,30 µg/ml of Sunitinib malate The solution was injected using a 20 µl fixed loop system and chromatograms were recorded. Calibration curve was constructed by plotting average peak area versus concentrations and regression equation was calculated for Sunitinib malate.
Page563 Determination of Sunitinib malate dosage form: Sample equivalent to 100mg was weighed accurately and transferred into a 100ml volumetric flask, dissolved and dilute up to mark with diluent. Take above solution 0.2 ml in 10 ml volumetric flask dilute up to mark with diluent (20ppm). Mix well and filter through 0.45µm filter. The solution was injected at above chromatographic conditions and peak areas were measured. The quantification was carried out by keeping these values to the straight line equation of calibration curve. The method was validated for accuracy, precision, specificity, and robustness. Accuracy: The accuracy of the method was determined by calculating recovery of Sunitinib malate by the spiked method. Known amount of Sunitinib malate was added to a pre quantified sample solution, and the amount of Sunitinib malate was estimated by measuring the peak areas and by fitting these values to the straight-line equation of calibration curve. Precision: The intraday and inter day precision study of Sunitinib malate was carried out by estimating the corresponding responses 5 times on the same day and on different days. The results are reported in terms of relative standard deviation. The Repeatability studies were carried out by estimating response of 5 different concentrations of Sunitinib malate and results are reported in terms of relative standard deviation (%RSD). Specificity: Commonly used excipients were spiked into a pre weighed quantity of drugs. The chromatogram was taken by appropriate dilutions and the quantities of drugs were determined. Robustness: Robustness of the method was studied by changing change in the chromatographic parameters: Effect of Variation in column oven temperature.± % 10 and the flow 0.9 and 1.1 ml/min instead of 1.0 ml/min. RESULTS AND DISCUSSION Optimization of mobile phase was performed based on asymmetric factor and peak area obtained for Sunitinib malate. The mobile phase phosphate buffer-methanol adjusted to ph 3.0 using ortho phosphoric acid was found to be satisfactory and gave symmetric peak for Sunitinib malate the retention time for Sunitinib malate was 2.567 min respectively (Figure 2). Figure 2: HPLC chromatogram of Sunitinib malate in optimized chromatographic conditions The calibration curve for Sunitinib malate was obtained by plotting the peak area of Sunitinib malate versus the concentration of Sunitinib malate over the range of 10-50 µg/ml, and it was found to be linear with r 2 = 0.995. The mean accuracy was found to be 99.5%. The results of accuracy were tabulated in table 2. The %RSD of precision was found to be less than 2%, that shows that the method was precise. The results were given in table 3. The validation parameters are summarized in (Table-1). The recovery Sunitinib malate was found to be 99.5% respectively. The system suitability test parameters are shown in (Table-1). The liquid chromatographic method was applied to the determination of Sunitinib malate in dosage form. The results for Sunitinib malate were comparable with the corresponding labeled amount. Finally the validated results were in accordance with the limits of ICH, that confirms that the method was accurate, precise, and reproducible which can be used for regular QC analysis of sunitinib.
Page564 2500000 2000000 1500000 y = 36245x + 34949 R² = 0.997 1000000 500000 0 0 2 4 6 Figure 3: Linearity curve for sunitinib maleate Table 1: Validation parameters and data for proposed method Validation parameter Results Sunitinib malate Linearity 10-30 µg/ml Regression coefficient (r 2 ) 0.9975 *Accuracy (% recovery) 99.5% ** System Precision (%RSD) 1.16 ** Method precision(%rsd) 1.39 ** Rugudness (%RSD) 0.97 Assay value (%) 99.2% System suitability parameters Tailing factor 1.7 Number of theoretical plates 2507 * Replicates of three concentration levels (in three determinations); ** Five repetitive injections of same homogeneous sample Table 2: Accuracy table of Sunitinib Maleate %Concentration Amount Amount (at specification Area Added Found % Recovery Level) (mg) (mg) 50% 724713 5 4.92 98.4% 100% 1460317 10.0 9.91 99.1% 150% 2236191 15.0 15.1 101.2% Mean Recovery 99.5% Table 3: Precision results Injection Area Injection-1 1405544 Injection-2 1434275 Injection-3 1404961 Injection-4 1432150 Injection-5 1438560 Average 1423098 Standard Deviation 16454.7 %RSD 1.16
Page565 CONCLUSION Proposed study describes a new RP-HPLC method for the estimation of Sunitinib malate using simple mobile phase with low buffer concentration compared to the reported method. The method gives short analysis time (<3 min). The method was validated and found to be simple, sensitive, accurate and precise. Percentage of recovery shows that the method is free from interference of the excipients used in the formulation. Therefore, the proposed method can be used for routine analysis of Sunitinib malate in dosage form. ACKNOWLEDGEMENTS The authors are very much thankful to Natco Pharma Ltd., (Hyderabad, India) for providing gift sample of Sunitinib maleate. REFERENCES 1. G.D. Demetri, A.T. van Oosterom, C.R. Garrett, M.E. Blackstein, M.H. Shah, J. Verweij, G. McArthur, I.R. Judson, M.C. Heinrich, J.A. Morgan, J. Desai, C.D. Fletcher, S. George, C.L. Bello, X. Huang, C.M. Baum, and P.G. Casali. Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumour after failure of imatinib: a randomised controlled trial. Lancet 368: 1329 1338 (2006). 2. R.J. Motzer, M.D. Michaelson, B.G. Redman, G.R. Hudes, G. Wilding, R.A. Figlin, M.S. Ginsberg, S.T. Kim, C.M. Baum, S.E. DePrimo, J.Z. Li, C.L. Bello, C.P. Theuer, D.J. George, and B.I. Rini. Activity of SU11248, a multitargeted inhibitor of vascular endothelial growth factor receptor and platelet-derived growth factor receptor, in patients with metastatic renal cell carcinoma. J. Clin. Oncol. 24: 16 24 (2006). 3. Benoit Blanchet et.al., Development and validation of an HPLC-UV-visible method for sunitinib quantification in human plasma, Clinica Chimica Acta, Volume 404, Issue 2, 27 June 2009, Pages 134 139 4. B.E. Houk, C.L. Bello, B. Poland, L.S. Rosen, G.D. Demetri, and R.J. Motzer. Relationship between exposure to sunitinib and efficacy and tolerability endpoints in patients with cancer: results of a pharmacokinetic/ pharmacodynamic meta-analysis. Cancer Chemother. Pharmacol. 66: 357 371 (2010). 5. T.J. Abrams, L.J. Murray, E. Pesenti, V.W. Holway, T. Colombo, L.B. Lee, J.M. Cherrington, and N.K. Pryer. Preclinical evaluation of the tyrosine kinase inhibitor SU11248 as a single agent and in combination with standard of care therapeutic agents for the treatment of breast cancer. Mol. Cancer Ther. 2: 1011 1021 (2003). 6. T.J. Abrams, L.B. Lee, L.J. Murray, N.K. Pryer, and J.M. Cherrington. SU11248 inhibits KIT and platelet-derived growth factor receptor beta in preclinical models of human small cell lung cancer. Mol. Cancer Ther. 2: 471 478 (2003). 7. Patton minkin et. al., Quantification of sunitinib in human plasma by high-performance liquid chromatography tandem mass spectrometry, Journal of Chromatography B, Volume 874, Issues 1 2, 15 October 2008, Pages 84 88. 8. Feng Qiu et al., Simultaneous determination of sunitinib and its two metabolites in plasma of Chinese patients with metastatic renal cell carcinoma by liquid chromatography tandem mass spectrometry, Biomedical Chromatography, Volume 27, Issue 5, pages 615 621, May 2013 9. Nienke A.G. Lankheet, Determination of Sunitinib and Its Active Metabolite N-Desethylsunitinib in Sweat of a Patient, Journal of Analytical Toxicology, Vol. 35, October 2011. 10. ICH Q2B: Validation of Analytical Procedures: Methodology (1997) pp. 1-8. 54878478451001262 Submit your next manuscript to IAJPR and take advantage of: Access Online first Double blind peer review policy No space constraints Rapid publication International recognition Submit your manuscript at: editorinchief@iajpr.com