B. Sandhya et al.

, IJSID, 2011, 1 (3), 441-450

ISSN:2249-5347

IJSID
International Journal of Science Innovations and Discoveries
Research Article
An International peer Review Journal for Science

Available online through www.ijsidonline.info

RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR THE ANALYISIS OF SUNITINIB IN PHARMACEUTICAL DOSAGE FORMS
B.Sandhya1, V.Chinnari Harika2, Bikshal Babu.Kasimalla3*, Reshma Syed3, Kalpana Pammi3
1Dept

of Life Sciences, Sims College, Guntur, AP, India, 2Dept of Food and Nutritional sciences, Acharya Nagarjuna University, Guntur, AP, India, 3R.V.Labs, Guntur, A.P ,India.

Received: 09.09.2011 Modified: 11.10.2011 Published: 29.12.2011

*Corresponding Author

ABSTRACT

A simple, selective, linear, precise and accurate RP-HPLC method was developed and validated for rapid assay of SUNITINIB in tablet dosage form. Isocratic elution at a flow rate of 1ml min-1 was employed on a symmetry C18 column at ambient temperature. The mobile phase consisted of Acetonitrile: Methonal: OPA 40:30:30(v/v). The UV detection wavelength was at 230nm.Linearity was observed in concentration range of 30-90ppm. The retention time for Sunitinib was 3.068 min. The method was validated as per the ICH guidelines. The proposed method can be successfully applied for the estimation of

Name: Bikshal Babu. Kasimalla Place: Guntur, AP, India E-mail: bikshalbabu@gmail.com

Sunitinib in pharmaceutical dosage forms. INTRODUCTION Key words: Sunitinib, HPLC Method, Development, 230nm.

INTRODUCTION

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INTRODUCTION

Sunitinib (marketed as Sutent by Pfizer, and previously known as SU11248) is an oral, smallmolecule, multi-targeted receptor tyrosine kinase (RTK) inhibitor that was approved by the FDA for the treatment of renal cell carcinoma (RCC) and imatinib-resistant gastrointestinal stromal tumor (GIST) on January 26, 2006. Sunitinib was the first cancer drug simultaneously approved for two different indications.[1]

IUPAC Name N-(2-diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-2,4-dimethyl-1Hpyrrole-3-carboxamide 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),[2] the RTK that (when improperly activated by mutation) drives the majority of gastrointestinal stromal cell tumors.[3] 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.[4][5]In addition, sunitinib inhibits other RTKs.[6] These include: like RCC, GIST does not generally respond to standard chemotherapy or radiation. Imatinib was the first cancer agent proven effective for metastatic 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.[7]

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B. Sandhya et al., IJSID, 2011, 1 (3), 441-450 Sunitinib offers patients with imatinib-resistant GIST a new treatment option to stop further disease progression and, in some cases, even reverse it. This was shown in a large, Phase III clinical trial in which patients who failed imatinib therapy (due to primary resistance, secondary resistance, or intolerance) were treated in a randomized and blinded fashion with either sunitinib or placebo.[7]The study was unblinded early, at the very first interim analysis, due to the clearly emerging benefit of sunitinib. At that time, patients receiving placebo were offered to switch over to sunitinib. In the primary endpoint of this study, median time to tumor progression (TTP) was more than 4-fold longer with sunitinib (27 weeks) compared with placebo (6 weeks, P<.0001). These are based on the assessments of an independent radiology lab assessment. The benefit of sunitinib remained statistically significant when stratified for a multitude of prespecified baseline factors.[7] Sunitinib is being studied for treatment

of meningioma which is associated with Neurofibromatosis.[8] In Nov 2010 Sutent gained approval from the European Commission for the treatment of 'unresectable or metastatic, well-differentiated pancreatic neuroendocrine tumors with disease progression in adults'.[9] In May 2011, the USFDA approved Sunitininb for treating patients with 'progressive neuroendocrine cancerous tumors located in the pancreas that cannot be removed by surgery or that have spread to other parts of the body (metastatic)'.[10] Sunitinib is approved for treatment of metastatic RCC. Other therapeutic options in this setting are sorafenib (Nexavar), temsirolimus (Torisel), interleukin-2 (Proleukin), everolimus(Affinitor), and bevacizumab (Avastin).RCC is generally resistant to chemotherapy or radiation. Prior to RTKs, metastatic disease could only be treated with the cytokines interferon alpha (IFN) or Interleukin 2 (IL2). However, these agents demonstrated low rates of efficacy (5%-20%). In a phase 3 study, median progression-free survival was significantly longer in the sunitinib group (11 months) than in the interferon alfa group (5 months), hazard ratio 0.42.[6][11] In the secondary endpoints, 28% of had significant tumor shrinkage with sunitinib compared to 5% with IFN. Patients receiving sunitinib had a better quality of life than IFN.At ASCO 2008, Dr Robert Figlin presented updated data from the final study analysis, including overall survival. The primary endpoint of median progression-free survival (PFS) remained superior with sunitinib: 11 months versus 5 months for IFN, P<.000001. Objective response rate also remained superior: 39-47% for sunitinib versus 8-12% with IFN, P<.000001.[12][13] Sunitinib was associated with somewhat longer overall survival, although this was not statistically significant. Sunitinib has been generally well tolerated. Adverse events were considered somewhat manageable and the incidence of serious adverse events low.[7][11]The most common adverse events
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B. Sandhya et al., IJSID, 2011, 1 (3), 441-450 associated with sunitinib therapy are fatigue, diarrhea, nausea, anorexia, hypertension, a yellow skin discoloration, hand-foot skin reaction, and stomatitis.[19] In the placebo-controlled Phase III GIST study, adverse events which occurred more often with sunitinib than placebo included diarrhea, anorexia, skin discoloration, mucositis/stomatitis, asthenia, altered taste, and constipation.[6][7] Dose reductions were required in 50% of the patients studied in RCC in order to manage the significant toxicities of this agent.Serious (grade 3 or 4) adverse events occur in 10% of patients and include hypertension, fatigue, asthenia, diarrhea, and chemotherapy-induced acral erythema. Lab abnormalities associated with sunitinib therapy include lipase, amylase, neutrophils, lymphocytes, and platelets. Hypothyroidism and reversible erythrocytosis have also been associated with

sunitinib.[6][20]Most adverse events can be managed through supportive care, dose interruption, or dose reduction.[7][11]This was the largest comparative trial in RCC to date, and sunitinib is the first agent to demonstrate an overall survival longer than 2 years in these patients. Hypertension was found to be a biomarker of efficacy in patients with metastatic renal cell carcinoma treated with sunitinib.[14] Patients with mRCC and sunitinib-induced hypertension had better outcomes than those without treatment-induced HTN (objective response rate: 54.8% vs 8.7%; median PFS: 12.5 months, 95% confidence interval [CI] = 10.9 to 13.7 vs 2.5 months, 95% CI = 2.3 to 3.8 months; and OS: 30.9 months, 95% CI = 27.9 to 33.7 vs 7.2 months, 95% CI = 5.6 to 10.7 months; P < .001 for all). The efficacy of sunitinib is currently being evaluated in a broad range of solid tumors, including breast, lung, thyroid and colorectal cancers. Early studies have shown single-agent efficacy in a number of different areas. Sunitinib blocks the tyrosine kinase activities of KIT, PDGFR, VEGFR2 and other tyrosine kinases that are involved in the development of tumours.A Phase II study in previously-treated patients with metastatic breast cancer found that sunitinib has significant single agent activity [15]A Phase II study of refractory non-small-cell lung cancer found that Sunitinib has provocative single-agent activity in previously treated pts with recurrent and advanced NSCLC, with the level of activity similar to currently approved agents. [16]In a Phase II study of patients with nonresectable neuroendocrine tumors (NET), 91% of patients responded to sunitinib (9% partial response + 82% stable disease) [17] EXPERIMENTAL Chemicals and reagents All HPLC SOLVENTS used like Acetonitrile, ammonium acetate which are of HPLC grade were purchased from E.Merck,

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B. Sandhya et al., IJSID, 2011, 1 (3), 441-450 Instrumentation and analytical conditions The analysis of the drug was carried out on Shimadzu HPLC model (VP series) containing LC-10AT (VP series) pump, variable wave length programmable UV/visible detector SPD-10AVP and rheodyne injector (7725i) with 20l fixed loop. Chromatographic analysis was performed using Gemini C-18 column with 250 x 4.6mm internal diameter and 5m particle size. Shimadzu electronic balance (AX-200) was used for weighing. Isocratic elution with ,Acetonitrile,methonal,OPA 40:30:30(v/v). was selected with a flow rate of 1.0 ml min- .The detection wavelength was set at 230 nm with a runtime of 3.068 min. The mobile phase was prepared freshly and it was degassed by sonicating for 5 min before use. The column was equilibrated for at least 30min with the mobile phase flowing through the system. The column and the HPLC system were kept at ambient temperature. Preparation of Stock, working standard solutions and Sample solutions 100mg of SUNITINIB was weighed and transferred (working standard) into a 100ml volumetric flask. The diluent methanol was added and sonicated to dissolve it completely and made up to the mark with the same solvent. Further 1ml of the above stock solution was pipetted into a 10ml volumetric flask and diluted up to the mark with diluent. The contents were mixed well and filtered through Ultipor N66 Nylon 6, 6 membrane sample filter paper. The calibration curve was plotted with the concentrations of the 30 to 90ppm working standard solutions. Calibration solutions were prepared and analyzed immediately after preparation. Table-1 Chromatographic conditions for SUNITINIB TEST RESULT H.P.L.C CONDITIONS 1 Elution ISOCRATIC 2 A.P.I CONC 30ppm 3 Mobile Phase Acetonitrile:methanol:OPA(40:30:30) 4 PH 4.8 5 Column C18 6 Wavelength 230 7 Flow 1ml\min 8 Runtime 10 Min 9 Retension Time 807346 10 Area 12361 11 Th.Plates 8284 12 Tailing Factor 1.68 13 Pump Presure 6.5 psi The formulation tablets of SUNITINIB were crushed to give finely powdered material. Powder equivalent to10 mg of drug was taken in 10 ml of volumetric flask containing 5 ml of mobile phase and
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B. Sandhya et al., IJSID, 2011, 1 (3), 441-450 was shaken to dissolve the drug and then filtered through Ultipor N66 Nylon 6,6 membrane sample filter paper. Volume of the filtrate was adjusted to the mark with the same solvent to obtain concentration of 30 ppm Method Validation procedure The objective of the method validation is to demonstrate that the method is suitable for its intended purpose as it is stated in ICH guidelines. The method was validated for linearity, precision, accuracy, specificity, and limit of detection, limit of quantification, robustness and system suitability. Linearity Table 2: Linearity of SUNITINIB S.NO CONC AREA 1 10ppm 4012 2 20ppm 8218 3 30ppm 12361 4 40ppm 16493 5 50ppm 20516 6 60ppm 24913 7 70ppm 28751 The developed method has been validated as per ICH guidelines (Zucman D, 2007). Working standard solutions of SUNITINIB in the mass concentration range of 10 ppm to 70 ppm was injected into the chromatographic system. The chromatograms were developed and the peak area was determined for each concentration of the drug solution. Calibration curve of SUNITINIB was obtained by plotting the peak area ratio versus the applied concentrations of SUNITINIB. The linear correlation coefficient was found to be 0.999

Figure 2: Calibration curve of SUNITINIBS

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B. Sandhya et al., IJSID, 2011, 1 (3), 441-450 Table.3 Linear Regression Data for Calibration curve Drug Concentration range Slope (m) Intercept (b) Correlation coefficient % RSD Precision Repeatability of the method was checked by injecting replicate injections of 30 ppm of the solution for six times on the same day as intraday precision study of SUNITINIB and the RSD was found to be 0.63 for intraday and 0.64for interday. INJECTION 1 2 3 4 5 6 Accuracy The accuracy of the method was determined by calculating recovery of SUNITINIB by the method of standard addition. Known amount of SUNITINIB (10ppm, 20ppm and 30ppm) was added to a pre quantified sample solution and the amount of SUNITINIB was estimated by measuring the peak area ratios and by fitting these values to the straight line equation of calibration curve. The recovery studies were carried out three times over the specified concentration range and amount of SUNITINIB was estimated by measuring the peak area ratios by fitting these values to the straight line equation of calibration curve. From the above determination, percentage recovery was calculated and the average recovery was found to be 99.23% Specificity The specificity of the method was determined by comparing test results obtained from analysis of sample solution containing excipients with that of test results those obtained from standard drug. Table 4: Precision parameters of SUNITINIB CONCENTRATION INTRA DAY 30ppm 12361 30ppm 12413 30ppm 12295 30ppm 12391 30ppm 12420 30ppm 12218 RSD 0.63 INTER DAY 12416 12512 12309 12425 12509 12361 0.64 SUNITINIB 70-10ppm 0.002422 0.103467 0.999 Intra day-0.63 Interday-0.64

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Figure 3: Typical chromatogram of SUNITINIB LOD and LOQ Limit of detection (LOD) and limit of quantification (LOQ) were calculated as 0.1ppm and 0.4ppm respectively as per ICH guide-lines. Results are shown in table 5. Table 5: Results of LOD and LOQ. Parameter LOD LOQ Robustness To determine the robustness of the method, two parameters from the optimized chromatographic conditions were varied. Results of Robustness are shown in table 6. Table 6: Robustness results Parameter Modification Standard No change M.PHASE Acetonitrile:Methanol:OPA 30:40:70 PH 5.2 WAVELENGTH 237nm System Suitability Parameter: Peak Area 21253 12457 12598 12490 % of change . 0.777 1.66 1.044 Measured 0.1ppm 0.3ppm

System suitability tests were carried out on freshly prepared standard stock solutions of SUNITINIB and it was calculated by determining the standard deviation of SUNITINIB standards by injecting standards in six replicates at 6 minutes interval and the values were recorded inTable7.
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B. Sandhya et al., IJSID, 2011, 1 (3), 441-450 Table7: System suitability parameters of SUNITINIB Parameters Values max (nm) 230nm Beers law limit (g/ml) 10-705ppm Correlation coefficient 0.999 Retention time 3.068 min Theoretical plates 8284 Tailing factor 1.68 Limit of detection 0.1ppm Limit of quantification 0.4ppm RESULT AND DISCUSSION Optimization of the chromatographic conditions The nature of the sample, its molecular weight and solubility decides the proper selection of the stationary phase. The drug SUNITINIB being non-polar is preferably analyzed by reverse phase columns and accordingly C18 column was selected. So the elution of the compound from the column was influenced by polar mobile phase. The concentration of the methanol and Acetonitrile were optimized to give symmetric peak with short run time based on asymmetric factor and peak area obtained. Different mobile phases were tried but satisfactory separation, well resolved and good symmetrical peaks were obtained with the mobile phase Acetonitrile, Methanol,OPA 40:30:30 (V/V). The retention time of SUNITINIB was found to be 3.068 min, which indicates a good base line. The RSD values for accuracy and precision studies obtained were less than 2% which revealed that developed method was accurate and precise. The system suitability and validation parameters are given in Table 7. The high percentage of recovery of SUNITINIB was found to be 99.23 indicating that the proposed method is highly accurate. Proposed liquid chromatographic method was applied for the determination of SUNITINIB in tablet formulation. The result for SUNITINIB was comparable with a corresponding labelled amount . The absence of additional peaks indicates no interference of the excipients used in the tablets. Table 8: Formulation results of SUNITINIB Formulation Sutent (capsule) Dosage 50mg Sample concentration 30ppm CONCLUSION %Estimation 99.74

A validated RP-HPLC method has been developed for the determination of SUNITINIB in tablet dosage form. The proposed method is simple, rapid, accurate, precise and specific. Its chromatographic run time of 3.068 min allows the analysis of a large number of samples in short period of time. Therefore, it is suitable for the routine analysis of SUNITINIB in pharmaceutical dosage form.
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B. Sandhya et al., IJSID, 2011, 1 (3), 441-450 REFERENCES 1. US Food and Drug Administration (2006). "FDA approves new treatment for gastrointestinal and kidney cancer". 2. Hartmann JT, Kanz L (November 2008). "Sunitinib and periodic hair depigmentation due to temporary c-KIT inhibition". Arch Dermatol 144 (11): 15256.doi:10.1001/archderm.144.11.1525. 3. "Prescribing information for Sutent (sunitinib malate)". Pfizer, Inc, New York NY. 4. Demetri GD et al. (2006). "Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumour after failure of imatinib: a randomised controlled trial". Lancet 368 (9544): 1329 1338. 5. "Phase II Trial of Sunitinib (SU011248) in Patients With Recurrent or Inoperable Meningioma" 6. "Pfizer Scores New Approval for Sutent in Europe". 2 Dec 2010. 7. FDA approves Sutent for rare type of pancreatic cancer

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