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Available online at www.scholarsresearchlibrary.com Scholars Research Library Der Pharmacia Lettre 2014 6 5:339-351 http://scholarsresearchlibrary.com/archive.html ISSN 0975-5071 USA CODEN: DPLEB4 339 Scholar Research Library A novel validated stability indicating RP-HPLC method development for the estimation of ceritinib in it’s bulk and finished dosage form as per ICH guidelines 1 Ch. Naveen Kumar 1 V. Prathyusha and 2 N. Kannappan 1 Teegala Krishna Reddy College of Pharmacy Department of Pharmaceutical Aanalysis Quality Assurance Medbowli Meerpet Saroornagar Hyderabad Telangana India 2 Department of Pharmacy Faculty of Engineering and Technology Annamalainagar Annamalai University ChidambaramTamilnadu India _____________________________________________________________________________________________ ABSTRACT A simple stability indicatingselective reproducible economic selectiveRP- HPLC method has been developed and validated as per ICH guidelines for the estimation of Ceritinib CER both in bulk drug and in capsule dosage forms in the presence of degradation products. Reversed-phase chromatography was performed on a Kromosil C 18 column with mobile phase Methanol: water 65:35 v/v at a flow rate of 1.0 ml min -1 . Detection was performed at 265nm and a sharp peak was obtained for CER at a retention time of 2.585 min. The method was validated for precision specificity and selectivity accuracy robustness detection and quantification limits and system suitability in accordance with ICH guidelines. Linear regression analysis data for the calibration plot showed there was a good linear relationship between response and concentration in the range 30- 150 ppm the regression coefficient was 0.998 and the detection LOD and quantification LOQ limits were found to be 2.97 and 9.92 μg/ ml -1 respectively. Statistical analysis had proved the method was precise reproducible selective specific and accurate for analysis of CER. In order to determine whether the analytical method and assay were stability-indicating CER was stressed under various conditions to conduct forced degradation studies. stability indicating forced degradation established studies show results that there is no interference of any degraded products or external environmental conditions and it did not interfere with the detection of CER and the performed assay is thus specific stability- indicating . The wide linearity range sensitivity accuracy short retention time and simple mobile phase imply the method is suitable for routine quantification of CER with high precision and accuracy. Keywords: Stability-indicating Reversed-phase chromatography Ceritinib forced degradation studies. _____________________________________________________________________________________________ INTRODUCTION Ceritinib CER is a 5-Chloro-N4-2-1-methylethylsulfonylphenyl-N2-5- methyl-2-1-methylethoxy-4-4- piperidinylphenyl 24- pyrimidinediamine. Empirical formula of C 28 H 36 N 5 O 3 ClS1. It is an Antineoplastic and kinase inhibitor indicated for the treatment of patients with anaplastic lymphoma kinase ALK-positive metastatic non-small cell lung cancer NSCLC who have progressed on or are intolerant to crizotinib 2. There are no novel analytical methods developed for the estimation of ceritinib in pharmaceutical dosage forms as it is newly approved by FDA April 29 th 20143. Except that the clinical and non clinical related studies 4 performed. Hence there was a need to develop simple preciseaccurate robust and conventional method for the determination of CER. The

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Ch. Naveen Kumar et al Der Pharmacia Lettre 2014 6 5:339-351 ______________________________________________________________________________ 340 Scholar Research Library aim of the present work is to develop an accurate selective precise and robust RP-HPLC method for the determination of CER both in bulk drug and in powder formulation. The proposed method was validated as per ICH guidelines 56 and its updated international convention regulatory discussions. MATERIALS AND METHODS Materials Pure Ceritinib CER was purchased from Novartis. Methanol and Water HPLC grade were used. Nylon 0.22μm- 45 mm membrane filter HPLC grade were used. And all other chemicals and reagents were of GR grade. Standard sample mobile phase and diluent preparation: Diluent: Methanol Mobile phase are used as diluents. Preparation of mobile phase:Accurately weigh and transfer about 1.5 grams of Potassium di-hydrogen phosphate in 500 mL of purified water added 0.5 mL of Triethylamine and adjusted the pH to 4.5 ±0.05 with dilute orthophosphoric acid and sonicated to enhance the solubility and degas. Preparation of standard stock solution Accurately weighed 10 mg CER was transferred to 10 ml volumetric flask dissolved in7 ml diluent and sonicate it. And it is further diluted up to make with solvent. Preparation of sample stock solution The capsule powder equivalent to 10 mg of CER was transferred into a 10ml volumetric flask and 1ml diluents were added. The content of the flask was sonicated for 10min and diluted to volume with the mobile phase. The above sample solution was filtered through Whatmann filter paper No. 22 prior to injection. To ensure the solution was filtered again through 0.22 μ filter paper. Method development A Waters HPLC module 2695 with photodiode array PDA detector and auto injector was used. Separation was performed on kromosil RPC18 4.5×150mm 5.0 μm maintained at room temperature. A variety of mobile phases were investigated in the development of an HPLC method suitable for analysis of CER in the bulk drug and in capsule powder. It consisting of different ratio of Methanol: water and MeOH: KH 2 PO 4 buffer pH 4.5. The mobile phase was filtered through nylon 0.22 μm - 45 mm membrane filter. Flow rate was adjusted to 1.0 ml min -1 and wavelength lamda max was observed at 265 nm. Method validation Linearity and range Solutions of different concentration 30- 150μg ml -1 are used for construction of calibration plots were prepared from the standard stock solution. The mobile phase was filtered through a 0.45 μ membrane filter and delivered at 1.0 ml min -1 for column equilibration the baseline was monitored continuously during this process. The detection wave-length was 265 nm. The prepared dilutions were injected in series peak area was calculated for each dilution and concentration was plotted against peak area. Data was analyzed through linear regression. Accuracy as Recovery Accuracy was determined by the standard addition method. Previously analyzed samples CER 90 μg ml -1 were spiked with 50 100 and 150 extra CER standard and the mixtures were analyzed by the proposed method. The experiment was performed in triplicate. Recovery SD and CV were calculated for each concentration. Precision Precision was determined as both repeatability Method precision and intermediate precision in accordance with ICH recommendations. The repeatability was checked by repeatedly injecting n 5 solution of CER 90μg ml -1 . The results were reported in terms of coefficient of variance CV should not be more than 2 . Intermediate precision was evaluated in terms of intra-day and inter-day precision. For both intra-day and inter-day variation replicate analysis of solutions of CER at three different concentrations 60 90 and 120μg ml -1 were carried out for six times. The results were reported in terms of coefficient of variance CV.

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Ch. Naveen Kumar et al Der Pharmacia Lettre 2014 6 5:339-351 ______________________________________________________________________________ 341 Scholar Research Library Specificity and selectivity The specificity of the method was established through resolution factor of the drug peak from the nearest resolving peak and also among all other peaks. Selectivity was confirmed through peak purity data using a PDA detector. To assess the method specificity capsule without CER placebo was prepared with the excipients as per commercial preparation and compared with respective CER standard to evaluate specificity of the method. Representative chromatograms of placebo and standard were compared for retention time resolution factor and purity. Robustness The robustness of the developed method was determined to assess the effect of small but deliberate variation of the chromatographic conditions on the determination of CER. Robustness was determined by changing ratio of mobile phase column own temperature wave-length and injection volume. Detection LOD and Quantification LOQ Limits LOD and LOQ were determined by the standard deviation Sy/x method. Blank samples were injected in triplicate and the peak area of this blank was calculated. LOD and LOQ were determined from the slope S of the calibration plot and the standard deviation of the response for the blank sample Sy/x by use of the formulae LOD 3.3 × Sy/x/S and LOQ 10 × Sy/x/S. System suitability As system suitability test was an integral part of chromatographic methods development and was used to verify that the system is adequate for the analysis to be performed the system suitability parameters for CER were evaluated. The suitability of the chromatographic system was demonstrated by comparing the obtained parameter values with the acceptance criteria of the ICH guidelines. Solution stability The stability of the drug in solution during analysis was determined by repeated analysis of samples during the course of experimentation on the same day and also after storage of the drug solution for 24 hrs under laboratory bench conditions 32 ± 1°C and under refrigeration 8 ± 0.5°C. Forced degradation study In order to determine whether the analytical method and assay were stability-indicating CER was stressed under various conditions to conduct forced degradation studies. Methanol was used as solvent in all studies as CER is freely soluble in methanol. In all cases contents equivalent to 10 mg CER was accurately weighed and solutions were prepared as previously described using suitable solvent to yield starting concentration 90 μg ml -1 of CER for analysis. Sample solution was exposed to hydrolytic acid and base thermal dry heat oxidative and photolytic stress conditions and the stressed samples were analyzed by the proposed method. Acid degradation To check the stability in acidic condition hydrochloric acid was used in different strength. Solution of CER is used for acid degradation. It was done by using 0.1N hydrochloric acid in methanol and the resultant solution was refluxed at 60 ºC for around 12 hours to facilitate acid degradation of CER. Alkali degradation To check the stability in alkaline condition sodium hydroxide was used. Solution of CER is used for alkali degradation. It was done by using 1N NaOH in methanol and the resultant solution was refluxed at 600 C for around 12hours to facilitate alkali degradation of CER. Wet heat Hydrolysis Solution of CER 90μg ml-1 for wet heat degradation study was prepared using HPLC grade water and the resultant solution was refluxed at 60 ºC for 12 hours to facilitate hydrolysis of CER. Dry heat Solution for dry heat study were prepared by exposing approximately 10 mg of powder in aluminum foil to dry heat in an oven at 60 ºC for 2hours to facilitates dry heat degradation of CER. The powder was removed from oven and the content of powder equivalent to 10 mg of CER was accurately weighed and transferred to volumetric flask to make the final concentration 90 μg ml-1 of CER.

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Ch. Naveen Kumar et al Der Pharmacia Lettre 2014 6 5:339-351 ______________________________________________________________________________ 342 Scholar Research Library Oxidation To check the stability in oxidative condition hydrogen peroxide was used in different strength. Solution of CER 90μg ml-1 for oxidation study was prepared using 30 H 2 O 2 in methanol and the resultant solution was refluxed at 60 ºC for 12 hours to facilitate oxidation of CER. Photo stability Sun light and UV light Powder was exposed to sunlight to determine the effects of light irradiation on the stability of CER. Approximately 100 mg of CER powder was spread on a glass dish in a layer that was less than 2 mm thick. Sample for photo stability testing is placed in direct sunlight exposed for 12 hours. The powder is removed from the sun light and the contents of powder equivalent to 10 mg of CER was accurately weighed and transferred to volumetric flask to make the final concentration 90 μg ml -1 of CER. Control samples which were protected with aluminum foil were also placed in the light cabinet and exposed concurrently. Following removal from the light cabinet all samples were prepared for analysis as previously described. Analysis of Ceritinib in powder Zykadia powder was used for analysis. Appropriate three different aliquots from sample solution 90 μg ml -1 were suitably diluted with mobile phase in such a way to get concentrations in a range of 30 to 150 ppm for CER. The finally prepared solutions were analysed under proposed chromatographic condition. The amount of CER present in sample solution was determined by fitting the area response into the regression equation of CER in the method. RESULTS AND DISCUSSION Method optimization Initial studies to develop the method involved the use of C18 column with various mobile phases ratio containing MeOH: KH2PO4 buffer. Almost every system studied while the separation of more polar compounds was in some instance obtained CER showed a retention time steady baseline required for the analysis of CER in presence of degraded impurities with an acceptable retention time. It was observed that satisfactory resolution of CER and its degradation products formed under various conditions and present in the mixture of stressed samples were achieved. The method was optimized to separate major degradation products formed under various conditions. The optimized chromatographic conditions are mentioned in Table 1. Resolution was also checked on mixture of the degradation samples to confirm the separation behaviour. It indicates that the gradient method was successful in separation of drug and all degradation products. A representative chromatogram is shown in Figure 1 which satisfies all the system suitability criteria better resolution of the peak from solvent peak with clear base line separation was found Table 1: showing Optimized method parameters Parameters Description Column kromosil RPC 18 4.5×150mm5.0 μm Column temperature 27 ± 1 0 C Mobile phase MeOH : H 2 O 65:35v/v Description Photodiode array detection at 265 nm Injection volume 20 μl Flow rate 1.0 ml min -1 Figure 1: Chromatogram of CER 1 μg m-1 standard with corresponding retention time at 265 nm by stability indicating RP-HPLC method

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Ch. Naveen Kumar et al Der Pharmacia Lettre 2014 6 5:339-351 ______________________________________________________________________________ 343 Scholar Research Library Ceritinib CER with corresponding retention time R t of 2.585min was detected at 265 nm. Method validation Linearity and range: Linearity of the method was evaluated at five concentration levels by diluting the standard stock solution to give solutions in the concentration range from 30 to 150 ppm. The results shown that an excellent correlation existed between the peak area and concentration of analyte. The calibration curve was prepared by plotting the area under the response from the detector AUC line versus the concentration and analyzed through linear regression Figure 2 3. The response for the drug was linear r 2 0.997 in the concentration range between 30 and 150ppm . The linearity was observed in the expected concentration range demonstrating its suitability for analysis in Table 1.2. Figure 2: Calibration curve of CER at 305 nm by stability indicating RP-HPLC method. Figure 3: Overlay diagram of Ceritinib linearity concentrations from 90-150ppm Accuracy Good recovery of the spiked drug was obtained at each added concentration indicating that the method was accurate. A known amount of CER 50 100 and 150 of standard powder was added to the pre analyzed solution of formulation. This solution was analyzed as previously described. The assay was repeated over 3 injections of each concentration to obtain data. The resultant CV for this study was found to be 2.0 with a corresponding percentage recovery value. The recoveries obtained by RP-HPLC method for CER shown in Table 2.

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Ch. Naveen Kumar et al Der Pharmacia Lettre 2014 6 5:339-351 ______________________________________________________________________________ 344 Scholar Research Library Table 1.2: Linearity data of Ceritinib by stability indicating RP-HPLC method LINEARITY Sl.No Conc μg/ml Rt Area 1 30 ppm 2.178 201932 2 60 ppm 2.179 338071 3 80 ppm 2.177 597859 4 120ppm 2.186 740654 5 150 ppm 2.202 950396 Co efficient of correlation R 2 0.998 Acceptance criteria: Coefficient of correlation shall be NLT 0.999 Table 2: Accuracy Recovery study of Ceritinib by stability indicating RP-HPLC method. ACCURACY Concentration at specification level Average Area Amount added mg Amount found mg Recovery Mean recovery 50 1508773 5 5.14 100.2 100.4 100 1866573 10 10.01 98.8 150 1942321 15 15.2 96.5 The accuracy study was performed for recovery of Ceritinib. The recovery was found to be 100.4 NLT 98 and NMT 102. Precision: Repeatability: The standard solution was injected for five times and measured the area for all five injections in HPLC. The RSD for the area of five replicate injections was found to be within the specified limits. Table 3: Method precision data of Ceritinib by stability indicating RP-HPLC method Sl.No R t Area 1 2.182 591196 2 2.177 594056 3 2.196 594419 4 2.178 596875 5 2.191 598538 Mean 595016 Standard deviation 2616.4 Rsd 0.5 Ruggedness intermediate precision: The standard solution was injected for five times and measured the area for all five injections in HPLC. The RSD for the area of five replicate injections was found to be within the specified limits. Table 4: Intermediate precision data of Ceritinib by stability indicating RP-HPLC method Sample R t Area 1 2.165 584681 2 2.181 589281 3 2.198 596719 4 2.186 597658 5 2.188 597800 Mean 593227 Standard deviation 5944.3 Rsd 1.0 The Method precision study was performed for the RSD of Ceritinib was found to be 0.3 and NMT 2. Specificity: The system suitability for specificity was carried out to determine whether there is any interference of any impurities in retention time of analytical peak. The study was performed by injecting blank.

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Ch. Naveen Kumar et al Der Pharmacia Lettre 2014 6 5:339-351 ______________________________________________________________________________ 345 Scholar Research Library Figure 4: Chromatogram showing blank mobile phase preparation Figure 5: chromatogram showing standard injection Robustness: The robustness was performed for the flow rate variations from 0.8ml/min to 1.2 ml/min and mobile phase ratio variation from more organic phase to less organic phase ratio for Ceritinib. The method is robust only in less flow condition and the method is robust even by change in the Mobile phase ±5. Table 5: Showing system suitability results for Ceritinib S. No Flow rate ml/min System suitability results USP Plate Count USP Tailing 1 0.7 4352 1.1 2 0.8 4024 1.2 3 0.9 3730 1.2 Detection limit: LOD’s can be calculated based on the standard deviation of the response SD and the slope of the calibration curve S at levels approximating the LOD according to the formula. The standard deviation of the response can be determined based on the standard deviation of y-intercepts of regression lines. Formula: Where - Standard deviation SD S - Slope.

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Ch. Naveen Kumar et al Der Pharmacia Lettre 2014 6 5:339-351 ______________________________________________________________________________ 346 Scholar Research Library Figure 6: Chromatogram Showing results for Limit of Detection. Table 6: showing values of LOD Drug name Standard deviationó Slopes LODμg Ceritinib 371827.90 563365963 2.97 Quantitation limit: LOQ’s can be calculated based on the standard deviation of the response SD and the slope of the calibration curve S according to the formula. Again the standard deviation of the response can be determined based on the standard deviation of y-intercepts of regression lines. Formula: Where - Standard deviation S - Slope. Figure 7: Chromatogram Showing results for Limit of Quantitation. Table.7: Showing results for Limit of Quantitation. Drug name Standard deviationó Slopes LOQμg Ceritinib 371827.90 563365963 9.92

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Ch. Naveen Kumar et al Der Pharmacia Lettre 2014 6 5:339-351 ______________________________________________________________________________ 347 Scholar Research Library System suitability: Table.8: Showing system suitability results for Ceritinib S. No Change in organic composition in the mobile phase System suitability results USP Plate Count USP Tailing 1 10 less 4331 1.20 2 Actual 4024 0.87 3 10 more 3693 1.26 Forced degradation of Ceritinib: 1. Acid degradation: Figure 8: Chromatogram Showing Acid degradation of ceritinib. Figure 9: Chromatogram Showing Purity plot. Figure 10: Chromatogram of spectrum Index plot. Table.9: Showing results for Acid degradation.

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Ch. Naveen Kumar et al Der Pharmacia Lettre 2014 6 5:339-351 ______________________________________________________________________________ 348 Scholar Research Library 2. Base degradation: Figure 11: Chromatogram Showing Base degradation of ceritinib. Figure 12: Chromatogram Showing purity plot. Figure 13: Chromatogram of spectrum Index plot. Table.10: Showing results for Base degradation of Ceritinib. 3. Thermal Degradation: Figure 14: Chromatogram Showing Thermal degradation of ceritinib. Figure 15: Chromatogram Showing purity plot.

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Ch. Naveen Kumar et al Der Pharmacia Lettre 2014 6 5:339-351 ______________________________________________________________________________ 349 Scholar Research Library Figure 16: Chromatogram of spectrum Index plot. Table.11: Showing results for Thermal degradation of Ceritinib. 4. Peroxide Degradation: Figure 17: Chromatogram Showing peroxide degradation of ceritinib. Figure 18: Chromatogram Showing purity plot. Figure 19: Chromatogram of spectrum Index plot.

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Ch. Naveen Kumar et al Der Pharmacia Lettre 2014 6 5:339-351 ______________________________________________________________________________ 350 Scholar Research Library Table.12: Showing results for Peroxide degradation of Ceritinib. 5. Photo Degradation: Figure 20: Chromatogram Showing photo degradation of ceritinib. Figure 21: Chromatogram Showing purity plot. Figure 22: Chromatogram of spectrum Index plot. Table.13: Showing results for Photo degradation of Ceritinib. CONCLUSION A new novel method was established for the estimation of ceritinib by RP-HPLC method. The chromatographic conditions were successfully developed by using Kromosil RPC18 4.6×150mm 5μm flow rate was 0.8ml/min mobile phase ratio was 65:35 v/v methanol: water MeOH and H 2 O detection wave length was 265nm. The instrument used was WATERS HPLC Auto Sampler Separation module 2690 PDA detector 2487 Empower- software version-2. The retention time was found to be 2.585min. The purity of ceritinib was found to be 99.6. The system suitability parameters for ceritinib such as theoretical plates and tailing factor were found to be 4024 and 0.87 the resolution was found to be 8.67. The analytical method was validated according to ICH guidelines ICH Q2 R1. The linearity study for ceritinib was found in concentration range of 30ppm to 150ppm and correlation coefficient r 2 was found to be 0.997. Recovery was found to be 100.4 RSD for repeatability was 0.5 and RSD for intermediate precision was 1.0. The precision study was precise robust and repeatable. LOD value was 2.97 and 6.60 and LOQ value was 9.92. Hence the suggested RP-HPLC method can be used for routine analysis of ceritinib in API and Pharmaceutical dosage form.

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Ch. Naveen Kumar et al Der Pharmacia Lettre 2014 6 5:339-351 ______________________________________________________________________________ 351 Scholar Research Library REFERENCES 11http://www.druginformation.com/RxDrugs/C/Ceritinib20Capsules.html 2http://www.pharma.us.novartis.com/product/pi/pdf/zykadia.pdf. 3http://www.drugs.com/newdrugs/fda-approves-zykadia-ceritinib-metastatic-non-small-cell-lung-cancer-035.html. 4http://www.rxlist.com/zykadia-drug/clinical-pharmacology.htm 5ICH Validation of analytical procedure: Methodology Q2A International Conference on Harmonization Geneva 1994. 6ICH Validation of analytical procedure: Methodology Q2B International Conference on Harmonization Geneva 1996.