International Journal of Research and Reviews in Pharmacy and Applied science.

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1 International Journal of Research and Reviews in Pharmacy and Applied science Corresponding Author B.Lakshmi 1, Prof.K.Saraswathi 2, Prof. T.V.Reddy 3 1Kallam Haranadha Reddy Institute of Technology,NH-5, Chowdavaram,Guntur, AP, India; 2S.V University, Tirupati, AP, India; 3Dept of chemistry, MallaReddy College Of Engineering, Secunderabad Contact Mail RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR THE ANALYISIS OF RUXOLITINIB IN PHARMACEUTICAL DOSAGE FORMS ABSTRACT: A simple, selective, linear, precise and accurate RP-HPLC method was developed and validated for assay of Ruxolitinibin 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 Methanol:0.1% OPA :Acetonitrile 50:2:48 (v/v/v). The UV detection wavelength was at 257 nm. Linearity was observed in concentration range of ppm. The retention time for Ruxolitinib was 4.80 min. The method was validated as per the ICH guidelines. The proposed method can be successfully applied for the estimation of Ruxolitinibin pharmaceutical dosage forms. Key words: RUXOLITINIB, HPLC, Linearity, Precision, Recovery, 257nm. Id: lakshmi_bumi@yahoo.com INTRODUCTION: 360

2 Ruxolitinib is a drug for the treatment of intermediate or high-risk myelofibrosis, a type of bone marrow cancer. It is also being investigated for the treatment of other types of cancer and for plaque psoriasis. It is a Janus kinase inhibitor with selectivity for subtypes 1 and 2 of this enzyme. The phase III Controlled Myelofibrosis Study with Oral JAK Innhibitor-I (COMFORT-I) and COMFORT-II trials showed significant benefits by reducing spleen size, relieving debilitating symptoms, and improving overall survival. In November 2011, ruxolitinib was approved by the U.S. Food and Drug Administration for the treatment of intermediate or high-risk myelofibrosis based on results of the COMFORT-I and COMFORT-II Trials. Figure 1 : Structure of Ruxolitinib Ruxolitinib is a prescription medication approved to treat people with intermediate-risk or high-risk myelofibrosis, a rare disease of the bone marrow. It is the first medication approved to treat this condition.myelofibrosis is a disorder of the bone marrow, the soft, spongy tissue found in the center of some bones. Normally, the bone marrow produces new blood cells, including: Red blood cells, which carry oxygen throughout the body, White blood cells, which fight infections, Platelets, which help the blood to clot. However, in people with myelofibrosis, this normal production of blood cells is disrupted. In addition, fibers inside the bone marrow thicken, resulting in scarring of the bone marrow, which further interferes with the ability of the bone marrow to make blood cells. The liver and spleen begin to produce blood cells to compensate for the lack of blood cells produced by the bone marrow. This causes the liver and spleen to enlarge. 361

3 Myelofibrosis is normally a progressive disorder, which means people usually get worse over time. Some people may progress to a serious form of leukemia. In the early stages of the disease, the main symptom is extreme fatigue, although not everyone will experience fatigue in the early stages of the disease. As the condition progresses, symptoms increase. EXPERIMENTAL: Chemicals and reagents All HPLC SOLVENTS used like Acetonitrile, Water, Methanol Ortho phosphoric Acid which are of HPLC grade were purchased from E.Merck, 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 20µl fixed loop. Chromatographic analysis was performed using Inertsil ODS C-18 column with 250 x 4.6mm internal diameter and 5µm particle size. Shimadzu electronic balance (AX-200) was used for weighing. Isocratic elution with THF: Methanol,Acetonitrile 10:40:50 (v/v/v) was selected with a flow rate of 1.0 ml min -1.The detection wavelength was set at 270nm with a runtime of 6 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 Ruxolitinibwas 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 the prepared solution filtered through Ultipor N 66 Nylon 6, 6 membrane sample filter paper. The calibration curve was plotted with the concentrations of the ppm working standard solutions. Calibration solutions were prepared and analyzed immediately after preparation. 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 The developed method has been validated as per ICH guidelines (Zucman D, 2007). Working standard solutions of Ruxolitinibin the mass concentration range of 10 ppm to 60ppm 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 Ruxolitinib was obtained by plotting the peak area ratio versus the applied concentrations of Ruxolitinib. The linear correlation coefficient was found to be

4 S.NO CONC ppm AREA Slope Intercept CC Table 1: Linearity of Ruxolitinib Figure 2: Calibration curve of Ruxolitinib 363

5 Drug Ruxolitinib Concentration range 10-60ppm Slope (m) 3571 Intercept (b) Correlation coefficient RSD 0.4 Precision Table.2: Linear Regression Data for Calibration curve Repeatability of the method was checked by injecting replicate injections of 60 ppm of the solution for six times on the same day as intraday precision study of Ruxolitiniband the RSD was found to be for intraday 0.32 and for inter day 0.54 INJECTION INTERDAY INTRADAY R.S.D =0.32 R.S.D =0.54 Table 3: Precision parameters of Ruxolitinib 364

6 Accuracy The accuracy of the method was determined by calculating recovery of Ruxolitinib(30,60,90ppm) by the method of standard addition. Known amount of Ruxolitinib was added to a pre quantified sample solution and the amount of Ruxolitinibwas 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 Ruxolitinib 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 and standard deviation of percentage recovery were calculated. Recovey Conc. of sample ppm Recovery % of recovery % of Average recovery 50% % % Table 4: Accuracy results of Ruxolitinib Figure 3: Typical chromatogram of Ruxolitinib 365

7 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. LOD and LOQ Limit of detection (LOD) and limit of quantification (LOQ) were calculated as 0.25 ppm and 0.8ppm respectively as per ICH guide-lines. Robustness To determine the robustness of the method, two parameters from the optimized chromatographic conditions were varied. Parameter Modification Peak Area % of change Standard M.PHASE Meoh:0.1%OPA: ACN :2:50 PH WAVELENGTH 255 nm Table 5: Robustness Results. Ruggedness Inter day variations were performed by using six replicate injections of standard and sample solutions of concentrations which were prepared and analyzed by different analyst on three different days over a period of one week. Ruggedness also expressed in terms of percentage relative standard deviation. 366

8 S.NO Concentration Area RSD 1 40ppm ppm ppm ppm ppm ppm Table 6: Ruggedness Results. System Suitability Parameter: System suitability tests were carried out on freshly prepared standard stock solutions of Ruxolitinib and it was calculated by determining the standard deviation of Ruxolitinib standards by injecting standards in six replicates at 6 minutes interval and the values were recorded. Parameters Values λ max (nm) 257nm Beer s law limit (μg/ml) 10-60ppm Correlation coefficient Retention time 4.80min Theoretical plates Tailing factor 1.05 Limit of detection 0.25ppm Limit of quantification 0.8ppm 367

9 Table 7: System suitability parameters of Ruxolitinibin 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 Ruxolitinib 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 Methanol:0.1% OPA :Acetonitrile 50:2:48 (v/v/v). The retention time of Ruxolitinibwas found to be 4.80 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 4. The high percentage of recovery of Ruxolitinibwas found to be indicating that the proposed method is highly accurate. Proposed liquid chromatographic method was applied for the determination of Ruxolitinibin tablet formulation. The result for Ruxolitinibwas comparable with a corresponding labelled amount (Table 8). The absence of additional peaks indicates no interference of the excipients used in the tablets. Formulation Tablet dosage Sample Drug estimated % of Drug estimated concentration JAKAFI 10mg 40ppm Table 8: Tablet estimation of Ruxolitinibin 368

10 CONCLUSION A validated RP-HPLC method has been developed for the determination of Ruxolitinibin tablet dosage form. The proposed method is simple, rapid, accurate, precise and specific. Its chromatographic run time of 10 min allows the analysis of a large number of samples in short period of time. Therefore, it is suitable for the routine analysis of Ruxolitinib pharmaceutical dosage form. REFERENCES 1. Shilling, A. D.; Nedza, F. M.; Emm, T.; Diamond, S.; McKeever, E.; Punwani, N.; Williams, W.; Arvanitis, A. et al (2010). "Metabolism, Excretion, and Pharmacokinetics of [14C]INCB018424, a Selective Janus Tyrosine Kinase 1/2 Inhibitor, in Humans". Drug Metabolism and Disposition 38 (11): Mesa, Ruben A.; Yasothan, Uma; Kirkpatrick, Peter (2012). "Ruxolitinib". Nature Reviews Drug Discovery 11 (2): Mesa, RA (2010). "Ruxolitinib, a selective JAK1 and JAK2 inhibitor for the treatment of myeloproliferative neoplasms and psoriasis". IDrugs : the investigational drugs journal 13 (6): Pardanani, A.; Tefferi, A. (2011). "Targeting myeloproliferative neoplasms with JAK inhibitors". Current Opinion in Hematology 18 (2): Harrison, C.; Kiladjian, J. J.; Al-Ali, H. K.; Gisslinger, H.; Waltzman, R.; Stalbovskaya, V.; McQuitty, M.; Hunter, D. S. et al (2012). "JAK Inhibition with Ruxolitinib versus Best Available Therapy for Myelofibrosis". New England Journal of Medicine 366 (9): Verstovsek, S.; Mesa, R. A.; Gotlib, J.; Levy, R. S.; Gupta, V.; Dipersio, J. F.; Catalano, J. V.; Deininger, M. et al (2012). "A Double-Blind, Placebo- Controlled Trial of Ruxolitinib for Myelofibrosis". New England Journal of Medicine 366 (9): Tefferi, A. (2012). "Challenges Facing JAK Inhibitor Therapy for Myeloproliferative Neoplasms". New England Journal of Medicine 366 (9): ASCO Annual Meeting 2011: JAK Inhibitor Ruxolitinib Demonstrates Significant Clinical Benefit in Myelofibrosis 9. FDA Approves Incyte's Jakafi(TM) (ruxolitinib) for Patients with Myelofibrosis" (Press release). Incyte. Retrieved