PVV Satyanaryana et al., IJSID, 2012, 2 (1), International Journal of Science Innovations and Discoveries

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1 ISSN: IJSID International Journal of Science Innovations and Discoveries An International peer Review Journal for Science Research Article Available online through RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR THE ANALYISIS OF RIVAROXABAN IN PHARMACEUTICAL DOSAGE FORMS P.V.V Satyanarayana* and Alavala Siva Madhavi Department of Chemistry, Acharya Nagarjuna University, Guntur, Andhra Pradesh, India Received: Accepted: *Corresponding Author Address: Name: PVV. Satyanaryana Place: Guntur, AP, India ABSTRACT A simple, selective, linear, precise and accurate RP-HPLC method was developed and validated for assay of Rivaroxabanin 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 THF:MEOH:ACN 10:40:50 (v/v/v). The UV detection wavelength was at 270 nm. Linearity was observed in concentration range of ppm. The retention time for Rivaroxabanwas 2.55 min. The method was validated as per the ICH guidelines. The proposed method can be successfully applied for the estimation of Rivaroxabanin pharmaceutical dosage forms. INTRODUCTION Keywords: Rivaroxaban, HPLC, Linearity, Precision, Recovery, 270nm INTRODUCTION 226

2 INTRODUCTION Rivaroxaban is an oral anticoagulant invented and manufactured by Bayer; in a number of countries it is marketed as Xarelto. [1] In the United States, it is marketed by Janssen Pharmaceutica. [2] It is the first available orally active direct factor Xa inhibitor. Rivaroxaban is well absorbed from the gut and maximum inhibition of factor Xa occurs four hours after a dose. The effects lasts 8 12 hours, but factor Xa activity does not return to normal within 24 hours so once-daily dosing is possible. Rivaroxaban is an oxazolidinone derivative optimized for inhibiting both free Factor Xa and Factor Xa bound in the prothrombinase complex. [5] It is a highly selective direct Factor Xa inhibitor with oral bioavailability and rapid onset of action. Inhibition of Factor Xa interrupts the intrinsic and extrinsic pathway of the blood coagulation cascade, inhibiting both thrombin formation and development of thrombi. Rivaroxaban does not inhibit thrombin (activated Factor II), and no effects on platelets have been demonstrated. [1] Rivaroxaban has predictable pharmacokinetics across a wide spectrum of patients (age, gender, weight, race) and has a flat dose response across an eightfold dose range (5 40 mg). [6] Clinical trial data have shown that it allows predictable anticoagulation with no need for dose adjustments and routine coagulation monitoring. [1] However, these trials have excluded patients with liver disease and end-stage liver disease; therefore, the safety of rivaroxaban in these populations is unknown. Rivaroxaban bears a striking structural similarity to the antibiotic linezolid: both drugs share the same oxazolidinone-derived core structure. Accordingly, rivaroxaban was studied for any possible antimicrobial effects and for the possibility of mitochondrial toxicity, which is a known complication of long-term linezolid use. Studies found that neither rivaroxaban nor its metabolites have any antibiotic effect against Gram-positive bacteria. As for mitochondrial toxicity, in vitro studies found the risk to be low, and not likely to be of clinical consequence because rivaroxaban is only meant (and approved) for shortterm use. [6] Figure.1 Structure Of Rivaroxaban EXPERIMENTAL Chemicals and reagents All HPLC SOLVENTS used like Acetonitrile,Water Methanol,Orthophosphoric 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 227

3 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 Rivaroxabanwas 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 Rivaroxabanin the mass concentration range of 1mg/ml to 0.2 mg/ml 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 Rivaroxabanwas obtained by plotting the peak area ratio versus the applied concentrations of Axitinib. The linear correlation coefficient was found to be Table 1: Linearity of Rivaroxaban S.NO CONC ppm AREA Figure 2: Calibration curve of Rivaroxaban 228

4 Precision PVV Satyanaryana et al., IJSID, 2012, 2 (1), Table.2: Linear Regression Data for Calibration curve Drug Axitinib Concentration range ppm Slope (m) Intercept (b) Correlation coefficient % RSD Repeatability of the method was checked by injecting replicate injections of 70 ppm of the solution for six times on the same day as intraday precision study of Rivaroxabanand the RSD was found to be Accuracy INJECTION INTERDAY INTRADAY S.D = R.S.D =0.056 S.D = R.S.D =0.066 Table 3: Precision parameters of Rivaroxaban The accuracy of the method was determined by calculating recovery of Rivaroxaban(50,75,100ppm) by the method of standard addition. Known amount of Rivaroxaban was added to a pre quantified sample solution and the amount of Rivaroxabanwas 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 Rivaroxabanwas 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. Figure 3: Typical chromatogram of Axitinib 229

5 Table 4: Accuracy results of Axitinib Recovey Conc. of sample ppm Recovery % of recovery % of Average recovery 50% % % 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 2 microgram/ml and 6 microliter respectively as per ICH guide-lines. Robustness varied. Ruggedness To determine the robustness of the method, two parameters from the optimized chromatographic conditions were 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. Table 5: Ruggedness Results. Parameter Modification Peak Area % of change M.PHASE THF10%:MeOH 60%:ACN30% PH WAVELENGTH 272 nm System Suitability Parameter: System suitability tests were carried out on freshly prepared standard stock solutions of Rivaroxaban and it was calculated by determining the standard deviation of Rivaroxaban standards by injecting standards in six replicates at 6 minutes interval and the values were recorded. Table 5: System suitability parameters of Axitinib Parameters Values λ max (nm) 270 nm Beer s law limit (μg/ml) ppm Correlation coefficient Retention time 2.55 min Theoretical plates Tailing factor 1.8 Limit of detection 2 ppm Limit of quantification 6 ppm 230

6 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 Rivaroxabanbeing 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,ACN,THF 40:50:10 (V/V/V). The retention time of Rivaroxabanwas found to be 2.55 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 Rivaroxabanwas found to be indicating that the proposed method is highly accurate. Proposed liquid chromatographic method was applied for the determination of Rivaroxabanin tablet formulation. The result for Rivaroxabanwas comparable with a corresponding labelled amount (Table 6). The absence of additional peaks indicates no interference of the excipients used in the tablets. Formulation Tablet dosage Sample concentration Amount of drug estimated XARELTO 10mg Table 6: Tablet estimation of Axitinib CONCLUSION A validated RP-HPLC method has been developed for the determination of Rivaroxabanin tablet dosage form. The proposed method is simple, rapid, accurate, precise and specific. Its chromatographic run time of 8 min allows the analysis of a large number of samples in short period of time. Therefore, it is suitable for the routine analysis of Rivaroxabanin pharmaceutical dosage form. REFERENCES 1. "Xarelto: Summary of Product Characteristics". Bayer Schering Pharma AG "FDA Approves XARELTO (rivaroxaban tablets) to Help Prevent Deep Vein Thrombosis in Patients Undergoing Knee or Hip Replacement Surgery". Janssen Pharmaceutica. 3. "Bayer's Xarelto Approved in Canada" (Press release). Bayer. 4. "Bayer s Novel Anticoagulant Xarelto now also Approved in the EU". Bayer. 5. Roehrig S, Straub A, Pohlmann J, et al. (September 2005). "Discovery of the novel antithrombotic agent 5-chloro-N-({(5S)- 2-oxo-3- [4-(3-oxomorpholin-4-yl)phenyl]-1,3-oxazolidin-5-yl}methyl)thiophene- 2-carboxamide (BAY ): an oral, direct factor Xa inhibitor". Journal of Medicinal Chemistry 48 (19): European Medicines Agency (2008). "CHP Assessment Report for Xarelto 7. A dose-ranging study evaluating once-daily oral administration of the factor Xa inhibitor rivaroxaban in the treatment of patients with acute symptomatic deep vein thrombosis: the Einstein DVT Dose-Ranging Study, Harry R. Buller, Blood September 15, 2008 vol. 112 no Interpretation of Benefit-Risk of Enoxaparin as Comparator in the RECORD Program: Rivaroxaban Oral Tablets (10 milligrams) for Use in Prophylaxis in Deep Vein Thrombosis and Pulmonary Embolism in Patients Undergoing Hip or Knee Replacement Surgery, David Van Thiel, CLIN APPL THROMB HEMOSTAugust 2009 vol. 15 no