International Journal of Chemical and Natural Science
Transcription
International Journal of Chemical and Natural Science
International Journal of Pharma Sciences Vol. 5, No. 3 (2015): 1030-1035 Research Article Open Access ISSN: 2320-6810 Analytical Method Development and Validation of RPHPLC Method for Simultaneous Estimation of Ranolazine and Dronedarone in Bulk Renuka Avvaru* and V. Prabakaran Department of Pharmaceutical Analysis and Quality Assurance, Krishna Theja Pharmacy College, Renigunta road, Tirupathi, A.P, India. * Corresponding author: Renuka Avvaru; email: [email protected] Received: 22 February 2015 Accepted: 25 March 2015 Online: 01 May 2015 ABSTRACT A simple, precise, accurate and rapid reverse phase high performance liquid chromatographic method was developed for simultaneous estimation of Ranolazine and Dronedrone in bulk. The separation was achieved by Xterra C18 column (150 mm × 4.6 mm; 5.0 μm). The estimation was carried out in isocratic program using buffer and acetonitrile (50:50 v/v) as mobile phase, at a flow rate of 1mL/min. diluent was water and methanol(60:40) and detection was carried out at 275nm. The retention time obtained for Ranolazine was 2.152min and Dronedrone was 6.213 min. The calibration curves were linear in the concentration range of 15-90µg/ml for Ranolazine and 318µg/ml for dronedrone correspondingly with correlation coefficient (r2) 0.999.The mean recoveries were found to be 99.97% for ranolazine and 100.02% for dronedarone. The proposed method has been validated as per ICH guidelines and successfully applied to the estimation of ranalazine and dronedarone in their combined form. Keywords: Ranolazine, Dronedarone, RP-HPLC simultaneous estimation. 1. INTRODUCTION Ranolazine is anti-anginal drug and chemically it is a piperazine derivative. IUPAC name of ranolazine is N(2,6-dimethylphenyl)-2-[4-[2-hydroxy-3-(2-methoxy phenoxy) propyl] piperazin-1-yl]acetamide. Ranolazine acts via altering the trans-cellular late sodium current. It is by altering the intracellular sodium level that ranolazine affects the sodium-dependent calcium channels during myocardial ischemia. Thus, ranolazine indirectly prevents the calcium overload the etiology of cardiac ischemia. Ranolazine is indicated for the treatment of chronic angina. Ranolazine may be used with beta blockers, nitrates, and calcium channel blockers, antiplatelet therapy, lipid-lowering therapy, ACE inhibitors, and angiotensin receptor blockers. Dronedarone, a benzofuran derivative, N-{2-butyl3 [4(3 di-butyl amino propoxy) benzoyl]-benzofuran-5yl} methanesulfonamide, is a potent drug mainly used for the indication of cardiac arrhythmias. Dronedarone is a multi-ion channel blocker, inhibiting the potassium currents involved in cardiac re- polarisation including http://ijps.aizeonpublishers.net/content/2015/2/ijps1019-1024.pdf IKr, IKs, IKur, and IK (Ach) and has been shown to be effective in the treatment of cardio- vascular hospitalization in patients with paroxysmal or persistent atrial fibrillation (AF) or atrial flutter (AFL). There were some estimation methods for single and for combination with other drugs but there is no method found for simultaneous estimation of ranolazine and dronidarone. [1-7] Hence the aim of present study is to develop simple, fast, accurate, precise and specific reversed phase high performance liquid chromatographic method for simultaneous determination of ranolazine and dronedarone in bulk. Figure-1a: Chemical structure of Ranolazine 1019 Renuka Avvaru et al. / Int J Pharma Sci. 2015, 5(3): 1030-1035 water bath for 5 minutes. Filter through 0.45 µ filter under vacuum filtration. Figure-1b: Chemical structures of Dronedarone 2. MATERIALS AND METHODS 2.1 Chemicals and solvents: Pure samples of Raolazine and Dronedarone were obtained respectively from Spectrum pharma research solutions, Hyderabad, India. Ammonium acetate, Methanol and water used are of HPLC grade. 2.2 Instrumentation: The chromatographic separations were performed using HPLC-Waters alliance (Model-2996) consisting of an inbuilt auto sampler, a column oven and 2965 PDA detector. The data was acquired through Empower-2software. The column used was Xterra C18 (150×4.6mm i.d, 5µm particle size) (make: Waters). Meltronics sonicator was used for enhancing dissolution of the compounds. Elico pH meter was used for adjusting the pH of buffer solution. All weighing was done on Sartorius balance (model AE-160). 2.3 Chromatographic conditions: Detector : 275nm Injection volume : 10µl Flow rate : 1.0ml/min Temperature : Ambient Temperature Run time : 9min Mobile phase : (pH 4.0) Ammonium acetate buffer and Acetonitrile taken in the ratio 50B:50A Diluent : Firstly dissolved in methanol and then made up with water 2.4 Preparation of buffer solution (pH 4): Accurately weighed and transferred 0.77gr of Ammonium acetate in 1000ml of Volumetric flask add about 900ml of milli-Q water added and degas to sonicate and finally make up the volume with water then PH adjusted to 4.0 with dil. Acetic acid solution. 2.5 Preparation of mobile phase: Mix a mixture of above Buffer 500 mL (50%), 500 mL of Acetonitrile HPLC (50%) and degased in ultrasonic Concentration of S.No Ranolazine in µg/ml 1 15ppm 2 30ppm 3 45ppm 4 60ppm 5 75ppm 6 90ppm Correlation Coefficient 2.6 Preparation of standard stock solutions: (600µg/ml and120µg/ml) Accurately Weighed and transferred 30mg of Ranolazine and 6mg of Dronidarone working Standards into 50ml clean dry volumetric flasks, add 3/4th volume of diluent, sonicated for 5 minutes and make up to the final volume with diluents. (Stock solution) 1ml from the above two stock solutions was taken into a 10ml volumetric flask and made up to 10ml. (60µg/ml and12µg/ml) 2.7 Preparation of sample solution: 5 tablets were weighed and calculate the average weight of each tablet. weight equivalent to 1tablet(1260mg) was transferred into a 500mL volumetric flask, 250mL of diluent was added and sonicated for 25 min, further the volume was made up with diluent and filtered. (Stock solution) From the filtered solution 0.4ml was pipette out into a 10 ml volumetric flask and made up to 10ml with diluent. (60µg/ml and12µg/ml) 2.8 METHOD VALIDATION: The developed method was validated as per the ICH (International Conference on Harmonization) guidelines with respect to System suitability, Precision, Specificity, Linearity, Accuracy, Limit of detection and Limit of quantification. 2.8.1 Linearity: Aliquots of 0.25, 0.5, 0.75, 0.1, 1.25 and 1.50 ml were taken from stock solution of concentration 600µg of ranolazine and 120µg of dronedarone, and then diluted up to 10ml with diluents such that the final concentrations were in the range 15-90µg for ranolazine and 3-18µg for dronedarone. Volume of 10µl of each sample was injected in five times for each concentration level and calibration curve was constructed by plotting the peak area versus drug concentration. The observations and calibration curve were shown in Table 1 and Fig. 2, 3. Table-1: Linearity data Ranolazine Area 156864 324871 466249 634275 783151 952249 http://ijps.aizeonpublishers.net/content/2015/3/ijps1030-1035.pdf Concentration of Dronedarone in µg/ml 3ppm 6ppm 9ppm 12ppm 15ppm 18ppm Dronedarone Area 169929 325400 479513 649881 826607 973167 0.999 1031 Renuka Avvaru et al. / Int J Pharma Sci. 2015, 5(3): 1030-1035 Figure-2: Ranolazine calibration curve Figure-3: Dronedarone calibration curve 2.8.2 Assay: Accurately weighed powder equivalent to 30mg of Ranolazine and 6mg of Dronedarone was transferred into a 50mL clean dry volumetric flask add diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. Drug Ranolazine Dronedarone % linearity level 50 100 150 (Stock solution) Further pipette 1ml of Ranolazine and Dronedarone of the above stock solution into a 10ml volumetric flask and diluted up to the mark with diluents it gives 60µg of Ranolazine and 12µg of Dronedarone. The results were shown in Table-2. Table-2: Ranolazine and Dronedarone assay Label claim mg/tab 750 150 Amount found mg/tab 751.95 150.195 Label claim(% ) 100.26 100.13 Table-3: Accuracy (Ranolazine) % recovery 98.27 101.09 100.97 99.79 100.06 98.29 99.97 100.46 100.77 http://ijps.aizeonpublishers.net/content/2015/3/ijps1030-1035.pdf S.D* % R.S.D 0.73 0.602 0.73 0.60 %mean recovery ± S.D % RSD 99.97% 1.06% 1032 Renuka Avvaru et al. / Int J Pharma Sci. 2015, 5(3): 1030-1035 % linearity level 50 % recovery 99.95 100.21 99.88 99.24 100.62 100.38 100.59 101.25 98.11 100 150 Accuracy (Dronedarone) 2.8.3 Accuracy: Accuracy of the method was done by recovery study. Sample solutions were prepared by spiking at about 50%, 100%, and 150% of specification limit to placebo and analyzed by the proposed HPLC method. Results are shown in Table-3. 2.8.4 Specificity: The specificity of the method was performed by injecting blank solution( without any sample) and then a drug solution of 10µl injected into the column, under optimized chromatographic conditions, to demonstrate the separation of both Ranolazine and Dronedarone from any of the impurities, if present. As there was no interference of impurities and also no change in the retention time, the method was found to be Specific. INJECTIONS 1 2 3 4 5 6 AVG S.D %R.S.D Drug Ranolazine Dronedarone Injections 1 2 3 4 5 6 1 2 3 4 5 6 %mean recovery ± S.D % RSD 100.02% 0.91% 2.8.5 Limit of detection (LOD) and Limit of quantification (LOQ): The parameters LOD and LOQ were determined on the basis of response and slope of the regression equation. The linearity for Ranolazine and Dronedarone was performed from 15-90µg/ml and 3-18µg/ml respectively. 2.8.6 System precision: Precision is the measure of closeness of the data values to each other for a number of measurements under the same analytical conditions. Standard solution of Ranolazine (60µg/ml) and Dronedarone (12µg/ml) were prepared as per test method and injected for 3 times. Results are shown in Table-4. Table-4: System precision AREAS(Ranolazine) 619758 620658 621384 618932 621554 620524 620468 992 0.16 AREAS(Dronedarone) 636653 646380 630940 651186 654454 638932 643091 9075.9 1.4 Table-5: Method precision % Assay 100.60 99.35 99.39 100.99 100.95 100.29 99.73 100.10 101.01 100.61 99.33 100.02 2.8.7 Method precision: Three samples were prepared and analyzed as per the test method on same day and three different days and calculated the % RSD for assay of five preparations. Results were shown in Table- 5. 2.8.8 Robustness: Robustness studies were carried out by variations in flow rate, mobile phase compositions and temperature. It was observed that the small changes in these operational parameters did not lead to changes of retention time of the peak interest. The degree of http://ijps.aizeonpublishers.net/content/2015/3/ijps1030-1035.pdf Mean S.D % R.S.D 100.26 0.73 0.73 100.13 0.602 0.60 reproducibility of the results proven that the method is robust. 2.8.9 System suitability test: The system suitability was determined by making six replicate injections from freshly prepared standard solutions. The observed RSD values were well within usually accepted limits (≤2%). Theoretical plates, tailing factor, resolution between Ranolazine and Dronedarone were determined. The results are all within acceptable limits summarized in Table-6. 1033 Renuka Avvaru et al. / Int J Pharma Sci. 2015, 5(3): 1030-1035 Drug Ranolazine Dronedarone Table-6: Characteristics of HPLC method Parameters defined Linearity range (µg/ml) Slope Intercept Regression coefficient(r2) LOD ( µg/ml) LOQ (µg/ml) Tailing factor Plate count Linearity range (µg/ml) Slope Intercept Regression coefficient(r2) LOD (µg/ml) LOQ (µg/ml) Tailing factor Plate count Obtained value 15-90µg/ml 10541 516.4 0.999 0.19 0.56 1.31 3397 3-18 µg/ml 54254 927 0.999 0.0150 0.0455 1.49 4346 Figure-4: Standard chromatogram of Ranolazine and Dronedarone 3. RESULTS AND DISCUSSION The nature of sample, its molecular weight and solubility decides the proper selection of stationary phase. The drugs Ranolazine and Dronedarone were preferably analyzed by reverse phase chromatography and accordingly C18 column was selected. The elution of the compounds from column was influenced by polar mobile phase. The ratio of ammonium acetate buffer to Acetonitrile was optimized to 50:50 to give well resolved and good symmetrical peaks with short run time. The retention time of Ranolazine and Dronedarone were found to be 2.152 and 6.213 min respectively. The calibration curve was linear over the concentration range of 15-90µg/ml (Ranolazine) and 318µg/ml (Dronedarone). The linearity of the method was statistically confirmed. RSD values for accuracy and precision studies obtained were less than 2% which revealed that developed method was accurate and precise. The system suitability parameters were given in table-5. The analytical recovery at five different concentrations of Ranolazine and Dronedarone was determined and the recovery results were in the range of 50-150µg/ml. Therefore proposed validated method was successfully applied to determine Ranolazine and Dronedarone in tablet dosage form. http://ijps.aizeonpublishers.net/content/2015/3/ijps1030-1035.pdf 4. CONCLUSION The developed method is accurate, simple, rapid and selective for the simultaneous estimation of Ranolazine and Dronedarone in pharmaceutical dosage form. The excipients of the commercial sample analyzed did not interfere in the analysis, which proved the specificity of the method for these drugs. The sample preparation is simple, the analysis time is short and the elution is by isocratic method. Hence the proposed method can be conveniently adopted for the routine quality control analysis in the combined formulation. 5. REFERENCES 1. 2. 3. 4. Ashish, Prakash and Sachin. (2010). Development and Validation of UV Spectrophotometric Method for the Estimation of Ranolazine in Bulk Drug and Pharmaceutical Formulation. IJCTR. 2(4): 1945-1948. Boovizhikannan, and Vijayaraj. (2012). “Estimation of Ranolazine Using Reverse Phase High Performance Liquid Chromatography Technique” AJPR. 2(6). Ramanaiah G.Ramachandran D, Srinivas, Jayapal G, Purnachanda Rao and Srilakshmi. (2012). "Development and Validation of Stability Indicating RP-LC Method for Estimation of Ranolazine in Bulk and Its Pharmaceutical Formulations." AJAC. 3: 378-384. Asifa N and Pavani H. (2014). Development and validation of HPLC method for simultaneous determination of Ranolazine and Dronedarone in bulk and pharmaceutical dosage forms. IJRPB. 1(4): 2320 – 3471. 1034 Renuka Avvaru et al. / Int J Pharma Sci. 2015, 5(3): 1030-1035 5. 6. 7. Naresh T, Shakil S, Surendranath, Ravi Kiran and Suresh Kumar. (2012). A Stability Indicating HPLC Method for Dronedarone in Bulk Drugs and Pharmaceutical Dosage Forms, AJAC. 2: 544-551. Kshitija K, Mrinalini D. (2014). Stability-indicating hptlc method for quantitative estimation of dronedarone hydrochloride in bulk and pharmaceutical dosage form, WJPPS. 3(6): 2163-2175. Shashikant B, Sanjay, Kapil P, Anil C, Vijayavitthal T. (2013). Stability Indicating RP-HPLC Method for the Determination of Dronedarone Hydrochloride and Its Potential Process- Related Impurities in Bulk Drug and Pharmaceutical Dosage Form. AJAC. 4: 323-335. © 2015; AIZEON Publishers; All Rights Reserved This is an Open Access article distributed under the terms of the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ***** http://ijps.aizeonpublishers.net/content/2015/3/ijps1030-1035.pdf 1035