Naga Jyothi. C et al, JPBMAL, 2015, 3(1): 242 246 ISSN: 2347-4742 Journal of Pharmaceutical and Biomedical Analysis Letters Journal Home Page: www.pharmaresearchlibrary.com/jpbmal Research Article Open Access Method Development and Validation of Nebivolol by RP-HPLC Naga Jyothi. C*, Gummi Vishwanth, P. Sunitha Dept. of Pharmaceutical Analysis and Quality Assurance, Teegala Krishna Reddy College of Pharmacy, Hyderabad, India A B S T R A C T An isocratic reversed-phase high performance liquid chromatographic method was established for the estimation of Nebivolol in tablet dosage form. The chromatographic conditions were successfully developedd for the estimation of Nebivolol by using Develosil C18 column (1504.6mm, 5µ), flow rate was 1ml/min, mobile phase ratio was (80:20 v/v) Acetonitrile: Phosphate buffer ph 6 (ph was adjusted with diluted phosphoric acid), detection wave length was found to be at 279nm. The Retention time was found to be 2.3mins. The % purity of Nebivolol was found to be 99.56%. Linearity was obtained in the concentration range of 20 µg/ml to 70 µg/ml with correlation coefficient of 0.999.. The mean recovery was found to be 100.52%. The Proposed RP-HPLC method was found to be accurate, precise, robust and specific and can be successfully applied for the routine analysiss of Nebivolol in tablet dosage form Keywords: Nebivolol, RP-HPLC, Validation. A R T I C L E I N F O CONTENTS 1. Introduction................................................................................242 2. Materials and Methods....................................................................... 243 3. Results and discussion....................................................................... 244 4. Conclusion................................................................................. 246 5. References.................................................................................246 Article History: Received 05 October 2014, Accepted 21 December 2014, Available Online 18 January 2015 *Corresponding Author Naga Jyothi. C Pharmaceutical Analysis and Quality Assurance, Teegala Krishna Reddy College of Pharmacy, Hyderabad, India Manuscript ID: JPBMAL2439 PAPER-QR CODE Citation: Naga Jyothi. C, et al. Method Development and Validation of Nebivolol by RP-HPLC. J. Pharm, Biomed. A. Lett., 2015, 3(1): 242-246. Copyright 2015 Naga Jyothi. C, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted ted use, distribution and reproduction in any medium, provided the original work is properly cited. 1. Introduction Nebivolol is a third-generation β 1 selective works by relaxing blood vessels and slowing heart rate to improve blood flow and decrease blood pressure [1,2]. It is Journal of Pharmaceutical and Biomedical Analysis Letters chemically1-(6-fluorochroman-2-yl)-(2-(6-fluorochroman -2-yl)-2hydroxyethyl) amino) ethanol. It is a white odourless powder used for the treatment of hypertention 242
and heart failure. Its mode of action is lowering blood pressure by reducing peripheral [3-6] vascular resistance, and significantly increases stroke volume with preservation of cardiac output. In the present work the simple, rapid, precise and accurate robust liquid chromatographic method was developed for the determination of Nebivolol tablets. Previously reported methods have one or other disadvantages and therefore a novel liquid chromatographic method was developed and validated as per ICH guidelines [7,8]. 2. Materials and Methods Aim: The main objective of this study is to develop an accurate and Economical method for the estimation of Nebivolol through RP-HPLC. Present study was performed by taking active pharmaceutical ingredient of Nebivolol and also Nebistar tablets (2.5mg). Samples: Nebivolol API Reagents: Methanol (HPLC grade), Water(HPLC grade), Potassium di hydrogen phosphate, Di potassium hydrogen phaosphate, phosphoric acid. Nebivolol is available (Label claim: 2.5 mg) with brand names Nebistar(Lupin). All chemicals were of analytical grade and used as received. Apparatus HPLC used was Azilent 1200 with PDA detector. Assay: Preparation of Nebivolol standard solution solution-1000μg/ml). Further pipette out 0.5 ml from the above stock solution into a 10 ml volumetric flask and was diluted up to the mark with mobile phase (50μg/ml or ppm). Mix well and filter through 0.45µm filter Preparation of Sample Solution solution-1000 μg/ml). Further pipette out 0.5ml of the above stock solution into a 10ml volumetric flask and was diluted up to the mark with mobile phase (50μg/ml or ppm). Mix well and filter through 0.45µm filter Chromatographic Procedure 10 L of the blank,standard and sample were injected into th e chromatographic system and recorded the chromatograms areas for the Nebivolol the peaks were used for calculating the % assay by using the formulae. Assay Calculation: Figure 1: Chemical structure of Nebivolol % = Journal of Pharmaceutical and Biomedical Analysis Letters 243. 100 100 Method Validation Accuracy The standard solutions of accuracy 80%, 100% and 120% were injected into chromatographic system. Linearity A stock solution of 1000µg/ml was prepared by using the premixed mobile phase as diluent. From this stock solution, different concentrations were prepared.i.e., 20, 30, 40, 50, 60, 70µg/ml respectively. Prepared dilutions were injected serially. Calibration curve was plotted by taking peak area and concentration of the prepared dilutions. Precision: Preparation of stock solution solution-1000μg/ml). Further pipette out 0.5 ml from the above stock solution into a 10 ml volumetric flask and was diluted up to the mark with mobile phase (50μg/ml or ppm). Mix well and filter through 0.45µm filter. Procedure: The standard solution was injected for five times and measured the area for all injections in HPLC. Intermediate Precision / Ruggedness To evaluate the intermediate precision (also known as ruggedness) of the method, precision was performed by different by analysts. Procedure 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 sample injections results should not be more than 2%. Limit of Detection (LOD) LOD s can be calculated based on the standard deviation of
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: LOD = 3.3 X σ Where σ - Standard deviation (SD) S - Slope Limit of Quantification LOQ s can be calculated based on the standard deviation of 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: LOQ = 10 X σ Where σ -Standard deviation, S -Slope 3. Results and Discussion A thorough study about Nebivolol drug and its estimation was done. Various trials were performed by using phosphate buffer, Acetonitrile, and various columns. Table 1: Optimized conditions Parameter Condition Mobile phase Acetonitrile:Phosphate buffer(p H 6) (80:20) Flow rate 1ml/min Column Ambient Temperature Figure 2: Optimized chromatogram Figure 3: Chromatogram showing blank preparation (mobile phase) Figure 4: Chromatogram showing Assay of Standard Figure 5: Chromatograms showing Assay of sample injection injection Journal of Pharmaceutical and Biomedical Analysis Letters 244
Calculation: % =. 100 =99.56% The retention time of Nebivolol was found to be 2.30mins. The % purity of Nebivolol in tablet dosage form was found to be 99.56%. Validation Accuracy The accuracy was determined at 3 different levels i.e 80%, 100% and 120%, and % recovery was calculated. Linearity The linearity study was performed for the concentration of 20ppm to 70ppm level. Each level was injected into chromatographic system. The area of each level was used for calculation of correlation coefficient. Correlation Table 2: Accuracy results % Concentration %Recovery 80% 100.65 100% 100.09 120% 100.82 Coefficient was found to be 0.999. Low values of standard deviation, standard error, etc serve as a proof to show that the calibration plot did not deviate from linearity. Figure 6: Showing Linearity curve Precision Determined RT and %RSD was calculated to prove that method is validated Table 3 S.No RT 1 2.29 2 2.29 3 2.29 4 2.31 5 2.31 6 2.32 Avg St. Dev %RSD Limit of Detection LOD s can be calculated based on the standard deviation of at levels approximating the LOD according to the formula. Formula: =. Standard deviation = 16053.32 Slope = 23930 =. = 3.3 X 16053.32/23930 = 2.21378 μg/ml. Limit of Quantification (LOQ) LOQ s can be calculated based on the standard deviation of according to the formula. Formula: = Standard deviation = 16053.32 Journal of Pharmaceutical and Biomedical Analysis Letters 245
Slope = 23930 = = 10 X 16053.32/23930 = 6.70844 Robustness The method is said to be robust if there is no effect at different conditions The Limit of Quantification for Nebivolol was found to be 6.70844. Figure 7: Chromatogram for flow rate 0.9ml/min Figure 8: Chromatogram for flow rate 1.0ml/min Table 4 Parameter (flow rate) RT Tailing factor 0.9ml/min 2.46 1.59 1ml/min 2.31 1.51 1.1ml/min 2.15 1.47 Figure 9: Chromatogram for flow rate 1.1ml/min 4. Conclusion The method was validated for system suitability, precision, accuracy, linearity, ruggedness. Therefore it was concluded that the proposed method can be used for routine analysis of 5. References 1. Judy WMC.Nebivolol: A third-generation β 1 blocker for hypertention. Cli.Therapeutics for 2009, 31(3): 447-62. 2. http://www.drugs.com/mtm/nebivolol.htm. 3. Sweetman SC, Eds.,In:Martindale;The complete Drug Reference,33 rd ed., The Pharmaceutical Press, London, 2002, 598. 4. The Merck Index, Thirteenth edition, Merck Res. Lab. Division of Merck and Co.Inc, Whitehouse station, New Jersy, USA., 2001, 1152, 1767. 5. Moffat A.C.,Osselton M.D.,Widdop B.Clarke's Analysis of Drugs and Poisons in nebivolol in tablet dosage form, which is simple, less time consuming using an economical column. pharmaceuticals, body fluids and postmortem material. 3 rd ed.pharmaceutical Press, London. 2004, pp.1322-23. 6. Sweetman SC, Martindale, The complete drug reference 34 th ed. Pharmaceutical Press, Great Britain, 2005, pp.650. 7. ICH Validation of analytical procedures: Text and methodology ( 2005) Q2 (R1), International Conference on Harmonization. 8. ICH Stability Testing of New Drug Substances and Products ( 2003) Q1A (R2), International Conference on Harmonization. Journal of Pharmaceutical and Biomedical Analysis Letters 246