DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION

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Page5521 Indo American Journal of Pharmaceutical Research, 2016 ISSN NO: 2231-6876 DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF ROSUVASTATIN

1 Page5521 Indo American Journal of Pharmaceutical Research, 2016 ISSN NO: DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF ROSUVASTATIN CALCIUM AND FENOFIBRATE IN BULK AND PHARMACEUTICAL DOSAGE FORM Swapnil Gaikwad *, Dr.L.A.Kawale, Kaveri Ahire, Sujata Yadav, Shubhangi Bodile Affiliation: Student at M. Pharm (Quality Assurance Techniques) M.V.P Samaj s College of Pharmacy, Nashik-2. ARTICLE INFO Article history Received 05/05/2016 Available online 31/05/2016 Keywords Rosuvastatin Calcium, Fenofibrate, HPLC, Stability Indicating. Corresponding author Swapnil Gaikwad M.V.P Samaj s college of pharmacy, Gangapur road, Nashik-2(422002) Maharashtra, India. ahire.kaveri17@gmail.com ABSTRACT A simple, rapid, economic, sensitive and precise RP-HPLC method has been developed for the simultaneous determination of Rosuvastatin calcium and Fenofibrate in bulk and pharmaceutical dosage form. The method was carried out using Greece C18 (4.6ID 250mm; 5μm) column and mobile phase comprised of methanol and water in proportion of ratio 90:10 v/v and degassed under ultrasonication. The flow rate was 1.0 ml/min and detection was carried out at 254 nm. The retention time of ROS and FEN were found to be min and min respectively. The described method shows excellent Linearity of ROS and FEN were in the range of 10 to 50 μg/ml and 67 to 335 μg/ml respectively. The % recoveries of ROS and FEN were found to be in between 97.94% to 99.81% for ROS and 97.18% to 98.49% for FEN respectively. The method was also applied for the determination of ROS & FEN in the presence of their degradation products formed under variety of stress conditions. Degradation products produced as a result of stress studies did not interfere with the detection of ROS and FEN. The correlation coefficient for both drug was and respectively. The limit of detection for ROS and FEN were and μg/ml respectively. The limit of quantification was and μg/ml respectively. The robustness study and percentage of assay of the formulation were found within limit as per ICH guidelines. Please cite this article in press as Swapnil Gaikwad et al. Development and Validation of Stability Indicating RP-HPLC Method for Simultaneous Estimation of Rosuvastatin Calcium and Fenofibrate in Bulk and Pharmaceutical Dosage Form. Indo American Journal of Pharmaceutical Research.2016:6(05). Copy right 2016 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 Page5522 INTRODUCTION Rosuvastatin Calcium: Figure No.1:Chemical Structures of Rosuvastatin calcium. IUPAC name : (3R, 5S, 6E)-7-[4-(4-fluorophenyl)-2- (N-methylmethanesulfonamido)- 6-(propan-2-yl) pyrimidin-5-yl]- 3, 5-dihydroxyhept-6-enoic acid. Generic Name : Rosuvastatin calcium Description : Off white to creamish white Crystalline powder Molecular Formula: (C22H27FN3O6S)2 Ca Molecular weight : g/mol Melting point: oc Solubility: Soluble in organic solvents such as methanol, ethanol. Category: Antihyperlipidemic Agent. Dosage forms: 5mg, 10mg, 20mg and 40mg (Tablet). Mechanism of Action: Rosuvastatin is a lipid lowering drug that reduce serum cholesterol levels by inhibiting a key enzyme (HMG CoA reductase) involved in the synthesis of cholesterol. Fenofibrate: Figure No.2: Chemical structure of Fenofibrate.

3 Page5523 IUPAC name : 1-Methylethyl 2-[4-(4-chlorobenzoyl) phenoxy] 2-methylpropanoate Generic Name : Fenofibrate Description : White to off-white crystalline powder Molecular Formula : C20H21ClO4 Molecular weight : g/mol Melting point : 79 to 82 oc Solubility : Practically insoluble in water, very soluble in methylene chloride. Category : Antihyperlipidemic Agent. Dosage forms : 40 mg, 120mg (Tablet) Mechanism of Action: Active moiety of Fenofibrate is Fenofibric acid. Lipid lowering effect of Fenofibric acid by the activation of peroxisome proliferator activated receptor α (PPARα). Through this mechanism, Fenofibrate increases lipolysis and elimination of triglyceriderich particles from plasma by activating lipoprotein lipase and reducing production of apoprotein C-III. MATERIALS AND METHODS Chemicals and Reagents Rosuvastatin calcium working standards were procured as gift sample from Macleods pharmaceuticals Ltd, Mumbai and Fenofibrate Wockhardt pharmaceuticals Ltd, Aurangabad. HPLC grade Methanol, Water (Milli Q) of Hexon Laboratories, INDIA. AR grade Hydrochlroic acid, Hydrogen peroxide, of Hexon Laboratories, INDIA. Sodium hydroxide of Merck. Tablet formulation Razel F 10 of Glenmark pharmaceuticals Ltd. were purchased from the local medical shop. Chromatographic Equipments and Conditions HPLC (P-3000-M Reciprocating with UV-3000-M) was used to developed and validate the method. The chromatographic separation was carried out by using (Greece C18(4.6 ID HPLC (Waters 2690 with PDA detector) was used to developed and validate the method. The chromatographic separation was carried out by using (Zodiac C mm,5μm) column, maintained at ambient temperature. Preparation of mobile phase and stock solutions: A mixture of Buffer and Acetonitrile in the ratio of (65:35). Filtered and degassed. (Buffer: 1ml o-phosphoric Acid in one liter of water.) In order to prepare stock solution, 10 mg ROS and 10 mg FEN were accurately weighed and transferred into two separate 100 ml volumetric flasks respectively, about 75 ml of diluent was added to each flask and sonicated to dissolve, diluted up to mark with the diluent to obtain 100 μg/ml concentration of ROS and 100 μg/ml concentration of FEN separately. Preparation of sample solution: Weigh and finely powder 20 tablets. Accurately weigh and transfer a quantity of powder sample equivalent to 10 mg of ROS and 67 mg FEN in to a 100 ml clean dry volumetric flask, add about 75 ml of diluent and sonicate to dissolve it completely and make volume up to the mark with the diluent. Filter the solution through 0.45μm membrane filter. Assay: The assay performed by the Marketed formulation of ROS and FEN (Razel F 10). The prepared standard and sample solution were injected in HPLC.

4 Page5524 Table No.1. Assay Method Development. Drug Label Claim (mg/tab) Conc.taken μg/ml) Area Amt. found (μg/ml) % Assay Rosuvastatin Calcium Fenofibrate Validation of method: All the analytical validation parameters were determined according to the ICH guidelines(q 2 R 1). 1) System Suitability 2) Linearity 3) Accuracy 4) Precision 5) Limit of detection and Limit of quantification 6) Robustness System Suitability: Six times standard solution was injected into HPLC system and the corresponding chromatograms were recorded. Retention time, peak area, theoretical plates, tailing factor, resolution, asymmetry were calculated. Linearity: Linearity was evaluated by analysis of working standard solution of ROS and FEN of five different concentrations. The range of linearity were from 10 to 50 μg/ml for ROS and 67 to 334 μg/ml for FEN The result shows that within the concentration range mentioned above, there was an excellent correlation between peak area ratio and concentration. Acceptance Criteria: The plot should be linear passing through the origin. Correlation Coefficient should not be less than Figure No.3: Linearity Plot For Rosuvastatin Calcium. Figure No. 4 : Linearity Plot For Fenofibrate.

5 Page5525 Accuracy( Recovery): Accuracy was determined by the range 50%-150% of the sample concentration. Calculated amount of ROS and FEN from standard stock solution was added in placebo to attain 50%,100% and 150% of sample concentration. Each sample was prepared in triplicate at each level. Accuracy was expressed as the percentage of analytes recovered by the assay. Lists the recoveries of the drug from a series of spiked concentrations. Acceptance Criteria: 1. Mean recovery should be in the range of %. 2. The RSD should not be more than 2.0%. Precision: Prepare six different test solution of the 100% test concentration from the same sample matrix. Inject duplicate injections of each test solution. In method precision, a homogenous sample of a single batch should be analyzed six times. This indicates whether a method is giving consistent results for a single batch. %RSD of sample solution were calculated. Acceptance criteria: % RSD Should be more than 2% for test results. Limit of detection( LOD) and Limit of quantification( LOQ): The LOD is the lowest concentration of the analyte that can be detected and LOQ is the lowest concentration that can be quantified with acceptable precision and accuracy. The limit of detection (LOD) and limit of quantitation (LOQ) were established at signal-to-noise ratio of 3:1 and 10:1 respectively. The LOD and LOQ of Rosuvastatin Calcium and Fenofibrate was experimentally determined by six injection of each drug. The LOD and LOQ of Rosuvastatin Calcium and was found to be μg/ml and μg/ml respectively. The LOD and LOQ of Fenofibrate was found to be μg/ml and μg/ml Respectively. Robustness: The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides indication of its reliability during normal usage. Carry out the following procedure individually by changing following variation in chromatographic conditions. 1) Change in flow rate of mobile phase to 0.90 ml/min. 2) Change in flow rate of mobile phase to 1.10 ml/min. Forced Degradation Study: Forced degradation studies are also known as stress testing, stress studies, stress decomposition studies, forced decomposition studies, etc. Forced degradation is a process that involves degradation of drug products and drug substances at conditions more severe than accelerated conditions and thus generates degradation products that can be studied to determine the stability of the molecule. Sample, Blank and placebo were exposed to the following different stress conditions. Acid Degradation Base Degradation Peroxide Degradation Photolytic Degradation Thermal Degradation Control sample: Weigh and finely powder 20 tablets. Accurately weigh and transfer a quantity of powder sample equivalent to 10 mg of Rosuvastatin calcium and 67 mg Fenofibrate into a 100 ml clean dry volumetric flask, add about 75 ml of diluent and sonicate to dissolve it completely and make volume up to the mark with the diluent. Filter the solution through 0.45μm membrane filter. Pipette 1 ml of the above stock solution into a 10 ml volumetric flask and dilute up to the mark with diluent. Figure No.5: Chromatogram of Control Sample.

6 Page5526 Acid Degradation sample: Weigh and finely powder 20 tablets. Accurately weigh and transfer sample equivalent to 10 mg of Rosuvastatin calcium and 67 mg Fenofibrate into a 100 ml clean dry volumetric flask, add about 75 ml of diluent and sonicate to dissolve it for about 30 minutes with intermittent shaking at controlled temperature. Then add 5 ml of 1N acid (Hydrochloric acid), refluxed for 60 minutes at 60 C, then cooled to room temperature, neutralize with 5 ml of 1N base (Sodium hydroxide) and make volume up to the mark with diluent and mix. Filter the solution through 0.45 μm membrane filter. Further pipette 1 ml of the above stock solution into a 10 ml volumetric flask and dilute up to the mark with diluent. Figure No.6: Chromatogram of Acid Degradation Sample. Base Degradation sample: Weigh and finely powder 20 tablets. Accurately weigh and transfer sample equivalent to 10 mg of Rosuvastatin calcium and 67 mg Fenofibrate into a 100 ml clean dry volumetric flask, add about 75 ml of diluent and sonicate to dissolve it for about 30minutes with intermittent shaking at controlled temperature. Then add 5 ml of 1N base (Sodium hydroxide), refluxed for 60 minutes at 60 C, then cooled to room temperature, neutralize with 5 ml of 1N base (Hydrochloric acid) and make volume up to the mark with diluent and mix. Filter the solution through 0.45 μm membrane filter. Further pipette 1 ml of the above stock solution into a 10 ml volumetric flask and dilute up to the mark with diluent. Figure No.7: Chromatogram of Base Degradation Sample. Peroxide Degradation sample: Weigh and finely powder 20 tablets. Accurately weigh and transfer sample equivalent to 10 mg of Rosuvastatin calcium and 67 mg Fenofibrate into a 100 ml clean dry volumetric flask, add about 75 ml of diluent and sonicate to dissolve it for about 30minutes with intermittent shaking at controlled temperature. Then add 2 ml of 30% Peroxide, refluxed for 60 minutes at 60 C, then cooled to room temperature and make volume up to the mark with diluent and mix. Filter the solution through 0.45 μm membrane filter. Further pipette 1 ml of the above stock solution into a 10 ml volumetric flask and dilute up to the mark with diluent.

7 Page5527 Figure No.8: Chromatogram of Peroxide Degradation Sample. Photolytic Degradation sample: Weigh and finely powder 20 tablets, this powder is exposed to UV light in a UV Cabinet for about 2 days. Accurately weigh and transfer a quantity of powder sample equivalent to 10 mg of Rosuvastatin calcium and 67 mg Fenofibrate into a 100 ml clean dry volumetric flask, add about 75 ml of diluent and sonicate to dissolve it for about 30 minutes with intermittent shaking at controlled temperature. Then make volume up to the mark with the diluent. Filter the solution through 0.45μm membrane filter. Pipette 1 ml of the above stock solution into a 10 ml volumetric flask and dilute up to the mark with diluent. Figure No.9: Chromatogram of Photolytic Degradation Sample. Thermal Degradation sample: Weigh and finely powder 20 tablets, this powder is exposed to heat at 105 C for about 2 days. Accurately weigh and transfer a quantity of powder sample equivalent to 10 mg of Rosuvastatin calcium and 67 mg Fenofibrate into a 100 ml clean dry volumetric flask, add about 75 ml of diluent and sonicate to dissolve it for about 30minutes with intermittent shaking at controlled temperature. Then make volume up to the mark with the diluent. Filter the solution through 0.45μm membrane filter. Pipette 1 ml of the above stock solution into a 10 ml volumetric flask and dilute up to the mark with diluent. Figure No.10: Chromatogram of Thermal Degradation sample.

8 Page5528 Acceptance criteria: 1. % Degradation of the drug sample should not be more than 30%. 2. Degradation product peak should be resolved from the drug peak. RESULT AND DISCUSSION Literature survey revealed that several methods have been reported for estimation of Rosuvastatin calcium and Fenofibrate individually or in combination with other drugs in pharmaceutical dosage forms. However, only single HPLC method has been reported so far for simultaneous estimation of Rosuvastatin calcium and Fenofibrate in combined dosage forms. Hence, in the present study, a new, sensitive and suitable reversed-phase high performance liquid chromatography method was developed and validated for the simultaneous estimation of Rosuvastatin calcium and Fenofibrate in pharmaceutical dosage form.the results of analysis in the method were validated in terms of accuracy, precision, specificity, linearity, limit of detection, limit of quantification and robustness. Table No. 2: System Suitability Test. Sr.No. Peak name Retention time Area Plate count Asymmetry 1 Rosuvastatin Calcium 2 Fenofibrate Table No.3: Linearity Result. Sr. No. Rosuvastatin Calcium Fenofibrate Conc. of ROS (μg/ml -1) ) Peak area Conc. of FEN (μg/ml -1) ) Peak area Correlation Coefficient(r 2 ) Correlation Coefficient (r 2 ) Slope (m) Slope (m) Intercept (y) Intercept (y) Table No.4: Accuracy Result. Drug Rosuvastatin Calcium Fenofibrate Sr. No. Conc. Level Conc.(μg/ml) Area Conc. Found Std. Stock Formulation (μg/ml) Solution Stock Solution % Recovery % % % % % % Aveg. % Recovery % RSD

9 Page5529 Table No.: 5 Precision Result. Injection Rosuvastatin Calcium (30ppm) Fenofibrate (201ppm) No. Retention Time (min) Area Retention Time (min) Area Mean S.D % RSD Table No.: 6 Robustness Result. Sr. Robustness Parameter Observation No. As such -10% Limits 1 % RSD of peak area Rosuvastatin NMT 2.0 Fenofibrate Theoretical Plate Rosuvastatin NMT Fenofibrate Tailing Factor Rosuvastatin NMT Fenofibrate Retention Time (min) Rosuvastatin Fenofibrate CONCLUSION Thus the proposed stability indicating RP-HPLC method for the simultaneous determination of Rosuvastatin Calcium and Fenofibrate in tablet dasage form was accurate, precise, linear, reliable, simple, economic and robust. The method has several advantages, including simple mobile phase, low solvent consumption, rapid analysis, simple sample preparation and improved selectivity well as sensitivity. The method can be used for routine analysis of marketed product of Rosuvastatin Calcium and Fenofibrate in combined tablet formulation. Abbreviations: RP-HPLC ROS FEN ICH PDP : Reverse Phase High Performance Liquid Chromatography : Rosuvastatin Calcium : Fenofibrate : Intarnational Council On Harmonization : Potassium dihydrogen phosphate REFERENCES 1. Devika G. S., Sudhakar M., Venkateshwarao V., RP-HPLC method for simultaneous estimation of Rosuvastatin Calcium & Fenofibrate in Tablets. International Journal of Pharmacy and Pharmaceutical Sciences 2011; 01(03): Sumaiatha M., Haritha K., Analytical method development and validation for the simultaneous estimation of Rosuvastatin and Fenofibrate in tablet dosage form by reverse phase high performance liquid chromatography. Indian Journal of Research in Pharmacy and Biotechnology 2013; 01(01): Sharma S., Bhandari P., Simultaneous estimation of Rosuvastatin calcium and Fenofibrate in bulk and tablet dosage form by UV-Spectrophotometry and RP-HPLC. Journal of Pharmacy Research 2012; 5(4): Karunakaran A., Subhash V., Chinthala R., Muthuvijayan J., Simultaneous Estimation of Rosuvastatin Calcium and Fenofibrate in Bulk and in Tablet Dosage Form by UV-Spectrophotometry and RP-HPLC. Stamford Journal of Pharmaceutical Sciences. 2011; 4(1): Zahi Mohammad, Stability-Indicating RP-HPLC Method Development and Validation for the Determination of Rosuvastatin (Calcium) In Pharmaceutical Dosage Form. International Journal of Pharmaceutical Sciences and Drug Research. 2014; 6(2): Sevda R. R., Ravetka A. S., UV Spectrophotometric estimation of Rosuvastatin Calcium and Fenofibrate in bulk Drug and Dosage Form using Simultaneous Equation Method. International Journal of ChemTech Research.2011;2(3): Elisavet M., Anastazia., Management of dyslipidemias with fibrates, alone and in combination with statins: role of delayedrelease fenofibric acid. Vascular Health and Risk Management.2010;4 (2): ICH guidelines: Guidance for industry; Q2B Validation of analytical procedures: Methodology,1996,1-10.

Page5530 9. Kulkarni G. T., Gowthamarajan K., Suresh B., Stability testing of pharmaceutical products- An overview, Indian J. Pharm Edu.,2004,38(4),194-199. 10.

10 Page Kulkarni G. T., Gowthamarajan K., Suresh B., Stability testing of pharmaceutical products- An overview, Indian J. Pharm Edu.,2004,38(4), ICH Topic Q1(A) Stability testing new drug substances and products, ICH Harmonised Tripartite Guideline, 2003, ICH-Q1B: Photo stability Testing of New Drug Substances and Products, FDA, Vol. 62, No. 95, 16 May1997; Indian Pharmacopoeia; Vol. III; Govt. of India, Ministry of Health and Family Welfare. New Delhi; Published by The Controller of Publications; 2007; British Pharmacopoeia; Vol. II; Published by The Stationery Office on behalf of Medicines & Healthcare Products Regulatory Agency (MHRA); 2010; Skoog D A, Holler F J, Timothy A, Principle of Instrumental analysis. Eastern Press; Bangalore, 2004, 5,

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF ROSUVASTATIN CALCIUM AND ASPIRIN IN

Page4417 Indo American Journal of Pharmaceutical Research, 2016 ISSN NO: 2231-6876 DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF ROSUVASTATIN CALCIUM