DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF ROSUVASTATIN CALCIUM AND ASPIRIN IN
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1 Page4417 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 ASPIRIN IN BULK AND PHARMACEUTICAL DOSAGE FORM Akshaykumar Khivasara*, Dr. L. A. Kawale, Lina Shirode, Nanda Badhe, Priyanka Ahire, Shivam Lale M.V.P Samaj s College of Pharmacy, Nashik-2, Maharashtra, India. ARTICLE INFO Article history Received 08/02/2016 Available online 29/02/2016 Keywords Rosuvastatin, Aspirin, RP-HPLC, Method Validation. Corresponding author Akshaykumar Khivasara M.V.P Samaj s college of pharmacy, Gangapur road, Nashik-2(422002). Maharashtra, India. shirodelina36@gmail.com ABSTRACT The objective of this present method is to develop simple, sensitive and precise HPLC method for the simultaneous determination of Aspirin and Rosuvastatin in pharmaceutical dosage form. The method was carried out using Greece C18 (4.6ID 250mm; 5μm) column and mobile phase comprised of methanol and PDP buffer adjusted to ph 2.8 with Ortho phosphoric acid in proportion of ratio 70:30 v/v and degassed under ultrasonication. The flow rate was 1 ml/min and detection was carried out at 243 nm. The Retention time of Aspirin and Rosuvastatin were found to be 2.84 and 3.46 respectively.the validation of method was carried using ICH guidelines. The method was validated in terms of specificity, linearity, precision, accuracy, specificity, limit of detection, limit of quantitation and robustness. Linearity of Aspirin and Rosuvastatin were in the range of 75 to 375μg/mL (r 2 = ) and 10 to 50μg/mL (r 2 = ) respectively. The % recoveries of Aspirin and Rosuvastatin were found to be in between % to % for Aspirin and % to % for Rosuvastatin respectively. The method was also applied for the determination of Aspirin & Rosuvastatin 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 Aspirin and Rosuvastatin and the assay can thus be considered stability indicating. The proposed method is suitable for the routine quality control analysis for simultaneous determination of Aspirin and Rosuvastatin in bulk and pharmaceutical dosage form. Please cite this article in press as Akshaykumar Khivasara et al. Development and Validation of Stability Indicating RP-HPLC Method for Simultaneous Estimation of Rosuvastatin Calcium and Aspirin in Bulk and Pharmaceutical Dosage Form. Indo American Journal of Pharmaceutical Research.2016:6(02). 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 Page4418 INTRODUCTION Aspirin Fig No. 1: structure of Aspirin. Description : It occurs as white powder. IUPAC name : 2-(acetyloxy)-benzoic acid. Molecular Formula : C 9 H 8 O 4 Molecular weight : g/mol Melting point : C Solubility : Slightly soluble in water. More soluble in basic solutions. Category : Antipyretic, analgesic, anti-inflammatory and antithrombotic agent. Mechanism of Action: It has anti-platelet effect by inhibiting the production of thromboxane, which under normal circumstances binds platelet molecule together to create a patch over damage of the walls within blood vessels. Chemically it is 2-acetoxybenzoic acid and is a non-steroidal anti-inflammatory drug (NSAIDs) and shows inhibition of the enzyme cyclogenase. Rosuvastatin Fig No. 2: structure of Rosuvastatin. Description : It occurs as off white to creamish white powder. IUPAC name : (3R, 5S, 6E)-7-[4-(4-fluorophenyl)- 2- (N-methyl methane sulfonamido)-6-(propan- 2-yl) pyrimidin-5-yl]- 3, 5-dihydroxyhept-6-enoicacid Molecular Formula : C22H28FN3O6S.Ca Molecular weight : g/mol Melting point : C Solubility : sparingly soluble in water and methanol, slightly soluble in ethanol, Soluble in acetonitrile. Category : HMGCoA reductase (class of statins) Mechanism of Action: It is a selective and competitive inhibitor of HMGCoA reductase (class of statins), which catalyzes the conversion of 3- hydroxy-3-methylglutaryl coenzymea to cholesterol precursor mevalonate and thus limits cholesterol synthesis. This action increases high-density lipoprotein level and decreases low density lipoprotein (LDL) level. [1]
3 Page4419 MATERIALS AND METHODS Table No. 1: List of instruments used. Sr.No. Instrument/equipment Model or manufacturer 1 UV-vis spectrophotometer Shimadzu (UV 2450) 2 HPLC Analytical technologies Ltd.(HPLC 3000 series) 3 Analytical balance PGB 1OO 4 Digital ph meter EU-Tech, ME Sonicator WUC-4L 6 Filter Millipore Table No. 2: List of reagents used. Sr. No. Chemical Grade 1 Water,Milli Q HPLC grade 2 Methanol HPLC grade 3 Hydrochloric acid AR grade 4 Hydrogen peroxide AR grade 5 Sodium hydroxide AR grade Table No. 3: Drugs used. Sr. No. Name of the drug Supplied by 1 Aspirin Gift sample from Emcure pharmaceuticals Ltd. 2 Rosuvastatin Gift sample from concept pharmaceuticals Ltd. Table No.4: Marketed formulation. Sr. No. Brand name Contents Company 1 Consivas ASP Aspirin 75mg Emcure Pharmceuticals Ltd. Rosuvastatin 10mg Method development by RP-HPLC The aim of the developed stability indicating RP-HPLC method was to resolve aspirin and rosuvastatin under system suitability conditions and at all forced degradation conditions. Desired HPLC method was optimized by testing number of mobile phases. Suitable wavelength was selected by scanning individual standard solutions of concentration 10 μg/ml for both drugs on UV - Vis spectrometer separately.the absorption spectra obtained was showing the wavelength at nm and nm for Aspirin and Rosuvastatin respectively. Varieties of mobile phases were checked that differ in terms of strength, ph, percentages, temperature, additives and solvent type. Best conditions were selected by considering minimizing peak tailing, improving peak symmetry, column efficiency, resolution and total analysis time. Compared to all conditions better separation was achieved at a mobile phase of Methanol: Buffer 20mM (PDP) (70:30). Finally, a ph of 2.8 was found to be suitable for the analysis of aspirin and rosuvastatin.room temperature was selected for the entire analysis as temperature was found to have a negligible effect on resolution and tailing factor. For both the drugs well resolved, retained and accepted system suitability was observed at these optimized conditions. [2] Preparation of standard stock solution: Accurately weighed Standard Aspirin 10 mg And Rosuvastatin 10 mg in 10 ml of Methanol it gives 1000 ug/ml Aspirin and 1000μg/ml Rosuvastatin. From aspirin Standard stock solution 0.75mL was taken and diluted upto 10mL it gives 75ppm. From Standard stock solution of rosuvastatin 0.10mL was taken and diluted upto 10mL it gives 10ppm.
4 Page4420 Aspirin: Rosuvastatin: Fig No 3: Absorption spectrum of Aspirin in methanol Showing λ max Fig No 4: Absorption spectrum of Rosuvastatin in methanol Showing λ max nm. Fig.No. 5: Standard chromatogram of Aspirin and Rosuvastatin. Optimized Chromatographic Conditions: Sample Name : Aspirin 75ppm + Rosuvastatin 10ppm Column : Greece C18 (4.6ID 250mm; 5μm) Wavelength : 243nm Mobile Phase : Methanol: Buffer 20mM (PDP) (70:30) Detector : U.V. Detector Sample volume : 20μl Flow rate : 1 ml/min Pressure : 9-10MPa Run Time : 5.50 min 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.
5 Page4421 Table No. 5: Results of system suitability. Sr. No. Peak name Retention time Area Resolution Plate count Asymmetry 1 Aspirin Rosuvastatin Analysis of marketed tablet formulation (assay): Preparation of sample solution: Weigh and finely powder not fewer than 20 tablets. Accurately weigh and transfer a quantity of powder sample equivalent to 75 mg of Aspirin and 10 mg Rosuvastatin 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. Procedure: Equal volumes (10μL) of standard and sample solution were injected separately after equilibrium of stationary phase. The chromatograms were recorded and the response i.e. peak area of major peaks were measured. The content of Aspirin and Rosuvastatin was calculated by comparing a sample peak with that of standard. [3] Validation of method: All the analytical validation parameters were determined according to the ICH guidelines (Q 2 R 1). 1) Linearity 2) Accuracy 3) Precision 4) Robustness 5) Limit of detection 6) Limit of quantification Linearity: Preparation of linearity solution: Linearity was performed by diluting standard stock solution to give final concentration in the range of 75 to 375 μg/ml for Aspirin and 10 to 50 μg/ml for Rosuvastatin. 10 μl of each concentration injected and calibration curve was constructed by plotting the peak area versus the drug concentration. Acceptance Criteria: The plot should be linear passing through the origin. Correlation Coefficient should not be less than [4, 5] Fig. No. 6: Linearity plot of aspirin.
6 Page4422 Fig. No. 7: Linearity plot of Rosuvastatin. Accuracy (Recovery) Preparation of sample Solution: Accuracy was performed in three different levels for Aspirin and Rosuvastatin. Spiked known quantity of Aspirin and Rosuvastatin Standard at 50%, 100% and 150% was added to a pre-quantified sample solution. Analyses of samples were done in triplicate for each level. From the results, % recovery was calculated. Acceptance Criteria: 1. Mean recovery should be in the range of %. 2. The Relative Standard Deviation 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. Acceptance criteria: %RSD NMT 2% for test results. Limit of Detection: The lowest conc. of the analyte in the sample that the method can detect but not necessarily quantify under the stated experimental conditions simply indicates that the sample is below or above certain level. Limit test prescribed as percentage or as parts per million. The limit of detection will not only depend on the procedure of analysis but also on type of instrument. Limit of Quantitation: The limit of quantitation (LOQ) is the lowest amount of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. It is expressed as the conc. of analyte (e.g., percentage, parts per billion) in the sample. The S/N ratio should not less than 10. 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. [6] 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
7 Page4423 Acid Degradation sample: Accurately weigh and transfer 75 mg of Aspirin and 10 mg Rosuvastatin drug powder 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 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 1mL of the above stock solution into a 10 ml volumetric flask and dilute up to the mark with diluent. Fig. No.8: Chromatogram of Acid Degradation sample. Base Degradation sample: Accurately weigh and transfer 75 mg of Aspirin and 10 mg Rosuvastatin drug powder 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. Fig.No.9: Chromatogram of Base Degradation sample. Oxidative Degradation sample: Accurately weigh and transfer 75mg of Aspirin and 10 mg Rosuvastatin drug powder 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 3% 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.
8 Page4424 Fig.No.10: Chromatogram of Oxidative Degradation sample. Photolytic Degradation sample: Both the drug powder were exposed to UV light in a UV Cabinet for about 2 days. Accurately weigh and transfer 75 mg of Aspirin and 10 mg Rosuvastatin drug powder 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. Fig.No. 12: Chromatogram of Photolytic Degradation sample. Thermal Degradation sample: Both the drug powder were exposed to heat at 80 C for about 2 days. Accurately weigh and transfer a quantity of powder sample equivalent to 75 mg of Aspirin and 10 mg Rosuvastatin 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. Fig. No. 13: Chromatogram of Thermal Degradation sample.
9 Page4425 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. [7-12] RESULT AND DISCUSSION One of the accurate and most widely used analytical methods for both quantitative and qualitative estimation of drugs is High performance liquid chromatography (HPLC). As per the literature survey, no stability indicating method was available for the simultaneous analysis of aspirin and rosuvastatin. Hence, a method was developed and it was successfully applied for the estimation of aspirin and rosuvastatin in the combined pharmaceutical dosage form. The method was proved to be accurate in terms of linearity and the results were shown in Table No. 6. Total validation results of the proposed method were listed in Table no.7 and the values were found to be within the range especially in terms of precision, repeatability and robustness. Table No. 6: Linearity results. Sr.No. Aspirin Rosuvastatin Concentration Area Concentration Area Correlation coefficient(r 2 ) Correlation coefficient(r 2 ) Slope(m) Slope(m) Intercept(y) Intercept(y) Table No. 7: Validation results. Sr.No. Parameter Results Limit Aspirin Rosuvastatin 1 Recovery in % Method precision Below 2 3 Limit of detection Limit of quantitation Robustness in % change i. % RSD in peak area response Below 2 ii. Theoretical plates NLT 2000 iii. Tailing factor NMT 2 iv. Retention time Formulation assay in % Solution stability in % 24 hrs 24 hrs CONCLUSION Current study provides that the RP-HPLC technique can be successfully used for the estimation of Aspirin and Rosuvastatin in their dosage formulations. The method shows good reproducibility and it is accurate, precise, specific and sensitive. A simple, specific RP-HPLC method was developed for the simultaneous estimation of Aspirin and Rosuvastatinin pharmaceutical dosage form and validated according to ICH Q2 (R1) guidelines. The method has several advantages, including simple mobile phase, low cost solvent, rapid analysis, simple sample preparation and improved selectivity as well as sensitivity. The regression co efficient (r 2 ) for each analyte is not less than which shows good linearity. The % recovery was in the acceptable range in tablet dosage forms suggests that the excipients present in the dosage forms have no interference in the determination. The %RSD was also less than 2% showing high degree of precision of the proposed method. It is also clear from the chromatograms that both the active ingredient peaks under all the stress conditions were found to be well separated from the degradation product (produced during forced degradation studies). Since the method does not require use of expensive reagent and also less time consuming, it can be performed routinely in industry for routine analysis of marketed product of Aspirin and Rosuvastatinin combined tablet formulation. Abbreviations: RP-HPLC: Reverse Phase High Performance Liquid Chromatography PDP: Potassium dihydrogen phosphate ICH: International Conference on Harmonization RSD: Relative Standard Deviation
10 Page4426 REFERENCES 1. Divya A, Reddy JR, Ramlingam P. Simultaneous Estimation of Aspirin and rosuvastatin calcium in Dossage form by RP-HPLC and its application in force degradation studies. J of Phar Res 2012; 5(11): Singh R. HPLC method development and validation- an overview. J Pharm Edu Res 2013;4(1): Christen GD. Analytical Chemistry.5th ed. John Wiley and Sons; pp , ICH, Q2 (R1); Validation of analytical procedures: text and methodology. International Conference on Harmonization, Geneva; 2005: Nash R Pharmaceutical Process Validation. 3rd ed. New York: Marcel Dekker Publication; p Solanki C, Patel NK. Development and validation of rp-hplc method for simultaneous estimation of Rosuvastatin calcium and aspirin in capsule dosage form. Int J of Pharm and Bio Sci 2012; 3(3): ICH-Q1B: Photo stability Testing of New Drug Sub-stances and Products, FDA, Vol. 62, No. 95, 1997; Charde MS, Kumar J, Welankiwar AS, Chakole RD. Review: Development of forced degradation studies of drugs. Int J of Adv in Pharm 2013; 2(3): George N. Forced Degradation as an Integral Part of HPLC Stability-Indicating Method Development 2010; 10(5). 10. Biradar SP, Kalyankar TM, Wadher SJ, Moon RS, Dange SS. Stability Indicating HPLC Method Development: Review. Asian J of Med and Anal Chem 2014; 01(01): Turbi ZM, Khatarbeh O. Stability-Indicating RP-HPLC Method Development and Validation for the Determination of Rosuvastatin (Calcium) In Pharmaceutical Dosage Form. Int J of pharm sci and drug res 2014; 6(2): Dumasia RD, Bhatt KK, Patel B. Development and Validation of stability indicating RP-HPLC method for estimation of Rosuvastatin calcium and aspirin. Pharma sci monitor 2012; 3(4):
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