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1 Research Article ISSN: Available online through Simultaneous equation method for the estimation of Atorvastatin calcium and Amlodipine besylate in bulk and in combined tablet dosage form using UV spectophotometry Kamepalli Sujana 1 *, NallamilliShravani 1, Medavankala Zabudal Vimmy Hamutal. 1* Department of Pharmaceutical Analysis, University College of pharmaceutical science, Acharya Nagarjuna University, Nagarjunanagar, Guntur , Andhra Pradesh (India Received on: ; Revised on: ; Accepted on: ABSTRACT A simple, accurate and precise method was developed for the simultaneous estimation of Atorvastatin Calcium (AVC, Amlodipine Besylate(AMB and in their combined dosage form by UV Spectrophotometric Method. Both drugs Atorvastatin Calcium and Amlodipine Besylate are soluble in methanol. The wave length at which maximum absorption takes place for Atorvastatin Calcium and Amlodipine Besylate were found to be 240 nm and nm respectively. Calibration curves were linear with correlation coefficient and over the concentration range of 5-30µg/mL for Atorvastatin Calcium and 3-5µg/mL for Amlodipine Besylate respectively. The developed methods were validated in terms of Accuracy, Precision, Limit of Detection and Limit of Quantification according to ICH guidelines. The LOD for AVC and AMB are 0.17µg/mL and 0.05µg/mL and the LOQ for AVC and AMB are 0.51µg/mL and µg/ml. Precision for both the drugs were indicated by %RSD and found to be within the acceptance limits. Thus the method can be successfully applied in routine work for the determination of Atorvastatin and Amlodipine in combined dosage form. KEYWORDS: Atorvastatin Calcium, Amlodipine Besylate, Simultaneous equation method, Method Validation. INTRODUCTION Atorvastatin calcium was chemically [R-(R*, R*]-2-(4-fluorophenyl-,-dihydroxy-5-(1-methylethyl-3-phenyl-4 [(phenyl amino carbonyl]- 1H-pyrrole-1-heptanoic acid, calcium salt (2:1 trihydrate. It was used as Anti hyperlipidemic, HMG CoA Reductase inhibitor and used for the prevention of cardiovascular disease 7. dihydropyridine benzenesulfonate. It was a Calcium channel blocker 7. A rapid and economical way for determining a new analytical methodfor the drug was in high demand. So development of a method which can be successfully applied in routine work for the determination of Atorvastatin Calcium and Amlodipine besylate in combined dosage form was the main aim. Figure 1. Structure of Atorvastatin Calcium Amlodipine besylate was chemically 2-[(2-Aminoethoxy methyl]-4- (2-chlorophenyl-3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl-1, 4- *Corresponding author. Kamepalli Sujana M.Pharm,Ph.D Department of Pharmaceutical Analysis, University College of Pharmaceutical Science, AcharyaNagarjunaUniversity, Nagarjunanagar, Guntur , Andhra Pradesh (India Figure 2 Structure of Amlodipine Besylate MATERIALS AND METHODS Instrumentation Shimadzu UV double beam spectrophotometer with 1cm path length supported by Shimadzu UV-probe software, version 2.21 was used for spectral measurements with 10mm matched quartz cells. Shimadzu balance (BL-220H was used for weighing. Chemicals and reagents: Pure drugs of Atorvastatin and Amlodipine, Commercial formulation of Atorvastatin and Amlodipine were available in the ratio of 3:2 (CAUDET as tablets, Methanol and Double distilled water.
2 Experimental condition Observation:? max was found to be nm for Amlodipine besylate According to the solubility characteristics, the common solvent for both the drugs were found to be Methanol. Hence the stock solution was prepared with methanol and further dilutions were made up with Distilled water. Preparation of stock solution (1000µg/mL: Accurately weighed quantity of pure AVC (10mg and pure AMB (10mg were transferred into two separate 10mL volumetric flasks, dissolved in methanol and made up to the volume 10mL with the same solvent. The stock solution was sonicated for 2min. Preparation of working standard solution (100µg/mL From the above stock solution 1mL each of AVC and AMB were taken and transferred to separate 10mL volumetric flasks and the volume was made up to 10 ml with distilled water. Determination of?max: λ max was the wavelength at which drug shows maximum absorption. 5 µg/ml solutions of the drugs in distilled water were scanned using UV-Visible spectrophotometer within the wavelength region of nm against distilled water as blank. Figure 5 overlay spectrum of AVC and AMB Observation: The isobestic point was found to be 246nm. Simultaneous Equation Method: 5µg/mL solutions of AVC and AMB were prepared separately in distilled water and the solutions were scanned against blank in the entire UV range to determine the λmax values. Clear peaks were observed at 240nm for AVC and 234.9nm for AMB. Hence these wavelengths were chosen as the λmax values for each drug respectively. Standard solutions of AVC and AMB in the concentration range of 5-30µg/mL and 3-5µg/mL respectively were prepared in distilled water and the absorbance of these solutions was measured at 240nm and nm. Calibration curves were plotted to verify the Beer s law and the absorptivity values were calculated at the respective wavelengths for both the drugs. Figure 3. UV spectrum of Atorvastatin calcium Observation:? max was found to be 240 nm for Atorvastatin calcium Figure 6 Linearity of Atorvastatin calcium Figure 4 UV spectrum of Amlodipine besylate Figure 7 Linearity of Amlodipine besylate
3 a For estimation of AMB: Table 2. Estimation of marketed formulation Drug Name Labelled claim(mg Test Simultaneous Concentration equation method (µg/ml % ± SD (n=6 b For estimation of AVC: A 1 and A 2 are absorbances of diluted mixture at 240nm and nm respectively. C x and C y concentrations of AVC and AMB respectively (g/100ml. ax 1, ax 2, ay 1, ay 2 are absorptivity of AVC and AMB at 240nm and nm respectively. Table 1: Simultaneous equation values Drug Absorbance Absorbance maxima(λ 1 maxima(λ 2 AVC % AMB % 3, 4, 5 Validation of developed simultaneous UV Method The method was validated with respect to linearity, accuracy, precision, LOD (Limit of detection, LOQ (Limit of quantitation. 1. Linearity Linearity was checked by diluting standard stock solution at six different concentrations. AVC was linear with the concentration range of 5-30µg/mL at 240nm and AMB showed the linearity in the range of 3 5µg/mL at 234.9nm for simultaneous equation method.the results were tabulated in table.3. Amlodipine Besylate 0.962(ax (ax 2 Atorvastatin Calcium 0.123(ay (ay 2 Formulation(Caduet 0.783(A (A 2 Table.3 Linearity values Parameter Simultaneous equation method AVC AMB Figure 8: Overlay spectrum of AVC, AMB, Formulation (Caduet Analysis of Tablet formulations: Ten tablets were accurately weighed and average weight was calculated. The tablets were triturated to produce a fine powder. An accurately weighed quantity of powder equivalent to 10 mg of AVC was transferred to 10 ml volumetric flask and dissolved in methanol by shaking and volume was made up to 10 ml with methanol. The solution was filtered through Whatmann filter paper No. 41 and aliquot portion of filtrate was diluted to obtain a solution of 5µg/mL of AVC and AMB respectively with distilled water. The absorbance of sample solution was measured at selected wavelengths. The content of AVC and AMB in sample solution of tablet was calculated by using SEM method (equations 1 and 2, Table No: 1 the analysis procedure was repeated six times 8. λmax(nm Linearity Range (µg/ml 5-30µg/mL 3-5 µg/ml Regression Equation(y=mx+c y=0.025x y=0.172x Slope (m Intercept ( c , 4, 5 2. Accuracy To check the accuracy of the developed method and to study the interference of excipients in formulation, analytical recovery experiments were carried out by using standard addition method in three different concentrations. From the total amount of drug found, the percentage recovery was calculated. This procedure was repeated for three times for each concentration. The % RSD was calculated and the results are shown in Table no.4 Table.4. %Recovery studies for AVC and AMB Conc. Spiked Amount Amount % (µg/ml level added found Recovery (% (mg (µg n=6 Atorvastatin Calcium Amlodipine Besylate
4 3. Precision The precision of the method was confirmed by repeatability and intermediate precision. The repeatability was performed by the analysis of formulation was repeated for six times.the amount of each drug present in the tablet formulation was calculated. The % RSD was calculated. The Intermediate precision of the method was confirmed by intraday and inter day analysis i.e. the analysis of formulation was repeated three times in the same day and on three successive days. The amount of drugs was determined and % RSD also calculated. The results were shown in Table no.5. Table.5. Precision Day of Analysis %Recovery ±SD;(n=3 %RSD Intraday Precision AVC (µg/ml Day ± ± ±0.03 Day ± ± ± Day2 99± ± ±0.04 RESULTS AND DISCUSSION The proposed method was based on spectrophotometric simultaneous estimation of AVC and AMB in UV region using distilled water as solvent.the absorbance spectral analysis shows the maximum absorbance (?max at 240 nm for AVC and 234.9nm for AMB. This method was based on simultaneous equation method which involves solving of simultaneous equations using absorptivity coefficient (A 1, A 2 values and absorbance at 240 and nm for estimation of AVC and AMB in standard and sample mixture. Beer s law obeyed in the concentration range of 5-30 µg/ml and 3-5µg/mL for AVC and AMB respectively. The correlation coefficients were found to be and which shows the good linear relationship for both components. The tablet assay results obtained by proposed method was very close to labelled claim and low value of Relative standard deviation, suggesting that the developed method has high precision. In order to check the accuracy of the developed method, known quantities of standard drugs of AVC and AMB in three different levels were added to its pre-analyzed tablet sample and analyzed by the developed method. The results of recovery studies are shown in Table.4. The mean percentage recoveries were found in the range of and it indicated the non interference of the excipients in the tablet formulation. AMB(µg/mL Day ± ± ±0.28 Day ± ± ± Day2 99.3± ± ±0.83 Inter day Precision AVC (µg/ml Day0,1, CONCLUSION: The proposed analytical UV spectrophotometric method was developed and validated thoroughly for quantitative determination of AVC and AMB in tablets. The developed method was found to be simple, rapid, accurate, precise and economical, which can be directly and easily applied to the analysis of AVC and AMB in pharmaceutical tablet formulations. AMB (µg/ml 99.13± ± ± Day0,1, Sensitivity The limit of detection (LOD and limit of quantitation (LOQ parameters were calculated using the following equations LOD=3.3s/ s LOQ = 10s/ s σ is standard deviation of y intercept of calibration times with the same concentration. Results were tabulated in table no.6 Table.6 LOD and LOQ Drug name Absorbance LOD LOQ AVC (µg/mL 0.51(µg/mL AMB (µg/mL 0.165(µg/mL 3. ACKNOWLEDGEMENT I am very thankful to principal, University College of Pharmaceutical Sciences, Acharya Nagarjuna University, Guntur for providing the laboratory facilities chemicals to carryout entire study. I am also thankful to Rainbow pharma training lab, Hyderabad, India, for providing Atorvastatin Calcium and Amlodipine Besylate working standard as gift sample. REFERENCES 1. Sharma.B.K, Instrumental methods of chemical analysis,24th edition, Goelpubkishers, 2005, Gurudeep.R.Chatwal, Sharm.K.Anand, HPLC, Instrumental methods of chemical analysis, 2010, ICH, Text on Validation of Analytical Procedures, ICH Q2A, International Conference on Harmonisation, IFPMA, Geneva, 1995, 2-3, A 1 to A ICH, Validation of Analytical Procedures Methodology, ICH Q2B, International Conference on Harmonisation, 1996, 1-
5 5. ICH Guidelines, Q2 (R1 Validation of Analytical Procedures Text and Methodology, 2005, p British pharmacopoeia 2011,volume 1, United States Pharmacopoeia 34 NF29,volume 2(1, , Indian pharmacopoeia 2010, volume 2, , Maryadele J.O. Neil, The Merck Index, An Encyclopedia OfChemical, Drugs and Biological,.Eds,13 th edition, Published by Merck Research Lab, Division of Merck and co. Inc., Whitehouse Station, NJ NJ Mishra.P, Alka Gupta, K Shah, Simultaneous estimation of Atorvastatin calcium and Amlodipine besylate from tablets, volume-69(6, 2007, Bharat G. Chaudhari*, Ashok B. Patel, Simultaneous spectrophotometric estimation of Atorvastatin calcium and Amlodipine besylate in tablet dosage forms, International journal of Chemtech research, volume-2(1,2010 Jan-Mar, RamyaGavini,Puranik.S. B, G. V. S. Kumar, Sridhar.K. A. and RamyaGavini, Simultaneous estimation of Amlodipine and Losartan by UV-method in bulk drug and tablet dosage formulation, Scholars Research Library Archives of Applied Science Research, volume-4(5,2012, KottaiMuthu.A, RattaiahGupta.T, Shailesh Sharma, Manavalan.R, Simultaneous estimation of Amlodipine and Atorvastatin in tablets using orthogonal function ratio spectrometry, Int.J.Chem.Sci,volume-6(4, 2008, Juyal.V, Chaudhary.M, Kumar.P, Gnanarajan.G, Yadav.P.K, Method development and validation for simultaneous estimation of Atorvastatin and Amlodipine in combination tablet dosage form by UV spectroscopy using multi-component ode of analysis, Journal of Pharmacy Research, vol 2, 2008 Dec. 15. Devi Ramesh, Ramakrishna.S, New spectrophotometric methods for simultaneous determination of Amlodipine Besylate and Atorvastatin Calcium in Tablet Dosage Form, International Journal of Pharmacy and Pharmaceutical Sciences, volume-2(4,2010, MahaAbdallaAlnowaiser, Simultaneous determination of Amlodipine and Atorvastatin in Caduet tablets using HPLC, American Journal of Applied Sciences, volume-10(8, 2013, Source of support: Nil, Conflict of interest: None Declared
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