Patel et al. / IJDFR volume 3 and issue 3, May-June, 2012

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1 Available online at ISSN Research Article INTERNATIONAL JOURNAL OF DRUG FORMULATION AND RESEARCH DEVELOPMENT AND VALIDATION OF SPECTROPHOTOMETRIC METHOD FOR SIMULTANEOUS DETERMINATION OF AMLODIPINE BESYLATE AND INDAPAMIDE IN THEIR COMBINED DOSAGE FORMULATION BY ABSORBANCE CORRECTION METHOD. Aanal B.Patel 1*, C. N. Patel 1 1 * - Department of Quality Assurance, Shri Sarvajanik Pharmacy College, Mehsana, Gujarat. Received: 20 May. 2012; Revised: 8 June 2012; Accepted: 26 June. 2012; Available online: 5 July ABSTRACT A simple, accurate and precise spectrophotometric method has been developed for simultaneous estimation of Amlodipine Besylate And Indapamide In combined dosage form. Simultaneous equation method is employed for simultaneous determination of Amlodipine Besylate And Indapamide in tablet dosage forms. In this method, the absorbance was measured at 360 nm for Amlodipine Besylate and 242 nm for Indapamide. Linearity were observed in range of μg/ml and 3-15 μg/ml for Amlodipine Besylate And Indapamide respectively. Recovery studies confirmed the accuracy of proposed method and results were validated as per ICH guidelines. The method can be used for routine quality control of pharmaceutical formulation containing Amlodipine Besylate And Indapamide. Key words Amlodipine Besylate, Indapamide, Simultaneous estimation, UV spectrophotometry INTRODUCTION Amlodipine Besylate (AMBL) is a calcium channel blocker. Amlodipine is a dihydropyridine calcium antagonist, with a long duration of action, used for the treatment of hypertension and is 3-Ethyl 5-methyl (4RS)-2-[(2-aminoethoxy) methyl]-4-(2-chlor 6- methyl-1,4 dihydropyridine-3,5-dicarboxylate benzenesulphonate. [Figure-1] with molecular formula of C 20 H 25 ClN 2 O 5.C 6 H 5 SO 3 H and molecular weight of g/mol.. Indapamide is a non-thiazide sulphonamide diuretic drug. A benzamide-sulfonamide-indole. It is called a thiazide-like diuretic but structure is different enough (lacking the thiazo-ring) and 4-chloro-N-(2-methyl-2,3- dihydroindol-1-yl)- 3-sulfamoyl-benzamide [Figure-2] with molecular formula of C 16 H 16 ClN 3 O 3 S. and molecular weight of g/mol. 90 Patel et al. / IJDFR volume 3 and issue 3, May-June, 2012

2 The literature reveals that several spectrometric methods available for individual AMLB and INDA [1, 2, 3, 4, 5, 6]. A number of HPTLC and HPLC method are reported for AMLB and INDA determination [7, 8, 9, 10,11]. but no method has been reported for their simultaneous estimation of AMLB and INDA in combined tablet dosage form by UV method. MATERIALS AND METHODS Apparatus A shimadzu model 1700(Japan) double beam UV/Visible spectrophotometer with spectral width of 2 nm, wavelength accuracy of 0.5 nm and a pair of 10 mm matched quartz cell was used to measure absorbance of all the solutions. Spectra were automatically obtained by UV-Probe system software (UV Probe version 2.31). An Electronic analytical balance (Acculab) and an ultrasonic bath were used in the study. Reagents and Materials Reference standard of AMLB and INDA were obtained from Zydus pharmaceutical respectively as a gift sample, whereas their formulation obtained from local market. Analytical grade Methanol obtained from Finar Chemicals (Mumbai, India). Preparation of standard stock solution An accurately weighed quantity of AMLB (10 mg) and INDA (10 mg) were transferred to a separate 10 ml volumetric flask and dissolved and diluted to the mark with methanol. Take 1 ml of above solution into 10 ml volumetric flask and dilute the mark with methanol to obtain standard solution having concentration of AMLB (100 μg/ml) and INDA (100 μg/ml). This solution was used as working standard solution. Method Ten tablets were weighed. They were triturated in glass mortar. Average weight was calculated. The tablet powder equivalent to 5 mg of Amlodipine besylate and 1.5 mg of Indapamide was transferred in 50 ml volumetric flask, dissolved and diluted up to mark with methanol. The solution was sonicated for 15minutes. Filter the solution through Whatman filter paper no.42 and discard first few drops of filtrate. Pipette out 1ml of the above solution in 10ml volumetric flask and diluted to mark with methanol. Absorbance of the resulting 91 Patel et al. / IJDFR volume 3 and issue 3, May-June, 2012

3 solution was measured at nm and nm against methanol.the proposed Absorbance correction method was successfully applied for determination of AMLB and INDA from Pharmaceutical formulation. The percentage of AMLB and INDA was found to be satisfactory, which is comparable with the corresponding claim amount. The concentration of AMLB and INDA can be obtained as = (1) =.... (2) Where, A 1 and A 2 are the absorbances of mixture at nm and nm respectively, ax 1 and ax 2 are absorptivities of AMLB at nm and nm respectively, ay 1 and ay 2 are absorptivities of INDA at nm and nm respectively. VALIDATION OF PROPOSED METHOD Linearity The calibration curves were plotted over a concentration range of μg/ml for AMLB and 3-15 μg/ml INDA. Accurately measured standard stock solutions of each AMLB (1, 2, 3, 4, 5, 6 ml) and INDA (0.3, 0.6, 0.9, 1.2, 1.5 ml) were transferred to a series of 10 ml volumetric flask separately and diluted up to the mark with methanol. The absorbance of solution was then measured at 360 nm and 242 nm. The calibration curves were constructed by plotting absorbance versus concentration and the regression equations were calculated. Precision Intraday Precision Mixed solutions containing µg/ml AMLB and 6-12 µg/ml INDA was analyzed 3 times on the same day and % RSD was calculated. Interday Precision 92 Patel et al. / IJDFR volume 3 and issue 3, May-June, 2012

4 Mixed solutions containing µg/ml AMLB and 6-12 µg/ml INDA was analyzed on 3 different day and % RSD was calculated. Accuracy The accuracy of the method was determined by calculating recoveries of AMLB and INDA in mixture by the standard addition method. Known amount of standard solutions of AMLB (0, 16, 20 and 24 µg/ml) and INDA (0, 4.8, 6.0 and 7.2 µg/ml) were added to a pre-quantified sample solution of 20 µg/ml AMLB and 6 µg/ml INDA mixtures. The absorbance of AMLB and INDA were recorded at λ 1 and λ 2. The percentage recovery was calculated by measuring the absorbance of both drug at their absorbance maxima and fitting these values into simultaneous equation. Each response was average of three determinations. Limit of detection and Limit of quantization The limit of detection (LOD) and the limit of quantization (LOQ) of the drug were derived by calculating the signal-to-noise ratio (S/N, i.e., 0.16 for LOD and 0.2 for LOQ) using the following equations designated by International Conference on Harmonization (ICH) guidelines [12]. LOD = 3.3 σ/s LOQ = 10 σ/s Where, σ = the standard deviation of the response and S = slope of the calibration curve. Analysis of MS and OLM in combined tablet Take 10 tablets and weighed. Find out average weight. Take tablet powder equivalent to 5 mg of AMLB and 1.5 mg of INDA was transferred in 100 ml volumetric flask, dissolved and diluted up to mark with methanol. The solution was sonicated for 30 minutes. Filter the solution through Whatman filter paper no.42 and discard first few drops of filtrate. Pipette out 1ml of the above solution in 10ml volumetric flask and diluted to mark with simple methanol. Absorbance of the resulting solution was measured at nm and nm against methanol, relative concentration of two drugs in the sample was calculated using above equation (1) and (2). 93 Patel et al. / IJDFR volume 3 and issue 3, May-June, 2012

5 a RSD = Relative standard deviation. b LOD = Limit of detection. c LOQ = Limit of quantitation d SD is Standard deviation and n is number of replicates. RESULT AND DISCUSSION In simultaneous equation method, the primary requirement for developing a method for analysis is that the entire spectra should follow the Beer s law at all the wavelength, which was fulfilled in case of both these drugs. The two wavelengths were used for the analysis of the drugs were 360 nm (λ max of AMLB) and 242 nm (λ max of INDA) at which the calibration curves were prepared for both the drugs. The overlain UV absorption spectra of AMLB (360 nm) and INDA (242 nm) in methanol is shown in Figure 3. The validation parameters were studied at all the wavelengths for the proposed method. Accuracy was determined by calculating the recovery and the mean was determined Table 2. The method was successfully used to determine the amounts of AMLB and INDA present in the tablet dosage forms. The results obtained were in good agreement with the corresponding labeled amount Table 3. Precision was calculated as repeatability and intra and inter day variations (% RSD) for both the drugs. CONCLUSION The developed simultaneous equation method is found to be simple, sensitive, accurate and precise and can be used for routine analysis of AMLB and INDA. The developed method was validated as per ICH guidelines [12]. Statistical analysis proved that the method is repeatable and selective for the analysis of AMLB and INDA in their combined pharmaceutical formulations. ACKNOWLEDGEMENT The authors are thankful to Zydus Pharmaceuticals, Ahemdabad, and Gujarat, India for providing gift sample of AMLB and INDA for research. The authors are highly thankful to Shri Sarvajanik Pharmacy College, Mehsana, and Gujarat, India for providing all the facilities to carry out the work. 94 Patel et al. / IJDFR volume 3 and issue 3, May-June, 2012

6 References [1] Garg G, Saraf S, Saraf S, Trends in Applied sciences research, 2008, 3(3), [2] Chitlange SS, Imran Md, Wankhede SB, and Sakarkar DM, Research Journal of Pharmacy and Technology, 2008, 1(2), 80. [3] Kakde RB, Kotak VH, Barsagade AG, Chaudhary NK, and Kale DL, Research J. Pharm. and Tech., 2008,1(4), [4] Kasture AV, Ramteke M, Indian journal of Pharmaceutical science, 2006, 68 (3), [5] Kadi Sarva Vishwavidyalaya, International Journal of Pharm Tech Research Jan-Mar 2009 Vo2, No.1, pp [6] P.V.Pawar et al. International Journal Of Pharmacy &Technology 2010, vol1. [7] Malesuik MD, Cardos SG, Bajerski L, Lanzanova FA, Journal of AOAC international, 2006,89(2), [8] Chitlange SS, Bagri K, and Sakarkar DM, Asian J. Research Chem., 2008,1(1), [9] Neha Manish Munot et al, Journal of Pharmacy Research 2010, 3(12), [10] Indian Pharmacopoeia, Volume 2, Ghaziabad: Govt. of India Ministry of Health & Family welfare, The Controller of Publication, 2007, , , A-84. [11] Pandya KK, Satia M, Gandhi TP, Modi I, Modi RI, Chakravarthy BK, layer C, J Chromatogr B Biomed Appl., 1995,667, [12] ICH Guideline Q2 (R1), Validation of Analytical Procedures: Text and Methodology, ICH, Geneva, Patel et al. / IJDFR volume 3 and issue 3, May-June, 2012

7 Table 1: Regression Analysis Data and Summary of Validation Parameters for the Proposed Method Sr. No Parameters AMLB INDA 1 Wavelength range (nm) Beer s law limit (μg/ml) Regression equation (y = mx + c) y = x y = x Slope Intercept Correlation Coefficient (r 2 ) System Precision (%R.S.D) a 7 1.Intraday Precision(n = 3) % % 2.Interday Precision(n = 3) % % 8 Accuracy (% recovery) (n = 3) % % 9 LOD b (μg/ml) LOQ c (μg/ml) Assay (±S.D.) d (n = 3) ± ± Patel et al. / IJDFR volume 3 and issue 3, May-June, 2012

8 Table 2: Recovery Data of Proposed Method Drug Amount taken Amount (μg/ml) added (μg/ml) Amount added (%) % Mean recovery (±S.D) (n = 3) AMLB ± ± ± INDA ± ± ±0.005 Table 3: Analysis of AMLB and INDA by Proposed Method Formulation Labeled claim (mg) % Label claim (±S. D.) (n = 3) Amlodac-D AMLB INDA AMLB INDA ± ± Patel et al. / IJDFR volume 3 and issue 3, May-June, 2012

9 Figure 1: The chemical structure of Amlodipine Besylate Figure 2: The chemical structure of Olmesartan Medoxomil Figure 3: Overlain absorption spectra of AMLB (360 nm) and INDA (242 nm) in methanol 98 Patel et al. / IJDFR volume 3 and issue 3, May-June, 2012