Research Note SIMULTANEOUS ESTIMATION OF S (-) AMLODIPINE AND HYDROCHLOROTHIAZIDE IN BULK AND TABLET DOSAGE FORM BY SIMULTANEOUS EQUATION METHOD
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1 Available online at International Journal of Pharmacy and Pharmaceutical Science Research Universal Research Publications. All rights reserved Research Note SIMULTANEOUS ESTIMATION OF S (-) AMLODIPINE AND HYDROCHLOROTHIAZIDE IN BULK AND TABLET DOSAGE FORM BY SIMULTANEOUS EQUATION METHOD MALLIKARJUNA RAO N.*, GOWRI SANKAR. D.. Research Scholar of Jawaharlal Nehru Technological University Kakinada (JNTUK), Department of Pharmaceutical Sciences, Kakinada, Andhra Pradesh, India Professor & H.O.D. Department of Pharmaceutical Analysis, A.U.College of pharmacy, Andhra University, Visakhapatnam. mallimpharmmba@gmail.com Abstract Received 7 March ; accepted April A simple, sensitive, accurate and reproducible method has been developed for simultaneous estimation of both the drugs by the Simultaneous equation method, using methanol as solvent. The two wavelengths 39 nm (λ max of AMLO) and 7 nm (λ max of HCTZ) were selected for the formation of Simultaneous equations. The two drugs follow Beer-Lambert s law over the range of.5 -.5µg/ml. for AMLO and 6-3µg/ml for HCTZ. Recovery study was performed to confirm the accuracy of the method. The recovery of the Amlodipine and Hydrochlorthiazide were found near to %. The Results were found to satisfactory and reproducible. The methods were validated as per ICH guidelines. The proposed methods have been applied successfully to the analysis of cited drugs in pharmaceutical formulations. Universal Research Publications. All rights reserved Key words: Amlodipine, Hydrochlorothiazide, simultaneous estimation, validation, Pharmaceutical formulations. INTRODUCTION Amlodipine (a dihydropyridine) is a calciumchannel blocker. It is given as besylate and used in the management of hypertension and angina pectoris. It is official in BP. Amlodipine (as besylate, mesylate or maleate), chemically (Fig. ) is 3 Ethyl 5 methyl (±) [( aminoethoxy) methyl] 4 ( chlorophenyl), 4 dihydro 6 methyl 3, 5 pyridine dicarboxylate, monobenzene sulphate.. Amlodipine is a dihydropyridine derivative with calcium antagonist activity. It is used in the management of hypertension, chronic stable angina pectoris and prinzmetal variant angina 3. Amlodipine acts by inhibiting the transmembrane influx of calcium ions into vascular smooth muscle and cardiac muscle and also acts directly on vascular smooth muscle to cause a reduction in peripheral vascular resistance and reduction in blood pressure 4. Hydrochlorothiazide (Fig.), 6 chloro 3, 4 dihydro H,, 4 benzo-thiadiazine 7 Sulphonamide, dioxide is a diuretic, which inhibits active chloride reabsorption at the early distal tubule via the Na Cl co transporter, resulting in an increase in the excretion of sodium, chloride and water 5. Literature survey reveals few analytical methods for the determination of Amlodipine alone and in combination with other drugs in pharmaceutical preparations and biological fluids, viz. Spectrophotometry 6-9, HPLC. and HPTLC -3.
2 Also there are some analytical methods reported for determination of HCTZ alone and in combination 4-8. Present work emphasizes on the quantitative estimation of Amlodipine and Hydrochlorothiazide in their combined dosage form by UV Spectroscopic methods. MATERIALS AND METHODS Instrumentation A Double beam UV Visible spectrophotometer (Jasco V 53) with mm matched quartz cells was used. All weighing were done on single pan balance (Shimadzu). Reagents and chemical AMLO and HCTZ reference standards were kindly provided by Cipla Pharmaceuticals, and Micro labs respectively. Analytical grade methanol was purchased from Merck Specialities Private Ltd., Mumbai. All the reagents were of analytical grade. Glass double distilled water was used throughout the experiment. Tablets were purchased from local market each containing 5 mg of AMLO and.5 mg of HCT. AMLO and HCTZ are available in the ratio of :.5 respectively in the formulation and were used in same ratio for preparation of calibration curves. Fig. : STRUCTURE OF AMLODIPINE BESYLATE Fig. : STRUCTURE OF HYDROCHLORTHIAZIDE Selection of solvent Solutions of s (-) amlodipine and hydrochlorothiazide were prepared in water, methanol, acetonitrile,. N hydrochloric acid,.n sodium hydroxide,.n sulphuric acid. Since absorbance of both drugs exhibited distinct λ max in methanol, the drug solutions were made in methanol for further studies. The λ max of s (-) amlodipine and hydrochlorothiazide were found to be 39nm and 7nm respectively (Fig.3). Preparation of standard solutions Standard stock solutions of s (-) amilodipine and hydrochlorothiazide were prepared by dissolving 5 and.5mg of the drug in methanol and the volume was made up to ml in a standard flask. From the stock solution a ranging from.5-.5µg/ml was prepared for s (-) amlodipine (Fig.4), (Table ). A ranging from µg/ml was made from the stock solution for hydrochlorothiazide (Fig.5), (Table ). Fig.3: UV spectrum spectra of s (-) amlodipine and hydrochlorothiazide combination in methanol Fig.4: Overlay spectra of standard s (-) amlodipine (.5.5µg/ml)
3 absorbance absorbance Absorbance LINEARITY s (-) Amlodipine s (-) amlodipine was found to be linear at a range of.5 -.5µg/ml. The absorbance of these solutions was noted at 39 and 7 nm respectively. Calibration curves Table : Absorbance of s (-) amilodipine at selected wavelengths (µg/ml) 39nm 7nm Fig.7: Calibration curve of s (-) amlodipine at 7 nm At wavelength 7nm, slope, intercept and correlation coefficient values were found to be.5,., and.9998 respectively (Fig.7). Hydrochlorothiazide Hydrochlorothiazide was found to be linear at a range of 6-3 µg/ml. The absorbance of these solutions was noted at 39 and 7 nm respectively. Calibration curves were plotted using Vs absorbance; at a wavelength of 7nm. The slope, intercept and correlation coefficient values were found to be.4,.54, and.997 respectively (Fig.8)..8.6 Fig.5: Overlay spectra of standard hydrochlorothiazide (6.- 3.µg/ml) were plotted using Vs absorbance; at wavelength of 39nm the slope, intercept and correlation coefficient values were found to be.89,.384, and.9956 respectively (Fig.6) Fig.8: Calibration curve of hydrochlorothiazide at 7nm Fig.6: Calibration curve of s (-) amlodipine at 39 nm At wavelength 39nm, slope, intercept and correlation coefficient values were found to be.5,.5, and.9988 respectively (Fig.9). 3
4 absorbance.3.5 ay = absorptivity of hydrochlorothiazide at 7nm. C amil = of amlodipine. C hct = of hydrochlorothiazide Fig.9: Calibration curve of hydrochlorothiazide at 39nm Table : Absorbance of hydrochlorothiazide at selected wavelengths (µg/ml) 39nm 7nm ANALYSIS OF FORMULATION Twenty tablets each containing 5mg of s (-) amlodipine and.5mg of hydrochlorothiazide were weighed and the average weight was calculated. A quantity equivalent to.5mg of drug was dissolved in methanol and the volume was made up to get the required s. Absorbance s were noted at 39nm and 7nm respectively (Fig.). The amount of s (-) amlodipine and hydrochlorothiazide was calculated using the simultaneous equation given below. The results were shown in table 3. At A = ax bc x + ay bc y A = ax bc x + ay bc y Aay A ay C amil ax ay ax ay A ax Aax C hct axay axay A = absorbance of formulation at 39nm. A = absorbance of formulation at 7nm. ax = absorptivity of s (-) amlodipine at 39nm. ax = absorptivity of s (-) amlodipine at 7nm. ay = absorptivity of hydrochlorothiazide at 39nm. Fig.: Spectra of s (-) amlodipine and hydrochlorothiazide in combination in formulation Table 3: Analysis of formulation Drug Amount (mg/tab) Labeled Found % label claim % RSD* s(-) amlodipine 5mg 4.98mg 99.%.6 hydrochlorothiazide.5mg.4mg 98.76%.4 RESULTS AND DISCUSSION Literature review indicated that, various methods have been reported for the analysis of s (-) amlodipine and hydrochlorothiazide in formulation by RP-HPLC, HPTLC. But no analytical methods were reported for the estimation of these drugs using UV spectroscopic by simultaneous equation method. The wavelength of s (-) amlodipine and hydrochlorothiazide was found to be 39 and 7 nm respectively. In these wavelengths absorbance s of s (-) amlodipine and hydrochlorothiazide and mixture was noted. The results showed an excellent correlation between absorbance s and of the drugs. Validation 4
5 parameters like accuracy and linearity showed low %RSD values which indicates that the method is sensitive. VALIDATED PARAMETERS Linearity : (s (-) amlodipine) (hydrochlorothiazide) Accuracy : 98.95%. (s (-) amlodipine) 99.5%. (hydrochlorothiazide) CONCLUSION Simple UV spectrophotometric methods were developed for the simultaneous determination of Amlodipine and Hydrochlorthiazide in bulk and tablet formulation without any interference from the excipients. To the best of our knowledge, the present study is the first report for the purpose. The present methods succeeded in adopting a simple sample preparation that achieved satisfactory extraction recovery and facilitated its application in coformulated formulation. It can therefore be concluded that use of these methods can save much time and money and they can be with accuracy. Abbreviations.AMLO : Amlodipine. HCTZ : Hydrochlorthiazide REFERENCES []. The Merck Index, 3rd ed. Whitehouse Station, New Jersey. 8: 56, 635. []. Martindale, Sweetman SC., The complete drug reference. 3 nd ed. Pharmaceutical Press. 999: 8, 97. [3]. 3.Oneil MJ, Smith A, Heckelman PE, Budawari S, The Merck Index, an Encyclopedia of Chemicals, Drugs and Biologicals, 3 rd ed.,merck and Co Inc., White House Station, New jersey. : 488, 865. [4]. [5]. Goodman and Gilman s, in: Gilman A G, Rall T W, Nies A S, Taylor P. The Pharmacological Basis of Therapeutics. (Eds.) Pergamon Press: Oxford; 996: 743. [6]. Devi Ramesh*, S Ramakrishna, New spectrophotometric methods for simultaneous determination of amlodipine besylate and atorvastatin calcium in tablet dosage forms, International Journal of Pharmacy and Pharmaceutical Sciences ISSN Vol, Issue 4, : 5 9. [7]. Priyanka R Patil*, Sachin U Rakesh, PN Dhabale, KB Burade, Simultaneous UV Spectrophotometric Method for Estimation of Losartan Potssium and Amlodipine Besylate in Tablet Dosage Form, Asian J. Research Chem. (): Jan. March, 9: [8]. Chandan Kumar Giri*, M.S. Kondawar and D.D Chougule, Simultaneous estimation of Nebivolol hydrochloride and Amlodipine besylate in combined tablet dosage form by Qanalysis method, International Journal of Pharma Research and Development online, 8. [9]. Bhatia N. M., Mohite A. S., Bhatia M. S, Simultaneous spectrophotometric estimation of atenolol and amlodipine besylate by multi wavelength method The Indian Pharmacist vol. 6, 7: []. Safeer K., Anbarasi B.*, N.Senthil Kumar3, Analytical Method Development and Validation of Amlodipine and Hydrochlorothiazide in combined dosage form by RP HPLC. International Journal of ChemTech Research Vol., No., Jan Mar : 5. [].Chandrashekhar T G, Rao P S N, Smrita K. Analysis of amlodipine besylate by HPTLC with fluorimetricdetection: a sensitive method for assay of tablets. J.Planar. Chromatogr. Mod. TLC. 994; 7: 458. []. Pandya K K, Satia M, Gandhi T P. Detection and determination of total amlodipine by high performance thin layer chromatography: A useful technique for pharmacokinetic studies. J. Chromatogr. B; Biomed. Appl. 995; 667: 35. [3]. Ilango K, Kumar P B, Prasad V RV. Simple and rapid high performance thin layer chromatographic determination of amlodipine in pharmaceutical dosage forms. Indian J. Pharm. Sci. 997; 59: 336. [4].Carlucci.G.and Palumbo. G. Simultaneous determination of losartan and hydrochlorothiazide in tablets by highperformance liquid chromatography, J. Pharm. Biomed. Anal., 3(), : [5]. Alaa. E.G. and Ahmed. A. Application of LC and HPTLCDensitometry for the Simultaneous determination of Benazepril Hydrochloride and Hydrochlorothiazide, J. Pharma.Biomed.Anal,5(), : [6]. Kartal. M. and Erk. N. Simultaneous determination of Hydrochlorothiazide and Amiloride Hydrochloride by Ratio Spectra Derivative Spectrophotometry and High Performance Liquid Chromatography, J. Pharma. Biomed. Anal., 9(3), 999: [7]. Palled. M. S. and Rajesh. P. M.N. RP HPLC Determination of Telmisartan in Tablet Dosage Forms, Indian J. Pharm.Sci.,.67, 5: 8. [8]. N.J.Shah et al, Development and validation of HPTLC method for Simultaneous estimation of Telmisartan and Hydrochlorothiazide in tablet dosage form, Indian Journal of Pharmaceutical Sciences, Mar 7:3. Source of support: Nil; Conflict of interest: None declared 5
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