Spectrophotometric Method for Simultaneous Estimation of Amlodipine Besylate in Pharmaceutical Formulation Naresh Kalra*, Suresh Choudhary Department of Pharmaceutical sciences, Alwar Pharmacy College, IET, North Extension M.I.A, Alwar, India ABSTRACT The present study deals with a rapid, simple, accurate, and rugged UV spectrophotometric methods have been developed for the estimation of some water insoluble calcium channel blockers using hydrotropic solubilization phenomenon in pure and as well as in dosage forms. The calibration graphs were linear in the 5 40 µg/ml concentration range (r>0.997) for Amlodipine. All the methods were validated as per USP guidelines. The detection was made on 350-200 nm in methanol and 0.1M sodium Benzoate observed maximum absorption at 237nm. Hence this method is simple, rapid, and accurate and can be successfully applied for routine analysis. Keywords: Spectrometric analysis; Amlodipine; Method validation; Hydrotrophy 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 chemically 3-ethyl-5-methyl-2-(2aminoethoxymethyl)- 4-(2-chlorophenyl)-1,4-dihydro-6-methyl pyridine-3,5- dicarboxylate mono benzene sulphonate 1. It is official in BP 2.Various methods have been employed to enhance the aqueous solubility and hydrotrophy is one of them. Maheshwari et al. have developed various analytical techniques employing hydrotropic solubilization phenomenon to analyze poorly watersoluble drugs like hydrochlorothiazide 3, aceclofenac 4 ofloxacin 5. Hydrotropic solution may be a proper choice to preclude the use of organic solvents because organic solvent having a problems related like toxicity, cost etc. Several analytical methods that have been reported for the estimation of amlodipine besylate in biological fluids and/or pharmaceutical formulations include UV spectrophotometric 6-10, and high-performance liquid chromatographic estimation (HPLC) 11, 12. Amlodipine is a calcium channel blocking agent. It inhibits the influx of extracellular calcium across the myocardial and vascular smooth muscle cell membranes. *Corresponding Author: Naresh Kalra Asst. Prof, Alwar Pharmacy College, North extension, IET MIA Alwar (Raj) India, M 09413787016 E-mail: neshu07@gmail.com The decrease in intracellular calcium inhibits the contractile processes of the myocardial smooth muscle cells, causing dilation of the coronary and systemic arteries, increased oxygen delivery to the myocardial tissue, decreased total peripheral resistance, decreased systemic blood pressure, and decreased after load. Another possible mechanism is that amlodipine inhibits vascular smooth muscle carbonic anhydrase I activity with consecutive ph increase which may be involved in intracellular calcium influx through calcium channels. Objective To develop an accurate, selective precise and specific method for estimation of water insoluble calcium channel blockers in bulk and as well as in dosage form. There is a broad scope for hydrotropic agent in quantitative estimation of poorly water soluble drugs. The present work is an attempt to study and develop spectroscopic methods employing hydrotropic solubilization phenomenon for quantitative estimation and validation of the method according to ICH guidelines 13. Estimation of Amlodipine in dosage form using hydrotropic solubilization technique MATERIALS AND METHODS: Instrument: Double beam UV-visible spectrophotometer (Shimadzu UV-1800) with 1 cm Advanced Journal of Pharmacie and Life science Research 16
Matched quartz cells, band pass 1.8 nm, Shimadzu AX -200 electronic weighing balance and Ultrasonicator, Enertech electronics Pvt. Ltd. Drug Sample: Amlodipine were obtained as gift sample from Sun Pharma limited, Bangalore. Chemicals and Reagent: Methanol A.R. Grade (Loba Chemie, Mumbai). & Sodium From SD fine Chemicals. Commercially available pharmaceutical dosage form Amdepin manufactured by Cadila pharmaceuticals limited, Cadila corporate campus, Sarkhej- Dholaka road, Bhatt, Ahmedabad-22500 were procured from local market for estimation. Estimation of amlodipine in pure and in dosage form in methanol: Preparation of standard stock solution of Amlodipine in Methanol Weighed accurately about 50mg of amlodipine and transferred into a 50ml standard flask dissolved and made unto the volume using methanol. This solution had a concentration of 1mg per ml of amlodipine (solution A). Accurately pipette out 5ml of solution A into a 50ml standard flask and the volume was made up to 50ml using methanol. The resulting solution had a concentration 100mg/ml of amlodipine (solution B).Accurately pipette out 4ml of solution B separately into 10ml of standard flask and the volume was made up to 10ml using methanol. The resulting solution had a concentration of 40mg/ml of amlodipine (solution C). Study of spectral characteristic of amlodipine in methanol After enabling the initial adjustment and blank correction, using methanol the solution C was scanned in the entire UV range from 350 to 200 nm. A broad band of absorption spectrum was observed with maximum absorption at 237nm. Preparation of calibration curve of amlodipine in methanol Accurately pipette out.5ml to 4.0ml of solution B into eight 10ml standard flask. Volume was made up to 10ml using methanol. The absorption of each solution was measured at 237nm against reagent blank. The readings were plotted by taking absorbance in Y-axis and concentration X-axis. Estimation of amlodipine from dosage form in methanol Twenty tablets containing each of 10mg of amlodipine were accurately weighed and finally now dried in a glass mortar. A weigh equivalent to 10mg of amlodipine was accurately transferred to a 10ml of standard flask 4ml of methanol was added and swirled gently a period of 10min.The clear supernatant solution was then transferred to 10ml standard flask through Whatmann No.1 filter paper.the residue was further extracted twice with 2ml each of methanol and passed the same filter paper and the volume was made up to 10ml with methanol. The resulting solution had a concentration of 1mg/ml (solution A). Accurately pipette 1ml of the above solution into a 10ml standard flask and made up to volume with methanol. The final solution had a concentration of 100mg/ml of amlodipine (solution B). Accurately pipette out 1ml of solution B into 10ml standard flask and the volume was made up using methanol to obtain concentration of 10mg/ml of amlodipine. The solution was measured at 237 nm and the concentration of amlodipine was calculated from the calibration graph Table 1. Estimation of amlodipine in dosage form using 0.1M sodium Benzoate as hydrotropic agent: Preparation of 0.1M Sodium Benzoate Weighed accurately about 0.0432gm of Sodium Benzoate and transferred into a 100ml standard flask dissolved and made up the volume using Distilled water. Preparation of standard stock solution of amlodipine in 0.1M Sodium Benzoate Weighed accurately about 50mg of amlodipine and transferred into a 50ml standard flask dissolved and made up the volume using 0.1M Sodium Benzoate. This solution had a concentration of 1mg per ml of amlodipine (solution A). Accurately pipette out 5ml of solution A into a 50ml standard flask and the volume was made up to 50ml using 0.1M Sodium Benzoate. The resulting solution had a concentration 100mg/ml of amlodipine (solution B) Accurately pipette out 4ml of solution B separately into 10ml of standard flask and the volume was made up to 10ml using 0.1M Sodium Benzoate. The Advanced Journal of Pharmacie and Life science Research 17
resulting solution had a concentration of 40mg/ml of amlodipine (solution C). Study of spectral characteristic of amlodipine in 0.1M Sodium Benzoate After enabling the initial adjustment and blank correction, using methanol the solution C was scanned in the entire UV range from 400 to 200 nm. A broad band of absorption spectrum was observed with maximum absorption at 364nm. Preparation of calibration curve of amlodipine in 0.1M sodium Accurately pipette out.5ml to 4.0ml of solution B into eight 10ml standard flask. Volume was made up to 10ml using 0.1M Sodium Benzoate.The absorption of each solution was measured at 364nm against reagent blank. Estimation of amlodipine from dosage form in 0.1M sodium Twenty tablets containing each of 10mg of amlodipine were accurately weighed and finally powdered in a glass mortar. A weigh equivalent to 10mg of amlodipine was accurately transferred to a 10ml of standard flask 4ml of 0.1M Sodium Benzoate was added and swirled gently for a period of 10min.The clear supernatant solution was then transferred to 10ml standard flask through Whatmann No.1 filter paper.the residue was further extracted twice with 2ml each of 0.1M Sodium Benzoate and passed the same filter paper and the volume was made up to 10ml with 0.1M Sodium Benzoate. The resulting solution had a concentration of 1mg/ml (solution A). Accurately pipette 1ml of the above solution into a 10ml standard flask and made up volume with 0.1M Sodium Benzoate. The final solution had a concentration of 100mg/ml of amlodipine (solution B). Accurately pipette out 1ml of solution B into 10ml standard flask and the volume was made up using methanol to obtain concentration of 10mg/ml of amlodipine. The solution was measured at 364 nm and the concentration of amlodipine was calculated from the calibration graph Table No. 1. RESULT AND STATISTICAL EVALUATION: The Weight of 20 tablets in methanol & 0.1M Sodium Benzoate is the 0.9844gm & 0.9844gm & Each tablet contains (label claim) amlodipine is 10mg. So that the Average weight of tablet is 0.0992 gm & 0.0992 in methanol & 0.1M Sodium Benzoate & the Weight of powder taken is 0.0992gm. So that the Average content of amlodipine /tablet determined by the proposed method is 10.082mg & 10.042 mg. & The Percentage of label claim for methanol and 0.1M Sodium Benzoate is 100.2% & 100.02%. Method validation 14,15 Linearity and range Calibration curve was prepared for amlodipine is methanol at 364nm and entire calibration data at the selected wave length as summarized in table No.2.These shows that amlodipine. The obeyed Beer s law in the concentration range 5 to 40mg/ml at 364nm Repeatability /precision Repeatability expresses the precision under the same operating condition area short interval of time. It is also termed as intraday precision. The precision of the analytical procedure is usually expressed as the standard deviation of a series of measurements. The results obtained from nine observations of the same concentration of sample solution the main volume obtained are tabulated in Table No.3. Robustness The robustness of the method was determined by using sodium from three different suppliers. SD fine chemicals, Mumbai. E Merck. Ltd, Mumbai, Thomas Baker chemicals Ltd.mumbai. Recovery studies Accuracy of the proposed method was determined by performing recovery studies. A fixed amount of drug from dosage form was taken and pure standard drug at three different concentrations within beer s range was added. The total concentration was found by the proposed method. The determination with each concentration was repeated three times and average percent recovery of the added standard was calculated and results are tabulated in Table No. 5. Advanced Journal of Pharmacie and Life science Research 18
Table 1: Results of analysis of amlodipine in dosage form Sr. Formulatio Method Label No. n concentration in µg Amount of drug found in µg* 1. AMDEPIN In Methanol 10 10.82 100.2 % label claim 2. AMDEPIN In 0.1M Sodium * Average of 5 experiments 10 10.042 100.02 Table 2: Calibration data of amlodipine at 237 nm and 364 nm Sr. No. Parameter Amlodipine at 237 nm Amlodipine at 364 nm 1. Beer s law limit (µg /ml) 5-40 5-40 2. Regression equation Y= 0.0235 X +0.0062 Y= 0.0135 X +0.0362 3. Slope 0.0062 0.0362 4. Intercept 0.0235 0.0135 5. Correlation of coefficient 0.998 0.997 Table 3: Precision data of Amlodipine in Methanol and in 0.1 M Sodium Sr. No. Sample concentration in µg /ml Concentration obtained by the method Mean Value In Methanol In 0.1M Sodium 01 9.567 9.543 02 9.453 9.675 03 9.458 9.456 04 9.234 9.345 05 10 9.457 9.789 06 9.875 9.034 07 9.436 9.340 08 9.345 9.134 09 9.651 9.456 Standard Deviation = 0.000108 Coefficient of Variance = 0.00547 Standard Error = 0.000629 In Methanol 4.186 5.234 In 0.1M Sodium Table 4: Robustness with Methanol and 0.1M Sodium from three different suppliers Sr. No. Methanol Drug taken in mg Drug obtained in mg* % of recovery 1. 10 10.005 100.31 2. 10 9.992 99.43 3. 10 9.891 99.54 0.1 M sodium Drug taken in mg Drug obtained in mg % of recovery 1. 10 10.09 100.01 2. 10 9.84 99.85 3. 10 9.23 99.24 Advanced Journal of Pharmacie and Life science Research 19
Table 5: Recovery Studies of Amlodipine in Methanol and in 0.1M Sodium Sr. No. Method Formulation Label claim in mg Concentration of pure drug in mg Concentration found in mg* % Recovery ±SD 1. Amlodipine in methanol 2. Amlodipine in 0.1M Sodium *Average of 5 determinations AMDEPIN AMDEPIN 10-9.563 95.63 10 5 15.021 99.64 10 10 19.652 99.82 10 15 24.291 99.72 10-10.232 101.03 10 5 15.340 102.04 10 10 19.831 99.90 10 15 25.001 100.01 Table 6: Calibration data and Molar absoptivity values of amlodipine Sr. No. Concentration in mg In Methanol at 237nm In 0.1M Sodium at 364nm Absorbance* E 1cm 1% Absorbance* E 1cm 1% 1. 5 0.1150 230.15 0.2150 431.21 2. 10 0.2301 231.33 0.4301 430.11 3. 15 0.3411 230.45 0.6411 430.21 4. 20 0.4611 230.44 0.8611 431.11 5. 30 0.5761 230.13 1.0761 430.10 6. 35 0.6911 231.14 1.2911 430.61 7. 40 0.8061 230.11 1.4061 430.11 8. Mean - 230.45-431.03 9. SD - 0.2842-0.592 10. CV - 0.0808-0.2681 11. ± SE - 0.1005-0.1872 *Average of 5 determinations ACKNOWLEDGEMENT: The authors are thankful to Sun Pharma limited, Bangalore for gift Sample of amalodipine & Management of alwar Pharmacy College for providing other necessary requirement for this work. REFERENCES: 1. RB Kakd, Spectrophotometric method for simultaneous estimation of amlodipine besylate and bisoprolol fumarate in pharmaceutical preparations. Research J. Pharm. and Tech.2008; 1(4): Oct.-Dec. 2. British Pharmacopoeia Vol. 1, Department of Health Social Services and Public Safety, H.M. Stationary Office London, 2004;126. 3. Maheshwari R K, Chaturvedi S C, Jain N K, Application of hydrotropic solubilization phenomenon in spectrophotometric analysis of hydrochlorothiazide tablets. Indian Drugs 2005; 42(8): 541-544. 4. Maheshwari R K. Application of hydrotropic solubilization in the analysis of aceclofenac. Asian J. Chem.18 (2):1572-1574. 5. Maheshwari R K, Chaturvedi S C, Jain N K. Application of hydrotrophy in spectrophotometric determination of pharmaceutical dosage forms. Indian Drugs 2005;42(11):760-763. 6. Jain N K, Agrawal R K, Singhai A K. Hydrotropic solubilization of nifedipine. Pharmzie 2005; 45:221-5. 7. Kasture AV and Ramteke M. Simultaneous UV-Spectrophotometric method for the Advanced Journal of Pharmacie and Life science Research 20
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