Publication Ref No.

: IJPRD/2009/PUB/ARTI/VOL-8/OCT/001

ISSN 0974 9446

DEVELOPMENT AND VALIDATION OF DERIVATIVE SPECTROPHOTOMETRIC METHOD FOR DETERMINATION OF ENTACAPONE IN PHARMACEUTICAL FORMULATION
Shailesh K. Koradia*1, Ashwin S. Agola2, Nurudin P. Jivani1, Ravi A. Manek1 and Shivanand Pandey1 1 Smt. R. B. Patel Mahila Pharmacy College, Atkot, Rajkot, Gujarat, INDIA. 2 N. R. Vekariya Pharmacy College, Junagadh, Gujarat, INDIA.

ABSTRACT A rapid, precise, accurate and specific first-order derivative spectrophotometric method was developed for the determination of Entacapone in pharmaceutical formulation. The technique was applied using 0.01M NaOH as diluent. The first-order derivative spectra were obtained at N = 10, = 4.0 nm, and determination were made at 375.0 nm. The method showed high specificity in the presence of formulation excipients and good linearity in the concentration range of 2-25 g/mL with correlation coefficient 0.9964. The detection limit and quantitation limit were found to be 0.42 g/mL and 1.28 g/mL, respectively. The recovery ranged between 96.11 and 100.61%. The proposed method is easy to apply, low-cost, does not use polluting reagents and require relatively inexpensive instruments. Key words: Entacapone, first-order derivative, method validation, tablets

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INTRODUCTION AND MATERIALS AND METHODS INTRODUCTION Entacapone is chemically known as 2-cyano-3-(5-dihydroxyamino-3,4-dioxo-1-cyclohexa-1,5-dienyl)N,N- diethyl-prop-2-enamide and belongs to the class of antiparkinson agent1. Entacapone is a selective and reversible inhibitor of catechol-O-methyltransferase (COMT), with mainly peripheral actions. It is used in the treatment of Parkinsons disease as an adjunct to Levodopa/Carbidopa therapy2. Literature survey revealed that High-Performance Liquid Chromatographic (HPLC)3 and Spectrophotometric4 methods for the determination of Entacapone in plasma and pharmaceutical formulation have been reported, respectively. The drug is not official in any pharmacopoeia. The validation procedures followed the International Conference on Harmonization (ICH) guidelines evaluating the parameters like linearity, precision, accuracy, detection limit and quantitation limit5. OBJECTIVE: A new, sensitive, accurate and precise first-order derivative spectrophotometric method has been developed for the routine determination of Entacapone in tablet formulation in the quality control department.

MATERIALS AND METHODS: Instruments UV-Visible double beam spectrophotometer (UV-2450, SHIMADZU Limited, Japan) with 1cm matched quartz cells, analytical balance (KEROY Pvt. Ltd.). Chemicals and Reagents Entacapone (Gift sample by Sun Pharmaceuticals Ltd. Mumbai), sodium hydroxide pellets (AR, CDH (P) Ltd., New Delhi), two brands of tablet formulations were procured from a local pharmacy and double distilled water.

Working standard solution of Entacapone Standard Entacapone (10 mg) was accurately weighed and transferred to 100 mL volumetric flask. It was dissolved properly and diluted up to the mark with 0.01M NaOH to obtain concentration of 100 g/mL. This solution was used as working standard solution. Spectrophotometric measurements The absorbance of the solutions containing Entacapone at 10 g/mL was determined in the UV range 500-300 nm (Fig. 1) using an appropriate blank. The first-order derivative spectra were obtained by 2 International Journal of Pharma Research and Development Online

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Publication Ref No.: IJPRD/2009/PUB/ARTI/VOL-8/OCT/001

ISSN 0974 9446

instrumental electronic differentiation in the range of 500-300 nm. The amplitude values obtained in the first-order derivative spectra were traced with N =10, = 4.0 nm and the amplitude at 375.0 nm were selected for the analysis of Entacapone (Fig. 2).

VALIDATION OF METHOD PARAMETERS: Specificity The specificity of the method was evaluated through the analysis of a market sample solution, which contains number of pharmaceutical excipients in their usual concentration. Linearity Aliquots of a 100 g/mL solution of Entacapone working standard were transferred to 10 mL volumetric flasks to obtain the final concentrations of 2.0, 5.0, 10.0, 15.0, 20.0, and 25.0 g/mL. Each solution was prepared in triplicate. The linearity was evaluated by linear regression analysis, which was calculated by the least square regression method (table 2). Precision A 20 mg of Entacapone standard was transferred to a 100 mL volumetric flask containing 50 mL of 0.01M NaOH and shaken for 15 minutes. The volume was completed with the same solvent. The solution was properly diluted to 10 g/mL. New solutions were prepared in 3 days for the interday and 3 times in a day for the intraday precision evaluation. Accuracy The accuracy of the method was evaluated through recovery test. From an Entacapone working standard solution of 100 g/mL, aliquots of 0.5, 1.0, and 1.5 mL were taken and transferred to 10 mL volumetric flasks containing 1.0 mL of the sample solution at 100 g/mL. The volume was completed with 0.01M NaOH to obtain the final concentrations of 15.0, 20.0, and 25.0 g/mL, respectively. Each solution was done in triplicate (table 3). Stability Sample solutions were kept at room temperature and 2-8 C (under refrigeration condition). The absorbance of the solutions kept under the two temperature conditions were measured periodically and compared. There was not much variation between the absorbance of the two solutions initially, but was observed after a few hours. From these observations, it was found that the stability of solution was more than 6 h at room temperature and 12 h at refrigeration condition. Assay of tablet sample A 20 mg of Entacapone tablet powder was transferred into a 100 mL volumetric flask containing 50 mL 0.01M NaOH and shaken for 15 minutes. The volume was completed with the same solvent. . The solution was filtered through Whatmann filter paper and diluted to obtain 10 g/mL concentration. The solution was measured and the 1D375 was obtained. Concentration of sample solution was calculated from calibration curve of Entacapone (table 4). 3 International Journal of Pharma Research and Development Online

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RESULT AND DISCUSSION: In the beginning of the development of this method, different solvents were tested such as water, methanol, acid and alkali. Alkali solution was considered the best since it had a great solubility and the absorbance of Entacapone was high. Besides, it does not offer toxicological risks. In order to choose the optimum conditions for the derivative method, different orders were tested, as well as different N (experiment points as smoothing function). The first-order demonstrated to be adequate and N = 10 the condition that allowed high amplitude and good spectra profile. Values of higher or lower than 10 produced noisy spectra and distorted the spectral resolution. The derivative wavelength difference () originated from this combination was 4.0 nm, according to the equipment used. The effect of wavelength scanning speed was studied and it was found that at high speed noisy spectrum was obtained and at low scanning speed, the noise decreased but a longer time was required for the measurements, so medium scanning speed was chosen to carry out the measurements. The Zero-order spectrum of Entacapone standard, first-order spectra of Entacapone standard, tablet and overlain are represented in fig. 1-4. Compared with conventional spectrophotometric determinations, derivative spectrophotometry has proved to be of great value in eliminating the interference from excipients and co-formulated drugs. The results from linearity are demonstrated in table 1.The data was validated by means of statistical methods. It was possible to calculate the detection and quantitation limits. The results found were LOD = 0.42 g/mL and LOQ = 1.28 g/mL. The precision (measurements of repeatability, intraday and interday) results showed good reproducibility with percent relative standard deviation (% RSD) is below 2.0 for all parameters. The evaluation of accuracy of the method, made through the recovery test, showed it is accurate, with recovery ranged between 96.11 and 100.61 % for 1D375 (table 3).

Figure 1: Zero-order spectrum of Entacapone standard (10 g/mL)

Figure 2: First-order derivative spectrum of Entacapone standard (10 g/mL) 4 International Journal of Pharma Research and Development Online

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Figure 3: First-order derivative spectrum of Entacapone tablet (10 g/mL)

Figure 4: Overlain first-order derivative spectra of Entacapone standard

Figure 5: Calibration curve of standard Entacapone by first-order derivative 5 International Journal of Pharma Research and Development Online

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Calibration curve for Entacapone

Y = 0.006X - 0.0035 r = 0.9964

2

0.16 Amplitude at 375.0 nm 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 0 5 10 15 20 25 30

Concentration in g/mL

Table 1: Results of standard curve of first-order derivative spectrophotometric method for Entacapone determination Feature Regression equation (Y) Slope (b) Intercept (a) Standard deviation of slope Standard deviation of intercept Correlation coefficient (r2)
1

D375

Y = 0.006X - 0.0035 0.0060 -0.0035 8.94 10-5 8.0 10-4 0.9964

Table 2: Calibration data of Entacapone standard at 375.0 nm Concentration (g/mL) 2.0 5.0 10.0 15.0 20.0 25.0 Amplitude at 375.0 nm Mean SD (n = 3) 0.0062 0.0004 0.0288 0.0010 0.0584 0.0015 0.0872 0.0015 0.1150 0.0025 0.1542 0.0019 % CV 1.67 2.01 1.78 1.76 1.25 1.19

Table 3: Accuracy data of Entacapone standard at 375.0 nm Formu Amount of Amount Amount of drug % recovery % 6 International Journal of Pharma Research and Development Online

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-lation

drug taken (g/mL) 10 10 10 10 10 10

Brand A Brand B

of drug added (g/mL) 5 10 15 5 10 15

found (g/mL) Mean SD 14.81 0.0878 19.64 0.0878 24.58 0.1476 15.00 0.0893 19.70 0.2032 24.81 0.1388

CV (n=6)

96.11 1.79 96.34 0.09 97.22 1.02 100.03 1.79 96.95 2.10 100.61 1.02

Table 4: Estimation of Entacapone in tablet formulation Formulation Brand A Brand B Label value (mg) 200 200 % label value found Mean SD (n = 3) 97.78 1.257 99.16 1.476 % CV 1.29 1.49

CONCLUSION: The first-order derivative spectrophotometric method is rapid, accurate and precise. The results of the analysis of the tablets by this method were reproducible reliable, and in good agreement with labeled claim of the drug. There was no interference from the excipients present in the tablets. ACKNOWLEDGEMENT: The authors would like to acknowledge Sun Pharmaceuticals Ltd., Mumbai, India, for providing pure drug sample of Entacapone for the development of this assay.

BIBLIOGRAPHY: 1. ONeil MJ, The Merck Index, Merck and Co., Inc., White House Station, NJ., 13th Ed, 2001:3630. 2. Sweetman SC, (Ed.) Martindale, The Complete Drug Reference, Pharmaceutical Press London, UK, 33rd Ed, 2002:1168. 3. Ramakrishna NVS, Vishwottam KN, Wishu S, Koteshwara M and Chidambara J, High-performance liquid chromatography for the quantification of Entacapone in human plasma, J. Chromatogr. B, 2005, 823(2): 189-194. 4. Koradia SK, Jivani NP, Malaviya SV, Chauhan SP, and Vaghani SS, Development and validation of spectrophotometric method for the estimation of Entacapone in pharmaceutical formulations, Int. J. Chem. Sci., 2009, 7(1): 349-352. 5. ICH Steering Committee, Validation of analytical procedures: Methodology, ICH Harmonized Tripartite Guidelines, 1996.

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