Pharma Science Monitor 5(2), Apr-Jun 2014 PHARMA SCIENCE MONITOR
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1 Impact factor: /ICV: Pharma Science Monitor 5(2), Apr-Jun 2014 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES Journal home page: SPECTROPHOTOMETRIC METHOD FOR ESTIMATION OF DABIGATRAN ETEXILATE IN BULK AND ITS PHARMACEUTICAL DOSAGE FORM Ankit Prajapati *, Sharad Kumar, Ashim Kumar Sen, Aarti Zanwar, AK Seth Department of Pharmacy, Sumandeep Vidyapeeth University, At & Po Pipariya, Ta- Waghodia, Dist. Vadodara (Gujarat) India ABSTRACT The present study describes a simple, accurate, precise and cost effective UV-Vis Spectrophotometric method for the estimation of Dabigatran Etexilate in bulk and it s pharmaceutical dosage form. Estimation of Dabigatran Etexilate was carried out in pharmaceutical dosage form by zero order spectrophotometric method using UV visible spectrophotometer. The solutions were prepared in ACN. Dabigatran Etexilate showed absorbance maxima at 313 nm. The linearity was found in the concentration range of 3-15 µg/ml for Dabigatran Etexilate. The slop, intercept and correlation coefficients were found to be 0.066, and for Dabigatran Etexilate at 313 nm, respectively. Values for LOD and LOQ were found to be µg/ml and µg/ml, respectively. Values for precision i.e. repeatability, intra day precision and inter day precision in terms of % RSD were found to be 0.48, 0.558, < 2.0 %. Accuracy of the method was confirmed by recovery studies and % recovery for formulation was determined and means recovery was found to be in the range of % %. The proposed method has been applied successfully for the analysis of the drug in pure and pharmaceutical dosage form. KEYWORDS: UV-Vis Spectrophotometer, Method validation, Dabigatran Etexilate. INTRODUCTION Dabigatran Etexilate chemically Ethyl 3-(1-{2-[({4[Amino({[(Hexyloxy)Carbonyl] Imino})Methyl]Phenyl}Amino)Methyl]-1-Methyl-1H-1,3-Benzodiazol-5-Yl}-N-(Pyridin-2 Yl) Formamido)Propanoate. Dabigatran etexilate is an oral prodrug that is metabolized by a serum esterase to dabigatran. It is a synthetic, competitive and reversible direct thrombin inhibitor. Inhibition of thrombin disrupts the coagulation cascade and inhibits the formation of clots. Dabigatran etexilate may be used to decrease the risk of venous thromboembolic events in patients who have undergone total hip or knee replacement surgery, or to prevent stroke and systemic embolism in patients with atrial fibrillation, in whom anticoagulation therapy is indicated. Literature survey reveals that only method like (HPLC & GC) in plasma determine its
2 Impact factor: /ICV: intermediates and metabolites. But to the best of our knowledge, there is one literature reported about the UV spectrophotometric method. The aim of the work was to develop and validate an analytical method by using UV-Vis Spectrophotometer for the estimation of Dabigatran Etexilate in bulk and pharmaceutical dosage forms. The formulation available in Indian market under the trade name of "PRADAXA" of different strength like 75 mg, 110 mg, 150 mg, manufactured by Boehringer Ingelheim. After development, analytical method was validated to ensure their quality and suitability as per ICH guideline. In the present research work a simple, accurate and economical UV-Vis Specrophotometric method has been developed for the estimation of Dabigatran Etexilate in Capsule dosage form and bulk drug substances. Figure 1: Structure of Dabigatran Etexilate MATERIAL AND METHODS Chemicals And Reagents Dabigatran Etexilate was obtained as gift sample and was used without further purification. All chemicals and reagents used were of analytical grade. Dabigatran Etexilate Capsules (PRADAXA 110) were purchased from market. Instruments The instruments used were UV 1800 Spectrophotometer (UV probe): Shimadzu, Digital balance : Ohaus gm, Sonicator: Ultrasonica cleaner - Toshcon Pvt. Ltd. RESULTS AND DISCUSSION Method Development Preparation of standard Stock Solution Accurately weighed 10 mg of standard Dabigatran Etexilate (DB) taken and transferred into a 10 ml volumetric flask and dissolved in acetonitrile (ACN). The flask was sonicated for 5 min and volume was made up to the mark with acetonitrile to give a solution of 1000 µg/ml. From
3 Impact factor: /ICV: this stock solution, 1 ml solution was pipetted out and placed into 10 ml volumetric flask. The volume was made up to mark with ACN to give a solution containing 100 µg/ml. Selection of Solvent Absorbance of the drug was higher and drug exhibited distinct λmax in ACN and hence ACN was selected as solvent for further studies. Selection of Wavelength Appropriate dilutions were prepared for drug and the solutions were scanned in the wavelength range of nm and the λ max was found to be 313nm against ACN as blank for DB. Figure 2: UV Spectra of DB in ACN at 313 nm Preparation of Calibration Curve Appropriate volume of aliquots from standard Dabigatran stock solution B was transferred to different volumetric flasks of 10 ml capacity. The volume was adjusted to the mark with ACN to obtain concentrations of 3, 6, 9, 12 and 15µg/ml. Absorbance of each solution against ACN as blank was measured at 313 nm for DB and the graph of absorbance against concentration was plotted and regression equation and correlation coefficient was determined.
4 Impact factor: /ICV: DABIGATRAN y = 0.066x R² = Absorbance Series Concentration μg/ ml Figure 3: Calibration curve of DB at 313 nm. Table 1 Regression analysis data and summary of the calibration curve for zero order spectrophotometric method PARAMETERS Drug DABIGATRAN ETEXILATE Detection wavelength 313 nm Beer s law limits (µg/ml) 3-15 Regression equation (y*) y = 0.066x Slope (m) Intercept (c) Correlation coefficient (R 2 ) *y = mx + c where, x is the concentration of Dabigatran in µg/ml and y is the absorbance at the respective wavelength. Preparation of Sample Solution Twenty capsules each containing 110 mg of Dabigatran Etexilate was weighed and powdered. An accurately weighed quantity of powder equivalent to 24.6 mg was dissolved in 10 ml ACN and sonicated for 15 min and volume was made up to 10 ml (1000 μg/ml). The solution was filtered through Whattman filter paper No. 41. Method Validation Linearity: To establish linearity of the proposed method, six separate series of solutions of DB (3-15 µg/ml
5 Impact factor: /ICV: in ACN) prepared from the stock solution and analyzed. Least square regression analysis was done for the obtained data and illustrated in Table: 2. Table 2 Determination of Linearity of DB (3-15 µg/ml) Sr. CONCENTRATION ABSORBANCE No. (μg/ml) (313 nm) ± SD* ± ± ± ± ± * Six separate series of solutions of Dabigatran (3-15 µg/ml in ACN) Sensitivity: The sensitivity of the proposed method for measurement of Dabigatran was estimated in terms of limit of detection [LOD] and limit of quantification [LOQ]. The LOD and LOQ were calculated by using the slope and SD of response (intercept). The mean slope value and SD of response was obtained after plotting six calibration curve. LOD and LOQ were found to be µg/ml and µg/ml, respectively. Precision: Repeatability studies were done by six times consequently taking absorbances of the standard solution containing 9 µg/ml of DB and the % RSD of the replicate absorbances was calculated. Values of repeatability in terms of %RSD were found to be < 2.0 %. Table 3 Determination of Repeatability of DB SR. CONCENTRATION ABSORBANCE MEAN NO. (μg/ml) (313 nm) ± SD % RSD ±
6 Impact factor: /ICV: Intra day precision studies were done by three times in a day taking absorbances of the standard solutions containing 6 µg/ml and 9 µg/ml of DB and the % RSD of the replicate absorbances was calculated. Values of Intra day precision in terms of %RSD were found to be < 2.0 %. Table 4 Determination of Intra Day Precision of DB SR. CONC. ABSORBANCE NO. (μg/ml) (313 nm) MEAN ± SD % RSD ± ± Inter day precision studies were done by once in three different day taking absorbances of the standard solution containing 6 µg/ml and 9 µg/ml of DB and the % RSD of the replicate absorbances was calculated. Values of Inter day precision in terms of %RSD were found to be < 2.0 %. Table 5 Determination of Inter Day Precision of DB ABSORBANCE (313 nm) MEAN SR. CONC. DAY ± NO. (μg/ml) % RSD SD ± ± Accuracy: The accuracy of the method was determined by calculating % recovery of DB by the standard addition method. Known amounts of standard solutions of DB was added at 50, 100 and 150 % level to prequantified sample solutions of DB. The amount of DB was estimated by applying obtained values to the respective regression line equations. The experiment was repeated for five times. The % recovery and %RSD were calculated Values of % recovery in terms of %RSD were found to be < 2.0 %.
7 Impact factor: /ICV: Table 6 Determination of Accuracy (% Recovery) of DB Amount Amount Sr. of of Final Amount % Mean % No. Level sample standard conc. recovered Recovery ± RSD (mg) added (μg/ml) (μg/ml) SD (mg) % % % 99.16% ± % % % ± % % % % ± % Assay of Formulation: From the sample solution, appropriate aliquots were pipetted out in to 10 ml volumetric flasks and dilutions were made with ACN to obtain final concentration of 15 μg/ml. Absorbances for these solutions were measured at 313 nm against ACN as blank for DB and % drug amount was found to in accepted criteria. Table 7 Determination of Assay of Formulation Formulation : PRADAXA DRUG LABEL CLAIM (mg) AMOUNT FOUND (mg) % DRUG FOUND DB
8 Impact factor: /ICV: CONCLUSION Hence it can be concluded that the developed UV spectroscopy method for estimation of Dabigatran Etexilate in capsule form is simple, economical, accurate and reproducible and will be conveniently adopted for the routine quality control analysis from its pharmaceutical formulations and bulk drug. ACKNOWLEDGEMENTS Authors are thankful to Principal and Management, Sumandeep Vidyapeeth, Department of Pharmacy, Vadodara, Gujarat for providing the facilities to conduct this research work. REFERENCES 1. Willard HH, Lynne L. Instrumental method of analysis. 7 th edition. New Delhi, CBS Publisher and Distributors, 1986; Kealey D, Haines PJ. Instant notes Analytical chemistry. 5 rd edition. London, BIOS Scientific Publishers Ltd, 2002; Khopkar SM. Basic concepts of Analytical chemistry. 3 rd edition. New Age Science Publisher, 2008; Kasture AV, Vadodkar SG, Mahadik KR, More HN. Pharmaceutical Analysis. Introduction to Pharmaceutical Analysis, 2 nd edition. New Delhi, Nirali Prakashan, 2008; United States Pharmacopoeia (USP-NF XXIV), Rockville MD 20852; United States Pharmacopoeial Convention Inc, 1985; Quality Assurance of Pharmaceuticals Geneva: World Health Organization Green JM. A Guide to Analytical Method Validation, J Amer Chem, 1996;68: Balaji N, P N. GC quantification of residual hexylmethane sulfonate in dabigatran etexilate mesylate. J Apply Che 2012;2(6): Delavenne X, Mismetti P. Rapid determination of apixaban concentration in human plasma by liquid chromatography/tandem mass spectrometry, Application to pharmacokinetic study. J Pharm Biomed Anal 2013;78: Larsson M, Logren U, Ahnoff M, Lindmark B, Abrahamsson P, Svennberg H, Persson BA, et al. Determination of melagatran, a novel, direct thrombin inhibitor, in human plasma and urine by liquid chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2002;766(1):47-55.
9 Impact factor: /ICV: Larsson M, Ahnoff M, Abrahamsson A, Logren U, Fakt C, Ohrman I, Persson BA, et al. Determination of ximelagatran, an oral direct thrombin inhibitor, its active metabolite melagatran, and the intermediate metabolites, in biological samples by liquid chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2003;783(2): Method validation guidelines (Q2R1) International Conference on harmonization. GENEVA; For Correspondence Ankit Prajapati aankit247@gmail.com
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