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1 Available online at International Journal of Trends in Pharmacy and Life Sciences Vol. 2, Issue: 1, 2016: METHOD DEVELOPMENT AND VALIDATION OF DABIGATRAN ETEXILATE MESYLATE BY RP-HPLC METHOD AND ITS DEGRADATION STUDIES A.Srinivas 1 *, K.Sridhar Reddy 2, G.Sai 3 1. St.John College of Pharmacy, Yellapur(V), Hasanparthy(M), Warangal(Dist), Telangana, India 2. SRR College of Pharm. Sciences, Valbhapur(V), Elkathurthy(M), Karimnagar(Dist), Telangana, India drampaty@gmail.com ABSTRACT A simple, precise, accurate, economical and reproducible RP-HPLC method for estimation of Dabigatran Etexilate Mesylate in capsule dosage form has been developed. Quantitative HPLC was performed with SHIMADZU LC 20ATwith Spin chrome Software with UV-Visible Detector (SPD-20A), PHENOMENEX Luna C 18, 5µm, 250 x 4.6mm (size) column was used in the study. The mobile phase of Methanol: Water (70:30) used in this study. The conditions optimized were: flow rate (1.2 ml/minute), wavelength (230 nm) and run time was 10 min, column temperature was maintained at 50 0 C. Retention time was found to be 4.60 min. The linearity was found to be in the concentration range of 0-25 g/ml. The developed method was evaluated in the assay of commercially available capsules Paradaxa containing Dabigatran Etexilate Mesylate. The amount of drug in capsule was found to be 75mg. Results of analysis were validated statistically and by recovery studies. The recovery studies % was indicative of the accuracy of proposed method. The precision was calculated as repeatability, inter and intraday variation (%RSD) for the drug. By using method, stability of the drug has been studied. Key Words: HPLC, method validation, Dabigatran Etexilate Mesylate, precision, stability studies *Corresponding Author: Dr. A.Srinivas, St.John College of Pharmacy, Yellapur(V), Hasanparthy(M), Warangal(Dist), Telangana, India Received: 04/01/2016 Revised: 25/01/2016 Accepted: 28/01/2016 INTRODUCTION It is necessary to find the content of each drug either in bulk or single or combined dosage forms for purity testing. It is also essential to know the concentration of the drug and it s metabolites in biological fluids after taking the dosage form for treatment. The scope of developing and validating an analytical method is to ensure a suitable method for a particular analyte more specific, accurate and precise 1. The main objective for that is to improve the conditions and parameters, which should be followed in the development and validation. According to the literature survey it was found that few analytical methods such as (RP- HPLC 2-4, Spectrophotometric 5-6 ) were reported for the estimation of Dabigatran Etexilate Mesylate. Also a few LC-MS 7, UPLC/MS 8 and were reported for the quantification of Dabigatran etexilate in Human plasma. The objective of the proposed method is to develop simple and accurate methods for the determination of Dabigatran Etexilate Mesylate by RP-HPLC method in pharmaceutical dosage forms & its stability indicative studies. Dabigatran etexilate mesylate is chemically 9 β-alanine, N-[[2-[[[4-[[[(hexyloxy) carbonyl]amino]phenyl]amino]methyl]-1-methyl-1h-benzimidazol-5-yl]carbonyl]-n-pyridinyl-, ethyl ester, Srinivas A et al. Int J Trends in Pharm & Life Sci. 2016: 2(1);

2 methanesulfonate. The empirical formula C 34 H 41 N 7 O 5.CH 4 O 3 S and the molecular weight is (mesylate salt), (free base). It is highly soluble in methanol and partly soluble in water. Dabigatran etexilate mesylate is a yellow-white to yellow powder. A saturated solution in pure water has a solubility of 1.8 mg/ml. It is freely soluble in methanol, slightly soluble in ethanol, and sparingly soluble in isopropanol 10 Dabigatran etexilate is an inactive pro-drug that is converted to dabigatran. Dabigatran, the main active principle in plasma, is a rapid-acting competitive and reversible direct inhibitor of thrombin Thrombin, a serine protease, is responsible for the conversion of fibrinogen to fibrin in the coagulation cascade. Inhibition of thrombin consequently prevents thrombus development. Dabigatran inhibits free thrombin, fibrin-bound thrombin and thrombin-induced platelet aggregation which results in a prolongation of a PTT (partial thromboplastin time), ECT (Ecarin clotting time), and TT (thrombin time). Fig. 1: Structure of Dabigatran Etexilate Mesylate MATERIALS AND METHOD Instruments and Reagents The chromatographic separation was performed on SHIMADZU LC 20AT reciprocating dual pump, UV-Visible Detector (SPD-20A), a reversed-phase PHENOMENEX Luna C 18, 5µm, 250 x 4.6mm (size) used for separation. Chromatographic data was acquired using Spin Chrome software & Hamilton injector used. Analytical balance (SHIMADZU), Ultra Sonicator (SOLTEC, SONICA Ultrasonic Cleaner) has been used in the work. Pure drug of Dabigatran etexilate was provided as a gift sample from SR Laboratories, Hyderabad, India. Paradaxa capsules were procured from local market Hyderabad. HPLC Grade Acetonitrile, Methanol and water were procured from Merck. Optimised Chromatographic conditions The mobile phase of Methanol:water = 70:30 were used in this study. The conditions optimized were: flow rate (1.2 ml/minute), wavelength (230nm) and run time was 10 min, column temperature was maintained at 50 0 C. Retention time was found to be 4.6 min. PHENOMENEX Luna C 18, 5µm, 250 x 4.6mm (size) column was used as stationary phase in the study. Srinivas A et al. Int J Trends in Pharm & Life Sci. 2016: 2(1);

3 Preparation of mobile phase Methanol and water were mixed in the ratio of 70:30 and filtered through 0.45 µm membrane filter and degassed in a sonicator for 10 minutes. Preparation of Standard solution The stock solutions of Dabigatran etexilate mesylate (1000 μg/ml) was prepared by dissolving an appropriate amount of analyte in diluent. Working standard solution was prepared by mixing above stock solution of Dabigatran with final concentration of 20 μg/ml, respectively. Preparation of Test solution Sample solution of a concentration of 20 μg/ml of Dabigatran was prepared by using diluents from stock solution (1000 μg/ml). Method Validation As per the ICH guidelines, the method validation parameters checked were linearity, accuracy, precision, limit of detection, limit of quantification. Linearity and Range: The linearity of the method was determined by preparing different concentration levels of solutions in the range from the stock solution (0-25 μg/ml). 20μl of each solution was injected into the chromatograph and calibration curve was plotted with peak are versus concentration of solution. The response for the drug was linear and the correlation coefficient value of Dabigatran etexilate mesylate was found to be Accuracy: The accuracy of the method was determined by recovery experiments. The recovery study was carried out by the standard addition method at three levels of 80, 100 and 120%. Each solution was injected in triplicate and the percentage recovery was calculated. Recovery was within the range of 100 ± 5% which indicates accuracy of the method. Precision: The precision of each method was ascertained separately from the peak areas & retention times obtained by actual determination of six replicates of a fixed amount of drug Dabigatran etexilate mesylate. (API). The percent relative standard deviations were calculated for Dabigatran etexilate mesylate. The intra & inter day variation of the method was carried out & the high values of mean assay & low values of standard deviation & % RSD (% RSD < 2%) within a day & day to day variations for Dabigatran etexilate mesylate. LOD and LOQ: These two parameters were calculated using the formula based on the standard deviation of the response and the slope. LOD and LOQ were calculated by using equations, LOD=3.3 s/s and LOQ=10 s/s, where s = standard deviation, S = slope of the calibration curve. Method Robustness: Influence of small changes in chromatographic conditions such as change in flow rate ( 0.2ml/min) and Wavelength of detection ( 2 nm) studied to determine the robustness of the method are also in favour of (% RSD < 2%) the developed RP-HPLC method for the analysis of Dabigatran etexilate mesylate ( API). Srinivas A et al. Int J Trends in Pharm & Life Sci. 2016: 2(1);

4 Forced degradation studies: The protocol was strictly adhered to for forced degradation of Dabigatran etexilate mesylate Active Pharmaceutical Ingredient (API). The API (Dabigatran etexilate mesylate) was subjected to stress conditions in various ways to observe the rate and extent of degradation that is likely to occur in the course of storage and/or after administration to body. This is one type of accelerated stability studies that helps us determining the fate of the drug that is likely to happen after long time storage, within a very short time as compare to the real time or long term stability testing. The various degradation pathways studied are acid hydrolysis, basic hydrolysis and oxidative degradation. Mother Sample: Before carry out the degradation studies the mother sample was prepared to find out the % degradation. It is prepared as the same method followed in sample or standard preparation. Acid Hydrolysis: An accurately weighed 10 mg. of pure drug was transferred to a clean & dry 10 ml volumetric flask. To which 1 N HCl was added & make up to the mark and kept for 8 hrs. from that 1 ml was taken in to a 10 ml volumetric flask & make up to the mark with diluent, then injected for HPLC analysis. Basic Hydrolysis: An accurately weighed 10 mg. of pure drug was transferred to a clean & dry 10 ml volumetric flask. To which 1 N NaOH was added & make up to the mark & kept for 8 hrs. from that 1ml was taken in to a 10 ml volumetric flask & make up to the mark with diluent, then injected for HPLC analysis. Oxidation with (3%) H 2 O 2 : An accurately weighed 10 mg. of pure drug was transferred to a clean & dry 10 ml volumetric flask. To which 3% H 2 O 2 was added & make up to the mark & kept for 8 hrs. from that 1 ml was taken in to a 10 ml volumetric flask & make up to the mark with diluent then injected for HPLC analysis. RESULTS AND DISSCUSION To develop a precise, linear, specific & suitable stability indicating RP-HPLC method for analysis of Dabigatran etexilate mesylate different chromatographic conditions were applied & the results observed are presented. Isocratic elution is simple, requires only one pump & flat baseline separation for easy and reproducible results. So, it was preferred for the current study over gradient elution. In case of RP-HPLC various columns are available, but here PHENOMENEX Luna C 18, 5µm, 250 x 4.6mm (size) column was preferred because using this column peak shape, resolution and absorbance were good. Mobile phase & diluents for preparation of various samples were finalized after studying the solubility of API in different solvents of our disposal (methanol, acetonitrile, water, 1M NaOH, IM HCl). The drug was found to be highly soluble in methanol and partly soluble in water. Using these solvents with appropriate composition newer methods can be developed and validated. The result shows the developed method is yet another suitable method for assay and stability studies which can help in the analysis of Dabigatran etexilate mesylate in different formulations. The precision of the method was demonstrated by intra-day and inter-day variation studies. For intra-day studies the drug having concentration value 80%, 100 % & 120% of the target concentration (n Srinivas A et al. Int J Trends in Pharm & Life Sci. 2016: 2(1);

5 MEAN AUC RESEARCH ARTICLE e-issn: ), were injected in triplicate into the HPLC system and for inter-day studies the drug at above three concentrations were injected in triplicate into the HPLC system for three days. Data were subjected to statistical treatment for the calculation of SD and %RSD. The value of %RSD for Dabigatran etexilate mesylate was found to be 0.86, 0.30, and 0.13 for intra-day studies. The values for inter-day studies were 0.87, 0.32, and 0.11 respectively. This shows that values are not more than 2%, indicates that the developed method is precise. The LOQ was found to be µg/ml and area and LOD was found to be µg/ml and area The results of the stress studies indicated the specificity of the method that has been developed. Dabigatran etexilate mesylate was degraded only in 3% H 2 O 2 & 1N Hcl, 1N NaoH conditions. Fig. 2: Chromatogram of Dabigatran etexilate mesylate (20 ppm) in optimized conditions (RT 4.60 min) Table 1: Linearity calculation CONC.(µg/ml) MEAN AUC (n=6) Linearity Graph y = 70244x R² = CONC Fig 3 : Linearity Graph Srinivas A et al. Int J Trends in Pharm & Life Sci. 2016: 2(1);

6 Table 2: Accuracy calculation Sample ID Concentration ( g/ml) Pure drug Formulation %Recovery of Pure drug Statistical Analysis S 1 : 80 % Mean= 96.38% S 2 : 80 % S.D. = S 3 : 80 % % R.S.D.= S 4 : 100 % Mean= 96.38% S 5 : 100 % S.D. = S 6 : 100 % % R.S.D.= S 7 : 120 % S 8 : 120 % S 9 : 120 % Mean= 96.49% S.D. = % R.S.D. = 0.01 HPLC Injection Replicates of Dabigatran Etexilate Mesylate Table 3 : Repeatability calculations Area Replicate Retention Time 4.63 Replicate Replicate Replicate Replicate Replicate Average Standard Deviation % RSD Table 4: Results of intra-assay & inter-assay Conc. Of (API) Observed Conc. Of Dabigatran Etexilate Mesylate (µg/ml) by the proposed method (µg/ml) Intra-Day Inter-Day Mean (n=6) % RSD Mean (n=6) % RSD Srinivas A et al. Int J Trends in Pharm & Life Sci. 2016: 2(1);

7 3.49 Intensity(mV) Intensity(mV) RESEARCH ARTICLE e-issn: Table 5: Robustness data of the proposed method VARIATION RETENTION TIME AREA FLOW RATE 1ml/min 1.4 ml/min WAVELENGTH 228nm 232nm Results of degradation studies: RetentionTime(min) Fig. 4: Chromatogram showing degradation in 0.1 N HCl RetentionTime(min) Fig.5: Chromatogram showing degradation in 0.1 N NaOH Srinivas A et al. Int J Trends in Pharm & Life Sci. 2016: 2(1);

8 Intensity(mV) 4.73 RESEARCH ARTICLE e-issn: RetentionTime(min) Fig.6: Chromatogram showing degradation in 3% H 2 O 2 Table 6: Results of Forced Degradation Studies of Dabigatran etexilate mesylate API. Stress condition Time Assay of active substance Assay of degraded products Mass Balance (%) Acid Hydrolysis (0.1 M HCl) 08 Hrs Basic Hydrolysis (0.I M 08 Hrs NaOH) Oxidation(3% H 2 O 2 ) 08 Hrs CONCLUSION The proposed method is simple, sensitive and reproducible and hence can be used in routine for determination of Dabigatran etexilate mesylate in bulk as well as in pharmaceutical formulations. Statistical analysis of the results has been carried out revealing high accuracy and good precision. REFERENCES: 1. Rockville MD, United States Pharmacopoeia; United States Pharmacopoeial Convention Inc, 25th Edn., 2002 ; 1 : Bernardi RM, Froehlich PE, Bergold AM. Development and validation of a stability-indicating liquid chromatography method for the determination of dabigatran etexilate in capsules. J AOAC Int. 2013; 96(1): Geetharam Y, Mallesh M, Ramamohan GV, Sridhar B, Srividya E, Subhash S. A validated stabilityindicating Liquid Chromatographic method for the determination of dabigatranetexilate (thrombin inhibitor) in capsules. Indo American Journal of Pharmaceutical Research Nadeem AK, Keyur BA, Namrata BS, Pooja Jairam M. A validated stability-indicating reversed phase High Performance Liquid Chromatographic method for the determination of dabigatranetexilatemesylate, Inventi:ppaqa, Srinivas A et al. Int J Trends in Pharm & Life Sci. 2016: 2(1);

9 5. Ankit P, Sharad K, Ashim KS. Spectrophotometric method for estimation of dabigatran etexilate in bulk and its pharmaceutical dosage form, pharma science monitor 5(2), Bharat P, Pravin R, Taslimahemad K, Pragnesh D. Study of Anticoagulant Dabigatran by Analytical Instrumentation, International Letters of Chemistry, Physics and Astronomy, 11(3) (2014) Zhe YH, Robert BP, Vanessa LH, S. Casey L. Conventional liquid chromatography/ triple quadruple mass spectroscopy based metabalite identification and semi quantitative estimation approach in the investigation of invitro dabigatran etexilate metabolite. Anal Bioanal Chem. Feb 2013; 405(5): Xavier D, Julie M, Silvy L, Patrick M, Thierry B. UPLC MS/MS assay for routine quantification of dabigatran A direct thrombininhibitor In human plasma, Journal of Pharmaceutical and Biomedical Analysis 58 (2012) Drug bank database is a detailed drug data with comprehensive drug target information Innovator Database about Dabigatran capsules Health Sciences University of Hokkaido, Department of Internal Medicine, Japan, Dabigatran etexilate, a thrombin inhibitor for the prevention of venous thrombo embolism and stroke, Curr Opin Investig Drugs ;8(9): Adis, Wolters Kluwer Business, Auckland, Dabigatran etexilate: a review of its use in the prevention of stroke and systemic embolism in patients with atrial fibrillation. Am J Cardiovasc Drugs. 2011; 11(1): Srinivas A et al. Int J Trends in Pharm & Life Sci. 2016: 2(1);