Method development and validation of dabigatran in pharmaceutical dosa

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Ajitha A et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-71 2019 01-08 1 IJAMSCR |Volume 7 | Issue 1 | Jan - Mar - 2019 www.ijamscr.com Research article Medical research Method development and validation of dabigatran in pharmaceutical dosage form by RP- HPLC method Ajitha A 1 P.Sandhya Rani 2 Chandu 2 1 Pharmaceutical Analysis and Quality Assurance CMR College of Pharmacy Medchal Hyderabad India-501401. Dept of Pharmaceutical Chemistry CMR College of Pharmacy Medchal Hyderabad India-501401. Corresponding Author: Ajitha A Email id: ajithaazhakesan27gmail.com ABSTRACT A simple precise accurate method was developed and validated by reversed phase high performance liquid chromatography method used for the estimation of Dabigatran in bulk and pharmaceutical dosage form. It is reversed phase liquid chromatography. The HPLC method has been carried out by using C18 150x4.6mm 5µm column. This method has been developed by using the mobile phase consisting buffer: Acetonitrile 65:35 and the flow rate of 1ml/min by the detection of UV at 330nm. The retention time of the dabigatran is 0.999 min. The runtime is 15min. the linearity was found to be over a concentration of 25-150 respectively. The accuracy was found to be 98.84 to 100.24. With a correlation coefficient of0.999.The proposed method can be used for the estimation of the drug in bulk and pharmaceutical formulation. The results of analysis have been validated satisfactorily using recovery studies. Keywords: RP- HPLC Dabigatran Method development. INTRODUCTION Method validation is the process of demonstrating that analytical procedures are suitable for their intended use and that they support the identity quality purity and potency of the drug substances and drug products. In normal phase mode the nature of stationary phase is polar and the mobile phase is non-polar. In this technique non-polar compounds travel faster and are eluted first because of the lower affinity between the non- polar compounds and stationary phase. Polar compounds are retained for longer time and take more time to elute because if their higher affinity with the stationary phase. Reversed phase mode is the most popular mode for analytical and preparative separations of compounds of interest in chemical biological pharmaceutical food and biomedical sciences. In this mode the stationary phase is non-polar hydrophobic packing with octyl and octadecyl functional group bonded to silica gel and the mobile phase is a polar solvent often a partially or fully aqueous mobile phase. Polar substances prefer the mobile phase and elute first. As the hydrophobic character of the solutes increases retention increases. Generally the lower the polarity of the mobile phase the higher is its eluent strength. The elution order of the classes of ISSN:2347-6567 International Journal of Allied Medical Sciences and Clinical Research IJAMSCR

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Ajitha A et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-71 2019 01-08 2 compounds is reversed thus the name reverse-phase chromatography DRUG PROFILE 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. Fig no. 1 Chemical structure of Dabigatran CAS Number 211915-06-9 Purity ≥98 Molecular weight 627.73 Molecular formula C 34 H 41 N 7 O 5 Physical state Solid Solubility Soluble in DMSO water and ethanol Storage store at 4 degree centigrade Melting point 180 ± 3 DSC: 10 K min-1 heating rate MATERIALS AND METHODS Dabigatran pure drug API and Dabigatran tablets CIPLA pharmaceutical laboratories. Distilled water Acetonitrile Glacial Acetic Acid. All the above chemicals and solvents are from Rankem. Instruments HPLC instrument used was of WATERS HPLC 2965 SYSTEM with Auto Injector and PDA Detector. Software used is Empower 2. UV-VIS spectrophotometer PG Instruments T60 with special bandwidth of 2mm and 10mm and matched quartz was be used for measuring absorbance for Dabigatran solutions Methanol Methanol is known as methyl alcohol. Methanol is easily available and in expensive compared to a Acetonitrile. Methanol is used as HPLC mobile phase for analytical and preparative analysis. As methanol mixes with water it forms adduct which has a viscosity even higher than that of water. Acetonitrile Acetonitrile is basically a polar solvent which is miscible with water but never the less has sufficient dispersive properties to elute substances from a liquid chromatography column by dispersive interactions with solute. Aceotonitrile used as HPLC mobile phase for analytical and preparative analysis. Water Double distilled water-HPLC grade is used as the mobile phase for analytical and preparative separations. Water for HPLC is purified and tested to ensure that it has low UV absorbance to provide most sensitive detection across all wavelengths.

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Ajitha A et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-71 2019 01-08 3 Chromatographic conditions Mobile phase used as buffer: Acetonitrile 60:40 Flow rate 1.0 ml/min column used as BDS C18 250x5mm 4.6μ Detection wavelength was 330nm column Temperature 30 0 C Injection Volume was 10µL Run time 15min. Diluent used was Acetonitrile and distilledHPLC grade water in 45:55 ratio RESULTS AND DISCUSSIONS Optimized method Fig 2. Optimized chromatogram of Dabigatran Assay Fig 2. Assay Chromatogram of Dabigatran Table no. 1.1 Assay data for Dabigatran Sample No Assay 1 100.92 2 99.28 3. 99.89 4. 100.25 5. 100.35 6. 99.83 AVG 100.09 STDEV 0.5570 RSD 0.56 System suitability All the system suitability parameters were in the range and satisfactory as per ICH guidelines

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Ajitha A et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-71 2019 01-08 4 Table no. 1.1 System suitability parameters for Dabigatran S no Peak Name RT A rea USP Plate Count USP Tailing 1 Dabigatran 2.634 3204012 3505 1.50 2 Dabigatran 2.650 3249713 4443 1.25 3 Dabigatran 2.650 3187847 4444 1.25 4 Dabigatran 2.650 3207954 4087 1.33 5 Dabigatran 2.654 3173777 4558 1.27 6 Dabigatran 2.664 3267995 4684 1.21 M ean 3215216 Std. Dev. 36387.32 RSD 1.13 Fig 3. System suitability Chromatogram of Dabigatran Specificity Fig4. Blank Chromatogram Linearity Table1.3Linearity Concentration and Responce for Dabigatran Linearity Level Concentration ppm Area 0 0 0 25 37.5 762156 50 75 1628358 75 112.5 2376430

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Ajitha A et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-71 2019 01-08 5 100 150 3079285 125 182.5 4014557 150 225 4710193 Fig5 linearity chromatogram of Dabigatran Precision Intermediate precisio Table 1.4 Intermediate precision data for Dabigatran n S.No Peak Area 1 3173094 2 3225208 3 3243260 4 3186601 5 3223210 6 3269874 AVG 3220208 STDEV 35724.7 RSD 1.11 Repeatability Table 1.5 Repeatability data for Dabigatran ility: S.No Peak Area 1 3248148 2 3195254 3 3214951 4 3226458 5 3229685 6 3212904 AVG 3221233 STDEV 17927.1 RSD 0.56 y 21035x + 892.57 R² 0.9992 0 1000000 2000000 3000000 4000000 5000000 0 50 100 150 200 250 Series1 Linear Series1

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Ajitha A et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-71 2019 01-08 6 Accuracy Table no1.6 Accuracy table for Dabigatran Level Amount Spiked μ g/m L Amount recovered μ g/m L Recovery Mean Recovery 50 75 74.13 98.84 99.92 75 75.72 100.97 75 75.24 100.33 100 150 149.23 99.49311 150 150.99 100.6658ö 150 150.14 100.0937 150 225 222.88 99.06 225 224.12 99.61 225 225.54 100.24 LOD: LOD Limit of detection: Ditection limit of the Dabigatran in this method was found to be 0.012µg/ml. Fig 6. LOD Chromatogram of Dabigatran LOQ Limit of quantitation : Quantification limit of the Dabigatran in this method was found to be 0.037µg/ml.

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Ajitha A et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-71 2019 01-08 7 Fig 7. LOQ Chromatogram of Dabigatran Robustness Table no. 1.7Robustness data of Dabigatran Parameter RSD Flow Minus 1.8 Flow Plus 0.1 Mobile phase Minus 1.9 Mobile phase Plus 0.0 Temperature minus 1.08 Temperature plus 1.7 CONCLUSION The proposed HPLC method was found to be precise specific accurate rapid and economical for simultaneous estimation of Dabigatran etexilate in capsule dosage form. The sample recoveries in all formulations were in good agreement with their respective Label Claims and this method can be used for routine analysis. It can be applied for routine analysis in laboratories and is suitable for the quality control of the raw materials formulations dissolution studies and can be employed for bioequivalence studies for the same formulation Acknowledgement The authors are thankful to Reference standards of drug samples and equipments were procured from CMR College of Pharmacy Hyderabad Telangana India. BIBLIOGRAPHY 1. Ankit Prajapati Sharad Kumar Ashim Kumar Sen Aarti Zanwar AK Seth Spectrophotometric method for estimation of dabigatran etexilate in bulk and its pharmaceutical dosage form. An international journal of pharmaceutical sciences 0.3397/ICV: 4.10. 2. Ankit Prajapati Sharad Kumar Ashim Kumar Sen Aarti Zanwar Seth A. K. Spectrophotometric method for estimation of dabigatran etexilate in bulk and its pharmaceutical dosage form. Pharma Science Monitor 52 2014 31.

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Ajitha A et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-71 2019 01-08 8 3. Dare M Jain R and Pandey A . Method validation for stability indicating method of related substance in active pharmaceutical ingredients dabigatran etexilate mesylate by reverse phase chromatography. Chromatogr Tech 62 2015 1000263. 4. Eman G. Nouman Medhat A. Al-Ghobashy Hayam M. Lotfy . Development and validation of LC–MSMS assay for the determination of the prodrug dabigatran etexilate and its active metabolites in human plasma 5. Mr. BRC Sekhar Reddy Dr. Nallagatla. Vijaya Bhaskar Rao. A stability indicating rp-hplc method for estimation of dabigatran in pure and pharmaceutical dosage forms. SPJPBS. 21 2014 080-092. 6. Mrinalini C.Damle Rupesh A. Bagwe . Development and validation of stability-indicating rp-hplc method for estimation of dabigatran etexilate. Journal of Advanced Scientific Research 53 2014 39-44. 7. S Roy B A Patel Hardik ghelani S J Parmar. Development validation of spectroscopic method for estimation of dabigatran etexilate mesylate in capsule dosage form. 8. International Journal of Pharmacy and Integrated Life Sciences V2-I10 PG 61-71. How to cite this article: Ajitha A P.sandhya Rani Chandu. Method development and validation of dabigatran in pharmaceutical dosage form by RP- HPLC method. Int J of Allied Med Sci and Clin Res 2019 71: 01-08. Source of Support: Nil. Conflict of Interest: None declared.

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