Method development and validation of dabigatran in pharmaceutical dosage form by RP- HPLC method Ajitha A ajithaazhakesan27@gmail.com Pharmaceutical Analysis and Quality Assurance, CMR College of Pharmacy, Medchal, Hyderabad, India-501401. India 11 08 2020 11 08 2020

www.ijamscr.com

Method development and validation of dabigatran in pharmaceutical dosage form by RP- HPLC method Ajitha A*<sup>1</sup>, P.Sandhya Rani<sup>2</sup>, Chandu<sup>2</sup>

*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: ajithaazhakesan27@gmail.com

Abstract

A simple precise, accurate method was developedand 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 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 C34H41N7O5
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.

 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 300C, Injection Volume was 10µL,Run time 15min. Diluent used was Acetonitrile and distilled(HPLC 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

Table no. 1.1 System suitability parameters for Dabigatran

S no P e a k N a me RT A r ea U S P P l a t e C ount U S P T ailing
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
Mean 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
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
Accuracy

Table no1.6 Accuracy table for Dabigatran

% Level Amount Spiked(μg/mL) Amountrecovered (μg/mL) % 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.

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

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.

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; 5(2), 2014, 31.

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, 6(2), 2015, 1000263.

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

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.2(1),2014, 080-092.

Mrinalini C.Damle*, Rupesh A. Bagwe . Development and validation of stability-indicating rp-hplc method for estimation of dabigatran etexilate. Journal of Advanced Scientific Research5(3), 2014, 39-44.

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.

International Journal of Pharmacy and Integrated Life Sciences V2-(I10) PG (61-71).