A Validated RP-HPLC method for related substances of Dabigatran etexilate mesylate
Abstract
The scope of this research work was to develop a reverse-phase liquid chromatographic method for the quantification of related impurities of dabigatran and to validate the method according to ICH guidelines. Chromatographic conditions were optimised with Poroshell SB C18, 150mm, 4.6mm, 2.7µm particle size column, mixer of Phosphate buffer with phosphoric acid in water and acetonitrile with percentage of 10:90 (v/v) as solvent-A and acetonitrile and buffer with a percentage of 70:30 (v/v) as solvent-B. Gradient compassion mode is employed for mobile phase delivery with a flow rate of 0.8 mL/min. Stationary phase was maintained at 35°C temperature and detection at 230 nm, with 10 µL of sample injection volume. Water and acetonitrile in the percentage 30:70(v/v) were used as a diluent. The developed RP-HPLC method was validated according to ICH guidelines. LOD and LOQ values for dabigatran its impurities were in the range from 33 to 55 ppm and 112 to 168 ppm correspondingly. Method validation results for all the parameters are meeting the ICH guidelines acceptance criteria for the parameters of robustness, ruggedness, linearity, reproducibility and recovery. The proposed method was found to be suitable for the quantitative determination of potential impurities in the bulk samples of Dabigatran etexilate mesylate API.
Keywords
Dabigatran, HPLC, UV Detector, Validation
Introduction
Dabigatran, marked with brand name Pradaxa (Lin, Wang, Zhang, & Guan, 2019), It is an anticoagulant drug, and it will avoid blood clots and stroke in patients with atrial fibrillation (Ankit, Kumar, Kumar, Aarti, & Seth, 2014; Patel, Ram, Khatri, Ram, & Dave, 2014). Particularly, used to avoid blood clots in surgery like hip or knee replacement and also in those with a history of prior clots. It is administrated orally (Delavenne, Moracchini, Laporte, Mismetti, & Basset, 2012; Kumar, Balaraju, Kumar, & A, 2015).
Common side effects include bleeding and gastritis. This drug is not suggested for pregnancy or breastfeeding women. It acts as a thrombin inhibitor (Eerenberg et al., 2011; Prathap, Dey, Johnson, & Arthanariswaran, 2013). USFDA approved dabigatran in 2010. Dabigatran is in the List of Essential Medicines of World Health Organization (Vidushi & Meenakshi, 2017). There are several methods available for the determination of potential impurities at API stage of dabigatran. But there is no single HPLC method available for these twelve impurities of dabigatran (Desai, Chavan, Amane, Shete, & Dhole, 2019).
Materials and Methods
Samples and reagents
The development samples of dabigatran and all impurities (From impurity-1 to impurity-12) were procured from Dr Reddy's Labs, PDLab R&D, and Srikakulam, India. Chemicals and reagents utilised for development are Orthophosphoric Acid (AR grade), and acetonitrile (HPLC grade) were purchased from Merck (India) Limited. Milli-Q grade purification system used for HPLC water. Chemical names of dabigatran and its impurities are tabulated in Table 1. Structures of dabigatran and its potential impurities are shown in Figure 1.
Instruments
Agilent Infinity 1260 series HPLC system and Sartorius and model MSA 225S-100-DA weighing balance is used for experimental analysis. Empower-3 software is used as data processing.
Chromatographic Conditions
Experimental analysis was performed on column Poroshell SB-18, 150mm, 4.6mm, 2.7µm particle size, a mixture of Acetonitrile and Phosphate buffer with phosphoric acid in water in the percentage of 10:90 (v/v) as solvent-A and mixture of Acetonitrile and Phosphate buffer in the percentage of 70:30(v/v) as solvent-B. Gradient method is optimised with Flow of 0.8 mL/mi. Column stationary phase has been maintained at 35°C temperature. Detection was set at l 230 nm, and the injection load was 10 µL. Water and acetonitrile in the percentage 30:70(v/v) were selected as a diluent.
Standard and Sample Preparation:
Related substance by HPLC was performed with 0.5 mg/mL test concentration. Resolution in related substance, all the twelve impurities are spiked 0.10% concerning 0.5 mg/mL test concentration.
Results and Discussion
Analytical method validation
Analytical method validation for the estimation related impurities by HPLC of Dabigatran etexilate mesylate API was performed by following Validation of Procedures of ICH guidelines.
|
S.NO |
Name of the impurity |
Chemical Name |
|---|---|---|
|
1 |
Impurity-1 |
ethyl 3-(2-(((4-carbamimidoylphenyl)amino)methyl)-1-methyl-N-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxamido) propanoate |
|
2 |
Impurity-2 |
ethyl 2-(((4-carbamimidoylphenyl) amino)methyl)-1-methyl-1H-benzo[d]imidazole-5-carboxylate |
|
3 |
Impurity-3 |
hexyl ((4-(((5-((3-amino-3-oxopropyl)(pyridin-2-yl)carbamoyl) -1-methyl-1H-benzo[d]imidazol-2-yl)methyl)amino)phenyl) (aimino)methyl)carbamate |
|
4 |
Impurity-4 |
2-(((4-(N-((hexyloxy)carbonyl) carbamimidoyl)phenyl)amino)methyl)-1-methyl-1H-benzo[d]imidazole -5-carboxylic acid |
|
5 |
Impurity-5 |
3-(2-(((4-(N-((hexyloxy)carbonyl) carbamimidoyl)phenyl)amino)methyl)-1-methyl-N-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxamido)propanoic acid |
|
6 |
Impurity-6 |
ethyl 3-(2-(((4-cyanophenyl) amino)methyl)-1-methyl-N-(pyridin-2-yl)-1H-benzo [d]imidazole-5-carboxamido) propanoate methyl 3-(2-(((4-(N |
|
7 |
Impurity-7 |
((hexyloxy)carbonyl)carbamimidoyl)phenyl)amino)methyl)-1-methyl-N-(pyridin-2-yl)-1H-benzo [d] imidazole-5-carboxamido) propanoate |
|
8 |
Impurity-8 |
diethyl 3,3'-((2,2'-((((6-oxo-1,6-dihydro-1,3,5-triazine-2,4-diyl) bis(4,1-phenylene)) bis (azanediyl)) bis(methylene))bis(1-methyl-1H-benzo[d]imidazole-2,5-diyl-5-carbonyl))bis(pyridin-2-ylazanediyl))dipropionate |
|
9 |
Impurity-9 |
ethyl 2-(((4-(N-((hexyloxy) carbonyl) carbamimidoyl) phenyl)amino)methyl)-1-methyl-1H-benzo[d]imidazole-5-carboxylate |
|
10 |
Impurity-10 |
4-(Methylamino)-3-nitro benzoic acid |
|
11 |
Impurity-11 |
Ethyl-3-(3-Amino-4-(methyl amino)-N-Pyridine-2-yl)-benzamido propanoate |
|
12 |
Impurity-12 |
Ethyl-3-(3-nitro-4-(methyl amino) benzyl)pyridine-2-yl)-amino) propionate |
|
13 |
Dabigatran |
Ethyl 3-(2-(((4-(N-((hexyloxy) carbonyl)carbamimidoyl) phenyl)amino)methyl)-1-methyl-N-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxamido)propanoate |
|
Name of the impurity |
LOD (ppm) |
LOQ (ppm) |
|---|---|---|
|
Impurity-1 |
55.4 |
168 |
|
Impurity-2 |
35.4 |
107 |
|
Impurity-3 |
44.5 |
135 |
|
Impurity-4 |
50.9 |
154 |
|
Impurity-5 |
37.0 |
112 |
|
Impurity-6 |
42.7 |
130 |
|
Impurity-7 |
43.1 |
130 |
|
Impurity-8 |
43.0 |
130 |
|
Impurity-9 |
38.6 |
117 |
|
Impurity-10 |
42.3 |
128 |
|
Dabigatran |
43.6 |
132 |
|
Impurity-11 |
33.5 |
102 |
|
Impurity-12 |
47.0 |
142 |
|
Impurity Name |
% RSD (n=6) |
% RSD (n=12) |
Correlation coefficient |
LOQ |
50% |
100% |
150% |
|---|---|---|---|---|---|---|---|
|
Impurity-1 |
3.6 |
2.8 |
0.9965 |
102.7 |
101.4 |
102.9 |
107.2 |
|
Impurity-2 |
4.3 |
2.7 |
0.9994 |
102.6 |
104.2 |
101.7 |
104.4 |
|
Impurity-3 |
2.3 |
2.8 |
0.9958 |
98.9 |
96.5 |
106.6 |
104.8 |
|
Impurity-4 |
4.3 |
3.8 |
0.9984 |
97.4 |
98.5 |
100.3 |
102.0 |
|
Impurity-5 |
4.6 |
2.9 |
0.9969 |
102.3 |
100.7 |
105.9 |
106.1 |
|
Impurity-6 |
2.2 |
2.5 |
0.9995 |
105.9 |
104.4 |
102.9 |
105.6 |
|
Impurity-7 |
3.5 |
1.5 |
0.9998 |
104.8 |
102.1 |
98.1 |
108.5 |
|
Impurity-8 |
3.8 |
3.0 |
0.9982 |
103.4 |
99.0 |
100.8 |
112.0 |
|
Impurity-9 |
3.8 |
3.5 |
0.9952 |
108.0 |
101.4 |
105.9 |
104.6 |
|
Impurity-10 |
2.6 |
1.3 |
0.9990 |
104.3 |
101.2 |
103.3 |
109.4 |
|
Dabigatran |
2.5 |
1.1 |
0.9992 |
105.9 |
NA |
NA |
97.3 |
|
Impurity-11 |
2.2 |
1.2 |
0.9981 |
102.7 |
101.0 |
100.6 |
104.1 |
|
Impurity-12 |
3.5 |
4.0 |
0.9966 |
107.1 |
101.3 |
100.4 |
108.4 |
|
Parameter |
Resolution Between Dabigatran & DBG3A methoxy impurity |
|---|---|
|
System Suitability |
2.4 |
|
Robustness |
|
|
Flow Variation (-10%) |
4.1 |
|
Flow Variation (+10%) |
2.2 |
|
Temperature Variation (300 C) |
2.3 |
|
Temperature Variation (400 C) |
3.3 |
Limit of Detection and Limit of Quantification
Limit of detection and Limit of quantification values for dabigatran and its 12 related impurities were established by preparing the known concentration solutions from their stock solutions that would give a signal of the peak to baseline noise ratio of 3:1 and 10:1 respectively. The LOQ concentrations were confirmed by verifying precision and accuracy at LOQ concentration. LOD and LOQ concentrations of impurities are as tabulated in Table 2. The % RSD of an area of all the impurities in six preparations at LOQ concentration were found within the specified limit, which confirms that the analytical procedure is precise at LOQ Concentration. The results are tabulated in Table 3. LOD and LOQ chromatograms are shown in Figure 4; Figure 3; Figure 2.
The percentage recovery of each impurity ranged from 95 to 108. Recovery all the impurities are well within the acceptance limit, which confirms that the method is accurate at LOQ level.
Precision
Precision is a term used to describe data from an experiment that has been repeated several times. Degree of scattering between a sequence of measurements attained from the various sampling of the homogeneous sample under the agreed conditions is defined as the precision of an analytical procedure. Repeatability of the related substance procedure was checked by six-fold analysis by adding all the twelve impurities at LOQ concentration as well as at 0.10% level in Dabigatran test sample. The study was also performed on a different day with a different analyst for the evaluation of inter-day and intra-day variation and analyst. The % RSD of all individual impurity areas in every six preparations were found well within the set acceptance limit, which confirms that the method is precise.
Linearity
Linearity for all the impurities was carried out from the limit of quantification (LOQ) to 150% concentration of the test concentration 0.05 mg/ml. Responses for all impurities were recorded and plotted the calibration curve for each impurity concentration versus response; the correlation coefficient achieved for each impurity was more than 0.999.
Accuracy
A known amount of each impurity is added in Dabigatran test sample, and the study was conducted to define the accuracy of the procedure for the quantification of all impurities. Experiments were performed in triplicate at LOQ, 0.05%, 0.1% and 0.15% of the test concentration (0.05 mg/ml) and calculated the recovery of all the fourteen impurities. The percentage recovery of each impurity was within the acceptance limit, which confirms that the proposed method was accurate. Spiked test chromatogram, as shown in Figure 5.
Robustness
To prove the robustness of the test procedure, method conditions were altered and estimated the change in the resolution of dabigatran and impurity-D. Experiments are performed by changing the Flow by ± 10% and column temperature by ±50C. Resolution between dabigatran and impurity-D has established the robustness of the method. Data is evaluated in the Table 4.
Stability of Solution
Dabigatran test sample solution stability was established by spiking all the impurities at 0.1% level to Dabigatran Drug Substance. All solutions which are prepared in the volumetric flask were tightly capped and kept at ambient temperature for 48 hours—estimated the level of all the impurities initially, after 24 hours and after 48 hours. Results were indicated the sample solution is stable up to 48 hours.
Mobile Phase solution stability was also established for Dabigatran related impurities with new sample solutions and holding the mobile phase for 48 hours. Freshly prepared sample solutions were analysed initially, after 24 hours and after 48 hours with the initially prepared mobile phase. Results were indicated the mobile phase solution is stable up to 48hours.
Conclusion
Developed quantitative related substance method for dabigatran is specific, linear, accurate, and precise. The analytical procedure was fully validated and found that the data generated in all the method validated parameters tested are found satisfactory. Newly developed HPLC method can be used to determine the related substance of regular Dabigatran commercial samples.