Basima Arous*, Mhd Amer Al-Mardini
Department of Pharmaceutical Chemistry and Quality Control, Faculty of Pharmacy, Damascus University, Damascus, Syria
*Corresponding Author E-mail: basima.arous@gmail.com
ABSTRACT:
Dabigatran is a novel anticoagulant drug acting as a direct and reversible thrombin inhibitor. The purpose of this work was to develop a sensitive and validated LC-MS method for the analysis of Dabigatran etexilate and estimation of its impurity profile in active pharmaceutical ingredient (API) and pharmaceutical dosage forms and to estimate its stability profile according to the ICH guidelines. The method enables to separate 22 impurities appeared in APIs from different resources and Pradaxa® the brand. Forced degradation under stress conditions were carried out in order to establish its stability profile. Dabigatran etexilate was subjected to hydrolysis (acidic and basic), photolysis, thermolysis, and oxidation as per ICH guidelines and found susceptible to all stress conditions. Eleven degradation products were successfully separated, identified and characterized by ESI-MS, then six of them were isolated by semi-preparative HPLC and subjected to NMR spectroscopy and FT-IR to characterize and elucidation of their most possible structures and to predict the possible degradation pathways of Dabigatran etexilate. Comparing Dabigatran etexilate impurity profile and stability profile lead to observe that nine impurities are degradation products resulted by the degradation of dabigatran etexilate, and that defined a considerable overlap can be observed between the impurities and degradation products.
KEYWORDS: Dabigatran etexilate, LC-MS, impurities, stability indicating, degradation products.
1. INTRODUCTION:
Dabigatran etexilate Figure 1, chemically known as (ethyl 3-{[(2-{[(4-{[(hexyloxy) carbonyl] carbamimidoyl} phenyl) amino] methyl}-1-methyl-1H-benzimidazol-5-yl) carbonyl] (pyridin-2-yl) amino} propanoate), is a pro-drug, rapidly converted to Dabigatran after oral administration. It is a novel anticoagulant drug invented and manufactured by Boehringer Ingelheim, acting as a direct, selective and reversible thrombin inhibitor. It is prescribed for the prevention of stroke and systemic thromboembolism after elective hip and knee replacement in patients with nonvalvular atrial fibrillation [1, 2].
Molecular Formula: C34H41N7O5
Molecular Weight: 627.75
Figure 1: Structure of the Dabigatran etexilate[3].
The presence of impurities in API can have a significant impact on the quality and safety of the drug products, so it is a mandatory requirement by regulatory authorities to identify and characterize all the unknown impurities that are present at a level more than 0.1% [4, 5], and as the API may undergo degradation, leading to the drug activity loss or to occurrence of adverse effects associated with degradation products, thorough knowledge of API’s stability profile is required. Stability testing provides evidence of the quality of API when exposed to the influence of environmental factors such as pH, temperature, humidity and light. The data from such studies enables storage conditions, re-test periods and shelf lives to be established. Stress testing helps to determine the intrinsic stability of the molecule by establishing the degradation pathways [6, 7]. According to the literature survey it was found that many analytical methods such as Spectrophotometric [8, 9], HPTLC [10], HPLC with Fluorescent Detection[11], RP-HPLC[12, 13], were reported for the estimation of Dabigatran etexilate in API and pharmaceutical dosage forms. Also there are many methods reported in the literature that can estimate Dabigatran etexilate and its process related impurities in API and pharmaceutical dosage forms[14-18]. In addition to several RP-HPLC [19-22] stability-indicating methods which have been reported for the stability of Dabigatran etexilate, but the stability profile of Dabigatran etexilate under various stress conditions has been recently investigated in two reports[23, 24]. One of them, Dabigatran stability study was performed using thermal stress condition, and the main degradation products formed were analyzed by LC-UV and LC-ESI-MS methods and structures were proposed also the degradation kinetics of the drug and cytotoxicity of the degraded samples were studied[23]. In the second report, the degradation behavior of Dabigatran under stress conditions were studied and the degradation products were identified by using high-resolution mass spectrometry in multistage mode (HR-MSn) [24].
Since Dabigatran etexilate is not yet official in any of the pharmacopoeia [25-27], and there isn't any study dealing with the relationship between stability profile and the identification and quantitative determination of related impurities in Dabigatran etexilate. The aim of this research was to develop a stability indicating method for determination of Dabigatran etexilate, identification, isolation and characterization of its impurities and degradation products (DPs) in order to identify its major degradation pathways, by using techniques of LC-MS, semi-preparative HPLC, NMR spectroscopy and FT-IR spectral data.
2. MATERIALS AND METHODS:
2.1. Chemicals and reagents:
Dabigatran etexilate standard and its measure impurities standards (ImpA: Ethyl 3-(2-((4-carbamimidoylphenylamino)methyl)-1-methyl-N-(pyridin-2-yl)-1H-benzo[d] imidazole-5-carboxamido) propanoate, Imp B: 3-[[[2-[[(4-Cyanophenyl)amino]methyl]-1-methyl-1H-benzimidazol-5-yl]carbonyl]pyridin-2-ylamino]propionic acid ethyl ester, Imp C: Ethyl-3-(1-{2-[({4-[amino ({[(methoxy)carbonyl]imino})methyl]phenyl} amino)methyl]-1-methyl-1H-1, 3-benzodiazol-5-yl}-N-(pyridin-2-yl)formamido) propanoate.) were purchased from Megafine Pharma (P) LTD. APIs were obtained from Megafine Pharma (P) Ltd and from Shandong Rongyuan Pharmaceuical Co, Ltd. HPLC acetonitrile, methanol, hydrochloric acid, sodium hydroxide, hydrogen peroxide, dimethylsulphoxide-d6 (DMSO-d6) and Formic acid were purchased from SIGMA-ALDRICH®. HPLC water was obtained by Siemens Water Technologies LaboStar. Pradaxa® capsules were purchased from Boehringer Ingelheim Pharma GmbH & Co.KGIngelheim am Rhein Germany.
2.2. Instruments:
The chromatographic analysis was performed with SHIMADZU LC prominence system (Shimadzu, Japan) provided with UV-Vis Detector SPD 20A, MS Detector 2020, two pumps A & B: LC/20AD, column oven CTO-20A, manual injector and with Shim-pack XR- ODS II (100 x 3.0 mm, 2.2µm particle size), System control and data analysis were carried out using LabSolutions CS (Schimadzu, Japan). KNAUER HPLC Smartline system with PDA detector (Germany). Jasco PU-2080 plus Semipreparative-HPLC (Jasco, Japan). Sartorius sensitive analytical balance (sensitivity of 10-4g). JEKEN Digital Ultrasonic Cleaner. Bechers, Volumetric flasks, Micropipettes and Glassware of different volumes from Marienfeld Company. Filters PVDF 0.45µm for HPLC purchased from TEKNOKROMA. The photostability study was carried out in a photostability chamber (Sanyo, Leicestershire, UK). The thermal stability study was carried out in a dry air oven (Memmert, Germany). NMR BRUKER 400 MHz Ultra shield TM instrument. FTIR Nicolet 6700 with the Detector DTGS Operating software (OMNIC version 7.3 Thermo Nicolet USA).
2.3. Chromatographic conditions:
The chromatographic separation was performed on Shim-pack XR- ODS II (100 x 3.0 mm, 2.2µm particle size) at a column temperature of 30°C, The mobile phase A was water with 0.1% of formic acid, while the mobile phase B was acetonitrile, the gradient program of the mobile phase was set as [Time(min)/ Pump B Value(%)] [0.01/15, 17/60, 25/80 and 30/15], The mobile phase was filtered using 0.45 µm disposable filter, and degassed by ultrasonic vibration prior to use. The flow rate was 0.3 ml/min. The injection volume was 10 µL and the detection was carried out at 225nm. Water, acetonitrile and methanol 50:40:10 (v/v) was used as a diluent. The analysis was performed in positive electro-spray/positive ionization mode ESI+, the ion source voltage was 5000 V, the source temperature was 450 °C, and the curtain gas flow was 15 psi. This LC-MS method was successfully developed and validated as per ICH guidelines [28] and according to USP 35 guideline recommendations[27].
2.4. Semi-preparative HPLC conditions:
A Lichrospher-RP-18 (250 x 3 mm, 5µm particle size) column with a mobile phase consisting of water and acetonitrile, the gradient program of the mobile phase was set as [Time(min)/ Pump B acetonitrile Value(%)] [0.01/70, 10/65, 20/20 and 30/70] at a flow rate of 1.1 ml /min, and the detection was carried out at 225nm.
2.5. NMR H1 & C13 spectroscopy:
H1 and C13 NMR spectra were recorded in DMSO-d6 at 25 °C. The NMR chemical shift values were reported on the δ scale in ppm, relative to TMS (δ = 0.00) as internal standard.
2.6. FT-IR spectroscopy:
The IR spectra for Dabigatran etexilate, and degradation products were recorded in the solid state as KBr dispersion, with Range 400-4000nm, the resolution was 4cm-1, scans were 32.
2.7. Degradation protocols:
The stress conditions employed for degradation studies as per ICH recommendation include photolytic, thermal, oxidation, and hydrolysis with acid and base. Degradation samples were prepared by diluting Dabigatran etexilate stock standard solution (2 mg/ml) in diluents to obtain a final concentration of (1mg/ml). The photolytic stress study was performed for 48 h at 200 W h/ m2 of UV light and 1.2 million lux hours of visible light. The thermal stress study was performed at 105°C for 7 days. The acid, base stress studies were performed with 0.1 N HCI for 12h and 0.1 N NaOH for 2 h at room temperature. The oxidation stress was done with 3% H2O2 solution over a period of 72 h at room temperature. All of the stressed samples were quantified against the Dabigatran etexilate reference standard. Each experiment was performed in triplicate and the working solutions were allocated in 10 ml hermetically sealed glass vials.
3. RESULTS:
3.1. Method development and validation
The main target of the chromatographic method is to achieve the separation of impurities and degradation products from Dabigatran etexilate in APIs and after stress conditions employed for degradation studies, respectively. The described LC-MS method was successfully developed and validated as per ICH and USP 35 guidelines. It was suitable for the separation of Dabigatran etexilate from most impurities and from the other degradation products and the retention time for Dabigatran etexilate was 8.984 min Figure 2, the impurities in APIs and the degradation products produced in the forced degradation were well-separated (Resolution > 2.0) from Dabigatran etexilate, the tailing factor for Dabigatran etexilate was 1.115, and the theoretical plates was 22909. The method was proved to be linear over the calibration range 200–1000 µg/ ml and the correlation coefficient was > 0.9998, and its accuracy, precision, repeatability and robustness were checked and the recovery were 103.25%, 99.80%, 103.90% and 101.65%respectively while the RSD was 0.267. The Detection limits (DL) and Quantification limits (QL) were 0.002 and 0.006 μg/ml respectively.
3.2. The impurity profile of Dabigatran etexilate:
Two samples of Dabigatran etexilate API from different sources, one Dabigatran etexilate working standard and Pradaxa® capsules were analyzed according to this new chromatographic method, the concentrations were calculated from the peak areas, the impurities were successfully separated from Dabigatran etexilate, and the results were shown in Figure 3 and Table 1.
Figure 2 Chromatogram of Standard mixture solution of Dabigatran etexilate and Impurities A, B and C.
Sample 1
Sample 2
Working Standard
Pradaxa® capsules
Figure 3 Chromatograms of Dabigatran etexilate and its impurities in samples of API from different sources, in one working standard and in Pradaxa® capsules.
3.3. Degradation of Dabigatran etexilate along with formation of Degradation Products:
The forced degradation was carried on Dabigatran etexilate, a significant degradation of Dabigatran etexilate was observed in thermolysis, photolysis, oxidation, acidic and the largest degradation was appeared in basic hydrolytic stress conditions. In total, eleven DPs were observed, six DPs were detected in the solutions subjected to acidic hydrolysis, seven DPs in the solutions subjected to base hydrolysis, three DPs appeared in each of thermal and oxidation conditions and one DPs appeared in photolysis, as it is shown in Figure 4 and Table 2. The degradation products are named “DPn”, where n accounts for the elution order.
Acidic Hydrolysis
Basic Hydrolysis
Oxidation
Thermal degradation
Photolytic degradation
Figure 4 Chromatograms of Degradation Products of Dabigatran etexilate.
Table 2: Summary of forced degradation studies of Dabigatran etexilate
|
Stress condition |
Time |
Assay of Dabigatran etexilate (% w/w) |
Total degradants (% w/w) |
Mass balance |
Commentaries |
|
Dabigatran etexilate before degradation |
- |
99.82 |
0.09 |
99.91 |
- |
|
Acid hydrolysis (0.1 N HCl) |
12 hours |
80.62 |
19.05 |
99.67 |
Degradation accompanied by appearance of DP2, DP3, DP6, DP7, DP9 and DP11 |
|
Base hydrolysis (0.1 N NaOH) |
2 hours |
3.21 |
96.24 |
99.45 |
Degradation accompanied by appearance of DP1, DP2, DP3, DP5, DP6, DP9 and DP10 |
|
Oxidation (3% H2O2) |
72 hours |
79.89 |
19.08 |
98.97 |
Degradation accompanied by appearance of DP2, DP6 and DP11 |
|
Thermal (105° C) |
7 days |
95.87 |
3.41 |
99.28 |
Degradation accompanied by appearance of DP4, DP6 and DP8 |
|
Photolysis (UV light) |
48 hours |
85.23 |
14.55 |
99.78 |
Degradation accompanied by appearance of DP6 |
Mass balance = assay %+ sum of all degradants%.
Mass balance (% assay + % total degradation products) of all the stressed samples of Dabigatran etexilate was obtained in the range of 98.97–99.78%. As shown in Table 2, loss of 19% of Dabigatran etexilate was observed after 12 hours in acidic conditions, while 96% was highlighted after 2 hours in basic conditions, which showed a strong impact on Dabigatran etexilates stability. Also the loss of Dabigatran etexilate achieved 19% after 72 hours of oxidation, 3% after 7 days in thermal conditions and 15% after 48 hours of photo degradation. The identification of Dabigatran etexilate and its degradation products was confirmed by ESI-MS using scan mode from 50-1000 mu to produce spectra of molecular weight as shown in Table 3.
Most possible structures were proposed for DPs by comparing their fragmentation patterns with that of Dabigatran etexilate.
DP4, Dp6, Dp7, DP9, DP10 and DP11 were isolated by semi-preparative HPLC and subjected to NMR and FT-IR studies for structure elucidation using conditions as mentioned in Materials & Methods section. Whereas structural elucidation of DPs has been accomplished from NMR, FT-IR and mass spectral data.
FT-IR spectra of DP4, DP6, DP7, DP9, DP10 & DP11 are shown in Figure 5.
Table 3: [M+H]+m/z of Dabigatran etexilate and its Degradation Products.
|
Stress condition |
Peak name |
Retention time RT(min) |
[M+H]+m/z |
|
Acid hydrolysis |
DP2 |
3.42 |
500 |
|
DP3 |
3.99 |
558 |
|
|
DP6 |
7.01 |
600 |
|
|
DP7 |
8.40 |
614 |
|
|
Dabigatran etexilate |
8.99 |
628 |
|
|
DP9 |
11.59 |
601 |
|
|
DP11 |
14.62 |
629 |
|
|
Base hydrolysis |
DP1 |
2.74 |
472 |
|
DP2 |
3.48 |
500 |
|
|
DP3 |
4.08 |
558 |
|
|
DP5 |
6.18 |
599 |
|
|
DP6 |
7.00 |
600 |
|
|
Dabigatran etexilate |
8.99 |
628 |
|
|
DP9 |
11.69 |
601 |
|
|
DP10 |
12.48 |
517 |
|
|
Oxidation |
DP2 |
3.43 |
500 |
|
DP6 |
7.02 |
600 |
|
|
Dabigatran etexilate |
9.01 |
628 |
|
|
DP11 |
14.58 |
629 |
|
|
Thermal degradation |
DP4 |
5.54 |
501 |
|
DP6 |
6.98 |
600 |
|
|
Dabigatran etexilate |
8.92 |
628 |
|
|
DP8 |
11.02 |
551 |
|
|
Photo degradation |
DP6 |
7.08 |
600 |
|
Dabigatran etexilate |
9.07 |
628 |
Dabigatran etexilate
DP4
DP6
DP7
DP9
DP10
DP11
Figure 5 FT-IR spectra of Degradation Products
Proton H1& carbon C13, NMR spectra of Degradation Products are shown in Figure 6.
H1 NMR of Dabigatran etexilate
C13 NMR of Dabigatran etexilate
H1 NMR of DP4
C13 NMR of DP4
H1 NMR of DP6
C13 NMR of DP6
H1 NMR of DP7
C13 NMR of DP7
H1 NMR of DP9
C13 NMR of DP9
H1 NMR of DP10
C13 NMR of DP10
H1 NMR of DP11
C13 NMR of DP11
Figure 6 NMR H1&C13 spectra of Degradation Products
4. DISCUSSION:
This rapid, simple, economic, precise, sensitive and accurate LC-MS method enables the separation of 22 process impurities with resolution >1.5, three of them are known and the 19 are unknown they were present in the Dabigatran etexilate APIs and in the Pradaxa® the brand, these impurities were detected and identified by ESI-MS. Eleven impurities were characterized in sample1, fourteen impurities in sample2, fifteen impurities in working standard and sixteen impurities were detected in Pradaxa® capsules. All the detected impurities, except impurity D, were acceptable according to USP35 and ICH guidelines none of them exceeded 0.10% the identification threshold, so it's not necessary to characterize any structure of the impurities or identify them. But Imp D which has 629.25 [M+H]+m/z exceeded 0.10% the identification threshold, so it was necessary to isolate it by using Semi-preparative HPLC
and characterize its structure according to MS, NMR H1, C13 and FTIR data.
Under influence of various stress conditions as per ICH guidelines, Dabigatran etexilate was susceptible to acid and base hydrolysis condition also to the oxidation, light and high temperature, it degraded under stress conditions giving rise to a new degradation products. The eleven degradation products were successfully characterized by use of mass spectrometry, six of them were isolated by semi-preparative HPLC and subjected to NMR and FT-IR studies, and the structural elucidation of DPs has been accomplished from NMR, FT-IR and mass spectral data.
By comparing the impurity profile of Dabigatran etexilate and its degradation products, the similarity in RT and [M+H]+ m/z between impurities and degradation products was observed and the impurities ImpA, ImpC, ImpF, ImpG, ImpH, ImpI, ImpM, ImpN and ImpD are degradation products DP2, DP3, DP4, DP5, DP6, DP7, DP8, DP9 and DP11, respectively. The NMR and FTIR data resulting from analyzing Imp D and DP11 support that.
DP1 appears at retention time 2.74min, in basic hydrolysis, with [M+H]+ 472m/z, unfortunately it couldn't be separated in sufficient amount to continue NMR & FT-IR studies, but its molecular weight suggest that it could be Dabigatran with molecular formula C25H25N7O3.
DP2 which appears in acidic and basic hydrolysis, also after oxidation, has the same retention time 3.42min, and the same [M+H]+ 500m/z as Imp A which is known and its standard is available. Their molecular formula is C27H29N7O3.
The same situation for Imp C and DP3 which appears in acidic and basic hydrolysis, they have the same retention time 4.08min, and the same [M+H]+ 558m/z. Their molecular formula is C29H31N7O5.
DP4 which appears in thermal degradation and Imp F have the same retention time 5.54min, and the same [M+H]+ 501m/z. Compared to that of Dabigatran etexilate, the NMR spectra of DP4 shows a lose in protons (13 protons), and carbons (7carbons), In addition the FT-IR spectrum shows a decrease in alkane (CH2-CH3) band at 2908 cm−1, also in C=O band at 1736 cm−1, and in NH2 band at 1648 cm−1, and C-N band at 1203 cm−1, while the O-H broad band appears at 3220 cm−1. According to that it was clear that Dabigatran etexilate has lost his hexyloxy carbonyl and NH2 has been substituted by OH, and the molecular formula of DP4 could be C27H29N6O4. .
Figure 7 Proposed degradation pathways of Dabigatran etexilate by acid hydrolysis
DP5 which appears in basic hydrolysis and Imp G both have the same retention time 6.18min, and the same [M+H]+ 599m/z, unfortunately it couldn't be separated in sufficient amount to continue NMR & FT-IR studies.
DP6 was the degradation product with the most frequent appearance, it appears in all stress conditions, it has the same retention time 7.01min, and the same [M+H]+ 600m/z as Imp H. Compared to that of Dabigatran etexilate, the NMR spectra of DP6 shows a loose in protons (4protons), and carbons (2carbons), In addition the FT-IR spectrum shows a decrease in alkane (CH2-CH3) band at 2837 cm−1, and the O-H broad band appears at 3299 cm−1. It was clear that the ethyl ester in Dabigatran etexilate has hydrolyzed and the molecular formula of DP6 could be C32H37N7O5.
DP7 which appears only in acidic hydrolysis and Imp I both have the same retention time 8.40min, and the same [M+H]+ 614m/z. It differ from Dabigatran etexilate only by losing two protons and one carbon according to their NMR spectrum, and the decreasing in alkane band in FT-IR spectrum at 2913 cm−1. Which means that DP7 derived from Dabigatran etexilate by losing methylene. It's molecular formula could be C33H39N7O5.
DP8 appears only in thermal degradation and it has the same retention time 11.02min, and the same [M+H]+ 551m/z as Imp M. Also it couldn't be separated in sufficient amount to continue NMR & FT-IR studies.
DP9 and Imp N both have the same retention time 11.59min, and the same [M+H]+ 601m/z. Dp9 appears in acidic and basic hydrolysis. And after comparing its NMR, MS and FT-IR spectrum with that of Dabigatran etexilate it was clear that there were a decrease in two carbons and five protons, also FT-IR spectrum shows bigger C=O stretching absorptions at 1737cm−1, C-O-H stretching absorptions at 1220cm−1 and O-H board stretching absorptions at 3358cm−1, also there were a decrease in NH2 stretching absorptions after 3358cm−1 and an increase in N-H band at 1517cm−1. Which means that the ethyl ester in Dabigatran etexilate was hydrolyzed, the NH2 has been substituted by O, and the C=N was converted to C-NH. It's molecular formula could be C32H36N6O6
Figure 8 Proposed degradation pathways of Dabigatran etexilate by base hydrolysis
Figure 9 Proposed degradation pathways of Dabigatran etexilate by Thermal and Photo degradation
Figure 10 Proposed degradation pathways of Dabigatran etexilate by Oxidation
DP10 which appears only in basic hydrolysis, has retention time 12.48min, and the [M+H]+ 517m/z. Compared to that of Dabigatran etexilate, the NMR spectra of DP10 shows a lose in protons (19protons), and carbons (9carbons). In addition the FT-IR spectrum shows a decrease in alkane (CH2-CH3) band at 2903 cm−1, also in C=O band at 1736 cm−1, and in NH2 band at 1648 cm−1, and C-N band at 1203 cm−1. A disappear of OH bands which was obviously related to substitute H by Na in basic medium lead to suggest a molecular formula C25H22Na2N6O4.
DP11 and Imp D both have the same retention time 14.58min, and the same [M+H]+ 629m/z and both were isolated by Semi-preparative HPLC and characterized according to MS, NMR H1, C13 and FT-IR data. By comparing the previous data with that belong to Dabigatran etexilate, it was clear that DP11 has the same carbons and one proton less than Dabigatran etexilate, the FT-IR shows an increase in N-H band at 1609 cm−1and in C=O band at 1731 cm−1, that lead to suggest a molecular formula C34H40N6O6. According to that, it was clear that C-NH2 bond in Dabigatran etexilate is very sensitive to oxidation and it converts easily to C=O, and for this reason Imp D was the most available Impurity in all APIs and Pradaxa® and in quantities exceeded the acceptable limits according to ICH.
The above results allowed to propose degradation pathways of Dabigatran etexilate, and the schematic representations of mechanism of formation of the degradation products under stress conditions are shown in Figures 7, 8, 9 and 10.
5. CONCLUSION:
Dabigatran etexilate is a sensitive drug susceptible to all environmental conditions. Impurity profile and forced degradation behavior of Dabigatran etexilate was studied as per ICH prescribed guidelines. Nine of the detected impurities were degradation products which confirms the close relationship between drug stability studies and the estimation of Impurity profiles, the data from such studies enables storage conditions, re-test periods and shelf lives to be established. A simple, rapid and selective stability indicating LC method has been developed and validated for the determination of Dabigatran etexilate and its impurities and degradation products. The developed method was found to be suitable for the drug quantification as well as for the impurity determination and stability indication.
6. ACKNOWLEDGEMENT:
The authors are thankful to the colleagues at the central laboratory in the faculty of science and the colleagues at the Atomic Energy Commission for their cooperation in carrying out this work.
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Received on 02.10.2017 Modified on 09.11.2017
Accepted on 18.12.2017 © RJPT All right reserved
Research J. Pharm. and Tech. 2018; 11(3): 1119-1130.
DOI: 10.5958/0974-360X.2018.00210.X