FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
1 Title of the invention - AN IMPROVED METHOD FOR THE QUANTITATIVE
DETERMINATION OF DABIGATRAN ETEXILATE MESYLATE
2. Applicant(s)
(a) NAME : ALEMBIC PHARMACEUTICALS LIMITED
(b) NATIONALITY: An Indian Company.
(c) ADDRESS: Alembic Campus, Alembic Road,
Vadodara-390, 003, Gujarat, India
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which is to be
performed:

FIELD OF THE INVENTION
The present invention relates to an improved reversed-phase liquid chromatographic (RP-LC) method for the quantitative determination of Dabigatran Etexilate mesylate.
The present invention further provides a stability indicating analytical method using the samples generated from forced degradation studies.
BACKGROUND OF THE INVENTION
Dabigatran Etexilate mesylate is chemically known as l-Methyl-2-[N-[4-(N-n-hexyloxycarbonylamidino)phenyl]- aminomethyl] benzimidazol-5-yl- carboxylic acid-N-(2-pyridyl)-N-(2-ethoxycarbonylethyl)-amide methane sulfonate. Dabigatran Etexilate mesylate is a direct thrombin inhibitor.

The product mixture of a reaction rarely is a single compound pure enough to comply with pharmaceutical standards. Side products and byproducts of the reaction and adjunct reagents used in the reaction will, in most cases, be present. At certain stages during processing of the Dabigatran Etexilate mesylate contained in the product mixture into an active pharmaceutical ingredient ("API"), the Dabigatran Etexilate mesylate must be analyzed for purity, typically by UPLC, HPLC or GC analysis, to determine if it is suitable for continued processing or ultimately for use in a pharmaceutical product.

The U.S. Food and Drug Administration's Center for Drug Evaluation and Research (CDER) has promulgated guidelines recommending that drug applicants identify organic impurities of 0.1% or greater in the active ingredient. "Guideline on Impurities in New Drug Substances," 61 Fed. Reg. 371 (1996); "Guidance for Industry ANDAs: Impurities in Drug Substances," 64 Fed. Reg. 67917 (1999). Unless an impurity has been tested for safety, is in a composition proven to be safe in clinical trials, or is a human metabolite, the CDER further recommends that the drug applicant reduce the amount of the impurity in the active ingredient to below 0.1%. In order to obtain marketing approval for a new drug product, manufacturers must submit to the regulatory authority evidence that the product is acceptable for administration to humans. Such a submission must include, among other things, analytical data showing the impurity profile of the product to demonstrate that the impurities are either absent, or present in a negligible amount. Therefore, there is a need for analytical methods to detect impurities to identify and assay those impurities.
Generally, impurities (side products, byproducts, and adjunct reagents) are identified spectroscopically and by other physical methods and then the impurities are associated with a peak position in a chromatogram (or a spot on a TLC plate). Thereafter, the impurity can be identified by its position in the chromatogram, which is conventionally measured in minutes between injection of the sample on the column and elution of the particular component through the detector, known as the "retention time" ("Rt"). This time period varies daily based upon the condition of the instrumentation and many other factors. To mitigate the effect that such variations have upon accurate identification of an impurity, practitioners use "relative retention time" ("RRt") to identify impurities.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a reversed-phase liquid chromatographic (RP-LC) method for the quantitative determination of Dabigatran Etexilate mesylate.

In another aspect, the present invention provides an HPLC method for Dabigatran Etexilate mesylate containing less than about 5% area by HPLC, preferably less than about 3% area by HPLC, more preferably less than 1% area by HPLC, of total impurities. In another aspect, the present invention further provides a stability indicating analytical method using the samples generated from forced Degradation studies. In yet another aspect, the present invention provides a simple, accurate and well-defined stability indicating and high performance liquid chromatography (HPLC) method for the determination of Dabigatran Etexilate mesylate in the presence of degradation products.
In one aspect, the HPLC method described in the present invention has the following advantages for determining the Dabigatran Etexilate mesylate and its related impurities:
i) All the impurities were well separated with a minimum resolution 2.58.
ii) Gradient profile to elute all related impurities and organic phase is 60% which
ensure the elution and detection of non polar impurities forming during the
process or stress study;
iii) The present method mobile phase pH is about 5.8 which is more stable in all
C18 HPLC columns;
iv) Consistency in specificity, precision & reproducibility with good peak shape;
and
v) The degradation impurities from stress studies are well separated from the
known impurities.
BRIEF DESCRIPTION OF DRAWINGS
Fig. 1 illustrates the HPLC chromatogram of spiked (impurity-I, impurity-II, impurity-III, impurity-IV, impurity-V, impurity-VI and impurity-VII spiked in Dabigatran Etexilate mesylate) sample.
Fig. 2 illustrates the spiked solution (Dabigatran etexilate mesylate + 0.15% of each Known impurity spiked solution) @ 230 nm.

DETAILED DESCRIPTION OF THE INVENTION
As used herein, "limit of detection (LOD)" refers to the lowest concentration of analyte that can be clearly detected above the base line signal, is estimated is three times the signal to noise ratio.
As used herein, "limit of quantization (LOQ)" refers to the lowest concentration of analyte that can be quantified with suitable precision and accuracy, is estimated as ten times the signal to noise ratio.
As used herein, "gradient elution" refers to the change in the composition of the gradient eluent over a fixed period of time, stepwise or at a constant rate of change, as the percentage of the first eluent is decreased while the percentage of the second eluent is increased.
As used herein, "gradient eluent" refers to an eluent composed of varying concentrations of first and second eluent.
The seven main known impurities of Dabigatran Etexilate mesylate are:
(i) 3-([2-[[(4-(N-n-hexyIoxycarbonyl carbamoyl)-phenylamino]-methyl]-l-methyl-lH-benzimidazole-5-carbonyl]-pyridin-2-yl-amino)ethyl propionate (Impurity-I) which has the following structure:


The impurity-I is detected and resolved from Dabigatran Etexilate mesylate by HPLC with a relative retention time (hereafter referred as RRT) of 0.81.
(ii) 3-([2-[[ (4-(N-2-ethylbutyloxycarbonyl carbamimidoyl) -phenylamino] -methyl] -1-methyl -lH-benzimidazole-5-carbonyl]-pyridin-2-yl-amino)ethyl propionate (Impurity-II), which has the following structure:

Impurity-II is detected and resolved from Dabigatran Etexilate mesylate by HPLC with a RRT of 0.92.
(iii) Ethyl 2-{[(4-[N-n-hexyloxycarbonylcarbamimidoyl)phenyI)amino]methyl}-l-methyl -lH-benzimidazole-5-carboxylate (Impurity-III), which has the following structure:

Impurity-III is detected and resolved from Dabigatran Etexilate mesylate by HPLC with a RRT of 1.23.

(iv) l-Methyl-2- [N-[4- (N-n-hexyloxycarbonylamidino)phenyl] -amino-methyl] benzimidazol -5-yl-carboxylic acid-N-(2-pyridyl)-N-(2-propoxycarbonylethyl)-amide (Impurity-IV), which has the following structure:

Impurity-IV is detected and resolved from Dabigatran Etexilate mesylate by HPLC with an RRTof 1.29.
(v) l-Methyl-2-[N-[4-(N-n-hexyloxycarbonylamidino)phenyl]-amino-methyl]
benzimidazol -5-yl-carboxylic acid-N-(2-pyridyl) -N-(2-butoxycarbonylethyl) -amide (Impurity-V), which has the following structure:

Impurity-V is detected and resolved from Dabigatran Etexilate mesylate by HPLC with a RRT of 1.40.
(vi) 1 -Methyl-2-[N-(4-amidinophenyl)-aminomethyl]benzimidazol-5-yl-carboxylic acid-N-(2-pyridyl)-N-(2-ethoxycarbonylethyl)-amide hydrochloride (Impurity-VI) , which has the following structure:

Impurity-VI is detected and resolved from Dabigatran Etexilate mesylate by HPLC with
aRRTof0.18.
(vii)_3-([2-[[(4-(N-n-hexyIoxycarbonyI carbamimidoyl)-phenyIamino]-methyI]-I-methyI-
lH-benzimidazole-5-carbonyl]-pyridin-2-yl-amino)propionic acid (Impurity-VII) which
has the following structure:

Impurity-VII is detected and resolved from Dabigatran Etexilate mesylate by HPLC with aRRTof0.14.
According to one aspect of the present invention, there is provided a reversed-phase liquid chromatographic (RP-LC) method for quantifying, by area percent, the amounts of Dabigatran Etexilate mesylate and all impurities, preferably, impurity-I, impurity-II, impurity-III, impurity-IV, impurity-V, impurity-VI and impurity-VII present in a sample of Dabigatran Etexilate mesylate.
According to another aspect of the present invention, there is provided a stability indicating analytical method using the samples generated from forced degradation studies.
According to another aspect of the present invention, there is provided an accurate and well-defined stability indicating HPLC method for the determination of Dabigatran Etexilate mesylate in the presence of degradation products.

Preferably, the method for determining the amount of impurities in a Dabigatran Etexilate mesylate sample comprises the steps of:
a) Combining a Dabigatran Etexilate mesylate sample with buffer and acetonitrile in the ratio of about 60:40 (v/v) to obtain a solution;
b) injecting the sample solution into a 150 mm x 4.6 mm, column with 3.5μm μm ZORBAX SB-Phenyl column;
c) gradient eluting the sample with 100% of eluent-A initial
d) Eluent A comprises of Buffer : Acetonitrile :: 60:40(v/v).
e) Eluent B comprises of Buffer : Acetonitrile :: 40:60(v/v)
f) Measuring of the amounts of Dabigatran and each impurity at 230 nm wavelength with a UV detector (having an appropriate recording device).
Preferably, the initial ratio of eluent A in step-(c) may be continued at the same ratio for 22 minutes then changed linearly to 100% eluent-B within 8 minutes followed by same ratio for 5 minutes. Again changed linearly to initial ratio within 3 minutes followed by same ratio for 7 minutes. The column temperature may be maintained at about 30°C.
The LOD /LOQ values of Dabigatran Etexilate mesylate and its related impurities, impurity-I, impurity-II, impurity-III, impurity-IV, impurity-V. impurity-VI and impurity-VII are summarized in Table 1.
Table 1

S.No Components LOD (%) LOQ (%)
1 Impurity-VII 0.0035 0.0107
2 Impurity-VI 0.0012 0.0037
3 Impurity-I 0.0029 0.0088
4 Impurity-II 0.0035 0.0105
5 Impurity-III 0.0045 0.0136
6 Impurity-IV 0.0036 0.0109
7 Impurity-V 0.0039 0.0119

1 I Dabigatran I 0.0032 I 0.0098
Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities and degradation products. The specificity of the LC method for Dabigatran Etexilate mesylate , Intentional degradation was attempted to stress conditions of acid hydrolysis (using 1M HC1), base hydrolysis (using 1M NaOH), and oxidative degradation (using 3.0% H2O2), to evaluate the ability of the proposed method to separate Dabigatran Etexilate mesylate from its degradation products. To check and ensure the homogeneity and purity of Dabigatran peak in the stressed sample solutions, PDA-UV detector was employed.
Preferably, the limit of detection (LOD) and limit of quantification (LOQ) were estimated by signal to noise ratio method, by injecting a diluted solution with known concentration. According to another aspect of the present invention, there is provided a chromatographic method to get the separation of all impurities and stress studies degradants from analyte peak. Satisfactory chromatographic separation was achieved using the mobile phase consists of buffer (Dissolve about 4.14 g sodium dihydrogen phosphate monohydrate in 1000 mL of water. Adjust pH to 5.80 + 0.05 with sodium hydroxide. Filter it through 0.45 u membrane filter and degas.(PALL, Ultipor N Nylon 6,6 membrane filter) and acetonitrile. In the optimized conditions, the Dabigatran Etexilate mesylate , impurity-I, impurity-II, impurity-III, impurity-IV, impurity-V, impurity-VI and impurity-VII were well separated with a resolution of 2.58 and the typical retention times (RT) of Dabigatran Etexilate mesylate, impurity-I, impurity-II, impurity-III, impurity-IV, impurity-V, impurity-VI and impurity-VII were about 21.98, 17.71, 20.17, 27.12, 28.29, 30.75, 3.92 and 3.16 minutes respectively, and typically shown in Figure 1. The system suitability results and the developed LC method was found to be specific for Dabigatran etexilate mesylate and its seven impurities, namely impurity-I, impurity-II, impurity-III, impurity-IV, impurity-V, impurity-VI and impurity-VII.
The system suitability values of Dabigatran Etexilate mesylate and its impurities were summarized in Table 2.

Table 2

Compound (n=l) Rt Rs N T m/z [M +H]
Impurity-VII 3.16 4501 1.20 600.7
Impurity-VI 3.92 3.99 7964 1.18 500.6
Impurity-I 17.71 37.69 17194 1.00 629.7
Impurity-11 20.17 4.15 16582 1.02 628.7
Dabigatran 21.98 2.58 13809 1.53 628.6
Impurity-III 27.12 8.96 83180 0.97 480.5
Impurity-IV 28.29 3.52 161986 1.00 642.6
Impurity-V 30.75 9.97 375295 0.99 656.6
*n=l: determination, Rt: retention time, Rs: USP resolution, N; number of theoretical plates (USP tangent method), T: USP tailing factor, m/z: mass number.
High level of degradation in test solution was observed using 3% hydrogen peroxide at RT for 2 hours, 1M sodium hydroxide initial and 1M HC1 at RT for 3.5 hours. Impurities observed in stress condition using PDA detector,. Major degradants were impurtity-VI and impurity-VII .Other unknown were also specific in this method .The peak test results obtained from PDA confirm that the Dabigatran peak is homogeneous and pure in all analyzed stress samples.
Experimental
The LC system, used for method development and forced degradation studies and method validation was Waters-Alliance (manufactured by Waters India Ltd) LC system with a photo diode detector. The out put signal was monitored and processed using Empower software system (designed by Waters India) on IBM computer (Digital Equipment Co). The chromatographic column used was a ZORBAX SB-Phenyl (150 mm x 4.6 mm), column with 3.5 urn particles. The mobile phase consists buffer (4.14 g sodium dihydrogen phosphate monohydrate in 1000 mL of water. Adjust pH to 5.80 + 0.05 with sodium hydroxide. Filter it through 0.45 u membrane filter and degas.(PALL5 Ultipor N

Nylon 6,6 membrane filter).), and solvent is acetonitrile. Eluent-A is Buffer : Acetonitrile :: 60:40(v/v) and Eluent-A is Buffer : Acetonitrile :: 40:60(v/v).
The flow rate of the mobile phase was kept at 1 .OmI/min. Beginning with the gradient ratio of 100% Eluent-A, system was continued at the same ratio for 22 minutes. The ratio was changed linearly 100% Eluent-B within 8 minutes and again system was continued at the same ratio for 5 minutes. The ratio was again changed linearly 100% Eluent-A within 3 minutes and again system was continued at the same ratio for 7 minutes. The column temperature was maintained at 30°C and the wavelength was monitored at a wavelength of 230 nm. The injection volume was 10 uL for related substances determination. Eluent B was used as diluent during the standard and test samples preparation.
Preparation of system suitability stock solution preparation.
10.5 mg each of impurity II standard was accurately weighed and transferred to the lOOmL volumetric flask(BOROSIL-Class-A), separately; 10ml of acetonitrile was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with diluent. Mix well and transfer 5 mL of this solution into a 10 mL volumetric flask and make up the volume with diluent.
Preparation of system suitability solution preparation.
35 mg of Dabigatran working standard was accurately weighed and transferred to the 50mL volumetric flask(BOROSIL-Class-A) , separately; add 1 mL system suitability stock solution was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with diluent.
Preparation of reference solution;
35 mg of Dabigatran etexilate mesylate working standard, weighed and transferred to the 50 mL volumetric flask(BOROSIL-Class-A) , separately; 30 mL diluent was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with diluent. Pipette out 1.0 mL from solution and transferred in to a lOOmL volumetric flask (BOROSIL-Class-A), and made up to mark with diluent. Further take ImL of this

solution into 10 mL volumetric flask(BOROSIL-Class-A) and make up with diluent. A working solution of 700μg/ml was prepared for related substances determination analysis.

We Claim,
1. A HPLC method for analyzing Dabigatran Etexilate mesylate, wherein the mobile phase comprises two or more liquids, including a first eluent A and a second eluent B, and the relative concentration of the liquids is varied to a predetermined gradient.
2. A HPLC method according to claim 1, wherein the first eluent A is Buffer : Acetonitrile :: 60:40(v/v).
3. A HPLC method according to claim 1, wherein the first eluent B is Buffer : Acetonitrile :: 40:60(v/v).
4. A HPLC method according to claim 1, wherein gradient of A eluent and B eluent in the ratio of 100% of eluent-A initial and continued at the same ratio for 22 minutes then changed linearly to 100% eluent-B within 8 minutes followed by same ratio for 5 minutes. Again changed linearly to initial ratio within 3 minutes followed by same ratio for 7 minutes.
5. A HPLC method according to claim 1, wherein UV detector is set to 230nm wavelength.
6. A HPLC method according to claim 1, wherein column is ZORBAX SB-Phenyl column 150 mm x 4.6 mm, 3.5μm.
7. A HPLC method for Dabigatran Etexilate mesylate containing less than about 5% area by HPLC, preferably less than about 3% area by HPLC, more preferably less than 1% area by HPLC, of total impurities.
8. A HPLC method determining the amount of impurities in Dabigatran Etexilate mesylate sample comprises the steps of:

i) Combining a Dabigatran Etexilate mesylate sample with buffer and acetonitrile in the ratio of about 60:40 (v/v) to obtain a solution;
ii) injecting the sample solution into a 150 mm x 4.6 mm, column with 3.5um Hin ZORBAX SB-Phenyl column;
iii) gradient eluting the sample with 100% of eluent-A initial and continued at the same ratio for 22 minutes then changed linearly to 100% eluent-B within 8 minutes followed by same ratio for 5 minutes. Again changed linearly to initial ratio within 3 minutes followed by same ratio for 7 minutes.
iv) Eluent A comprises of Buffer : Acetonitrile :: 60:40(v/v).
v) Eluent B comprises of Buffer : Acetonitrile :: 40:60(v/v)
vi) Measuring of the amounts of Dabigatran and each impurity at 230 nm wavelength with a UV detector (having an appropriate recording device).
9. A HPLC method according to previous claims, wherein buffer is 4.14 g sodium dihydrogen phosphate monohydrate in 1000 mL of water and adjusts to pH to about 5.80.