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 ß-Alanine  N-[[2-[[[4-[[[(hexyloxy)carbonyl] amino]iminomethyl] phenyl]amino]methyl]-1-methyl-1H-benzimidazol-5-yl]carbonyl]-N-2-pyridinyl- ethyl ester  methanesulfonate.
Dabigatran Etexilate mesylate is a direct thrombin inhibitor.

Structure of Dabigatran etexilate mesylate

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 3.26.
ii) Gradient profile to elute all related impurities and organic phase is 70% 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 (KSM-I  Stage-IIA  Stage-II  Propionate ester  methyl Ester  Amidine impurity  Amide Diester  Diester impurity  Etexilate impurity spiked in Dabigatran Etexilate mesylate) sample.

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 nine main known impurities of Dabigatran Etexilate mesylate are

(i) Ethyl 3-[{[3-amino-4-(methylamino)phenyl]carbonyl}(pyridin-2-yl)amino]propanoate
(KSM-I) which has the following structure


The KSM-I is detected and resolved from Dabigatran Etexilate mesylate by HPLC with a relative retention time (hereafter referred as RRT) of 0.48.

(ii) Ethyl-3-[[[2-[[(4-[imino(ethoxy)methyl]phenyl)amino]methyl]-1-methyl-1H-
benzimidazol-5-yl]carbonyl]pyridine-2-ylamino]propionate  hydrochloride
(Stage-IIA)  which has the following structure

Stage-IIA is detected and resolved from Dabigatran Etexilate mesylate by HPLC with a RRT of 0.77.

(iii) Ethyl-3-[[[2-[[(4-midinophenyl)amino]methyl]-1-methyl-1H-benzimidazol-5-yl]carbonyl]pyridine-2-ylamino] propionate  hydrochloride (Stage-II)  which has the following structure

Stage-II is detected and resolved from Dabigatran Etexilate mesylate by HPLC with a RRT of 0.54 .

(iv) Ethyl 3-(pyridin-2-ylamino) propionate (propionate ester)  which has the following
structure

Propionate ester is detected and resolved from Dabigatran Etexilate mesylate by HPLC with an RRT of 0.35.

(v) Methyl-3-[[[2-[[(4-midinophenyl)amino]methyl]-1-methyl-1H-benzimidazol-5-yl]carbonyl]pyridine-2-ylamino] propionate (Methyl ester)  which has the following structure

Methyl ester is detected and resolved from Dabigatran Etexilate mesylate by HPLC with a RRT of 0.46.

(vi) Ethyl 2-{[(4-carbamimidoylphenyl)amino]methyl}-1-methyl-1H-benzimidazole-5-
carboxylate (amidine impurity)   which has the following structure

Amidine impurity is detected and resolved from Dabigatran Etexilate mesylate by HPLC with a RRT of 0.52.

(vii)Ethyl-3-[[[2-[[(4-(ethoxycarbonyl)phenyl)amino]methyl]-1-methyl-1H-benzimidazol-5-yl]carbonyl]pyridine-2-ylamino]propionate  hydrochloride
(amide Diester impurity) which has the following structure


Amide Diester impurity is detected and resolved from Dabigatran Etexilate mesylate by HPLC with a RRT of 0.85.
(viii) Ethyl 2-({[4-(ethoxycarbonyl)phenyl] amino}methyl)-1-methyl-1H-benzimidazole-5-carboxylate (Diester impurity) which has the following structure

Diester impurity is detected and resolved from Dabigatran Etexilate mesylate by HPLC with a RRT of 0.88.

(ix) Ethyl 2-{[(4-[N-n-hexyloxycarbonylcarbamimidoyl)phenyl)amino]methyl}-1-methyl-1H-benzimidazole-5-carboxylate (Etexilate impurity) which has the following structure

Etexilate impurity is detected and resolved from Dabigatran Etexilate mesylate by HPLC with a RRT of 1.04.

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  KSM-I  Stage-IIA  Stage-II  Propionate ester  Methyl Ester  Amidine impurity  Amide Diester  Diester impurity and Etexilate impurity 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 eluent A and acetonitrile in the ratio of about 20:30 (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 a mixture of buffer and acetonitrile in the ratio of 85:15 (v/v) initial and progressively increased to 30:70(v/v) in 45 minutes .
d) Preparing eluent A by dissolving 4.14 g of Sodium dihydrogen phosphate in 1000 ml of water  dissolve and adjust pH = 5.8 with sodium hydroxide solution. Filter it through 0.45 µ membrane filter and degas.
e) Measuring of the amounts of Dabigatran and each impurity at 225nm wavelength with a UV detector (having an appropriate recording device).
Preferably  the initial ratio of eluent A and acetonitrile in step-(c) may be continued at the same ratio for 5 minutes then changed linearly to 55:45 (v/v) within 35 minutes followed by same ratio for 5 minutes. Again changed linearly to 30:70 (v/v) within 45 minutes followed by same ratio for 5 minutes. After 3 minutes the initial gradient of 85:15 is for 7 minutes to be conditioned for every analysis. The column temperature may be maintained at about 50°C.

The LOD /LOQ values of Dabigatran Etexilate mesylate and its related impurities  KSM-I  Stage-IIA  Stage-II  Propionate ester  Methyl Ester  Amidine impurity  Amide Diester  Diester impurity and Etexilate impurity are summarized in Table 1.
Table 1

S. No Components LOQ (%) LOD (%)
1 KSM-I 0.009 0.003
2 Stage-IIA 0.010 0.003
3 Stage-II 0.007 0.002
4 Propionate ester 0.018 0.006
5 Methyl Ester 0.010 0.003
6 Amidine impurity 0.008 0.003
7 Amide diester 0.006 0.002
8 Diester impurity 0.003 0.001
9 Etexilate impurity 0.007 0.002
10 Dabigatran etexilate mesylate 0.010 0.003

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 HCl)  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 (4.14 g of Sodium dihydrogen phosphate dissolved in 1000 mL of water. Dissolve and adjust pH = 5.8 with sodium hydroxide solution)In the optimized conditions the Dabigatran Etexilate mesylate   KSM-I  Stage-IIA  Stage-II  Propionate ester  Methyl Ester  Amidine impurity  Amide Diester  Diester impurity and Etexilate impurity were well separated with a resolution of 3.26 and the typical retention times (RT) of Dabigatran Etexilate mesylate   KSM-I  Stage-IIA  Stage-II  Propionate ester  Methyl Ester  Amidine impurity  Amide Diester  Diester impurity and Etexilate impurity were about 39.58  19.09  30.39  21.56  13.79  18.31  20.64  33.53  34.71 and 41.20 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 nine impurities  namely KSM-I  Stage-IIA  Stage-II  Propionate ester  Methyl Ester  Amidine impurity  Amide Diester  Diester impurity and Etexilate impurity.
The system suitability values of Dabigatran Etexilate mesylate and its impurities were summarized in Table 2.
Table 2
Compound (n=1) Rt Rs N T
Propionate ester impurity 13.79 30667 1.09
Methyl Ester impurity 18.31 15.86 88815 1.11
KSM-I 19.09 3.26 111007 1.13
Amidine impurity 20.64 6.03 88310 1.12
Stage-II 21.56 3.46 120672 1.11
Stage-IIA 30.39 34.85 236927 1.04
Amide Diester impurity 33.53 12.73 323921 1.03
Diester impurity 34.71 4.67 293773 1.04
Dabigatran 39.58 17.27 282730 1.08
Etexilate impurity 41.20 4.91 215688 1.08

*n=1: determination  Rt: retention time  Rs: USP resolution  N: number of theoretical plates (USP tangent method)  T: USP tailing factor  m/z: mass number.

The pattern of elution of isomer of Dabigatran with m/z value of 628.7(M*) at RRT of about 0.98 and elution of an impurity with m/z value of 629.7(M+1) at RRT of about 0.95 is observed in innovator tablet as well as Dabigatran Etexilate mesylate samples.
High level of degradation in test solution was observed using 3% hydrogen peroxide at 60°C for 2 hours  1M sodium hydroxide at 60°C for 2 hours and 1M HCl at 60°C for 2 hours. Impurities observed in stress condition using PDA detector . Major degradants were stage-II and unknown impurities .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.
*M is the molecular weight of Dabigatran.

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 µm particles. The mobile phase consists buffer (4.14 g sodium dihydrogen phosphate dissolved in 1000 ml of water and adjust pH = 5.8 with sodium hydroxide solution)  and solvent is acetonitrile. The flow rate of the mobile phase was kept at 1.0ml/min. Beginning with the gradient ratio of mobile phase buffer and solvent(acetonitrile) 85:15(v/v)  system was continued at the same ratio for 5 minutes. The ratio was changed linearly 55:45(v/v) within 35 minutes and again system was continued at the same ratio for 5 minutes. The ratio was again changed linearly 30:70(v/v) within 45 minutes and again system was continued at the same ratio for 5 minutes. After 3 minutes the initial gradient of 85:15 is for 7 minutes to be conditioned for every analysis. The column temperature was maintained at 50°C and the wavelength was monitored at a wavelength of 225 nm. The injection volume was 10 µL for related substances determination. Eluent A was used as diluent during the standard and test samples preparation.

Example 1

Preparation of system suitability stock solution preparation

7.5mg each of KSM-I  Stage-IIA  Stage-II  Propionate ester  Methyl Ester  Amidine
impurity  Amide diester  Diester impurity  Etexilate impurity and 5 mg of Dabigatran working standard were accurately weighed and transferred to the 50mL volumetric flask(BOROSIL-Class-A)   separately; 30ml of acetonitrile was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with diluent.

Example 2

Preparation of system suitability solution preparation

10mg of Dabigatran working standard was accurately weighed and transferred to the 10mL volumetric flask(BOROSIL-Class-A)   separately; add 1 mL system suitability stock solution and 6 mL acetonitrile was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with diluent.

Example 3

Preparation of reference solution-(a)

1mL of system suitability stock solution transferred to the 10mL volumetric flask(BOROSIL-Class-A)   separately; 6 mL acetonitrile was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with diluent.

Example 4

Preparation of reference solution-(b)

50mg of Dabigatran working standard  weighed and transferred to the 50mL volumetric flask(BOROSIL-Class-A)   separately; 30 mL acetonitrile was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with diluent.
Pipette out 1.0mL from solution and transferred in to a 100mL volumetric flask (BOROSIL-Class-A)  and made up to mark with diluent. Further take 1mL of this solution into 10mL volumetric flask(BOROSIL-Class-A)  add 6.0 ml acetonitrile and make up with diluent.
A working solution of 1000µg/ml was prepared for related substances determination analysis.