INTRODUCTION:

Dabigatran Etexilate Mesylate is chemically β-Alanine, N-[[2-[[[4-[[[(hexyloxy)carbonyl]amino]phenyl]amino] methyl]-1-methyl-1H-benzimidazol-5-yl]carbonyl]-N-pyridinyl-, ethyl ester, methanesulfonate. Dabigatran Etexilate Mesylate is an orally available mesylate salt form of the etexilate prodrug of dabigatran and a direct thrombin inhibitor with anticoagulant activity. Thrombin, a serine protease, is responsible for the conversion of fibrinogen to fibrin in the coagulation cascade. Inhibition of thrombin consequently prevents thrombus development. Dabigatran inhibits free thrombin, fibrin-bound thrombin and thrombin-induced platelet aggregation, which results in a prolongation of a PTT (partial thrombo plastin time), ECT (Ecarin clotting time), and TT (thrombin time).

It is used in embolism associated with atrial fibrillation, cardioversion of atrial fibrillation/flutter, thromboprophylaxis in orthopaedic surgery, cerebral embolism, and treatment of acute venous thromboembolism. DEM is available in the form of Capsules with the Brand name of PRADAXA (Marketed by Boehringer Ingelheim, India) with strengths 75 and 150 mg. The literature survey reveals that various analytical methods like Spectro photometric and HPLC were reported for the determination of DEM in formulations, but there were no reported HPLC methods in bulk form. Moreover, the available HPLC methods for the determination of dabigatran in formulations were time consuming for elution and uneconomical. Hence an attempt was made to develop, a simple, precise, accurate, robust, and economical RP-UPLC method for the estimation of DEM in bulk drug.

According to ICH (International Conference on Harmonization) drug stability test guidelines Q1A (R2), the stability-indicating assay method (SIAM) is employed for the analysis of stability samples^1,2. The analysis of stability test samples should be done by using validated SIAM after subjecting a drug to a variety of stress conditions such as hydrolysis, oxidation, photo stability and thermal degradation³. The ICH guidelines Q6A provides guidance on specifications⁴ and also the requirement of stability-indicating assays under Universal Tests/Criteria for drug substances and drug products. Apart from ICH, the United states-Food and Drug Administration (US-FDA) draft guidelines of 1998 also provides guidance on stability testing of drug substances and drug products⁵. The requirement of stability testing of well-established or existing drug substances and products is also provided in World Health Organization (WHO) guidelines⁶. There are few analytical methods reported in literatures for analysis of DEM which includes HPLC⁷, UPLC MS/MS⁸ in human plasma, LC/MS based metabolite identification and semi quantitative estimation approach in the investigation of in vitro dabigatran etexilate mesylate metabolism

MATERIALS AND METHODS:

Acquity UPLC H Class - PDA detector with auto sampler, Acquity UPLC BEH Column C18 (100x2.1, 1.7μm particle size) equipped with Empower-3 software.

Chemicals and solvents:

Dabigatran Etexilate Mesylate was obtained as a gift sample from MSN Laboratories, Hyderabad, India and was used without further purification. All chemicals and reagents used were of HPLC grade. HPLC grade acetonitrile, formic acid and water were procured from Merck Pharmaceuticals Private Ltd., Mumbai, India. Formic acid was purchased from Qualikems, Vadodara, India.

Preparation of standard stock solution:

Dabigatran Etexilate Mesylate is accurately weighed (20.24) and dissolved in 100ml of HPLC grade acetonitrile: water (50:50) to get 200 ppm stock solution. Working standard solutions were further diluted with diluent (Acetonitrile: Water 50:50 v/v) to get concentrations of 20-120 ppm. Each of these drug solutions (3μl) were injected into the column, the peak area and retention times were recorded.

Method development and optimization:

A gradient elution was necessary for optimizing separation of degradation products formed under variety of stressed conditions. Among the various trials for separation of degradation products by gradient programming, the best resolution was achieved with initial run of Formic acid: ACN in the ratio of 90:10 (v/v) for 1 min. then changed to gradient T/%B 0/10, 0.3/10, 3.5/95, 5/95, 5.2/10, 7/10 The Injection volume was 3μl and mobile phase flow rate was 0.4 ml/min. The column was maintained at 40 ^OC temperature and detection wavelength was 220 nm.

System Suitability Test:

The system suitability tests were performed to ensure that the proposed UPLC method was suitable for intended analysis. The parameters of these tests are column efficiency (number of theoretical plates) tailing of chromatographic peak, repeatability as percentage of RSD peak area for six injections and reproducibility as percentage RSD of retention time. These results are listed in table-1.

Table-1 : System suitability result for DEM

Parameter	Obtained value	Acceptance criteria
Tailing factor	1.46	NMT 2
Theoretical plates	23304	NLT 10000
%RSD of 6 injections	0.08	NMT 2 %

Validation of method:

The developed optimized chromatographic method was validated for different parameters such as linearity, accuracy, precision, specificity and selectivity.

Linearity:

The response of DEM was found to be linear (r2 =0.996) in the concentration range between 20-120 ppm and each of this concentration was injected in triplicate into the HPLC column. The results of the linearity studies are shown in Table 2.

Table-2 Linearity study data

Linearity Test:
S.No.	Ppm	Peak Area
1	20	563256
2	40	1145463
3	60	1716311
4	80	2214635
5	100	2749720
6	120	3311921
correlation coefficient		0.99975
Slope		55474

Fig-2 Linearity study data

Specificity and Selectivity:

The specificity of the UPLC method was established through study of resolution factor of the drug peak from nearest resolving peak. Selectivity was established through determination of purity of each peak using PDA detector. In purity plots, the purity angle of each peak was found to be less than purity threshold thus indicates method is selective. The purity plot of Dabigatran Etexilate Mesylate in presence of degradation products.

Specificity Chromatograms of standard and blank were recorded and chromatogram of blank did not show any peak at the retention time of analyte. This shows that the method is specific.

Table-3: Specificity data

Specificity
	Standard	Sample
RT	2.736	2.741
Area	2740793	2744764

Limit of detection and limit of quantification:

The limit of detection (ICH Q2B,1996) which represents the concentration of the analyte at S/N ratio of 0.03 ppm and the limit of quantification (ICH Q2B,1996) which represent the concentration of analyte at S/N ratio of 10 ppm were determined experimentally for the proposed method and the results are shown in table-4.

Table-4: Limit of detection and limit of quantification

LOD and LOQ
Ppm	Area	s/n
10ppm	288890	47.51
5ppm	142603	37.17
2 ppm	65579	22.69
1 ppm	34370	10.96
0.5 ppm	16233	5.39
0.25 ppm	8598	3.73
0.0125 ppm	4231	1.34
0.06 ppm	2543	1.11
0.03 ppm	995	0.92

Precision:

Precision studied were performed under different conditions Intra-day and Inter-day. Intra-day study was performed by analyzing three different concentrations of drugs for three times on the same day. Inter-day precision was performed by analyzing three different concentration of drug for three different days. The results of precision study are shown in table-5.

Table-5: Intra-day and inter-day precision results

	Intra-day		Inter day
No. of Injections	Retention time	Peak Area	Retention time	Peak Area
1	2.735	2746951	2.744	2737704
2	2.733	2746538	2.736	2737798
3	2.729	2745668	2.734	2736267
4	2.735	2747247	2.735	2737004
5	2.732	2746466	2.735	2736776
6	2.728	2747663	2.732	2737193
Mean	2.732	2746755	2.736	2737123
%RSD	0.109	0.025	0.152	0.021

Accuracy:

The accuracy of the method was evaluated by recovery study of DEM at three concentration levels (80%, 100% and 120 %). A study was carried out in triplicate at 2, 4 and 6 μg/ml in UV and 20, 40 and 60µg/ml in UPLC. A fixed amount of pre-analysed sample and standard drug was added and recovery was studied for the quantification of the DEM. The percentage recovery and mean % recovery were calculated and presented in table-6.

Table-6 Accuracy data

S. No.	Recovery at 80%		Recovery at 100%		Recovery at 120%
	Standard	Sample	Standard	Sample	Standard	Sample
1	2187023	2216335	2702412	2764066	3261860	3325185
2	2188942	2217179	2703901	2765433	3262050	3326792
3	2187910	2216640	2702471	2764689	3260422	3324129
Mean	2187958	2216718	2702928	2764729	3261444	3325368
%RSD	0.044	0.019	0.031	0.025	0.027	0.041
% of Recovery	100.23		99.02		99.71
Average % of Recovery	99.65

CONCLUSION:

In this study, the degradation behavior of Dabigatran Etexilate Mesylate was established according to the ICH recommended stress conditions. The degradation of drug occurred extensively in hydrolytic, photolytic and oxidative conditions whereas mild degradation of it was seen to thermal stress. The results of the stress testing according to ICH guidelines reveal that method is selective and stability-indicating. The developed method is simple, accurate, precise, specific and is able to separate drug from degradation products. The method is proposed for the analysis of stability samples generated during stability studies on drug and its formulations.

REFERENCES:

1. ICH. Stability Testing of New Drug Substances and Products. International Conference on Harmonization, IFPMA. Geneva: 1993.

2. Bakshi M, Singh S. J Pharm Biomed Anal 2002, 28, 1011-1040.

3. ICH. Stability Testing of New Drug Substances and Products (Q1AR). International Conference on Harmonization, IFPMA. Geneva: 2000.

4. ICH, Specifications: Test procedures and Acceptance Criteria for new drug substances and new drug products: chemical substances. International conference on Harmonization, IFPMA, Geneva, 1999.

5. FDA, Guidance for industry: Stability testing of drug substances and drug products (Draft guidance), Food and Drug Administration, Rockville, MD, 1998.

6. WHO, Guidelines for stability testing of Pharmaceutical products containing well established drug substances in conventional dosage forms, in WHO expert committee on specifications for Pharmaceutical preparations. Technical report series 863, World Health Organization, Geneva, 1996, 65-79.

7. Bernardi RM, Froehlich PE, Bergold AM. J AOAC Int 2013, 96(1), 37-41.

8. Xavier D, Julie M , Silvy Laporte, Patrick M, Thierry B. J Pharm Biomed Anal 2012, 25(58), 152-156.

Received on 07.04.2018 Modified on 30.04.2018

Asian J. Research Chem. 2018; 11(5): 787-790

DOI: 10.5958/0974-4150.2018.00138.4