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Chromatographia

, Volume 79, Issue 3–4, pp 169–178 | Cite as

Liquid Chromatographic Method Development for Forced Degradation Products of Dabigatran Etexilate: Characterisation and In Silico Toxicity Evaluation

  • Debasish Swain
  • Prinesh N. Patel
  • Gangam Nagaraj
  • Kona S. Srinivas
  • Mahesh Sharma
  • Prabha Garg
  • Gananadhamu SamanthulaEmail author
Original

Abstract

A simple and accurate UHPLC method was developed for Dabigatran etexilate (DAB) using Acquity CSH C18 (100 mm × 2.1 mm × 1.7 µm) column with mobile phase containing ammonium acetate (pH 5.0) and methanol in gradient program. This method was used to study the forced degradation products of DAB. The drug was found to degrade in all hydrolytic and oxidative conditions resulting in formation of four major degradation products (DP1 to DP4). DAB and its degradation products were well separated using the proposed method. The degradation products were characterized using LC-Q-TOF/ESI/MS/MS and accurate mass measurements for obtaining the structural information. The degradation pathway leading to the formation of the degradation products was also proposed. In silico tools like TOPKAT and DEREK software were used to evaluate the toxicity associated with DAB and its degradation products. The developed method was validated as per ICH Q2 (R1) guidelines. The method showed linear response over a concentration range of 45–105 µg mL−1 with correlation coefficient of 0.9998. The accuracy of the method was observed within the acceptable limits of 98–102 %. The precision of the method was good as indicated by %RSD values less than 1.0. The proposed method was found to be robust when deliberate changes were made in pH of buffer, column temperature and flow rate of the mobile phase. The developed method finds its application as a stability indicating assay method for the determination of DAB.

Keywords

Dabigatran etexilate UHPLC LC-Q-TOF/ESI/MS/MS Forced degradation In-silico toxicity 

Introduction

Dabigatran etexilate (DAB), (Z)-ethyl 3-(2-(((4-(N′-((hexyloxy) carbonyl) carbamimidoyl) phenyl) amino) methyl)-1-methyl-N-(pyridin-2-yl)-1H-benzo[d] imidazole-5-carboxamido) propanoate, is the prodrug of dabigatran, a novel oral anticoagulant belonging to the class of direct thrombin inhibitors. It is being widely studied for various clinical applications [1]. DAB has been approved in EU and several other countries for prophylaxis of thromboembolism in patients undergoing total knee or hip replacement [2]. DAB has also shown certain positive affects in reducing the risk of cardiovascular stroke in patients with atrial fibrillation, when compared with warfarin [3]. DAB provides an excellent advantage where coagulation monitoring is not required unlike warfarin which requires frequent monitoring of bleeding [4].

A few analytical methods were reported for the estimation of DAB includes the determination of DAB in bulk drug and pharmaceutical dosage forms using spectrophotometric techniques [5], high-performance liquid chromatography (HPLC) [6], stability indicating methods using HPLC [7, 8], quantification of DAB in human plasma using liquid chromatography–tandem mass spectrometry (LC–MS) and metabolite identification using LC–MS [9, 10]. A forced degradation study was also carried out, but characterisation of all the DPs and the degradation pathway were not attempted [11].

Forced degradation is a process where the drug and drug products are subjected to conditions more severe (elevated temperature, pH and light) than the accelerated conditions of stability testing for generation of degradation products. Presence of degradation products may pose severe risk to health of the patients. Hence, forced degradation study of drug substance is very important to predict the likely degradation products, which can in turn help establish the degradation pathways and intrinsic stability of the molecule [12, 13]. The characterization of degradation products and evaluation of toxicities associated with drug and drug degradation products play a crucial role in establishing the overall therapeutic benefit of the drug [14, 15]. In the present study, the authors developed LC method and characterized the degradation products of DAB by LC/ESI/QTOF/MS/MS. In-silico toxicity was also evaluated for the degradation products.

Experimental

Chemicals and Reagents

Pure dabigatran etexilate was obtained as gift sample from Ranbaxy Laboratories, Gurgaon, India. HPLC grade acetonitrile (ACN) and methanol (MeOH) were bought from Merck, Mumbai, India. All reagents (ammonium acetate, sodium hydroxide, hydrochloric acid and 30 % Hydrogen peroxide) of analytical grade were purchased from S. D. Fine Chemicals, Mumbai, India. HPLC grade water was obtained from a Millipore Milli-Q system (Millipore, Milford, MA, USA).

Instrumentation

Waters UPLC® system (Waters Corp., Milford, MA, USA) equipped with an auto sampler and quaternary gradient pump with an in-line degasser was used. The photodiode array (PDA) detector was employed throughout the analysis. The chromatographic data were acquired using Empower 3 software. LC/ESI/QTOF/MS/MS experiments were carried out using an Agilent 1200 infinity series LC instrument (Agilent Technologies, USA) coupled to a quadrupole time-of flight mass spectrometer (Q-TOF, LC/MS, Agilent Technologies, USA) equipped with an electrospray ionization (ESI) source and data acquisition under the control of Mass Hunter Workstation software. Photostability studies were carried using Osworld Photostability Chamber (Osworld OPSH-G-16-GMP series, Osworld Scientific Pvt. Ltd., India). The thermal degradation studies were carried out in the Osworld laboratory oven (Osworld Scientific Pvt. Ltd., India). Power Sonic-405, an ultra-sonicator (Hwashin Technology Co., Seoul, South Korea) was used to dissolve the samples and Eutech pH meter (Eutech Instruments, Singapore) was used to adjust the pH of the mobile phase.

Chromatography and Mass Spectrometric Conditions

The chromatographic separation was carried out on Acquity CSH C18 (100 mm × 2.1 mm × 1.7 µm) column with mobile phase containing ammonium acetate (pH 5.0) and methanol in gradient elution program at a flow rate of 0.3 mL min−1 with λ max at 260 nm. The injection volume used for the analysis of samples was 2 µL and the run time for the chromatographic analysis was 15 min. The typical operating source conditions for MS scan of DAB in positive ESI mode were optimized as follows: the fragmentor voltage was set at 144 V, the capillary at 3500 V, the skimmer at 65 V, and nitrogen was used as the drying (325 °C, 10 L min−1) and nebulising (40 psi) gas. The scan range and scan rate were 100–1000 Da and 5 spectra/s, respectively. Ultrahigh pure nitrogen gas was used as collision gas.

Forced Degradation Studies

These studies were performed using 1000 µg mL−1 solution of DAB. Initially degradation was started with mild conditions, which were subsequently made stronger to get sufficient degradation. The forced degradation was performed as per ICH guidelines Q1A (R2) [12]. For acid hydrolysis the drug solutions were refluxed in 1 N HCl for 30 min. The neutral hydrolysis of DAB was conducted in water refluxed for 4 h. For alkaline conditions the drug solution was exposed to 0.1 N NaOH at room temperature for 15 min. The oxidative stress study was performed using 10 % H2O2 for 5 h at room temperature. The thermal degradation studies were done by exposing the solid drug to 100 °C for 5 days. The photostability studies were carried out by exposing solid and solutions of the drug samples to UV light of 200 Whm−2 and 1.2 × 106 lux hours of fluorescent light at 40 °C and 75 % RH as per ICH guidelines Q1B [13]. The samples were collected at specified time points and were diluted with mobile phase at about 75 µg mL−1 of DAB and filtered through 0.22 µm membrane syringe filter. The acid and alkaline hydrolysis samples were neutralized before dilution.

In Silico Toxicity Prediction

A number of software tools are available for toxicity prediction, covering a range of toxicity endpoints. To assess the toxicity of the DAB and its degradation products, expert systems like TOPKAT (Discovery Studio 2.5, Accelrys, Inc., San Diego, CA, USA) and DEREK (Nexus v2.0, Lhasa Ltd., Leeds, UK) were used. TOPKAT (Toxicity Prediction by Komputer Assisted Technology) estimates the toxicity of a compound quantitatively whereas DEREK (Deductive Estimation of Risk from Existing Knowledge) assesses the results qualitatively. Using well established Quantitative Structure Toxicity Relationship (QSTR) models, TOPKAT predicts toxicological end points in term of probability values. Probability values from 0.0 to 0.30 are considered as low probabilities for any toxicological end point whereas probability values greater than 0.70 are considered as high probabilities [16].

Unlike TOPKAT which uses built QSTR models to predict toxicity, DEREK is knowledge and rule-based toxicity prediction software. To predict toxicological endpoint, DEREK uses the rules which have been derived by the collective literature knowledge and expert advice. It includes more than 50 structural alerts covering a wide range of toxicological endpoints in humans, other mammals and bacteria. These set of rules connects a structural alert (toxicophore) to a particular toxicity. Whenever a query molecule is submitted in DEREK for toxicity prediction, it first searches the knowledge base for ant toxic structural alert. Then it assigns it depending on the availability of the alert. For any toxicity prediction DEREK provides nine different types of confidence level [17].

Results and Discussion

Optimisation of Chromatographic Method

The objective of the method was to separate all the DPs from each other and DAB. Waters Acquity CSH C18 column (100 mm × 2.1 mm, 1.7 µm) was found to be suitable for the optimum separation of the peaks. During optimization process several gradient chromatographic conditions were tried using MeOH/water and ACN/water in different proportion. It was observed that DP 4 merged when using ACN. Hence, MeOH was used as an organic modifier for optimization of the method. However, DPs showed asymmetrical peak shapes due to the absence of the buffer/mobile phase additives. Therefore, LC–MS compatible volatile buffers/additives were tried with formic acid, ammonium formate and ammonium acetate to improve the resolution and peak shape. Well resolved peaks with acceptable symmetry were achieved by employing a mobile phase composed of (A) ammonium acetate buffer (10 mM, pH 5.0) and (B) methanol in a gradient program as follows (Tmin/%B): 0/30, 0.3/30, 10/75, 12/90, 12.5/30, 16/30 and flow rate of 0.3 mL min−1. The temperature of the column oven was set at 25 °C.

Forced Degradation Behavior of DAB

The degradation of DAB was studied using UHPLC under various stress conditions. The drug degraded extensively under alkaline hydrolysis. DAB also degraded under acidic, neutral and oxidative conditions, whereas it was found to be stable in other stress conditions. The chromatograms of all stress degradation samples are given Fig. 1a–d. Four DPs (DP1 to DP4), were identified and characterized using LC/ESI/QTOF/MS/MS experiment and accurate mass measurements (Table 1).
Fig. 1

Overlaid chromatograms showing the separation of degradation products in a acid hydrolysis, b alkaline hydrolysis, c neutral hydrolysis and d oxidative degradation conditions

Table 1

Elemental composition of DAB and its degradation products (DPs)

DAB and its DP’s

Molecular formula [M + H]+

Calculated m/z

Observed m/z

Error (ppm)

MS/MS fragment ions

DAB

C34H41N7O5 +

628.3242

628.3219

3.6605

526,434,365,332,306,289,189,172

DP 1

C27H29N7O3 +

500.2405

500.2393

2.3988

400,306,289,172

DP 2

C24H29N5O4 +

452.2292

452.2292

0.0000

350,324,289,189,172,148

DP 3

C32H37N7O5 +

600.2929

600.2918

1.8324

472,434,350,332,324,306,289, 189,172

DP 4

C34H40N6O6 +

629.3082

629.3118

−1.4301

527,484,435,365,333,289,172

Hydrolysis

Acidic degradation study was started with mild conditions in 0.1 N HCl at 80 °C. No degradation peak was observed; hence the study was performed with harsher environment of 1 N HCl for 30 min. The drug showed around 20 % degradation (Fig. 1a). Similarly, alkaline hydrolytic study was started with milder condition in 0.1 N NaOH at RT with sample collection at different time points. The chromatogram showed optimum degradation of the drug after 15 min in room temperature (Fig. 1b). Degradation was observed after exposing the drug in water at 80 °C for 4 h (Fig. 1c).

Thermal Conditions

Dabigatran etexilate was found to be stable under thermal degradation conditions even after exposing the pure solid drug to dry heat at 100 °C for 5 days. This indicated that the drug is stable to elevated temperature under solid state.

Photolysis

The drug DAB proved to be stable under photolytic stress conditions after exposing solution state and solid state samples to Fluorescent and UV light.

Oxidative Stress

Oxidative degradation study was started with 3 % H2O2 for 48 h at room temperature. The drug was found to be stable. The study was then carried out under harsh conditions employing 10 % H2O2 and standing for 5 h, two degradation products were observed (DP1 and DP4), which showed that the drug was susceptible to oxidation at intense oxidative conditions (Fig. 1d).

Degradation Pathway of DAB

The degradation pathway followed by DAB is outlined in Scheme 1. In neutral hydrolysis and oxidative stress conditions, the carbamic ester bond of DAB was cleaved to yield DP 1 through formation of unstable intermediate carbamic acid. DP2 was formed only in acid hydrolysis as a result of hydrolytic cleavage of amide linkage. The degradation product DP 3 was obtained as a major degradation product under base hydrolysis because of the susceptibility of ester bonds to alkaline hydrolysis. DP3 was also formed in minor quantity under acid hydrolytic conditions. DP 4 was formed due to the conversion of imidamide bond to amide under acid and neutral hydrolytic conditions and also oxidative stress conditions.
Scheme 1

Degradation pathway of DAB

Mass Spectrometric Study of DAB

The MS/MS spectrum of protonated DAB (Rt = 12.4 min) with m/z 628 showed the product ions of m/z 526 (loss of C6H14O from m/z 628), m/z 434 (loss of C10H14N2O2 from m/z 628), m/z 365 (loss of C8H7N3O from m/z 526), m/z 332 (loss of m/z C10H14N2O2 from m/z 526), m/z 306 (loss of m/z C7H12O2 from m/z 434), m/z 289 (loss of m/z NH3 from m/z 306), m/z 172 (loss of m/z C7H5N2 from m/z 289) (Fig. 2, Scheme 1). The formation of fragment m/z 172 indicated the presence of benzimidazole and the fragment m/z 289 showed that the above moiety is attached to a 4-aminobenzimidamide portion in the structure of DAB. These structures and their corresponding losses were confirmed from the accurate mass measurements.
Scheme 2

Mass spectral fragmentation of DAB and its degradation products

Characterisation of Degradation Products

All the degradation products formed were subjected to Online LC/ESI/QTOF/MS/MS experiments to characterize all the DPs formed under various stress conditions. The most probable structures for all the DPs were proposed based on the m/z values of their [M + H+] ions and MS/MS data in combination with elemental compositions derived from accurate mass measurements.

DP 1

The LC/ESI/QTOF/MS/MS spectrum of [M + H+] ion (m/z 500) of DP 1 at Rt = 5.6 min (Fig. 2), shows structure with fragment ions of m/z 400 (loss of C5H8O2 from m/z 500), m/z 365 (loss of C7H9N3 from m/z 500), m/z 306 (loss of C10H14N2O2 from m/z 500), m/z 289 (loss of NH3 from m/z 306) and m/z 172 (loss of C7H5N2 from m/z 289) (Scheme 2). The fragmentation pattern indicated the chemical structure of DP1 to be as ethyl 3-(2-(((4-carbamimidoylphenyl) amino) methyl)-1-methyl-N-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxamido) propanoate.
Fig. 2

LC/ESI/QTOF/MS/MS spectra of DAB and its degradation products

DP 2

DP 2 [M + H+, m/z 452] formed under acidic degradation study and eluting at Rt = 9.1 min, showed the MS/MS spectra (Fig. 2) containing fragment ions at m/z 350 (loss of C6H13O from the m/z 452), m/z 324 (loss of C7H12O2 from m/z 452), m/z 289 (loss of H5NO from m/z 324), m/z 189 (loss of C7H9N3 from m/z 324), m/z 172 (loss of C7H5N2 from m/z 289) and m/z 148 (loss of C9H8N2 from m/z 323) (Scheme 2). DP 2 was identified to be (Z)-2-(((4-(N′-((hexyloxy) carbonyl) carbamimidoyl) phenyl) amino) methyl)-1-methyl-1H-benzo[d] imidazole-5-carboxylic acid.

DP 3

The LC/ESI/QTOF/MS/MS spectrum of [M + H+] ion (m/z 600) of DP 3 (Rt = 9.4 min), (Fig. 2), with an elemental composition of C32H38N7O5 + showed fragment ions at m/z 472 (loss of C7H12O2 from m/z 600), 434 (loss of C8H10N2O2 from m/z 600), 350 (loss of C14H22N2O2 from m/z 600), 332 (loss of C6H14O from m/z 434), 324 (loss of C8H8N2O from m/z 472), 306 (loss of CO2 from m/z 350), 289 (loss of NH3 from m/z 306 and m/z 172 (loss of C7H5N2 from m/z 289) (Scheme 2). Based on the fragmentation pattern, DP 3 chemical structure can be assigned as, (Z)-3-(2-(((4-(N′-((hexyloxy) carbonyl) carbamimidoyl) phenyl) amino) methyl)-1-methyl-N-(pyridin-2-yl)-1H-benzo [d] imidazole-5-carboxamido) propanoic acid.

DP 4

DP 4 [M + H+, m/z 629] is formed under acid, neutral hydrolytic conditions and oxidative stress conditions (Rt = 11.7 min). The MS/MS spectra of DP4 (Fig. 2) showed fragment ions at m/z 527 (loss of C6H14O from m/z 629), 484 (loss of C7H15NO2 from m/z 629), 365 (loss of C7H5NO from m/z 484), 333 (loss of C10H14N2O2 from m/z 527), 289 (loss of C17H27N2O5 from m/z 307) and 172 (loss of C7H5N2 from m/z 289) (Scheme 2). DP 4 chemical structure was proposed as ethyl 3-(2-(((4-(((hexyloxy) carbonyl) carbamoyl) phenyl) amino) methyl)-1-methyl-N-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxamido) propanoate based on mass spectrometric fragmentation and accurate mass measurement experiments.

In Silico Toxicity Prediction

Toxicity prediction results using TOPKAT have been given in Table 2. By evaluating the prediction results, it is clear that the toxicity profile of the drug and its degradation product is quite similar which can be linked to the presence of benzimidazole moiety in all molecules. However, they deviate for some of the animal models. Unlike other molecules, DP 2 and DP 3 show zero probability for Ames mutagenicity (v3.1) and DP 1 is only molecule which shows developmental toxicity potential (DTP) (v3.1) toxicity. All compounds show exactly similar toxicity profile for NTP carcinogenicity call (male mouse) (v3.2), FDA carcinogenicity female rat non vs carc (v3.1), FDA carcinogenicity female rat single vs mult (v3.1), and skin irritation (v6.1) models. The qualitative results from DEREK software have been summarized in Table 3. The results show that all the compounds have hepatotoxicity. The structure alerts for hepatotoxicity is benzimidazole which is present in all the molecules and is responsible for their toxicity (details given in Table 3). No other toxicological end point was predicted for any molecule.
Table 2

Quantitative toxicity prediction of the drug and its degradation products by TOPKAT analysis

Model

Drug

DP 1

DP 2

DP 3

DP 4

NTP carcinogenicity call (male rat) (v3.2)

1

0.597

1

1

0.98

NTP carcinogenicity call (female rat) (v3.2)

0

0.501

0.841

0.996

0

NTP carcinogenicity call (male mouse) (v3.2)

1

1

1

1

1

NTP carcinogenicity call (female mouse) (v3.2)

0

0.725

0.002

0

0.036

FDA carcinogenicity male rat non vs carc (v3.1)

1

0.999

0.295

1

0.999

FDA carcinogenicity male rat single vs mult (v3.1)

0

0

0.544

0

0

FDA carcinogenicity female rat non vs carc (v3.1)

0

0

0

0

0

FDA carcinogenicity female rat single vs mult (v3.1)

0

0

0

0

0

FDA carcinogenicity male mouse non vs carc (v3.1)

0.04

0.042

0.006

0.01

0

FDA carcinogenicity male mouse single vs mult (v3.1)

0

0

0

0

0

FDA carcinogenicity female mouse non vs carc (v3.1)

0.911

0.999

0.024

0.41

0.997

FDA carcinogenicity female mouse single vs mult (v3.1)

0

0

0

0

0

Weight of evidence carcinogenicity call (v5.1)

0

0

0.004

0

0

Ames mutagenicity (v3.1)

0.998

1

0

0

0.877

Developmental toxicity potential (DTP) (v3.1)

0

0.921

0

0

0.003

Rat oral LD50 (v3.1) (g kg−1)

312.0 mg kg−1

201.8 mg kg−1

734.9 mg kg−1

134.3 mg kg−1.

1.8 g kg−1

Rat maximum tolerated dose-feed/water (v6.1) (mg kg−1)

280.2

22.6

120.9

10.7

103.0

Rat maximum tolerated dose-gavage (v6.1) (mg kg−1)

775.8

62.7

334.7

29.6

285.3

Rat inhalational LC50 (v6.1) (g m−3 H−1)

641.5 mg m−3 H−1

98.5 mg m−3 H−1

863.2 mg m−3 H−1

580.8 mg m−3 H−1

10 g m−3H−1

Chronic LOAEL (v3.1) (mg kg−1)

152.8 μg kg−1

441.1 μg kg−1

6.9 mg kg−1

934.3 μg kg−1

77.2 μg kg−1

Skin irritation (v6.1)

0

0

0

0

0

Skin sensitization NEG v SENS (v6.1)

0.438

0.995

1

0.917

0.28

Skin sensitization MLD/MOD v SEV (v6.1)

1

1

1

1

0.986

Ocular irritancy SEV/MOD vs MLD/NON (v5.1)

0

0

0

0

0

Ocular irritancy SEV vs MOD (v5.1)

1

1

0.041

1

1

Ocular irritancy MLD vs NON (v5.1)

1

0.703

0.698

1

1

Aerobic biodegradability (v6.1)

0

0

0

0

0

Fathead minnow LC50 (v3.2) (μg L−1)

489.4 pg L−1

198.9 ng L−1

246.2 ng L−1

2.3 ng L−1

584.6 pg L−1

Daphnia EC50 (v3.1) (mg L−1)

113.8

50.8

49.5

127.4

44.9

Table 3

Qualitative toxicity prediction of the drug and its degradation products by DEREK analysis

Drug and degradation product

Hepatotoxicity

Structural alert

Open image in new window

Comments

This alert describes the hepatotoxicity of benzimidazole derivatives. In humans, benzimidazoles (Mebendazole, albendazole, omeprazole and thiabendazole) were reported to cause various hepatic injuries including abnormal liver function tests and cholestasis. The mechanism of toxicity is not clear, but inhibition of microtubule polymerisation, and the formation of quinone-imine reactive metabolites have been proposed

Drug

DP 1

DP 2

DP 3

DP 4

Method Validation

Specificity

The specificity of the developed method was established by evaluating the peak purity of DAB and all the degradation products using photodiode array detector followed by confirmation with mass spectrometry. All the peaks were found to be pure which shows the method to be selectively stability indicating.

Linearity

The method was found to be linear over a concentration range of 45–105 µg mL−1 (60–140 % of the nominal concentration). Each concentration was injected in triplicate (n = 3). Standard calibration curve was plotted by taking concentration of DAB (µg mL−1) on x-axis and mean area response (AU) on y-axis. The proposed method was found to be linear with correlation coefficient of 0.9998 and linear regression equation of y = 18,247x – 13,710.

Accuracy

The accuracy was established by the addition of known quantities of standard to the synthetic mixture of excipients which are taken in the formulation of the drug product. Each solution was injected in triplicate and the percentage recovery was calculated. The percentage recovery was found to be 99–101 %. Results are given in Table 4.
Table 4

Accuracy data of DAB

Spiked concentration (µg mL−1)

Amount found in µg mL−1 (mean ± SD;  %RSD)

Mean recovery (%)

45

45.00 ± 0.15; 0.02

100.0

75

75.30 ± 0.40; 0.04

100.4

105

104.82 ± 0.17; 0.17

99.8

Precision

The repeatability of the method was established by analyzing six injections of the standard drug at 100 % level and the %RSD was found to be 0.2. The intermediate precision of the method was investigated by analyzing the drug at three different concentrations (60, 100 and 140 % of the nominal concentration) on different days (interday precision), different column (with different lot number), different analyst and different instrument within the same laboratory.  %RSD was determined for the concentration of DAB found at each level as shown in Table 5. The developed method has good precision as low %RSD values were obtained.
Table 5

Precision data of DAB

Concentration of DAB (µg mL−1)

Concentration of DAB found (µg mL−1) ± SD;  %RSD

Day 1

Day 2

Different analyst

Column with different lot number

Different instrument

45

45.03 ± 0.12; 0.12

45.10 ± 0.40; 0.40

45 ± 0.11; 0.24

44.9 ± 0.09; 0.21

44.9 ± 0.12; 0.28

75

75.07 ± 0.09; 0.09

75.13 ± 0.17; 0.17

74.9 ± 0.13; 0.18

74.9 ± 0.04; 0.05

74.9 ± 0.06; 0.07

105

105.03 ± 0.02; 0.02

105.07 ± 0.31; 0.07

105 ± 0.39; 0.37

104.7 ± 0.07; 0.07

104.8 ± 0.22; 0.21

Robustness

Robustness is a measure of reliability of the method to small and deliberate changes made to the parameters of the developed analytical method. This study involved small changes in pH (5.0 ± 0.2 units), column temperature (25 ± 5 °C) and flow rate (0.3 ± 0.05 mL min−1). The peak area of the injections (n = 3) was taken as a measure for calculation for determining the robustness of the method. The %RSD for the pH change and flow rate was less than 1 %. In addition, there was no significant variation in the assay of the components indicating the method to be robust as shown in Table 6.
Table 6

Robustness data of DAB

Parameter

Condition

Tailing

Theoretical plates

Resolution

% Assay of drug

pH of buffer

4.8

1.21

341252

1.75

100.01

5.2

1.19

365241

1.84

100.03

Column temperature

20.0 °C

1.25

395687

1.79

100.16

30.0 °C

1.11

432548

1.82

100.11

Flow rate

0.25 mL min−1

1.22

401248

1.83

99.97

0.35 mL min−1

1.18

421587

1.89

99.99

Conclusions

An UHPLC method was developed and all the degradation products were well separated from DAB and also each other. Hence, this method can be used as stability indicating method for determination of DAB. The degradation behavior of the drug was studied under hydrolytic, oxidative, thermal and photolytic conditions. The drug was found to be susceptible to hydrolytic (acidic, alkaline and neutral) and oxidation stress conditions. DAB degraded under acidic hydrolysis to yield three DPs, two DPs in neutral and one each under alkaline hydrolysis and oxidation conditions. All the four stress degradation products were characterized using LC/ESI/QTOF/MS and their probable structures were proposed. The degradation products were evaluated for their toxicities in various models using the TOPKAT and DEREK in silico toxicity prediction tools.

Notes

Acknowledgments

The authors wish to thank Dr. Ahmed Kamal, Project Director, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad for his constant encouragement. The authors are thankful to Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, New Delhi, India for providing research fellowship.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Debasish Swain
    • 1
  • Prinesh N. Patel
    • 1
  • Gangam Nagaraj
    • 1
    • 2
  • Kona S. Srinivas
    • 2
  • Mahesh Sharma
    • 3
  • Prabha Garg
    • 3
  • Gananadhamu Samanthula
    • 1
    Email author
  1. 1.Department of Pharmaceutical AnalysisNational Institute of Pharmaceutical Education and Research (NIPER)HyderabadIndia
  2. 2.Daiichi Sankyo Life Science Research Centre (RCI)GurgaonIndia
  3. 3.National Institute of Pharmaceutical Education and Research (NIPER)MohaliIndia

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