White Analytical Chemistry Aligned Validation and Stability Indicating RP-HPTLC Method for the Determination of Dabigatran Etexilate in Pharmaceutical Formulation

INTRODUCTION

A common oral direct thrombin inhibitor used to prevent and treat thromboembolic disorders, dabigatran etexilate.¹ is especially useful for lowering the risk of stroke in people with non-valvular atrial fibrillation. The chemical structure of Dabigatran Etexilate is shown in Figure 1.Dabigatran Etexilate is a recommended treatment option in many clinical settings because it offers similar anticoagulant efficacy to warfarin without requiring regular international normalised ratio (INR) monitoring. However, the most commonly reported side effects are bleeding and gastrointestinal disturbances, which are mainly attributed to the tartaric acid in the capsule formulation, which enhances drug absorption by lowering gastric pH.Ensuring the safety and quality of pharmaceutical goods requires effective impurity monitoring. Several process- and degradation-related impurities of dabigatran have been reported, arising from raw materials, intermediates, synthesis, or storage conditions. According to ICH guidelines.², these impurities should be identified and quantified to ensure product quality, efficacy, and patient safety. Only a small number of publications provide trustworthy stability-indicating RP-HPTLC methods for the detection of related chemicals, even though various analytical techniques have been described for the assay determination of dabigatran. A comparison of details in reported analytical methods and present proposed method is provided in Table 1.

RP-HPTLC³ is a simple, rapid, economical, and sensitive chromatographic technique widely used for pharmaceutical analysis. It is well suited for routine quality control and impurity profiling because of its high sample throughput, low solvent consumption, excellent separation efficiency, and ease of operation. For the examination of Dabigatran Etexilate and its associated impurities, it is crucial to create a validated, stability-indicating RP-HPTLC approach.

As a result of the enhanced focus on Green Analytical Chemistry (GAC).⁴Comprehensive approaches to evaluate the ecological sustainability of analytical procedures have been developed. Among these, the Analytical GREEnness Metric Approach (AGREE).⁵, Red Analytical Performance Index (RAPI).⁶, and Blue Applicability Grade Index (BAGI).⁷ are well known evaluators for several facets of sustainability and analytical performance. Unlike conventional validation parameters that primarily focus on analytical performance, these assessment tools provide a comprehensive evaluation by integrating environmental, practical, and analytical considerations

The combined application of AGREE, RAPI, and BAGI offers a comprehensive evaluation of analytical methods because each tool addresses a distinct aspect of method assessment. BAGI focuses on the practical applicability of analytical methods for routine laboratory use, RAPI integrates analytical performance with environmental sustainability, whereas AGREE mainly assesses method adherence to the Green Analytical Chemistry. The combined use of all three assessment tools offers a more balanced evaluation of analytical methods.

White Analytical Chemistry (WAC) concept integrates analytical efficiency, environmental sustainability, and practical applicability into a unified framework. Therefore, this forms the prime reason to evaluate developed method using White Analytical Chemistry principles through AGREE, RAPI, and BAGI assessment tools.

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MATERIALS AND METHODS

Chromatographic analysis was conducted by HPTLC from CAMAG.⁸with Linomat 5 as sample dispenser, TLC Scanner 4, developing chamber with twin-troughs, UV chamber for visualisation, and winCATS software (Version 1.4.3). Aluminium-backed silica gel 60 F254plates of HPTLC (10 × 10 cm, 250 µm thick; Merck from Germany) made up the stationary phase. A Hamilton 100 µL syringe was sample applier. The forced deterioration investigations were conducted using a hot-air oven and a temperature-controlled water bath.

Chemicals and reagents

The reference standard for dabigatran etexilate mesylate (>99.0% purity) was purchased from Micro Labs in Bengaluru, India. We purchased commercial dabigatran etexilate capsules (Pradaxa®) from a nearby drugstore. We bought hydrogen peroxide, hydrochloric acid, sodium hydroxide, glacial acetic acid, and spectroscopic-grade solvents from Loba Chemicals Pvt. Ltd., Mumbai, India. A Merck Milli-Q Direct-Q3 water purification system was used to obtain high-purity deionised water.⁹

Chromatographic Development

Before being used, HPTLC plates were activated at 60 °C for 15 minutes and prewashed with methanol. Using a CAMAG Linomat 5 applicator, samples were applied as 4 mm bands 15 mm from the plate's lower edge. In a pre-saturated twin-trough chamber, plate development was carried out for 30 minutes to a migration distance of 70 mm. Following drying, a CAMAG TLC Scanner 4 was used to perform densitometric scanning at 254 nm, and winCATS software.¹⁰ was used to record the chromatograms. The optimised chromatographic conditions established during method development are illustrated in Figure 2.

Preparation of Standard and Sample Solutions

A stock solution of the drug was made by dissolving ten milligrams of dabigatran in ten millilitres of methanol (1 mg/mL). The working standard solution (1000 ng/µL) was made by diluting 500 µL of the stock solution to 10 mL with methanol. The capsule sample solution was prepared similarly after the appropriate sample preparation and dilution.

Forced Degradation Studies

In compliance with the guidelines for stability-indicating analytical methods, forced degradation studies.¹¹ were conducted under acidic (0.1 M HCl), alkaline (0.1 M NaOH), oxidative (3% H2O₂), thermal (100 °C), and photolytic (direct sunlight, 6 h/day for 7 days) stress conditions to assess the developed method's stability-indicating capability.

Method validation

Linearity

The working standard solution was diluted with ethanol in the appropriate aliquots to achieve concentrations of 100–800 ng/spot. After applying one microlitre of each solution to RP-HPTLC plates that had already been coated, the plates were developed, dried, and densitometrically scanned at 254 nm¹²Plotting peak area versus concentration allowed for the construction of calibration curves. Table 2 presents the linearity data, while Figures 3 and 4 display the related densitogram and calibration curve.

Limit of Detection (LOD)

The concentration of analyte that is lowest yet detectable but not quantifiable is known as the limit of detection (LOD).¹³ The following formula was used to compute it. LOD = (3.3 × σ)/S

Limit of Quantitation (LOQ)

The quantitative measurement of lowest analyte concentration with reasonable accuracy andprecisionis limit of quantitation, or LOQ.¹⁴ The following equation was used to compute it. (10 × σ)/S = LOQ

Precision

The 100% concentration in six replicates on the same day (intra-day precision) and on different days (inter-day precision) was analysed to assess precision.¹⁵ The TLC plate was treated with a 1 µL aliquot, which was then developed, dried, and scanned at 254 nm. The resulting peak regions were utilised to assess precision. Table 4 and Figure 6 display the relevant precision results and densitogram.

Accuracy

The standard addition method was used to evaluate accuracy at concentration levels of 50%, 100%, and 150%.¹⁶ Table 4 and Figure 5 show the accuracy findings and matching densitogram, respectively.

Robustness

Small intentional changes in the mobile phase composition, Sample application volume, and detecting wavelength were considered to study robustness.¹⁷ Table 5 displays the robustness study's findings.

Analytical Procedure for Commercially Available Formulation

Ten capsules, each containing 110 mg, were weighed and powdered. Ten milligrams of dabigatran were extracted using methanol, sonicated for twenty minutes, and then diluted to ten millilitres. The filtered solution was added to the TLC plate in a 1 µL aliquot, developed, dried, and scanned at 254 nm. To determine the drug content the following formula was used :

%. Assay = (Amount Recovered/Label Claim × 100)¹⁸

Forced Degradation Study Procedure

To assess the stability of the devised approach in compliance with ICH Q1A(R2).¹⁹ recommendations, stress studies were conducted under acidic, alkaline, oxidative, hydrolytic, thermal, and photolytic conditions. To identify degradation products, the stressed and unstressed samples were analysed using RP-HPTLC, and the resulting chromatograms and densitograms were documented.

RAPI Tool Evaluation

The Red Analytical Performance Index (RAPI) is a comprehensive assessment tool that evaluates by integrating analytical performance, environmental impact, occupational safety, reagent hazards, waste generation, and energy consumption. The colour-coded scoring system enables comparison of analytical methods based on both sustainability and analytical quality. Higher RAPI scores indicate superior analytical performance and environmental sustainability.²⁰

AGREE Tool Evaluation

Based on the 12 principles of Green Analytical Chemistry, the Analytical GREEnness Metric Approach (AGREE).²¹ assesses analytical techniques based on factors like sample preparation, reagent toxicity, solvent consumption, energy needs, waste production, occupational safety, and automation. A circular pictogram with a score between 0 and 1 is produced by the software; higher values suggest more adherence to the principles of Green Analytical Chemistry.

BAGI Tool Evaluation

By considering analytical performance, operational simplicity, cost-effectiveness, instrument availability, sample throughput, environmental impact, and overall reliability, the Blue Applicability Grade Index (BAGI) assesses how practically applicable analytical methods are. Higher BAGI ratings are more appropriate for sustainable laboratory applications and routine quality control.⁷

Statistical Analysis

The experiment was conducted in triplicates for every sample or as specified for each validation parameter. To assess the accuracy and resilience of the developed approach, the relative standard deviation (%RSD) was calculated. Utilising linear regression analysis, the calibration curve and correlation coefficient (R²) were established.

Ethics Statement

This study did not require ethical approval because it solely used commercially available pharmaceutical formulations for analytical evaluation and did not use biological materials, animals, or human subjects.

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RESULTS AND DISCUSSION

Linearity

The new RP-HPTLC method exposed good linearity over the investigated (100–800 ng/spot) range with a correlation value (R²) of 0.9994, proving relationship between concentration and peak area as very much intact (Table 2).

Precision

The developed RP-HPTLC technique demonstrated good repeatability and moderate precision with %RSD values of 1.433 and 1.608 for intra-day and inter-day respectively both below the allowable limit of 2% (Table 3).

Accuracy

The assessment of accuracy of the devised method was led by using the standard addition method at concentrations of 50%, 100%, and 150%. The percentage recoveries varied from 98.17% to 100.08%, indicating that the technique satisfies the ICH Q2(R2) suggested acceptance requirements.

Robustness

With %RSD values ranging from 0.69% to 1.02% after slight intentional changes in chromatographic conditions, the new approach was confirmed to be robust. All the obtained values were below 2% exposing robustness and dependability of the method (Table 5).¹⁶

Assay

Commercial dabigatran capsules were successfully assayed using the suggested RP-HPTLC method, producing an assay value of 100.82% that is within the acceptable pharmacopeial limit of 98–102%. The method's usefulness for regular pharmaceutical quality control is confirmed by these results (Table 6).

RAPI Tool Evaluation

The devised RP-HPTLC method achieved 72.5 as score from RAPI (Figure 12), indicating good analytical performance with favourable environmental sustainability, supporting its suitability for routine pharmaceutical analysis.

BAGI Tool Evaluation

With a BAGI score of 80.0 (Figure 12), the developed method showed good operational efficiency, analytical applicability, and suitability for regular quality monitoring of pharmaceutical formulations containing dabigatran etexilate.

AGREE Tool Evaluation

The suggested RP-HPTLC method's environmental sustainability was confirmed by an AGREE score of 0.81(Figure 12), which demonstrated outstanding concurrence with the standards of Green Analytical Methodology.

Forced degradation studies

The new RP-HPTLC approach successfully separated dabigatran etexilate from its break down products under acidic, alkaline, hydrolytic, peroxide, thermal, and UV conditions, according to forced degradation studies, validating the method's capacity to indicate stability. Table 7 displays the % degradation and matching Rf values found under each stress situation. Figures 7–11 display representative chromatograms produced under the different stress settings.

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CONCLUSION

For dabigatran etexilate’s quantitative determination in pharmaceutical formulations, a quick, reproducible, precise, robust, stability-indicating, and eco-friendly RP-HPTLC approach was successfully initiated and validated. According to ICH Q2 (R2) validation rules, the method exposed outstanding linearity, precision, accuracy, robustness, sensitivity, and assay performance. Additionally, the suggested method's environmental sustainability and practical application were validated by greenness assessment utilising RAPI, BAGI, and AGREE. Thus, routine quality control, stability investigations, and sustainable pharmaceutical analysis can all benefit from the suggested approach.

Acknowledgement

The management and authorities of Acharya Nagarjuna University, Guntur, and Chalapathi Drug Testing Laboratory, Lam, Guntur, are acknowledged by the authors for their encouragement and help in conducting the research.

Funding

The research, writing, and publication of this paper were not funded by the authors.

Conflict of Interest

No conflict of interest was declared by authors.

Author Contributions

K.E. Pravallika planned and supervised the study. D. Mounika performed the experimental work, and manuscript drafting. Ravi Parimi contributed to data collection, data analysis and data interpretation. All the authors reviewed and gave their consent for the final manuscript.