Introduction

Dabigatran is an anticoagulant which is used to treat and prevent blood clots. The FDA approved Dabigatran etexilate mesylate on October 19, 2010. The mechanism of action involves binding to thrombin: dabigatran reversibly binds to the active site on the thrombin molecule, preventing thrombin-mediated activation of coagulation factors. Fibrin-bound inactivation: Even when thrombin is fibrin-bound, dabigatran can still inactivate it. This property reduces thrombin-mediated inhibition of fibrinolysis, potentially enhancing fibrinolysis. This prodrug degrades in the liver and plasma cells to its active form in the presence of esterase .. Thrombin is quickly and reversibly suppressed competitively by DAB.^1-3

A thorough review of the literature found that few analytical techniques are available for determining DAB using UV spectroscopy, RP-HPLC, or HPLC.^4-8

Figure 1: Structure of DABClick here to view Figure

Ultraviolet-visible (UV-Vis) spectroscopy is a commonly used technique in many scientific fields, from chemical research and quality control in the beverage industry to bacterial culture, medication identification, and nucleic acid purity checks and quantification ^9-12.  Based on the UV-visible spectrophotometer instrumentation, the intensity of light can be reasonably expected to be quantitatively related to the amount of light absorbed by the sample. A single beam spectrophotometer measures the intensity of light before and after it passes through the sample.¹³ A double beam spectrophotometer splits the light into two beams: one passes through the sample, and the other passes through a reference.¹⁴

A biological assay of plasma with a drug typically involves measuring the concentration of the drug in plasma to assess its pharmacokinetics, bioavailability, and therapeutic efficacy. Bioanalytical method validation (BMV) is a critical process in pharmaceutical research and development. It ensures that the methods used to measure drugs and their metabolites in biological matrices (like blood or urine) are reliable, accurate, and consistent. The validation process typically involves evaluating parameters such as accuracy, precision, specificity, linearity, and stability.¹⁵

Biological sample extraction techniques are essential for isolating specific components from biological samples for analysis. This method involves adding a precipitating agent (like acetonitrile or methanol) to the sample to denature and precipitate proteins, leaving the analytes in the supernatant.

Chromogenic assays are detection methods that rely on a color change to obtain qualitative and quantitative results. These assays are widely used in biochemical and pharmaceutical research. A chromogen is a chemical compound that can be converted into a colored compound, or chromophore, through a chemical reaction. Chromogens can be colorless or have a faint color.¹⁶

Materials and methods

Dabigatran etexilate mesylate standard drug, Sodium Nitrite, Ammonium Sulphmate, 1-Naphthol, Ethanol, Acetone, Plasma procured from the blood bank.Instruments used for analysis: Double Beam UV-visible spectrophotometer ELICO SL-210, Centrifuge REMI R-4C.

Preparation of Standard Stock Solution (1000µg/ml)

Weighed accurately 10mg of pure DAB and transferred it into a 10ml volumetric flask. Dissolved the drug with ethanol and make up the volume up to the mark. The resultant solution of standard stock was 1000µg/ml.

Preparation of Working Standard Solution (100µg/ml)

Pipetted out 1ml of stock solution into 10 volumetric flasks and marked up to the volume with ethanol. The resultant solution of the working standard was 100µg/ml.

Preparation of Standard solution (10 µg/ml)

Pipetted out 1.0ml from working standard solution into 10ml volumetric flask and made up to the mark with ethanol.

Preparation of Spiked sample and Extraction

1 ml of plasma was pipetted out into a test tube and 1 ml of 10ppm drug was added to the above plasma sample. Then 2 ml of acetone was transferred to the test tube. The solution was vortexed for 1min. After vortexing the contents were transferred into a centrifugation tube at 14,000rpm for 10 minutes. The supernatant layer was collected into a 10-ml vol flask.

Preparation of blank plasma

1ml of plasma was measured and transferred to a 10ml volumetric flask followed by the addition of 9ml of ethanol.

Preparation of 2M HCl

Pipetted out 4.3ml of conc. HCl (37%) into a 25ml volumetric flask made up of water up to the mark. The resultant solution was found to be 2M HCl.

Preparation of 0.025% Sodium Nitrite

We weighed accurately 25mg of Sodium Nitrite and transferred it into a 100 volumetric flask. Dissolved with water and  made up to the mark with water. The resultant solution was found to be 0.025% Sodium Nitrite.

Preparation of 0.5 % Ammonium Sulphamate

We weighed accurately 0.5g of Ammonium Sulphamate in 100 ml of the volumetric flask, diluted with water, and the remaining volume was brought up to mark with diluent. The resultant solution was to be 0.5% Ammonium Sulphamate.

Preparation of 0.250% 1-Naphthol

Weighed accurately 250mg of 1-Naphthol and diluted it with ethanol and the remaining solution was brought up to the mark with ethanol in a 100 ml volumetric flask. The resultant solution found was to be 0.250%1-Naphthol.

Preparation of Reagent Blank

1ml of plasma was measured and transferred to a 10ml volumetric flask followed by the addition of 1ml of HCl, 1ml of Sodium Nitrite, 1ml of Ammonium Sulphamate, and 1ml of 1-naphthol in 10ml volumetric flask. Water was added to the remaining solution in a 10ml volumetric flask.

Quantification Of Dabigatran in Spiked Plasma Formulation in Bulk Samples Using 1 – Naphthol

 Extraction Procedure

The bioanalytical method used a protein precipitation extraction procedure.^17-22 Plasma was taken from the refrigerator and allowed to thaw at room temperature on the analysis day. 1 ml of Human plasmawas taken into a test tube and 1 ml of 10ppm drug was added to the above plasma sample. Then 2 ml of acetone was transferred to the test tube. The solution was vortexed for 1min. After vortexed the contents were transferred into a centrifugation tube at 14,000rpm for 10 minutes. The supernatant layer was collected into a 10-ml vol flask. 2 ml of the supernatant solution was transferred to a 10ml volumetric flask. 1 ml of 2MHCl and 1 ml of Sodium Nitrite (0.025%) were added to the above solution. After 2mins 1ml of Ammonium Sulphamate (0.5%) was added to the flask. After 3mins 1ml of 1-Naphthol (0.25%) was added. The solution was brought up to the mark with ethanol. ^23-29 The resultant solution was scanned in the 400–800 nm range using UV–visible spectroscopy against a reagent blank. It has been found that the optimal wavelength, which corresponds to green chromogen, was 449 nm. ^30-32 The recovery studies were found to be 98.24%.

Validation Parameters as Per M10 Guidelines^33-39

Selectivity

Selectivity is the capacity of an analytical technique to recognize and quantify an analyte in the presence of other chemicals or any substance that disturbs the biological matrix.

 Specificity

The ability of a bioanalytical approach to differentiate between and recognize products made from various materials, particularly related molecules, is known as specificity.

Matrix effect

A change in the analyte reaction caused by intervening and sometimes undetectable components in the sample matrix is known as a matrix effect.

Preparation of low QC (5ppm) and high QC (45ppm)

Three different 10 ml vol flasks were pipetted with 0.5 ml and 4.5ml of the standard DAB solution and 1ml of plasma from three different sources, the drug solution was received after centrifugation. The supernatant layer was collected and three replicates were prepared for each concentration followed by reagents added. The solution was made up to the mark with water. UV-visible spectroscopy was used to scan the resulting solution at 449 nm against the corresponding reagent blank.

Calibration curve

The calibration curve illustrates the relationship between an analyte’s real concentration and the testing platform’s reaction to it. The calibration curve is made up of the calibration standards, which are created by combining a certain quantity of analytes or analytes into a matrix.

 Procedure for establishing of Calibration curve

A series of 10 ml volumetric flasks were filled with aliquots of the standard drug solution of DAB in the following sizes: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5 ml. Each 10ml vol flask received a spike of 1ml plasma. They added 1 ml of 2MHCl, 1 ml of 0.5% ammonium sulfamate, 1 ml of 0.02% sodium nitrite, and 1 ml of 0.25% 1-naphthol. Ethanol makes up the difference in the solution. The equivalent reagent blank was used to compare the green chromogen of dab absorbance at 449 nm. The calibration curve’s linearity (absorbance vs. concentration) for dab was examined in pure solution using concentration ranges of 5, 10, 15, 20, 25, 30, 35, 40, and 45 µg/ml.

Accuracy and Precision

Accuracy and precision should be assessed using identical runs and data.

Preparation Accuracy and Precision samples

A series of 10 ml volumetric flasks were filled with aliquots of the standard drug solution of DAB in the following sizes: 0.5 ml, 1.5 ml, 3.0 ml, and 4.0 ml. Each 10ml vol flask received a spike of 1ml plasma. The drug solution was received after centrifugation. The supernatant layer was collected followed by reagents added. The solution was made up to the mark with water. The LLQC, LQC, MQC, and HQC were observed and recorded in the results. UV-visible spectroscopy was used to scan the resulting solution at 449 nm against the corresponding reagent blank. The mean ± standard deviation was determined for five QC samples.

Stability

Stability studies must be conducted to make sure that choices regarding sample preparation, production, analysis, and storage conditions do not affect the analyte concentration. The material’s endurance in the matrix is evaluated using QCs at both low and high concentrations.

Benchtop stability

Both high and low QCs should be thawed on the tabletop for the same amount of time and at the same temperature as the research materials.

Preparation of low QC (5µg/ml) high QC (45µg/ml) sample

A 10 ml vol flask that had been previously spiked with plasma was filled with 0.5 ml and 4.5ml of the standard drug sol (100µg/ml). The supernatant layer was collected followed by reagents were added for each concentration. The remaining volume was filled with water to reach the mark. For a longer period, the three low-concentration replicas were created and kept in a freezer at -20°C. A UV scan of the solution at 449 nm was performed after a specific amount of time to document any changes.

 Reinjection reproducibility

Following storage, a run that includes a minimum of five replicas of all medium, low, and high QCs as well as calibration standards is reinjected to evaluate injection repeatability. The viability of the treated samples is determined by the reinjected QCs’ accuracy as well as precision.

Preparation of low QC (5µg/ml) and high QC (45µg/ml) sample

A 10 ml vol flask that had been previously spiked with plasma was filled with 0.5 ml and 4.5ml of the standard drug sol (100µg/ml). The supernatant layer was collected followed by reagents were added for each concentration. The remaining volume was filled with water to reach up to the mark. UV-visible spectroscopy was used to scan the resulting solution at 449 nm against the corresponding reagent blank. The samples were reinjected to know any deviations in the prepared sample for analysis.

Validation Parameters for UV Spectroscopy in Spiked with Plasma as Per M10 Guidelines

Bioanalytical Extraction Procedure

The bioanalytical method used a protein precipitation extraction procedure. Plasma was taken from the refrigerator and allowed to thaw at room temperature on the analysis day. 1 ml of Human plasmawas taken into a test tube and 1 ml of 10ppm drug was added to the above plasma sample. Then 2 ml of acetone was transferred to the test tube. The solution was vortexed for 1min. After vortexed the contents were transferred into a centrifugation tube at 14,000rpm for 10 minutes. The supernatant layer was collected into a 10-ml vol flask. 2 ml of supernatant solution was transferred to a 10-ml volumetric flask. The solution was brought up to the mark with ethanol. The resultant solution was scanned in UV spectroscopy against a blank at 325nm. ^32-39

Calibration curve

Procedure for establishing of calibration curve

A series of 10 ml volumetric flasks were filled with aliquots of the standard drug solution of DAB in the following sizes: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 ml. Each 10ml vol flask received a spike of 1ml plasma. The calibration curve’s linearity (absorbance vs. concentration) for dab was examined in pure solution using concentration ranges of 5, 10, 15, 20, 25, 30, 35, and 40 µg/ml.

Accuracy and Precision

Preparation Accuracy and Precision samples

A series of 10 ml volumetric flasks were filled with aliquots of the standard drug solution of DAB in the following sizes: 0.5 ml, 1.5 ml, 3.0 ml, and 4.0 ml. Each 10ml vol flask received a spike of 1ml plasma. The drug solution was received after centrifugation. The supernatant layer was collected. The solution was made up to the mark with water. The LLQC, LQC, MQC, and HQC were observed and recorded in the results. UV spectroscopy was used to scan the resulting solution at 325 nm against the corresponding blank. The mean ± standard deviation was determined for five QC samples.

Matrix effect

Preparation of low QC (5ppm) and high QC (45ppm)

A 10 ml volumetric flask was pipetted with 0.5 ml and 4.5ml of the standard DAB solution and 1 ml of plasma, the drug solution was received after centrifugation. The supernatant layer was collected and three replicates were prepared. The solution was made up to the mark with water. UV-visible spectroscopy was used to scan the resulting solution at 325 nm against the corresponding blank.

Bench-top (short-term) stability

Preparation of low QC (5µg/ml) and high QC (45µg/ml) sample

A 10 ml vol flask that had been previously spiked with plasma was filled with 0.5 ml and 4.5ml of the standard drug sol (100µg/ml). The supernatant layer was collected. The remaining volume was filled with water to reach the mark. For a longer period, the three low-concentration replicas were created and kept in a freezer at -20°C. A UV scan of the solution at 325 nm was performed after a specific amount of time to document any changes.

Reinjection reproducibility

Preparation of low QC (5µg/ml) and high QC (45µg/ml) sample

A 10 ml vol flask that had been previously spiked with plasma was filled with 0.5ml and 4.5ml of the standard drug sol (100µg/ml). The supernatant layer was collected. The remaining volume was filled with water to reach up to the mark. UV-visible spectroscopy was used to scan the resulting solution at 325nm against the corresponding blank. The samples were reinjected to know any deviations in the prepared sample for analysis.

Results

Validation parameters of dabigatran in spiked plasma as per M10 guidelines

Calibration curve

Table 1: Calibration range of DAB in spiked plasma with ethanol

Figure 2: Calibration Curve of DAB spiked with plasma at 449nmClick here to view Figure

Figure 3: Calibration Curve of DAB spiked with plasma at 325nmClick here to view Figure

Precision

Table 2: Between-run precision data of dabigatran for the day-1 and 2 at 449nm

 Table 3: Between-run precision data of dabigatran for the day –1 and 2 at 325nm

Standard deviation (SD) = S=∑(x_i−x)² / n−1

%RSD = (standard deviation / |mean|) * 100%

Accuracy and precision were validated as per M10 guidelines. The accuracy at each concentration level should be within ±15% of the nominal concentration, except at the LLOQ, where it should be within ±20%. The precision (%CV) of the concentrations determined at each level should not exceed 15%, except at the LLOQ, where it should not exceed 20%.

Accuracy

Table 4: Within-run precision data of dabigatran for each run at 449nm.

 Table 5: Within-run precision data of dabigatran for each run at 325nm.

 Matrix effect

Table 6: Matrix effect of dabigatran with Low QC and High QC sample at 449nm

Table 7: Matrix effect of dabigatran with Low QC and High QC sample at 325nm

Benchpoint stability studies

Table 8: Stability study of dabigatran at LQC (5ppm) and HQC (45ppm) at 449nm.

 Table 9: Stability study of dabigatran at LQC (5ppm) and HQC (45ppm) at 325nm

Reinjection reproducibility

Table 10: Reinjection reproducibility data at 449nm and 325nm

 Discussion

Chromatography and UV-visible spectroscopy methods both demonstrate strengths in the quantitative determination of apixaban, dabigatran, rivaroxaban, edoxaban, and their metabolites in biological samples. The chromatographic method provides a broad calibration range of 10–750 µg/mL, which is suitable for diverse concentrations, along with superior accuracy (>92%) and precision (<3.7% and <3.6%), ensuring high reliability and reproducibility. In contrast, the UV-visible spectroscopy method offers a narrower calibration range of 5–45 µg/mL, with comparatively lower accuracy and precision (11–15%), limiting its application in critical analyses. However, spectroscopy excels in stability and matrix effect studies (below 15%), combined with minimal solvent consumption, making it an eco-friendly and cost-effective choice. While chromatography stands out for its high sensitivity and applicability in complex analyses, spectroscopy is a practical alternative for routine tasks, emphasizing simplicity and efficiency. The bioanalytical method shows adequate accuracy, precision, stability, and minimal matrix effects, making it appropriate for the intended applications.

Conclusion

A validated UV-visible spectrophotometric method has been developed for quantifying dabigatran etexilate mesylate (DAB) in human plasma and pharmaceutical dosage forms. The method demonstrates reliability and practicality, meeting all regulatory requirements for selectivity, sensitivity, linearity, accuracy, precision, and stability. Its advantages include low solvent concentration, lack of extraction stages, specificity, sensitivity, and cost-effectiveness, making it suitable for regular use in quality control applications. Despite slightly elevated %RSD values compared to typical HPLC methods, the technique remains within acceptable limits for bioanalytical applications.

Acknowledgement

The author would like to thank RBVRR women’s college of pharmacy for granting the Master’s research work. The author is profoundly grateful to the principal and head of the department of pharmaceutical analysis for their guidance during the research procurement.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest-

The authors do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

Informed Consent Statement-

This study did not involve human participants, and therefore, informed consent was not required.

Clinical Trial Registration

This research does not involve any clinical trials.

Permission to reproduce material from other source

Not Applicable 

Author Contributions

Khagga Bhavyasri: Conceptualization, Supervision, Project Administration.

Thandra Ambika: Data Collection, Methodology, Writing-Original Draft, Review & Editing

Mogili Sumakanth: Funding Acquisition, Resources. 

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Abbreviations

Dabigatran etexilate mesylate (DAB), Ultra violet visible (UV) spectroscopy, Reverse phase high performance liquid chromatography (RP-HPLC), High performance liquid chromatography (HPLC), Hydrochloric acid (HCl), standard deviation relative (RSD), Quality control (QC), Limit of Quantification (LOQ), Limit of Detection (LOD).