Method Development and Validation for the Assay of the Anticoagulant dosage form by HPLC
Kailash Pati Pandey*, K Saravanan
Bhagwant University, Sikar Road, Ajmer, Rajasthan, India.
*Corresponding Author E-mail: kailashpatipandey20@gmail.com
ABSTRACT:
Anticoagulants are class of dosage form used in treatment of thrombotic disorders, which prolong the clotting time. This study includes quantifying Edoxaban in solid oral dosage form. An accurate RP-HPLC method was developed to determine Edoxaban content with specific stability indicating method. Edoxaban well resolve with C18 column using isocratic elution with acetonitrile and Triethylamine buffer (pH 5.5), at a flow rate of 1 mL/min appeared at retention time about 3.5 min and was quantified at its λmax (290nm). It exhibited a accuracy and linearity over the concentration range of 14.910µg/ml to 89.460µg/ml of the test concentration. Forced degradation study was performed to prove the specificity of the method and degradation was achieved and proven stability indicating method. The developed method has been validated inline to ICH guidelines and found to be simple, precise, linear and accurate. Thus the proposed RP-HPLC method was used in routine quality control analysis for content determination.
KEYWORDS: Anticoagulant, RP-HPLC, Method development, Validation, Forced degradation.
INTRODUCTION:
Edoxaban: (class: Novel Oral Anti-Coagulants (NOACs) (1); IUPAC name: N- (5-Chloropyridin-2-yl)-N′-[(1S,2R,4S)-4-(N,N-dimethylcarbamoyl)-2-(5-methyl-4,5,6,7 tetrahydro[1,3]thiazolo[5,4-c]pyridine-2-carboxamido)cyclohexyl] oxamide; used to treat the people with atrial fibrillation (a heart rhythm disorder)1 to lower the risk of stroke caused by a blood clot.) In addition, edoxaban is indicated following hip or knee replacement surgery to prevent deep vein thrombosis (DVT)2, a specific type of blood clot, which can result in blood clots in The lungs (pulmonary embolism)
During the process of development of the drug product in generic pharmaceutical industries, development of an accurate and efficient analytical method for determining the quality of the product is a key activity3.
Fig. 1: The structure of Edoxaban.
Development of Analytical Method for Quantitative Determination of Edoxaban Tablets 60 mg using C18 column4. In addition, Edoxaban is yet to be official in any of the pharmacopoeia. Therefore, a highly selective, rapid, and stability-indicating RP- HPLC method should be developed5.
Method Development:
Edoxaban Tablets content can be successfully determined using RP-HPLC which is found to be the most sensitive and repeatable method available. Various columns from different makes were tried, but the Inertsil ODS column1 showed the most suitable results in a peak shape, resolution, and reproducibility. The significant work on creating a method of lower run time. From 50% level to 150% level, to achieve the recovery (95%-105%). The buffers used in the Water and Acetonitrile mixture were different. The TEA was optimized at 0.05 % with Water: Acetonitrile in the ratio 65:356.
MATERIALS AND METHODS:
Acetonitrile, hydrochloric acid, sodium hydroxide, and hydrogen peroxide (all of AR grade) were purchased from Merck (India). Analyses were performed using HPLC grade water (Millipore Inc., USA).
Instrumentation and Analytical Condition:
Chromatographic condition WATERS HPLC Auto Sampler separation photodiode array detector7 Chromeleon-software was carried. The chromatographic separation was performed using Inertsil ODS -3 5µm C18 100 Å (100 × 4.6 mm) HPLC column and the sample compartment temperature was maintained at 35°C. Mixture of 2000 ml of water and 1.0 ml Triethylamine, adjusted the pH to 5.5 with Orthophosphoric acid, filtered through 0.45 μm membrane filter (0.45 μ, Millipore) and degassed in sonicater8.
A premixed mobile phase consisting of 65:35 acetonitrile and buffer (pH 5.5). To check the peak purity of the stressed sample photodiode array detector was used.
Preparation of Analytical Solutions:
The diluent for the preparation of the analytical solution of 2h was a mixture of 65:35 (v/v) of water and acetonitrile. Separation of the blank, and six replicates of standard and single test solutions (60µg/mL) was done separately under the optimized chromatographic conditions9.
RESULTS AND DISCUSSION:
Development and Optimization of Chromatographic Conditions: The objective of method development was to quantify the Edoxaban Method development trials were conducted on different stationary phases and quantity of Organic/inorganic buffer, pH of the mobile phase, sample temperature and column oven temperature. Satisfactory resolution achieved, and good peak shape of Edoxaban was observed on Inertsil ODS -3 5µm C18 100 Å (100 × 4.6 mm) HPLC with column flow rate 1.0 ml/min, wavelength 290 nm, and column oven temperature 35°C. The mobile phase was consisting 1.0 ml of Triethylamine mixed 2000 ml of water and pH was adjusted to 5.5 with Orthophosphoric acid mixed with acetonitrile in isocratic elution mode. The column efficiency/theoretical plates should be more than 2000, and tailing factor should be not more than 2.0 and %RSD for six replicate injections of standard solution should be not more than 2.0% were finalized as system suitability criteria.
Method Validation:
Specificity: Specificity is the method ability to quantitate of the analyte response in the presence of their degradation impurities and the matrix. Stress testing of drug product can aid in identifying degradation products that form during stability studies, which can be used to establish degradation pathway and inherent stability of the molecule and validate stability-indicating capability of the analytical method used.
The specificity of the developed RP-HPLC method for Edoxaban Tablets the in presence of its degradation impurities and placebo matrix. Forced degradation studies were also conducted on Edoxaban tablets to give an insight into the stability indicating property and specificity of the proposed method. The degradation analysis includes exposing the sample to different stress conditions like heat (105˚C for 7 days), acid hydrolysis (2M HCl at 60 °C for 120 minute), base hydrolysis (5 M NaOH for 2hrs at room temperature) and oxidation (30%v/v H2O2 for 24 hrs at room temperature). Both alkali solution and acid solution exhibited substantial degrading property as illustrated in Fig. 3, 4. Photodiode array (PDA) detector was used to confirm the Edoxaban peak homogeneity and purity in all stressed sample solutions. These results of forced degradation studies are given in Table 1.
Fig. 2: The sample chromatogram
Fig. 3: The placebo chromatogram
Force degradation study:
The force degaradation study was tabulated below which shows stability indicating method for Edoxaban Tablets.
Table 1: Forced degradation study results of Edoxaban Tablets
|
Stressed condition |
% Assay |
% Degradation |
Purity Match |
|
Control |
99.9 |
NA |
1000 |
|
Acid hydrolysis (2M HCl at 60 °C for 2 hrs. |
93.4 |
6.5 |
1000 |
|
Base hydrolysis(5 M NaOH for 2 hrs at room temperature) |
94.8 |
5.1 |
1000 |
|
Oxidation (30%v/v H2O2 for 24 hrs at room temperature) |
99.6 |
0.3 |
1000 |
|
Thermal degradation (105˚C for 2 days) |
99.8 |
0.1 |
1000 |
Fig. 3: The Acid hydrolysis degradation chromatogram
Fig.4: The Base hydrolysis degradation chromatogram
Fig.4: The Oxidation degradaation chromatogram
Fig.5: The Thermal degradaation chromatogram
Linearity:
The Edoxaban is linear over the range from 14.910 µg/ml to 89.460 µg/ml Correlation coefficient found for Edoxaban is 1.0000.
Table. 2: Linearity results
|
Linearity Level |
Concentrations |
Area Response |
|
1 |
14.910 |
232277 |
|
2 |
29.82 |
464155 |
|
3 |
59.64 |
928709 |
|
4 |
71.568 |
1114541 |
|
5 |
89.460 |
1393164 |
|
Correlation Coefficient |
1.0000 |
|
|
Slope (M) |
15573.5760 |
|
|
Intercept (y) |
-69.9832 |
|
|
Statistical Y-Intercept |
0.0000 |
|
Fig. 6: The Linearity graph
Precision:
The precision of the method is the degree of agreement between the results. The precision of the method was studied for system precision, method precision, and intermediate precision. A standard solution of Edoxaban was injected six times to determine the system precision of the method, and %RSD was calculated for Edoxaban in table 3. Method precision study shows relative standard deviation of result for Edoxaban are 0.3% and Intermediate precision study shows relative standard deviation of result for Edoxaban are 0.5 % respectively table 4.
Table. 3: System Precision results
|
Sl. No. |
Standard Area Response |
|
1 |
924898 |
|
2 |
925676 |
|
3 |
924923 |
|
4 |
926798 |
|
5 |
924711 |
|
6 |
924731 |
|
Average |
925290 |
|
STD Deviation |
819.6504 |
|
% RSD |
0.1 |
Table. 4: Method Precision and Intermediate Precision results
|
Sl. No. |
Method precision |
Intermediate precision |
|
|
Assay (%) |
|
|
1 |
99.9 |
99.2 |
|
2 |
100.1 |
99.5 |
|
3 |
99.3 |
100.2 |
|
4 |
99.9 |
99.7 |
|
5 |
99.6 |
99.0 |
|
6 |
100.2 |
100.0 |
|
Average |
99.8 |
99.6 |
|
STD Deviation |
0.3327 |
0.4604 |
|
% RSD |
0.3 |
0.5 |
Table 5 Accuracy
|
Accuracy Level |
Wt. Taken (mg) |
mg Spiked |
mg recovery |
% Recovery |
Avg % Recovery |
% RSD |
|
50 |
59.58 |
59.52 |
59.3 |
99.7 |
99.3 |
0.3 |
|
50 |
60.55 |
60.49 |
60 |
99.2 |
||
|
50 |
59.89 |
59.83 |
59.3 |
99.1 |
||
|
100 |
119.92 |
119.8 |
119.9 |
100.1 |
100.0 |
0.5 |
|
100 |
121.24 |
121.12 |
120.5 |
99.5 |
||
|
100 |
119.76 |
119.64 |
120.1 |
100.4 |
||
|
100 |
119.48 |
119.36 |
118.6 |
99.4 |
99.6 |
0.6 |
|
100 |
121.33 |
121.21 |
120.1 |
99.1 |
||
|
100 |
119.98 |
119.86 |
120.1 |
100.2 |
||
|
150 |
181.21 |
181.03 |
180.2 |
99.5 |
99.7 |
0.4 |
|
150 |
180.22 |
180.04 |
178.9 |
99.4 |
||
|
150 |
180.33 |
180.15 |
180.3 |
100.1 |
||
|
Overall |
99.6 |
0.5 |
||||
Accuracy (Recovery):
The accuracy of the method for Edoxaban was determined by analyzing Edoxaban sample solutions at three different concentration levels of 50% to 150%. The recovery of all these was found to be in between the predefined acceptance criterion of 95.0% - 105.0%.
Stability of Analytical Solution:
The solution stability study of Edoxaban, after 48 h at 25 °C and 2-8 °C temperature and no continuous increasing or decreasing trend was observed in the % content of Edoxaban.
Robustness:
The method was robust for ± 10% variation in flow rate of mobile phase, ± 5°C variation in column oven temperature, ± 1% absolute organic phase variation (mobile phase-B) in isocratic program variations. The effect of column temperature on the resolution was studied at 30 °C and 40 °C instead of 35 °C. It indicating the robustness of the method.
CONCLUSION:
A rapid, specific, sensitive and precise reverse-phase HPLC method for the quantitative determination of process-related and degradation impurities of Edoxaban, an anticoagulant drug, is described. The developed RP-HPLC method was successfully applied to the analysis of Edoxaban drug substances. A forced degradation study was carried out under acidic, alkaline, peroxide, and thermal conditions to demonstrate the stability-indicating nature of the developed RPHPLC method. The developed method was validated as per ICH guidelines and specific, precise, sensitive and robust. The developed and validated method is stability indicating method that can be used to analyze routine and stability samples of Edoxaban Tablets dosahe form.
CONFLICTS OF INTEREST:
The authors declare no conflict of interest.
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Received on 17.05.2025 Revised on 13.09.2025 Accepted on 20.11.2025 Published on 27.01.2026 Available online from February 02, 2026 Asian Journal of Pharmaceutical Analysis. 2026; 16(1):9-13. DOI: 10.52711/2231-5675.2026.00002 ©Asian Pharma Press All Right Reserved
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