RP-HPLC Method Development and Validation of Dissolution Method for Anticoagulant Dosage Form

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;

utilized for treating individuals with atrial fibrillation (a heart rhythm disorder) (1). Furthermore, edoxaban is prescribed after hip or knee replacement surgery to avert deep vein thrombosis (DVT) (2), a particular kind of blood clot that may lead to clots in the lungs (pulmonary embolism). During the development of the drug product within generic pharmaceutical industries, creating a precise and effective analytical method for assessing the quality of the product is a crucial task (3,4). Edoxaban is available in an immediate release tablet form, with a half-life of 10-14 hours, and it attains its peak plasma concentration within 1-2 hrs.

Fig. 1: The structure of Edoxaban.

Dissolution is considered one of the most important quality control tests performed on pharmaceutical dosage form and is now developing into a tool for predicting bioavailability, and in some cases, replacing clinical studies to determine bioequivalence. Dissolution behaviour of drug has a significant effect on their pharmacological activity.

Fig. 1: The flow of dissolution process

METHOD DEVELOPMENT

Chromatographic method for Edoxaban Tablets was successfully developed to determine the content of Edoxaban using RP-HPLC. Objective was to develop on suitable method with shorter runtime, reproducible, rugged and simple which can be intended for routine analysis and can be successfully validated as per ICH guideline (5, 6). Below trials were taken to optimise the method:

• Various columns from different makes, different dimension was tried
• Buffers with variable pH
• Buffer and organic composition varied
• Flow rate varied
• Variable Column oven temperature tried

Based on the above outcomes optimal chromatography was concluded with standard and test concentration of 66.6 µg/mL. Method found capable of quantify the content at the stated concentration.

MATERIALS AND METHODS 

Acetonitrile, potassium dihydrogen phosphate, Sodium hydroxide, DM water Triethylamine  (all of AR grade) HPLC grade water (Millipore Inc., USA), Sonicator.

Analytical Condition and Instrument Methodology

Buffer preparation: Added 1 mL of Triethylamine in 2000 mL of millli Q water and mixed well. pH of the buffer was adjusted to 5.50.

Mobile Phase Preparation: Mixed well buffer and acetonitrile in the ratio of 65:35 v/v and degassed by sonication.

Diluent preparation: Mixed well water and acetonitrile in the ratio of 50:50 v/v and degassed by sonication.

Standard preparation: Standard stock was prepared using diluent and further dilution was made by dissolution medium to make the concentration 66.6 µg/mL.

Chromatographic condition:

The chromatographic separation was performed using Waters HPLC 2695 Alliance System, photodiode array detector, Chromeleon-software was used as CDS.

Selection of dissolution media:

The solubility of a drug is crucial in regulating its dissolution from the dosage form. Compounds that possess high solubility typically demonstrate increased dissolution rates. The aqueous solubility of a drug is a significant determinant of its dissolution rate. Consequently, it is essential to evaluate the aqueous solubility of the drug substance across the physiologically relevant pH range of 1.2-7.5 to anticipate the impact of solubility on dissolution. To understand the solubility profile saturation solubility at controlled temperature was done.

Table. 1: Saturation solubility

The Biopharmaceutical Classification System serves as a scientific framework for categorizing drug substances according to their aqueous solubility and inherent permeability. When combined with the dissolution characteristics of drug products, the BCS considers three primary factors that influence the rate and extent of drug absorption from immediate-release solid oral dosage forms. Solubility, permeability, and dissolution and Edoxaban belongs to class IV.

Dissolution profile was generated throughout physiological pH and based on the reproducibility and discrimination pH 6.0 phosphate buffer selected as media. Different speed (rpm), basket, paddle was tried, based on the outcome 50rpm, paddle dissolution type was finalized.

Table. 2: Dissolution parameter

RESULTS AND DISCUSSION

The objective of method development was to determine release of Edoxaban in Edoxaban Tablets. Reversed-phase high-performance liquid chromatography (RP-HPLC) is a commonly employed separation technique where a non-polar stationary phase interacts with a polar mobile phase, leading to the separation of molecules with different hydrophobic properties. 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 no more than 2.0% were finalized as system suitability criteria (7-9).

Selection of dissolution media one of the key factors to evaluate the dosage form and developing an In vitro and in vivo correlation. In vitro research plays a vital role in preliminary assessments, recognizing possible drug candidates, and comprehending molecular mechanisms within a regulated setting. Subsequently, these results are frequently complemented by in vivo investigations using animal models or, ultimately, clinical trials involving humans to assess the overall effects on the body and any potential adverse effects. The oral bioavailability of medications is a critical factor that influences the outcome of drugs administered orally and is mainly dictated by the speed and degree of drug absorption through intestinal barriers (10, 11).

Method Validation:

Specificity:

Specificity is the ability of the method to assess unequivocally the analyte in the presence of components which may be expected to be present. The specificity of the developed RP-HPLC method for Edoxaban was determined in the presence of Blank and placebo. No interference observed at the retention time of Edoxaban from blank and placebo.

Fig. 2: The chromatogram of placebo

Fig. 3: The chromatogram of Test

Results obtained for the selectivity:

Linearity

The Edoxaban is linear over the range from 16.683 µg/ml to 100.100 µg/ml (About 25% level to about 150% of test concentration). Correlation coefficient found for Edoxaban is 0.9995.

Table. 3: Linearity of Edoxaban

Fig. 4: The Linearity plot

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. Method precision study shows relative standard deviation of result for Edoxaban are 1.4 % and Intermediate precision study shows relative standard deviation of result for Edoxaban are 1.1 % respectively.

Table. 4: Precision results

Acceptance criteria: % RSD should not be more than 5.0% 

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% of each in triplicate. The recovery of all these was found to be in between the predefined acceptance criterion of 95.0% - 105.0%.

Table. 5: Precision results

Acceptance criteria: 

Recovery at each, mean recovery and overall recovery should be in range 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 % release of Edoxaban.

The study was performed on the both standard and test solution at specific interval of time and observation are tabulated below.

Table. 6: Solution stability results of standard

Table. 7: Solution stability results of Test solution