Development and Validation of a Green RP-HPLC Method for Pharmaceutical Drug Analysis and Greenness Assessment of Edoxaban

The most commonly used method of analysis is high-performance liquid chromatography (HPLC)[1]. It is a quick, accurate, and simple analysis method [1], [2]. The goal of this study was to quantify and identify chemicals and contaminants in pharmaceutical products. With a high pressure of up to 400 atmospheres, HPLC is an enhanced form of column chromatography [3]. RP-HPLC is a popular technology that is rapid and precise. The stationary phase is composed of highly nonpolar materials, such as C18, C12, or C8 columns [4], [5]. Polar liquids, such as water, acetonitrile, and methanol, are used in the mobile phase. Since most materials are polar, they elute quickly in a polar mobile phase [6].

The guidelines for validating the HPLC method were created in accordance with international regulatory bodies and were provided by the International Council of Harmonization (ICH) Q2 (R2). The standard values of each parameter are provided [7].

Edoxaban tosylate monohydrate belongs to the direct/novel oral anticoagulant (DOAC/NOAC) class [8], [9], [10], [11], [12]. Coagulation-related, selective factor Xa inhibitor. It is used in deep vein thrombosis (DVT), pulmonary embolism (PE), nonvalvular atrial fibrillation (NAVF), and systemic embolism (SE) to reduce complications. The antagonist of edoxaban is vitamin K [12].

Fig 1 Structure of Edoxaban

The Edoxaban structure contains a diamine group. which are biologically charged and protonated. Therefore, Edoxaban has a high polarity. The IV class designation of edoxaban indicates limited permeability and solubility [10]. The solubility of the medication in organic solvents, which are more environmentally friendly than others, was a major challenge when devising a new method for validating Edoxaban. This was the main motivation for this Green Chemistry procedure.

Anastas and Warner proposed 12 green chemistry tenets [13], [14], [15], [16], [17]. can minimize or reduce the use of elements that eventually cause environmental harm. A large amount of solvent should be employed during the technique development [15], [16]. Because ACN and MeOH are more polar and have longer wavelengths, they are frequently used as hazardous solvents. According to the solvent selection guide, ACN is categorized as a high-risk solvent [14]. It has detrimental effects on the environment and is extremely hazardous to the analysts [17].

The literature review revealed that only a few green methods have been developed for the determination of edoxaban. We created a system based on green principles that was accurate, precise, robust, linear, and compliant with the ICH Q2 (R2) guidelines. The selection of a highly polar and environmentally friendly solvent was crucial in method development.

Metric tools were available to assess the greenness of the method and provide scores that indicate the greenness of the approach. It has 12 principles [17].

MATERIALS AND METHOD

MATERIALS

 Edoxaban was  gifted by MORPEN Laboratories Ltd.. In India, SUPEXA OD 15 tablets were purchased from a local pharmacy. HPLC Grade water was purchased from Qualigens, Thermo Fisher Scientific, Mumbai, India. Methanol was purchased from Qualigens, Thermo Fisher Scientific, Mumbai, India. Ethanol was purchased from Changshu Song Sheng, China. Glacial acetic acid was purchased from Qualigens, Thermo Fisher Scientific, Mumbai, India. Propan-2-ol was purchased from Finar Limited, Ahmedabad, India. Ammonium formate was purchased from Qualigens, Thermo Fisher Scientific, Mumbai, India. Nylon syringe filters and nylon 6,6 membrane filters were purchased from Ultipor, Pall  Life Science, India.

INSTRUMENTS

HPLC System Agilent-HPLC system Model: 1100, Pump – Isocratic, DE43629962, Detector – UV detector, JP43826546,  Software – EZ Chrome Elite, Column - C₁₈ ( Thermo Scientific) (4.6 × 250 mm, 5μm), UV spectroscopy Shimadzu UV 1800 series(Shimadzu) UV-1700 Double beam with Software UV Probe 2.33, pH meter (Digisun electronics)  pH System 7007, UltraSonicator (Servewell Instrument)   RC-SYSTEM MU-1700,  Analytical balance (ESSAE) DS-852J Series (Micro Analytical Balance).

METHOD

Optimization of chromatographic condition

Table 1 Optimization of chromatographic condition

Solubility studies

Prior to developing a procedure, solubility studies should be performed. To identify the appropriate solvent, the solubility of the drug was tested in various solvents. Edoxaban is a BCS class IV medication that changes with pH and has poor solubility [18], [19]. It is somewhat soluble in water and ethanol and barely soluble in ACN [20]. The solubility increases as the pH decreases and vice versa [18], [19], [20].

Selection of mobile phase

Edoxaban in its pure form was injected into the HPLC system and run in various solvent systems. Each mobile phase was permitted to equilibrate with the stationary phase until a stable baseline was achieved. Various mobile phases, such as methanol: water, isopropyl alcohol: ammonium formate, and isopropyl alcohol: water: glacial acetic acid (GAA) were tested. To achieve a stable peak in each mobile phase, different ratios of Glacial Acetic Acid: Ethanol were tested, sonicated in an ultrasonic bath, and filtered through a 0.45 μm filter paper. Following trials, a final solvent combination of methanol, ethanol, water, and glacial acetic acid, in the ratio of 70:14.5:15:0.5, produced a sharp peak and good resolution.

Preparation of standard stock solution

Edoxaban tosylate standard (30 mg) was precisely weighed and transferred to a 100 ml volumetric flask. The mixture was shaken thoroughly after adding approximately 90 mL of diluent. The samples were sonicated them for two minutes, and the volume was adjusted accordingly. The final concentration of the stock solution was 300 μg/ml.

Preparation of standard solution

2 ml of the stock solution should be pipetted out, transferred to a 20 ml volumetric flask, diluted to the appropriate level with diluent, well shaken, sonicated for approximately two minutes, and filtered through a 0.2 µm syringe filter before injection. The standard solution's final concentration is 30 μg/ml.

Marketed Tablet Test Preparation

Ten SUPEXA OD 15 tablets were weighed individually, and each tablet was crushed using a mortar and pestle. Tablet powder (API) equal to the standard concentration was weighed, dissolved in 100 mL of diluent, and thoroughly shaken. After two minutes of sonication, the mixture was passed through a 0.2 µm membrane syringe filter.

Validation of Edoxaban

According to ICH guideline Q2 (R2), validation is a crucial step in demonstrating the accuracy, precision, linearity, specificity, system appropriateness, LOD, LOQ, and robustness of the method [7], [21]. The standard solution for the main parameter was 30 μg/ml. Three distinct concentrations of the reference solution were prepared at 80%, 100%, and 120% for accuracy.

Specificity

The method's specificity was proven when it was recorded and compared that there was no interference between the test sample and the standard. The tailing factor (T), resolution factor (Rs), and retention time (R) were examined. This study demonstrated that the developed approach was tailored for Edoxaban [22].

System suitability

It examines several factors, including the theoretical plate (S), tailing factor (T), resolution (RS), capacity factor (K), repeatability, and relative retention. quick injection of the reference solution was performed. when the ICH-compliant system suitability parameter was used [23].

Linearity

A curve was used to assess the linearity and range of the five levels of samples prepared: 15, 22.5, 30, 37.50, and 45 μg/ml. The edoxaban concentration was plotted on the x-axis, and the peak area was calculated on the y-axis. R² is known [7],[24].

Accuracy

This indicates the closeness of the mean test to the actual edoxaban concentration. In accordance with the ICH guidelines, the concentrations were measured at 80%, 100%, and 120% in triplicate to determine the mean of each concentration. The edoxaban test was performed [7].

Precision

Both intraday and interday precision were performed in accordance with ICH guidelines. Inter-day precision refers to the analysis of a sample at two separate times on two different days. Intraday precision was determined by analyzing the sample twice on the same day. The RSD was limited to 2% [25].

LOD and LOQ

The LOD is the lowest analyte concentration that can be detected but not quantified under certain conditions [22].The lowest analyte concentration that can be measured under specific conditions is known as the LOQ [7].

Robustness 

This slightly alters the chromatographic state. This change in chromatographic conditions was followed by the injection of a standard solution [7]. The mobile phase flow rate was varied by ± 2%. ± 2% variation in wavelength. The outcome should differ from the initial run by less than 2%.

RESULT AND DISCUSSUION

Selection of wavelength

The UV spectrum of 5 μg/ml edoxaban in methanol was scanned at wavelengths between 200 and 400 nm. The greatest absorption was found at 291 nm (fig. 2) for Edoxaban, and this wavelength was chosen for further operation.

Fig 2 Wavelength of Edoxaban at 291 nm.

Method development

To achieve efficient separation with a short run time and sharp peak in accordance with ICH guidelines, the number of solvent composition was examineds. to develop an environmentally friendly analytical technique. This method was developed in Trial 4.

Trial 1: In trial 1, the mobile phase was IPA and buffer (0.01 ammonium formate) in an 80:20 ratio. The column was Hypersil, C18 (250 mm × 4.5 mm × 5 μm), with a flow rate of 1.5 ml/min and a run time of 12 min. TP and TF did not meet the acceptance criteria. Fig 3

Fig 3 Trial 1

Trial 2: IPA, water, and glacial acetic acid were obtained in an 80:19:1 ratio. An Epic C12 column (250 × 4.5 × 5 μm) was used. The injection volume was 20 μl, the flow rate was 1 ml/min, and TP and TF did not meet the acceptance criteria. Fig. 4

Fig 4 Trial 2

Trial 3: In three tests, the mobile phase was methanol, ethanol, water, and glacial acetic acid in a ratio of 60:10:30:0.1; the column was Hypersil, C18 (250 mm × 4.5 mm × 5 μm); the flow rate was 1.5 min/ml, but it should have been 1.1 ml/min; the run time was 15 min; the peak was eluted at a longer retention time; TF was not found to be within the current conditions. Fig 5

Fig 5 Trial 3

Trial 4 : The chromatographic technique is finalized and optimized in trial 4. Methanol, water, ethanol, and glacial acetic acid in a ratio of 70:15:14.5:0.5 should be the mobile phase. Choose the Hypersil, C18 column (250 x 4.5 x 5 μm) with a flow rate of 1.5 ml/min, an injection volume of 20 μl, a run time of 7 minutes, and ambient temperature. Peak shape is good, and TP & TF (Asymmetry) are seen within acceptable standards. This approach has been devised, however it needs to be validated before being used. Fig. 6

Fig 6 Trial 4 final method development was selected.

Specificity

The chromatograms of the Edoxaban test solution, standard, and mobile phase blank were used to compare specificity [22], [25]. In the HPLC system, solutions were injected, and the chromatogram is shown in fig. The blank does not interfere with the Edoxaban peak's retention time in either the test or standard solution. Edoxaban retention times in the test and standard solutions match one another [26]. Therefore, specificity makes acceptable.

Fig7 Blank solution.

Fig 8 standard solution.

Fig 9 Test solution.

Linearity

Within a certain range, linearity yields results that are directly proportional to concentration. Create five levels of samples with varying concentrations, ranging from 50% to 150% [7], [26]. The approach is linear and linearity is supported because the linear regression equation and R2 were y = 893240x + 4000486 and R2 = 0.9998, respectively, and the correlation coefficient was found to be within the acceptance standards [22].

Fig 10 linearity curve

Table 1. linearity of Edoxaban.

Con. (ppm or μ g/ml)	Area
15.00	13838594
22.50	20313520
30.00	27354643
37.50	33979710
45.00	40502011
Correlation coefficient	NLT 0.995
	0.99992
Intercept	400486
Slope	893240

Accuracy

analytical technique to verify the drug's authenticity. This involves comparing the standard and theoretical concentrations obtained from the Edoxaban sample. In accordance with ICH guidelines, prepare three standard solution concentrations, such as 80%, 100%, and 120%, and determine the triple mean for each concentration [7], [21]. In the range of 97.70% to 99.92%, our analytical approach demonstrates a remarkable percentage recovery [25]. The procedure is accurate and the accuracy is justified because both the mean recovery and the %RSD of the recovery were observed within the acceptance requirements.

Table 2 Accuracy result of Edoxaban.

Precision

The ICH guidelines were followed while measuring intraday precision [7]. Obtain two sets in one day. The devised approach yielded a value of 0.41% RSD.

Table 3. Intra day Precision of Edoxaban.

Two times on two separate days, interday precision was measured. RSD was determined to be 0.84%.

Table 4 Inter day precision of Edoxaban.

Since the procedure is accurate in terms of repeated analysis in a single day, intraday and interday precision are justified [22], [25]. The overall percentage RSD for results of sets 1 and 2 done in a single day and two consecutive days was observed within acceptance requirements.

LOD and LOQ

0.56 μg/ml is the lowest limit of concentration (LOD) that can be detected but not quantified. 1.78 μg/ml is the lowest quantifiable limit of concentration (LOQ). They precisely and properly quantify [7], [26].

Table 5 LOD and LOQ of Edoxaban

Con. (ppm or μ g/ml)	Area
15.00	13838594
22.50	20313520
30.00	27354643
37.50	33979710
45.00	40502011
STEYX	159272.15
SLOPE	893240
LOD (μ g/ml)	0.59
LOQ (μ g/ml)	1.78

Robustness

Robustness was the analytical parameter since there was no significant change in the system suitability parameter when the method's internal parameters were deliberated. Modify the mobile phase flow rate by ± 2%. Wavelength variation of ± 2% [22].

table 6 Robustness of Edoxaban

Assay of Marketed Tablet

Assay the marketed Edoxaban tablet solution twicely. Sample shows the good % Assay of the drug.

Table 7 Assay of Marketed Tablet