RP-HPLC method development and validation of Albendazole and its impurity

: Oxibendazole is a type of benzimidazole that is commonly used as an antiparasitic medication for both humans and animals. However, it is also a significant impurity found in albendazole, and it is crucial to follow the ICH Q3B criteria when analysing oxibendazole impurities. Therefore, it is recommended to use a simple, fast, sensitive, and precise RP-HPLC approach to identify oxibendazole impurities in bulk and pharmaceutical formulations of albendazole.To separate the oxibendazole impurity, acetonitrile and 10 nM potassium phosphate were used as a mobile phase. Orthophosphoric acid was used to accurately adjust the pH of the mobile phase to 2.03, ensuring optimal conditions. A nucleosil C18 column (250 x 4.6 mm, 5 µm) was used for the separation process, and it effectively provided the necessary separation. The gradient elution was set at a wavelength of 235 nm and a flow rate of 1 mL/min. The analytical technique was successfully designed and validated. The AQbD technique was used to optimize the analytical conditions for the suggested methodology, and the Design Expert 13® trial version was used for the central composite design optimization of analytical conditions. The procedure's linearity was verified using a regression coefficient of 0.999 within a working range of 0.5 to 3 μg/ml. Accuracy research showed results of 99.94 – 100.10% at 50, 100, and 150% levels of the working concentration. The oxibendazole impurity's average retention time was found to be 6.40 minutes, with a relative standard deviation of less than 2%, indicating high accuracy. The limits of detection (LOD) and quantification (LOQ) were found to be 0.073 and 0.091 μg/ml, respectively. Following the ICH Q2(R1) criteria, other validation criteria, such as robustness, were also evaluated. In conclusion, the proposed approach is suitable for analysing albendazole and oxibendazole in bulk and pharmaceutical formulations, making it ideal for detecting oxibendazole impurities.


INTRODUCTION
Thorough testing and analysis are necessary parts of the verification process to ensure that albendazole formulations are free from contaminants that could endanger user health.It is important to continuously evaluate the stability of these formulations over time to further verify their safety and effectiveness and to ensure that no new contaminants arise during storage or ageing.The quality of these formulations is influenced by the materials and procedures used during the production process.These products may contain various contaminants such as synthetic precursors, side reaction products, unreacted raw materials, intermediates, and degradation products [1].However, the benefits of delivering these contaminants are offset by the toxicological adverse effects they typically induce.It is therefore crucial to keep a constant watch on these contaminants to maintain a high-quality industrial environment.Oxibendazole (Albendazole Impurity I EP) has a chemical makeup of methyl ester of 5propoxy-1H-benzo The anthelmintic drug oxibendazole is used to treat parasitic infestations such as roundworms, strongyles, threadworms, pinworms, and lungworms in horses and other domestic animals.Different analytical methods have been developed for detecting the residues of this drug in bulk medications, milk, and human plasma as reported in literature studies [3].
The current study aimed to identify the oxibendazole impurity in albendazole solid formulation using RP-HPLC technology.Quality by Design (QbD) methodology was used to optimize the analytical conditions, which focuses on the quality expected during the planning phase.AQbD is an instrument that reports quality from the planning stage by relying on predetermined goals, risk evaluations, and risk management [Figure 2].By using QbD, fewer experiments are needed to establish a technique.Additionally, the analytical technique provides a better understanding of how variables can affect significant metrics [4][5].
Fig. 2 The schematic representation of the analytical method QbD approach

Material and methods
For the analysis of the impurity and albendazole the Ashirwad Analytical from Panchkula, India provided a free sample of albendazole and the impurity was provided by the Pharma affilaites, Panchkula, India.Only HPLCgrade chemicals were used, which were all procured from Merk India.The solvents and solutions were sonicated and filtered through a membrane filter before being employed.The Jasco HPLC System and a PDA detector were used for the chromatographic process.The sample was injected into the analytical column (C18 Columns 250 x 4.6 mm, 5µm) through a rheodyne sample injection port.The output response was recorded using the Borwin-PDA program (Version 1.5).

Initial characterization of impurity and API
The solubility of both oxibendazole impurity and albendazole was determined in various solvents, including water, methanol, and ethyl acetate.Fourier-transform infrared spectroscopy (FTIR) was used to analyze the functional groups present in the compounds and confirm their identity [ Fig 3].

Mobile phase preparation
To prepare the mobile phase, a mixture of acetonitrile, 10 nM potassium dihydrogen phosphate, and orthophosphoric acid was made in a 40:60 v/v ratio until the pH reached 3.5.The mobile phase was then passed through a 0.45 µm membrane paper filter and ultrasonically sonicated for ten minutes [6].To prepare a 1000 μg/ml concentration of the drug, 10mg of the albendazole was mixed with 10 ml of 1% methanolic sulfuric acid to form the standard drug stock solution.From this stock solution, we produced additional doses ranging from 5 to 25 μg/ml.A similar procedure was followed to prepare the dilutions of the impurities [7].

Detection of wavelength
Additional dilutions of the standard stock solution were made with the mobile phase and the UV spectrum was scanned to acquire spectra.It was found that both albendazole and its primary impurity, Oxibendazole, exhibited significant absorption at 235 nm [8].

Optimization of Analytical Conditions
During the screening process, critical parameters such as the column temperature, mobile phase ratio, buffer pH, flow rate, and injection volume were identified.The central composite design (CCD) was then utilized to maximize the separation of oxibendazole impurities [9].The independent variables, which included the mobile phase's pH (X2) and composition (X1), were examined for their impact on the dependent variable.Table 1 shows the independent factors used in the experiment [10].The experimental design was developed for the separation process, the independent variables were the type of column used, the flow rate of the mobile phase, and the system's temperature.The number of theoretical plates (Y2) indicates how effective the separation process is, while the retention time (Y1) is the amount of time taken for a component to elute from the column.The recovery (Y3) of a compound from the sample post-column passage is used to measure its effectiveness [11].To get the best composition predictions, an overlay plot and desirability function are used.The anticipated analytical conditions were determined using Design-Expert Software and numerical optimization techniques [12].
The Design-Expert-13® program was used to statistically analyze the experimental design's outcomes.Statistical validation involved assessing the distribution of the correlation coefficient F-value (R2), adjusted R-squared (R2 Adj), predicted R-squared (R2 Pred), PRESS statistics, and adequate precision (AP) produced by the ANOVA provision to determine the model's appropriateness and reliability.A model was considered significant if the Fvalue was p <0.05.It was considered acceptable if the difference between R2 Adj and R2 Pred was less than 0.2.The PRESS statistics were used to determine the measure of fit, where a smaller PRESS value was preferred [13][14].
Design Expert software-13® was used to generate diagnostic graphs to compare externally studentized residuals with planned and actual plots for the impurity experimental design.The plots were examined to determine if the points fell within the designated limits [15][16].[17][18] To conduct chromatographic separation, albendazole and oxibendazole were used as working standard solutions.

Selection of mobile phase and chromatographic conditions
The experiments were carried out by changing the ratios of acetonitrile to buffer, and by using a buffer with different pH values, to obtain the required system suitability characteristics.The optimal mobile phase was determined to be acetonitrile: 10 nM potassium dihydrogen phosphate with a pH of 3.5, adjusted with orthophosphoric acid in a 40:60 v/v ratio.After 11 trials, it was found that this mobile phase showed high resolution and adequate peak characteristics at a flow rate of 1 ml/min [19].

Chromatograms and system suitability parameters
The mobile phase was carefully applied to the column, ensuring full saturation and resulting in continuous back pressure at the optimal flow rate.Following this, a precisely measured working standard solution containing 1 μg/ml oxibendazole and 200 μg/ml albendazole was injected into the system [20][21].

Linearity
Six replicates of each concentration were extracted and introduced into the system from the standard albendazole stock solution.The linearity of the correlation between the drug's peak area and concentration was established over the concentration range of 100-600 μg/ml [23].Similarly, from the oxibendazole standard stock solution, six duplicates of each concentration were extracted and introduced into the apparatus.It was observed that the relationship between the drug's peak area and concentration was linear over the concentration range of 0.5-3 μg/ml [24].

Precision
The precision of the method was determined by evaluating intra-day and inter-day variability.For intra-day variability, three concentrations of albendazole (200, 400, and 600 µg/ml) were assessed with three replicates each, and the percent relative standard deviation was calculated.Inter-day variability was evaluated over three consecutive days with the same concentrations and replicates, and the percent relative standard deviations were calculated accordingly [25].Similarly, for oxibendazole, three duplicates of three distinct concentrations (1, 2, and 3 μg/ml) were examined in one day, and the percent relative standard deviation was calculated.Inter-day variability was also investigated over three consecutive days with three different concentrations and three replicates, and the percent relative standard deviations were calculated [26].

Accuracy
To establish the accuracy of the method, a recovery study was conducted for both drug and impurity.Pure albendazole was added to sample solutions at selected levels (50%, 100%, and 150%), with a basic concentration of 200 µg/ml.Additionally, standard oxibendazole impurity (0.3, 0.6 & 0.9 μg/ml) was spiked, and chromatograms were obtained after injecting in triplicates.The concentration of drug and impurity was calculated using the linearity equation of albendazole and oxibendazole [27][28].

Limit of Detection (LOD)
To determine the sensitivity of the proposed method, the lowest concentrations in a sample that can be detected but not always measured -LOD and LOQ -were analysed.LOD represents the lowest concentration in a sample that can be accurately measured, while LOQ represents the lowest amount of analyte in a sample that can be accurately measured.The drug and impurity concentrations in the lower part of the calibration curve were used to calculate LOD and LOQ using the equation 3.3 x σ/S and 10 x σ/S, respectively [29].

Robustness
The robustness of the method was tested by varying chromatographic conditions, such as flow rate, pH, and wavelength, and observing their effects.

FTIR study
To characterize the oxibendazole, the first step was to record the FTIR (Fourier Transform Infrared Spectroscopy) spectrum of the impurity.This was done by making a pellet using a small quantity of impurity powder and KBr, which was then placed between two discs.Once this was done, the prepared KBr disc was placed in the sample holder and analysed to obtain the FTIR spectra of the impurity (oxibendazole).

Experimental design for optimization of analytical method conditions
Throughout the optimization investigation, a range of measurements and analyses were conducted to assess the recoveries, theoretical plates, and retention time.The software algorithm recommended a total of eleven trial runs, which were conducted across three separate levels of the design matrix.The outcome of these experimental runs enabled us to create a robust mathematical model, which accurately describes the results obtained from the trials.
[Table 2].The study assigned independent variables, such as pH and mobile phase composition, to levels +1, 0, and -1, and examined their effects with response variables.Highly significant models were constructed for each response variable (p < 0.05).3]

Optimization of analytical conditions
The program is designed to improve the performance of chromatography by adjusting the mobile phase ratio and pH level.This process helps to maximize the retention time, theoretical plates, and recovery of target molecules.
The program selects the most appropriate combination based on the specific requirements.For instance, after testing various combinations, it was found that using a mobile phase ratio of 0.499 and a pH level of 2.030 resulted in 6.17

Chromatogram and system suitability parameters
The column was saturated with the mobile phase and carefully monitored the pressure to ensure the appropriate flow rate.Then the working standard solution, containing oxibendazole at a concentration of 1 μg/ml and albendazole at 200 μg/ml was introduced into the system.The results of the system's applicability were then recorded and are presented in detail in Table 4 and Fig. 8.

Linearity
Six replicates were taken from the standard stock solution of albendazole (100-600 μg/mL) and oxibendazole (0.5-3 μg/mL) at each concentration and injected into the system.Calibration curves were drawn to establish the linear relationship between the concentrations and peak areas of the drug and impurity.[

Precision
The method's precision was evaluated by testing intra-day and inter-day variability using three concentrations and three replicates of albendazole (200, 400, and 600 µg/mL) and oxibendazole (1, 2, and 3 μg/mL).The percent relative standard deviation was calculated.

Accuracy
The method's accuracy was determined by conducting a recovery study of a drug and its impurity.Pure albendazole was added to sample solutions at different levels (50, 100, and 150%) with a basic concentration of 200 µg/ml.Standard oxibendazole impurity was also added, and chromatograms were obtained after injecting three replicates.The linearity equation of albendazole and oxibendazole was used to calculate the concentrations of the drug and impurity.[Table 7-8]

LOD and LOQ
The limit of detection (LOD) and limit of quantification were calculated using the equations 3.3 x σ/S and 10 x σ/S, respectively.The method was able to detect the lowest amounts of albendazole and oxibendazole, which were found to be 111.25 μg/ml and 0.223 μg/ml, respectively.Additionally, the limit of quantification for the drug and impurity were determined to be 337.12μg/ml and 0.667 μg/ml, respectively.

Robustness
The robustness of the method was tested by varying chromatographic conditions, such as flow rate, pH, and wavelength, and observing their effects.

Discussion
In this investigation, a quality-by-design enabled approach was used to analyse the oxibendazole impurity in bulk and albendazole formulation.The optimization process involved evaluating the theoretical plates, recovery reactions, and retention time.Based on the observed data, the DOE program made predictions using statistical analysis.The accuracy and reliability of the model were evaluated by comparing the projected values with the actual observed values.This allowed for additional experimental conditions to be adjusted and refined to enhance retention time, theoretical plate count, and recovery percentage in subsequent experiments.With an error percentage of less than 5%, the model exhibited strong predictability.The retention period of the oxibendazole impurity increased with decreasing mobile phase pH, possibly because oxibendazole is a basic impurity (pKa 4.56), and partial ionization occurs at pH values below 3.0.The retention period of oxibendazole is significantly decreased at high pH levels because of non-ionization, particularly when the mobile phase's pH value is two units over its pKa value.Changing the mobile phase ratios led to a considerable variation in the impurity's region time.Raising the mobile phase ratio or the ratio of 10 nM potassium dihydrogen phosphate also extended the impurity's retention period.buffers is increased.The non-ionizable forms become more hydrophobic, resulting in substantial retention on the non-polar stationary phase.The mobile phase's pH shift had the least impact on the percentage recovery.At 235 nm, it was discovered that the retention times of albendazole and oxibendazole in the current solvent, as well as the composition of the mobile phase, were respectively 6.399 ± 0.114 and 11.095 ± 0.087.The technique exhibited high linearity between the concentration range of 100-600μg/ml and 0.5-3μg/ml for albendazole and oxibendazole, respectively.Good recovery was demonstrated by the approach at concentrations of 50, 100, and 150% In summary, a validated RP-HPLC technique has been devised for the analysis of oxibendazole impurity in albendazole formulation and bulk.The created approach is fast, accurate, and precise since it is based on quality by design.Based on the statistical assessment of the method's good linearity and its validation for many parameters, we concluded that the suggested methodology may be used for the rapid and accurate assessment of oxibendazole impurity in pharmaceutical formulations.

Conclusion
The experimental methods and analytical techniques used in this study were successfully validated in accordance with ICH recommendations.To validate the proposed method, recovery studies and preliminary analysis of a standard sample were conducted.The study developed a validated RP-HPLC method for the measurement of oxibendazole impurity in bulk and pharmaceutical dosage forms.The suggested method was found to be rapid, accurate precise, and easy to use.Furthermore, the method demonstrated excellent precision and reproducibility, making it suitable for routine quality control analysis.The method's sensitivity was also satisfactory, as it could detect even trace amounts of oxibendazole impurity in pharmaceutical formulations.

Fig. 5
Fig. 5 Effect on (a) retention time (b) theoretical plates (c) and (d) percent recovery as a function of mobile phase ratio and mobile phase pH The model's residual mean square and mean square were both greater than F, indicating an excellent fit to the data.Using DOE software, values compared the anticipated retention, theoretical plate, and recovery values and found that the model predicted them with good accuracy, with only a 5% error rate.[Table3]

Fig. 6
Fig. 6 The actual vs predicted values plots of (a) retention time (b) theoretical plates (c) recovery minutes of retention time, 4861.570theoretical plates, and 98.30% recovery.To achieve these optimal values, the values were converted to the translation factor coded level and lowered the pH to 2.03.Based on these calculations, it was recommended to use a mobile phase ratio of 40:60 (acetonitrile and 10 nM potassium dihydrogen phosphate) to achieve the best results.[Fig 7]

Fig. 7 3 . 4
Fig.7 Mobile phase ratio (0.499) pH 2.030 which provided the retention time of 6.17 min, theoretical plates 4861.570, and the % recovery of 98.30% at a 95% confidence interval with a desirability value of 1

Table . 1
Conversion of factor levels into units

Table . 2
Experimental runs and response variables oxibendazole impurity

Table . 4
Predicted values for response variables and data from experiments Recovery, Theoretical Disks, and retention time

Table . 6
System suitability study

Table . 6
Inter-day precision results of albendazole and impurity

Table . 7
Accuracy study of albendazole

Table 8 .
Accuracy study of oxibendazole

Table . 10
Summary of analytical method validation parameters