Materials
Methanol of HPLC grade (Finar Ltd, India) was obtained from Chemoquip Ltd, Nairobi. Analytical grade concentrated hydrochloric acid (HCl), sodium lauryl sulfate (SLS), and albendazole and mebendazole working standards were provided by the Drug Analysis and Research Unit (DARU) of the Department of Pharmaceutical Chemistry, University of Nairobi. Commercial pharmaceutical products containing albendazole or mebendazole active pharmaceutical ingredient (API) were acquired from wholesalers in the Central Business District (CBD) and the outskirts of the city of Nairobi, Kenya. Throughout the period of the study, nine albendazole and two mebendazole brands were analyzed.
Instrumentation
All weights were taken using a Sartorius top-loading electronic weighing balance (Sartorius GMBH, Germany). Absorbance readings were read on a Genesys 10S UV-Vis Spectrophotometer (ThermoFisher Scientific, China).
A Merck Hitachi HPLC machine (Hitachi Ltd, Tokyo, Japan), with a Varian HPLC column, 250 × 4.0 mm, 5 μm LiChristopher 100-5 RP 18 end capped, kindly availed by the National Quality Control Laboratory (NQCL), Nairobi, Kenya, was used for the orthogonal analysis of commercial samples. It was equipped with an L-7100 low pressure quaternary pump, an L-m7200 autosampler, an L-7400 variable UV detector set at 308 nm, an L-7350 thermostatic column oven maintained at 40 °C, and an L-7000 computer interphase.
Method development
Key considerations
A method from literature developed by Agrawal et al. [7] was adapted for the analysis of both albendazole and mebendazole at a single wavelength using a common solvent. The key considerations in development of the method included choice of solvent in which both albendazole and mebendazole exhibited adequate solubility, determination of optimal wavelength of analysis, and determination of suitable working concentration. To make the analytical process simple, analysis needed to be performed at a common wavelength at which both APIs showed adequate absorbance with minimal interference from excipients, related substances, and degradation products possibly present in analytical samples. A working concentration within the linear range of absorption signal of both APIs also had to be determined.
Determination of a suitable solvent
Two solvents were investigated for the dissolution of both the bulk APIs and the commercial samples. These were 0.1 M HCl containing 0.05% SLS and 0.1 M methanolic HCl. The former had been used in the Agrawal et al. method [7] while the latter was used by Al-Kurdi et al. [5].
Choice of wavelength of analysis
To decide on a single wavelength for both analytes, the UV spectra of each API at a nominal concentration of 12 μg/mL in 0.1 M methanolic HCl were run independently between 200 and 400 nm. The two spectra were then overlaid. Two wavelengths (233 and 294 nm) were initially chosen for further investigation.
Choice of working concentration
The appropriateness of a concentration of 12 μg/mL as used by Agrawal et al. [7] was investigated and was found to fall within the linear range for both APIs.
Adapted method
After the preliminary investigations (“Determination of a suitable solvent,” “Choice of wavelength of analysis,” and “Choice of working concentration” sections), optimal conditions for the method were suggested as UV absorbance of a 12-μg/mL solution of each API in 0.1 M methanolic HCl and measured at 294-nm wavelength. This method was taken through a validation process to assess its suitability.
Method validation
Linearity and range
A 1.0-mg/mL stock solution of each of the two APIs was prepared by weighing 50 mg of the respective API into a 50-mL volumetric flask, dissolving in minimum 0.1 M methanolic HCl, and the solution made to volume with the same solvent. Working solutions were prepared by transferring aliquots of the stock solution into 25-mL volumetric flasks and making to volume using 0.1 M methanolic HCl producing ten solutions of 4, 8, 12, 16, 20, 24, 28, 32, 36, and 40 μg/mL nominal concentrations. This represented a range of 33.3 to 333.3% of the working concentration. The absorbances of these solutions were read at 294 nm, and the data obtained plotted using a Microsoft Excel spreadsheet and subjected to linear regression analysis.
Precision
Repeatability and intermediate precision were determined in this study as outlined in the sections “Repeatability” and “Intermediate precision.” Reproducibility was not determined as the study did not involve a collaborating laboratory.
Repeatability
About 50 mg of each API was weighed into a 50-mL volumetric flask, dissolved in minimum 0.1 M methanolic HCl, and made to volume with the same solvent. A 0.3-mL aliquot of this solution was transferred to a 25-mL volumetric flask and made to volume with the same solvent to give a final solution. Absorbance of the test solution was determined on the same day six times at 294 nm. The standard deviation, relative standard deviation, and coefficient of variation (COV) of these data were then calculated.
Intermediate precision
The procedure for the determination of repeatability (“Repeatability” section) was followed after several days.
Accuracy
The accuracy of the method was established by adding a known amount of the analyte (API) to a solution of a commercial product whose API concentration was 80, 100, and 120% of the working concentration (12 μg/mL) [9]. The percentage recovery of the analyte in each solution was then determined. The determinations were done in triplicate.
Orthogonal HPLC analysis
To compare the reliability and accuracy of the developed method with that of a validated method in routine use, the HPLC procedure for the analysis of albendazole as described in the USP 2018 was used. The suspension dosage form of one of the commercial products was analyzed. The results obtained from both methods were then compared.
Specificity
The process of testing for accuracy (“Accuracy” section) involving the analysis of the API in the presence of excipients, possible related compounds, and degradation products was additionally used to assess the specificity of the developed method.
Sensitivity
As a measure of sensitivity, the limits of detection and quantitation (LOD and LOQ) were determined by computing the standard deviation (σ) of the response and the slope (S) of the linearity plot [9]. The standard deviation was determined by measuring the absorbance of the blank (0.1 M methanolic HCl) six times and calculating the standard deviation of the responses.
The LOD and LOQ were calculated using Eqs. 1 and 2:
$$ \mathrm{LOD}=3.3\upsigma /\mathrm{S} $$
(1)
$$ \mathrm{LOD}=10\upsigma /\mathrm{S} $$
(2)
Analysis of commercial samples
The linear plots used in the determination of linearity and range (“Linearity and range” section) were also used as the calibration curves for content determination of both APIs.
Sample preparation
Tablet dosage forms
Twenty tablets were accurately weighed and pulverized to a fine powder. An amount of the powder equivalent to 50 mg of the respective API was accurately weighed into a 50-mL volumetric flask. About 25 mL of 0.1 M methanolic HCl was added and the mixture shaken to dissolve. The solution was ultrasonicated for 5 min and made to volume with the same solvent and the solution filtered. A 0.3-mL aliquot of the filtrate was pipetted into a 25-mL volumetric flask and made to volume with the same solvent. The absorbance of this solution was read at 294 nm. The samples were prepared in triplicate.
Suspension dosage forms
An amount of the suspension equivalent to 50 mg of the respective API (as determined by the use of a density bottle) was accurately weighed into a 50-mL volumetric flask. A minimum amount of 0.1 M methanolic HCl was added and the flask shaken to dissolve. The solution was ultrasonicated for 5 min and made to volume with 0.1 M methanolic HCl and the solution filtered. A 0.3-mL aliquot of the filtrate was pipetted into a 25-mL volumetric flask and made to volume with the same solvent. The absorbance of this solution was read at 294 nm. The samples were prepared in triplicate.
