Development and Validation of Stability-Indicating High Performance Liquid Chromatographic Method for Determination of Tetramisole Hydrochloride in Pharmaceutical Formulation

Objective: Development of simple and selective stability-indicating HPLC method for determination of tetramisole hydrochloride in presence of its degradation products. Method: Analysis was performed on SUPELCO C18 column (250mm length, 4.6mm width, 5μm particle size) using acetonitrile : methanol : water (50:33:17, by volume) as a mobile phase. The analysis process was carried out at ambient temperature with flow rate of 1 mL/min and the separated peaks were detected by UV at 212 nm. Drug was exposed to forced degradation condition like hydrolysis, oxidation, thermal and photolysis. Results: Method can well resolve all degraded product as compare to tetramisole hydrochloride. The method has been linear for the range of 1 – 6 μg/mL with r2 0.9997. Conclusion: This work focused on study of the stability of tetramisole hydrochloride and its behavior when exposed to different stress conditions. The proposed method was successfully applied for determination of tetramisole hydrochloride in pharmaceutical preparation without interference from excipients or degradation products and the method was found to be valid according to International Conference on Harmonization (ICH) guidelines.


Preparation of Standard Solutions and Construction of Calibration Graph
Stok solution of tetramisole hydrochloride (100 µg/mL) was prepared by dissolving 10 mg of tetramisole hydrochloride powder in 50 mL of the mobile phase and complete the volume to 100 mL with the mobile phase.Working standard solutions of tetramisole hydrochloride (1 -6 µg/mL) were prepared by dilution of aliquots of the stock solutions with the mobile phase.Into HPLC column, 20 µl from each solution was injected and eluted by the action of the mobile phase under the previously described chromatographic conditions.Calibration graph was constructed by plotting the peak area against the corresponding concentrations and the linear regression equations was derived.

Application on Pharmaceutical Dosage Form
Accurate weight of Anthimizole ® 10 % powder equivalent to 10 mg of tetramisole hydrochloride was obtained and transferred into 100-mL volumetric flask.Add 75 mL of the mobile phase and shake vigorously for 10 min then filter the solution through membrane filter.Completed the volume to 100-mL with mobile phase.From the obtained solution prepare serial dilution covering concentration range and inject them into HPLC.Calculate the concentrations of tetramisole hydrochloride in pharmaceutical samples by using regression equation.

Forced Degradation Studies of Tetramisole Hydrochlorid
Acidic hydrolysis was carried out using 2 mL of 0.1 N hydrochloric acid for 24 hr.at ambient temperature.Basic hydrolysis were carried out using 2 mL of 0.1 N sodium hydroxide for 4 hr.at ambient temperature, neutralization of the samples with alkali or acid was performed before dilution.Study of neutral hydrolysis was carried out by using 2 mL of deionized water for 6 hours at 90°C.Oxidative degradation was carried out using 1 mL of 3 % H2O2 for 6 hours at room temperature.Photosensitivity of the drug was studied by exposing the drug solution to sunlight for 48 hours, for thermal study 0.2 g of the powder sample was placed in a vial in a controlled-temperature oven at 100 °C for 6 hours.Tetramisole hydrochloride at a concentration of 5 μg/mL was used in all the degradation studies.From the above resultant stressed solutions 20 μl was injected onto the column, and the chromatograms were obtained under the previously described conditions.

Optimization of HPLC Method
Three parameters should be studied and optimized carefully in order to enhance the resolution and sensitivity of the HPLC method.The first parameter is the mobile phase selection.Several mobile phase compositions were tried including acetonitrile : methanol : water in different ratios.A good separation and satisfactory peak symmetry for tetramisole hydrochloride was achieved by using acetonitrile : methanol : water (50:33:17, v/v/v) as a mobile phase at flow rate 1 mL/min.The second parameter is the choice of the sutible stationary phase.Different columns were also tried such as SUPELCO ® C18 column and Discovery ® HS C18 column.Good separation was carried out on SUPELCO C18 column.The third parameter is the choice of the optimum wavelength at which the determination of tetramisole htdrochloride was done.Several wavelengths were tried but 212 nm which is the λmax of tetramisole htdrochloride was chosen as this wavelength gave the best results regarding accuracy and sensitivity.Figure 2 shows a typical chromatogram for tetramisole hydrochloride with retention time of 4.05 min.

Degradation Behaviour
The instability of the drug may lead to loss of its activity and increase the risk of excepted adverse effects through formation of degradation products, so different stress conditions were applied to evaluate the stability of tetramisole hydrochloride.

Hydrolytic conditions:
Upon exposing the drug to stress hydrolytic conditions the drug showed 22.63% degradation towards acidic hydrolysis with appearing of one degradation peak at retention time 2 min.(Figure 3), and showed 20.35 % degradation towards basic hydrolysis with appearing of one degradation peak at retention time 2.91 min.(Figure 4), but no degradation products were found when the drug exposed to neutral hydrolysis.Photolytic studies: When drug solution was exposed to sun light for 48 hours the drug showed 12.25 % degradation with appearing of one degradation peak at retention time 2.15 min.(Figure 6).
Thermal studies: There was no degradation of solid tetramisole hydrochloride on exposure to heat at 100°C for 24 hrs, which indicated that the drug was thermally stable.Results of stability studies of tetramisole hydrochloride are shown in Table 1.

System Suitability Test
System suitability of HPLC method was achieved by calculating some parameters including number of theoretical plates (N), capacity factor (k), resolution factor (R) and tailing factor (T). Results of all parameters were good and indicative of the specificity of the method for evaluating of the stability of tetramisole hydrochloride when compared to USP reference values [14], as shown in Table 2.

Method Validation
The proposed HPLC method was validated in terms of linearity, specificity, precision, accuracy and robustness according to ICH guidelines [15].

Linearity and range.
A linear correlation was obtained by plotting the peak area versus drug concentrations in µg/ml in the range of 1 -6 µg/mL with coefficient of determination [r 2 ] = 0.9997 Accuracy.In order to ensure the accuracy of the proposed HPLC we calculate the mean percent recovery of three determination for three different concentrations of pure tetramisole hydrochloride and it was found to be 100.20.

Precision.
Method precision was checked by measuring % RSD of three different concentrations of tetramisole hydrochloride repeated three times within the same day (repeatability) and in three different days (intermediate precision).Small values of % RSD indicating high precision of the method.
Specificity.The specificity of the developed method was assessed by resolving tetramisole hydrochloride from its possible degradation products.
Robustness.Analytical procedure robustness was studied by measuring the capacity of the method to remain unaffected by small changes in method parameter such as small changes in flow rate (±0.1 mL/min), mobile phase ratio and wavelength of detection (± 1 nm).The peak area or retention time of tetramisole hydrochloride not affected by these small changes in parameter which confirms the robustness of the method.The summary of these parameters and other validation and regression parameters are shown in Table 3.

Application of the Proposed Method to Marketed Formulation
The proposed method was successfully applied for the determination of the drug in its marketed formulation Anthimizole ® powder.There was no interference from the excipients commonly present in the assayed powder.The results obtained from the application of standard addition technique confirming that the proposed HPLC method could be determine tetramisole hydrochloride in marketed formulation without any interference from excipient or additives as shown in Table 4.

Statistical Analysis
By comparing the results obtained from the developed HPLC method with that of the reported method [6], we found that the t and F values obtained are less than the tabulated ones which indicate that there is no significant differences between the developed HPLC method and the reported method regarding both accuracy and precision as shown in Table 5.

CONCLUSION
This work focuses on the study of the stability of tetramisole hydrochloride and its degradation behavior under different stress conditions.The developed HPLC methods gave accurate, precise and selective results for determination of tetramisole hydrochloride in the presence of its degradation products without prior separation and can be applied for routine analysis and for checking quality of pharmaceutical preparations during storage of the drug.Moreover, the proposed HPLC method uses simple reagents, with minimal preparation procedures and short run time encouraging application of the method in routine analysis.

Figure 3 .Figure 4 .
Figure 3. HPLC chromatogram for acidic hydrolysis of tetramisole hydrochloride after exposure to 0.1 N HCl for 24 hr.at ambient temperature

Figure 5 .Figure 6 .
Figure 5. HPLC chromatogram of oxidative degradation behavior of tetramisole hydrochloride after exposing to 3 % H2O2 for 6 hour at ambient temperature

Table 1 .
Summary of Forced Degradation Studies

Table 2 .
Results of System Suitability for the Determination of Tetramisole hydrochloride by the Proposed HPLC Method

Table 3 .
Validation Sheet and Regression Parameters of Tetramisole hydrochloride the Proposed HPLC Method a Peak area of tetramisole b Concentration of tetramisole

Table 4 .
Standard Addition Technique of the Proposed HPLC Method Pharmaceutical

Table 5 .
Statistical comparison between the results obtained by applying the proposed methods and the reported method for determination of tetramisole hydrochloride in Anthimizole® veterinary powder a The values in the parenthesis are the tabulated values of t and F at (P = 0.05) Nassar et al. / Forced Degradation Study of Tetramisole 8 / 8