Stability Indicating Method Development and Validation of Cenobamate in Bulk and Dosage form by Liquid Chromatography

A simple, Precise, Accurate method was developed for the estimation of Cenobamate by RPHPLC technique. Chromatographic conditions used are stationary phase symmetry C18 (150 mm* 4.6 mm 5 μm), mobile phase Acetonitrile: 0.01NKH2PO4in the ratio of 55:45 and flow rate was maintained at 1.0ml/min, detection wave length was 272.0 nm; column temperature was set to 30 o C. Retention time was found to be 2.908 min. System suitability parameters were studied by injecting the standard six times and results were well under the acceptance criteria. Linearity study was carried out between 25% to150 % levels, R 2 value was found to be as 0.999.Precision was found to be 0.5 for repeatability and 0.8 for intermediate precision. LOD and LOQ are 0.01μg/ml and 0.03μg/ml respectively. By using above method assay of marketed formulation was carried out 100.32% was present. Degradation studies of Cenobamate were done, in all conditions purity threshold was more than purity angle and within the acceptable range.


Diluent
Based up on the solubility of the drugs, diluent was selected, Acetonitrile and buffer taken in the ratio of 50:50

Preparation of Standard stock solutions
Accurately weighed 25mg of Cenobamate transferred 50ml and volumetric flasks, 3/4 th of diluents was added and sonicated for 10 minutes. Flasks were made up with diluents and labeled as Standard stock solution (500µg/ml of Cenobamate).

Preparation of Standard working solutions (100% solution)
1ml of Cenobamate from each stock solution was pipetted out and taken into a 10ml volumetric flask and made up with diluent. (50µg/ml of Cenobamate).

Preparation of Sample stock solutions
5 tablets were weighed and the average weight of each tablet was calculated, then the weight equivalent to 1 tablet was transferred into a 100 ml volumetric flask, 50ml of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters (500 µg/ml of Cenobamate).

System suitability parameters
The system suitability parameters were determined by preparing standard solutions of Cenobamate (50 ppm) and the solutions were injected six times and the parameters like peak tailing, resolution and USP plate count were determined.
The % RSD for the area of six standard injections results should not be more than 2%.

Specificity
Checking of the interference in the optimized method. We should not find interfering peaks in blank and placebo at retention times of these drugs in this method. So this method was said to be specific.

Robustness
Small deliberate changes in method like Flow rate, mobile phase ratio, and temperature are made but there were no recognized change in the result and are within range as per ICH Guide lines.
Robustness conditions like Flow minus (0.27 ml/min), Flow plus (0.33 ml/min), mobile phase minus, mobile phase plus, temperature minus (25°C) and temperature plus (35°C) was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. %RSD was within the limit.

Assay Methodology
Assay of the marketed formulation was carried out by injecting sample corresponding to equivalent weight into HPLC system.

Oxidative Degradation studies
To 1 ml of stock solution of Cenobamate 1 ml of 20% hydrogen peroxide (H 2 O 2 ) was added separately. The solutions were kept for 30 min at 60 0 c. For HPLC study, the resultant solution was diluted to obtain (50 ppm) solution and10µlwereinjected into the system and the chromatograms were recorded to assess the stability of sample.

Acidic Degradation Studies
To 1 ml of stock solution Cenobamate 1ml of 2N Hydrochloric acid was added and refluxed for 30mins.The resultant solution was diluted to obtain (50 ppm) solution and10µl solutions were injected into the system and the chromatograms were recorded to assess the stability of sample.

Alkaline Degradation Studies
To 1 ml of stock solution Cenobamate 1 ml of 2N sodium hydroxide was added and refluxed for 30mins at 60 0 c. The resultant solution was diluted to obtain (50 ppm) solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Thermal Degradation Studies
The standard drug solution was placed in oven at 105 0 C for 6h to study dry heat degradation. For HPLC study, the resultant solution was diluted to (50 ppm) solution and10 µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Photo Stability Degradation studies
The photochemical stability of the drug was also studied by exposing the (500 ppm) solution to UV Light by keeping the beaker in UV Chamber for 7 days or 200 Watt hours/m 2 in photo stability chamber . For HPLC study, the resultant solution was diluted to obtain (50 ppm) solutions and 10µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Hydrolytic Degradation Studies
Stress testing under neutral conditions was studied by refluxing the drug in water for 6hrs at a temperature of 60ºc. For HPLC study, the resultant solution was diluted to (50 ppm) solution and 10µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Optimized Chromatographic Condition
Based on drug solubility and pKa Value following conditions has been used to develop the method estimation of Cenobamate. Chromatographic conditions used are stationary phase symmetry C18 (150 mm*4.6 mm 5 µm), Mobile phase Acetonitrile:0.01N kh2po4 in the ratio of 55:45 and flow rate was maintained at 1.0ml/min, detection wave length was 272.0 nm, column temperature was set to 30 o C. The retention time was found to 2.908 min. Optimized chromatogram is shown in Fig. 2.

System Suitability Parameters
The system suitability parameters were evaluated from standard Chromatograms obtained by calculating the % RSD of retention time, tailing factor, theoretical plates and peak areas from five replicate injections are within range and Results were shown in Table 2.

Specificity
The chromatogram of Blank, placebo and drugs are shown in Fig. 3, 4, 5.

Repeatability
Six working sample solutions of 60ppm are injected and the % Amount found was calculated and %RSD was found to be 0.5 and data is shown in Table 3.

Intermediate precision
Six working sample solutions of 60 ppm are injected on the next day of the preparation of samples and the percentage amount found was calculated and %RSD was found to be 0.8 and Table 4 shows the data of intermediate precision

Linearity
To demonstrate the linearity of assay method, inject 6 standard solutions with concentrations of about 12.5 ppm to 75 ppm of Cenobamate. Plot a graph to concentration versus peak area. Slope obtained was y = 45610x + 24737 and Correlation Co-efficient was found to be 0.999 and Linearity plot was shown in Fig. 6 and data is shown in Table 5.

Accuracy
Three Concentrations of 50%, 100%, 150% are Injected in a triplicate manner and % Recovery was calculated as 99.38. Accuracy data are depicted in Table 6.
LOD: Detection limit of the Cenobamate in this method was found to be 0.89 µg/ml.

LOQ:
Quantification limit of the Cenobamate in this method was found to be 2.68 µg/ml.

Robustness
Small Deliberate change in the method was made like Flow minus, flow plus, Mobile phase minus, Mobile phase plus, Temperature minus, Temperature Plus. %RSD of the above conditions is calculated and expressed in Table 7.

ASSAY OF MARKETED FORMULATION
Standard solution and sample solution were injected separately into the system and chromatograms were recorded and drug present in sample was calculated using before mentioned formula. Table 8 shows the Assay data of Formulation.

Degradation Studies
Degradation studies were performed with the formulation and the degraded samples were injected. Assay of the injected samples was calculated and all the samples passed the limits of degradation and data shown in Table 9 and chromatograms are shown in Fig. 7-12.

CONCLUSION
An antiepileptic medication Cenobamate is used to treat partial onset seizures. The separation was achieved by using stationary phase symmetry C 18 (150 mm*4.6 mm 5 µm), mobile phase Acetonitrile: 0.01NKH 2 PO 4 in the ratio of 55:45 and flow rate was maintained at 1.0ml/min, detection wave length was 272.0 nm; column temperature was set to 30 o C. Conditions were finalized as optimized method with a retention time of 2.908min. System suitability parameters were studied by injecting the standard six times and results were well under the acceptance criteria. Linearity study was carried out between 25% to 150% levels, R 2 value was found to be as 0.999. Precision was found to be 0.5 for repeatability and 0.8 for intermediate precision.
LOD and LOQ are 0.89µg/ml and 2.68µg/ml respectively. By using above method assay of marketed formulation was carried out 100.32% was present.
Degradation studies of Cenobamate were done, in all conditions purity threshold was more than purity angle and within the acceptable range. Method was found to be Precise, Accurate, simple and easy to perform and it follows all the ICH guideline Q2 (R1).

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.