Analytical Method Development and Validation of Brivaracetam in Pharmaceutical Dosage Form

Table 1: Optimized Chromatographic Conditions

Preparation of Potassium Phosphate buffer PH- 3

To prepare Potassium phosphate buffer solution, add 13.6gm of phosphate buffer in 1000ml water. Adjust this solution to pH by using diluted HCL.

Preparation of mobile phase:

Mix a mixture of above Methanol 700ml (70%) and 300 ml potassium dihydrogen phosphate (30%) and degas in ultrasonic water bath for 5 minutes. Filter through 0.45 µ filter under vacuum filtration.

Standard Solution Preparation:

Accurately weigh and transfer 25 mg of Brivaracetam working standard into a 25 ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution).Further pipette 0.3ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents. (30ppm)

VALIDATION PARAMETERS:

ASSAY:

Standard Solution Preparation:

Accurately weigh and transfer 25 mg of Brivaracetam working standard into a 25 ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 0.3ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents. (30ppm)

Sample Solution Preparation:

Accurately weigh and transfer equivalent to 25 mg of Brivaracetam equivalent weight (50mg) of the sample into a 25 ml clean dry volumetric flask add about 7ml of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. Further pipette 0.3ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents.

Inject 10 L of the standard, sample into the chromatographic system and measure the areas for the Brivaracetam peaks and calculate the % Assay by using the formulae.

Preparation of drug solutions for Linearity (Level 1-V):

To make 10 ppm, 20 ppm, 30 ppm, 40 ppm, and 50 ppm concentrations of brivaracetam solutions, pipette out 0.1, 0.2, 0.3, 0.4, and 0.5 ml of stock solution and mix up to 10 ml with diluents. Measure the peak area after injecting each level into the chromatographic apparatus. Plot the peak area against concentration on a graph. Further pipette 0.3ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents.

Inject 10 L of the standard, sample into the chromatographic system and measure the areas for the Brivaracetam peaks and calculate the % Assay by using the formulae.

Precision:

The standard solution was injected for six times and measured the area for all six injections in HPLC. The %RSD for the area of six replicate injections was found to be within the specified limits.

Intermediate Precision/Ruggedness:

The standard solution was injected for six times and measured the area for all six injections in HPLC. The %RSD for the area of six replicate injections was found to be within the specified  limits.

ACCURACY:

Preparation of 50%, 100% and 150% standard stock solution:

Accurately weigh and transfer 12.5mg, 25mg and 37.5mg of Brivaracetam working standard into a 25 ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution).Pipette 0.3ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents.

Inject the standard solution, Accuracy -50%, Accuracy -100% and Accuracy -150% solutions.

Calculate the Amount found and Amount added for Brivaracetam and calculate the individual recovery and mean recovery values.

LIMIT OF DETECTION:

Preparation of Brivaracetam solution:

Preparation of 0.04µg/ml solution:

Accurately weigh and transfer 25 mg of Brivaracetam working standard into a 25 ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Pipette 1 milliliter of the stock solution mentioned above into a 10 milliliter volumetric flask, then use diluents to dilute it to the appropriate level. Pipette 0.1 ml of the stock solution mentioned above into a 10 ml volumetric flask, then use diluents to dilute it to the appropriate level. Pipette 0.4 ml of the stock solution mentioned above into a 10 ml volumetric flask, then use diluents to dilute it to the appropriate level. (0.04 ppm)

LIMIT OF QUANTIFICATION:

Preparation of Brivaracetam solution:

Preparation of 0.1µg/ml solution:

Accurately weigh and transfer 25 mg of Brivaracetam working standard into a 25 ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution).Pipette 1 ml of the stock solution mentioned above into a 10 ml volumetric flask, then use diluents to dilute it to the desired level. After pipetting 1 milliliter of the previously indicated stock solution into a 10 milliliter volumetric flask, dilute it to the proper concentration using diluents.
After pipetting 0.1 ml of the previously described stock solution into a 10 ml volumetric flask, dilute it to the proper concentration using diluents. 0.1 parts per million.

ROBUSTNESS:

As part of the Robustness, deliberate change in the Flow rate, Mobile Phase composition, Temperature Variation was made to evaluate the impact on the method.

1. The flow rate was varied at 1.4 ml/min to 1 ml/min.

        Brivaracetam standard solution (30µg/ml) was made and analyzed using different flow rates and method flow rates. The Organic composition in the Mobile phase was varied from 40% to 60%.A standard solution containing 30 µg/ml of Brivaracetam was made and examined using both the method's true mobile phase composition and the modified mobile phase composition.

3.RESULTS:

Figure 1: Chromatogram showing Blank

Table 2: Area of different concentration of Brivaracetam

Figure 2: Calibration graph for Brivaracetam

Table 3: Results of system suitability parameters

1. ASSAY:

Standard and sample solution injected as described under experimental work. The corresponding chromatograms and results are shown below.

Figure 3: Chromatogram for Standard

Figure 4: Chromatogram for Sample

TABLE 4: ASSAY TABLE

LINEARITY:

The plot of Concentration (x) versus the Average Peak Area (y) data of Brivaracetam is a straight line and the linearity range was found to lie from 10µg/ml to 50µg/ml of Brivaracetam with

Slope (m) = 51179

Intercept (c) = 48225 and

Correlation coefficient (R²) = 0.998

PRECISION:

Precision of the method was carried out for both sample solutions as described under experimental work. The corresponding results are shown below.

Table 5: Results of Precision for Brivaracetam

%RSD for sample should be NMT 2 which is within the limits hence          method is precise.

INTERMEDIATE PRECISION (ruggedness)

There was no significant change in assay content and system suitability parameters at different conditions of ruggedness like day to day and system to system variation

Table 6: Results of Intermediate precision for Brivaracetam

%RSD of six different sample solutions should not more than 2 which is within the limit, hence the method is rugged.

ACCURACY:

Sample solutions at different concentrations (50%, 100%, and 150%) were prepared and the % recovery was calculated.

Table 7: Accuracy (recovery) data for Brivaracetam

*Average of three determinations 

The percentage recovery was found to be within the limit (98-102%).The results obtained for recovery at 50%, 100%, 150% are within the limits.

LIMIT OF DETECTION FOR BRIVARACETAM

The lowest concentration of the sample was prepared with respect to the base line noise and measured the signal to noise ratio.

Limit of Detection for Brivaracetam is 0.04µg/ml and Limit of Quantitation for Brivaracetam is 0. 1µg/ml respectively.

ROBUSTNESS:

The standard and samples of Brivaracetam were injected by changing the conditions of chromatography. There was no significant change in the parameters like resolution, tailing factor, asymmetric factor, and plate count.

Table 8: Results for variation in flow for Brivaracetam

Table 9: Results for variation in mobile phase composition for Brivaracetam