Analytical Method Validation

The principal purpose of analytical method validation is to confirm that the analytical procedure employed for a specific test is suitable for its intended use. 

The typical analytical performance parameters, which are used for the purpose of method validation:

1. Specificity (Selectivity)

2. Precision

3. Linearity

4. Accuracy & Recovery

5. Sensitivity

6. System suitability

7. Ruggedness

1. Specificity / Selectivity:

The analyte should have no interference with other materials, which present in the test sample (e.g. Excipients, decomposition products). So, specificity can be defined as the ability of the assay method to remain unaffected. Otherwise, Some information may be deduced from the linearity & accuracy checks.

2. Precision:

Precision is the measure of how close the data values are to each other for a number of measurements under the same analytical conditions. This was calculated by the standard deviation of the repeatability from the results of 5 different samples of the same batch.   

3. Linearity:

The dimensionality/Linearity  of associate degree analytical procedure is its ability to get take a look at results, which are directly proportional to the concentration of analyte in the sample. dimensionality/ Linearity is set by evaluating a minimum of 5 completely different concentrations of the expected concentration vary (e.g. 0%-160%). dimensionality is performed by the subsequent studies, such as –

     a. Linearity of the active agent with different concentrations.

     b. Linearity of active agent of different concentration with fixed concentration of formulation placebo

     c. Linearity of fixed active agent with different concentration of formulation placebo.

                                                        

Linearity/dimensionality of active ingredient with completely different concentration:

For this purpose six different concentration of solutions ranging from 0-160% are prepared and their respective peak area is detected by HPLC and it is observed that the peak area of the sample solution increases with the increase of its concentration. Then a graph of % age of Nominal Concentration Vs Peak Area is plotted and found that all the points lie in a straight line showing the increase of peak area with the increase of concentration solution. The data of analysis and the graph plotted is shown bellow:

Linearity of active agent of different concentration with fixed concentration of formulation

Placebo:

In order to observe linearity of active ingredient of different concentration with fixed concentration of formulation placebo, six different samples are prepared where the concentration of active covered is 0-160% of test solution containing fixed concentration of formulation placebo.

c.  Linearity of fixed active agent with different concentration of formulation placebo

For the analysis of this purpose, six different samples are prepared where the concentration of active is fixed & the concentration of formulation placebo is different.

4. Accuracy & Recovery

Accuracy is the measure of how close the experimental value is to the true value or an accepted reference value. It is calculated as the percentage of recovery by the assay of the known added amount of analyte in the sample.

5. Sensitivity:

Sensitivity is the capacity of the procedure to record small variations in concentration. Limit of detection and limit of determination are dependent on the sensitivity.

a. Limit of detection:

It is the lowest concentration of analyte in a sample that can be detected but not necessarily quantities under the experimental conditions. Limit of detection is a measure of response to the analyte which is the double of the amplitude of the base line noise.

b. Limit of determination:

It is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under the experimental conditions. Limit of determination is a measure of response to the analyte that is six times of the amplitude of the base line noise i.e., 3 x limit of detection.

      a) Thiamine Mononitrate: 0.006 mg/ml

      b) Pyridoxine Hydrochloride: 0.003 mg/ml

6. System suitability:

It is a test of analysis by which any apparatus or set of apparatus reads exactly or not can be determined. For this purpose 10 replicate samples are analyzed.

7. Ruggedness:

The ruggedness was determined by using the data obtained by the analysis performed by different analyst. Each analyst prepared 5 samples of the same batch.

alyst prepared 5 samples of the same batch.