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.
