2. Analytical Method Validation
What is Validation ?
The Objective of validation of an analytical method is to demonstrate
that it is suitable for it’s intended purpose.
OR
It is the process by which it is established, by laboratory studies, that
the performance characteristics of the method meet the requirements
for the intended analytical applications.
3. Analytical Method Validation
• Different Types of Validation characteristics
Precision
Accuracy
Specificity
Linearity
Range
Ruggedness
Robustness
4. Analytical Method validation
• Considerations & requirements prior to Method Validation
Instruments / Equipments are validated and calibrated.
Reference Standards and Reagents are standardized.
Analysts are trained and qualified.
Availability of Test methods, glassware's etc.
Availability of test material.
Approved protocols.
5. Analytical Method validation
System Suitability :
System suitability testing is an integral part of analytical procedures.
The tests are based on the concept that the equipment, electronics,
analytical operations and samples to be analyzed constitute an integral
system that can be evaluated as such.
System suitability test parameters to be established for a particular
procedure depend on the type of procedure being validated.
6. Analytical Method validation
• Methodology :
Prepare the solution and evaluate system suitability parameters
as per the test method .
Based on the evaluation of system suitability parameters at
each
validation parameter,system suitability criteria can be
established.
Acceptance Criteria :
As per the test method.
7. Analytical Method Validation
• Precision :
• Definition :
It is degree of agreement among individual test results when the method is
applied repeatedly to multiple samplings of a homogeneous sample.
• The precision of test method is usually expressed as the standard deviation or
relative standard deviation of a series of measurements.
• It is a measure of either the degree of reproducibility or repeatability of the
analytical method under normal operating conditions.
8. Analytical Method Validation
Methodology :
1. Prepare the sample as per the test method and analyze.
2. Perform the the analysis on six determinations at 100% level of test
concentration.
Acceptance Criteria:
Relative standard deviation; NMT 2.0%
9. Analytical Method validation:
• Accuracy:
Definition:
It is the closeness of agreement between true value and measured value.
Methodology for Accuracy :
1. If the test method is dropping of tablet ,spike the active ingredient
from 50%,75%,100%,125% and 150% of the test concentration with
placebo equivalent to the test concentration(Subtract the weight of
active ingredient) and perform the analysis.
2. In case of crushing tablet method ,weigh the sample equivalent to
50%,75%,100%,125% and 150% of the test concentration and perform the
analysis.
10. Analytical Method validation:
Methodology for Accuracy Cont….. :
3 .Perform the analysis on three different determinations at each spike level..
Acceptance Criteria:
% Recovery should be NLT 97.0% and NMT 103.0%.
11. Analytical Method Validation
Specificity :
Definition :
The ability to assess unequivocally the analyte in the presence of components
that may be expected to present, such as impurities, degradation products and
matrix components.
Methodology:
Specificity shall be demonstrated by performing Placebo / blank interference
and forced degradation studies.
12. Analytical Method Validation
Methodology cont…..:
1. Blank interference :
Prepare blank solution as per test method and analyse as per test method.
2. Placebo interference (In case of Drug products) :
Prepare the placebo solution equivalent to the test concentration (Subtract the
weight of active ingredient) and analyse as per test method.
3. Spike the known impurities to the test preparation at 0.3% or specification
limit level.
4. Force Degradation studies :
Degrade the sample forcefully under the various stress conditions like Light,
heat, humidity, acid/base/water hydrolysis and oxidation and ensure the
degradation from 1% to 50%.
13. Analytical Method Validation
Methodology Cont……:
a) Light : Expose the Drug product, drug substance and placebo to UV light for
about 200watts/M2 and 1200 Klux for visible light.
b) Heat : Expose the Drug product, drug substance and placebo at 105 °C for
about 12 hours. Prepare the sample and placebo solution as per test method and
analyse.
c) Humidity : Expose the Drug product, drug substance and placebo for about
80% RH at about 25°C for about one week. Prepare the sample and placebo solution
as per test method and analyse.
d) Acid /Base : Prepare the acid or base solution of 0.1N and reflux the sample and
placebo with 50ml of acid/base solution for about 1 hour at 60°C. Neutralize the
solution and dissolve the contents in diluent as per test method. Change the strength
of acid and base or refluxion time to ensure the desired degradation.
14. Analytical Method Validation
Methodology Cont….:
e) Water : Reflux the sample / placebo with 100ml water for 12 hour at 60°C
Dissolve the contents in diluent as per test method. Change the refluxion
time so as to ensure the desired degradation
f) Oxidation : Reflux for 12 hour at 60°C. Dissolve the contents in diluent as per test
method. Change the refluxion time so as to ensure the desired degradation.
Note : Based on the physio-chemical properties and literature stress conditions can
be decided.
Acceptance Criteria :
1. Peak purity of analyte peak should be confirmed.
2. Degradation of active analyte peak should be from 1% to 50%.
15. Analytical Method Validation
• Linearity :
Definition:
The linearity of an analytical method is it’s ability to elicit test results that are directly or by
a well defined mathematical transformation, proportional to the concentration of the
analyte in a sample within the given range.
Methodology :
Prepare the analyte solutions from 50% to 150% (or as per protocol) of the
target concentration and establish the calibration curve by analyzing the
sample as per test method.
Linearity shall be established across the range. Minimum five concentration across the
range are recommended.
16. Analytical Method Validation
• Linearity cont……
Calculate the co-efficient of co-relation, Y-intercept and slope of regression
line.
If linearity is not meeting the acceptance criteria, establish the range of
concentration in which it is linear.
Acceptance criteria:
Co efficient of co relation should be NLT 0.999.
17. Analytical Method Validation
• Range :
The range of an analytical method is the interval between the upper and lower
levels of analyte that have been demonstrated to be determines with a suitable
level of precision, accuracy and linearity using the test method.
The range of the test method is validated by verifying that the analytical method
provides acceptable precision, accuracy and linearity when applied to samples
containing analyte at the extremes of the range as well as within the range.
Range can be established during linearity and accuracy study,
Acceptance Criteria:
Same as applicable for Precision, Linearity and accuracy.
18. Analytical Method Validation
Robustness :
Definition:
It is a measure of method's ability to remain unaffected by small but
deliberate variations in method parameters.
The following are the typical method parameters need to change
deliberately and verify during method validation :
Flow rate (+/- 0.2ml/minutes)
Mobile phase composition (+/- 10% of organic phase)
Column oven temperature (+/- 5°C)
pH of buffer in mobile phase (+/- 0.2 units)
Filter suitability (At least two filters)
19. Analytical Method Validation
Methodology :
a) Mobile phase variation: Prepare the mobile phases by changing organic
phase to ±10% of the mobile phase composition.
b) Flow rate : Change the flow rate by ± 0.2 ml/minutes of the target flow
rate mentioned in test method.
c) Temperature of the Column : Change the temperature the column by ±
5.0°C of the target temperature mentioned in Test method.
d) pH of the buffer of mobile phase : Prepare the mobile phases by changing
the pH of buffer by ± 0.2 units of the pH mentioned in the test method.
20. Analytical Method Validation
Methodology :
E) Filter Suitability : Prepare the test solution as per test method and filter through
two different types filters. Analyse the sample as per test method and compare
the results against unfiltered / centrifuged sample.
Acceptance Criteria :
For Variations :
1. System suitability should meet the acceptance criteria as per test method.
For filter suitability :
Similarity factor should be from 0.98-1.02
21. Analytical Method Validation
Ruggedness :
Definition:
It is a measure of method reproducibility under variable conditions within
specified test parameters of test method.
The following are the typical method parameters need to tested during
method validation :
Analyst-to-Analyst variability.
Column-to-Column variability.
System-to-System variability.
Different days.
Different Laboratories.
Stability of Solutions and mobile phase. (At least for 48 hours)
22. Analytical Method Validation
• Methodology :
• Perform the precision as mentioned in precision of test method
by using different systems, different columns, different analyst ,
in different Laboratories and on different day.
• The method is to be consider rugged for individual variation, if it meets
the precision of test method and method transfer acceptance criteria.
23. Analytical Method Validation
Methodology :
Perform the bench top and refrigerator stability of the standard and test solution
for 24hrs and 48 hrs ,calculate the results each time against freshly prepared
standard solution.If 24hrs stability is not meeting the acceptance criteria,
perform the stability studies on hourly basis on bench top and in refrigerator.
Acceptance Criteria :
The % Difference should not be more than 2.0% for an assay from initial value.
Similarity factor of standard should be NLT 0.98 and NMT 1.02.
