This document provides an overview of analytical method validation. It defines validation as demonstrating a method is suitable for its intended purpose. Key validation characteristics discussed include precision, accuracy, specificity, linearity, range, detection limit, quantitation limit, ruggedness and robustness. The document describes the methodology for evaluating each characteristic, such as spiking known concentrations of analytes and establishing acceptance criteria. It emphasizes that validation confirms a method consistently produces results meeting pre-defined standards of quality.

1. ANALYTICAL METHODANALYTICAL METHOD
VALIDATION FOR RSVALIDATION FOR RS
Bhanu Prakash . N
Analytical R&D
Email: bhanu.analytical@gmail.com

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
 Detection Limit
 Quantitation Limit
 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
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 repeadtly 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 impurity stock solution of known concentration (individual / Mix).
While preparation of stock solution consider the purity of analytes, target
concentrations and concentration matrix.
2. Spike the portion of the stock solution to test sample ( Drug product /Drug
substance) so that the concentration of the impurity would be 0.3%(or
specification limit) of the test concentration as per the test method.(100%
level).
3. Perform the the analysis on six determinations at 100% level of test
concentration.
Acceptance Criteria:
Relative standard deviation; NMT 15.0%

9. Analytical Method validation:
• Accuracy:
Definition:
It is the closeness of agreement between true value and measured value.
Methodology for Accuracy :
1. Prepare the impurity stock solution of known concentration (individual/Mix).
2. Spike the known portion of the stock solution to test sample ( Drug product/Drug
substance) so that the concentration of the impurity would be 0.3% (or
specification limit) of the test concentration as per the test method.(100% level).

10. Analytical Method validation:
Methodology for Accuracy Cont….. :
3. In the same manner as above, spike the impurity solution at different levels e.g
50%,75%,125%,150% (or specified in protocol) so that the concentration of the
impurity will be 0.15%,0.225%,0.375% and 0.45% respectively, of the test
concentration.Perform the analysis as per the test method and calculate the %
recovery.
4 .Perform the analysis on three different determinations at each spike level.
Acceptance Criteria:
% Recovery should be NLT 85.0% and NMT 115.0%.

11. Analytical Method Validation
• Detection Limit:
Definition:
It is lowest amount of analyte in a sample that can be detected but not
necessarily quantitated under the stated experimental conditions.
Methodology:
Following are different approaches
1) Visual Evaluation Method.
Prepare the sample solutions with known lowest possible concentrations
of analyte and establish the minimum concentration at which the analyte
can be reliably detected by analysing as per test method .

12. Analytical Method Validation
2) Based on Signal to Noise Ratio Method.
Prepare the analyte solutions with known lowest possible concentrations
of analyte and analyse as per test method .
Consider the concentration as a Limit of detection which gives signal to noise ratio
between 3 : 1 or 2 : 1
3) Based on the standard Deviation of the Response and the Slope:
 Prepare the blank solution as per test method and inject six times into the
chromatographic system.
Similarly prepare the linearity solution staring from lowest possible
concentration of analyte to 150% (or as per protocol) of target concentration and
establish the linearity curve.

13. Analytical Method Validation
The detection limit (DL) may be expressed as
3.3 x standard deviation of the response of the blank
DL = ----------------------------------------------------------------------------
Slope
The slope shall be estimated from the calibration curve of the
analyte.

14. Analytical Method Validation
• Quantitation Limit:
Definition:
It is lowest amount of analyte in a sample which can be quantitatively
determined with acceptable accuracy and precision.
Methodology:
Following are different approaches
1) Visual Evaluation Method.
Prepare the sample solutions with known lowest possible concentrations
of analyte and establish the minimum concentration at which the analyte
can be reliably quantified by analysing as per test method .

15. Analytical Method Validation
2) Based on signal to noise ratio method.
Prepare the analyte solutions with known lowest possible concentrations of
analyte and analyse as per test method .
Consider the concentration as a Limit of Quantification which gives signal
to noise ratio between 10 : 1.
3) Based on the standard Deviation of the Response and the Slope:
 Prepare the blank solution as per test method and inject six times into the
chromatographic system.
Similarly prepare the linearity solution staring from lowest possible concentration
of analyte to 150% (or as per protocol) of target concentration and establish the
linearity curve.

16. Analytical Method Validation
The Quantification limit (QL) may be expressed as
10 x standard deviation of the response of the blank
QL = ----------------------------------------------------------------------------
Slope
The slope shall be estimated from the calibration curve of the
analyte.
Perform the Precision and accuracy at the level of limit of quantification by
spiking LOQ concentration on placebo / Drug
product / Drug substance.
For detail methodology and acceptance criteria refer Precision and accuracy of
test method.

17. 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.

18. 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. 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 20%.

19. 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. Prepare the sample and placebo solution
as per test method and analyse.
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.

20. 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. Placebo / Blank should not elute at the retention time of analyte peak.
2. Peak purity of analyte peak should be confirmed.
3. Degradation of active analyte peak should be from 1% to 20%.

21. 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 limit of quantification level to 150% (or as
per protocol) of the target concentration and establish the calibration
curve by analysing the sample as per test method.
Linearity shall be established across the range. Minimum five concentration across
the range are recommended

22. 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 0.997.

23. 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.

24. 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)

25. 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.

26. 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.
2. RRT’s of impurity peaks should be comparable with RRT’s mentioned in test
method.
For filter suitability :
Difference between the % impurity should not be more than 0.04 against the
unfiltered / centrifuged sample.

27. 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)

28. 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.

29. 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 0.04% for individual known impurity
and 0.1% for total impurities from initial value.
Similarity factor of standard should be NLT 0.95 and NMT 1.05.

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