Analytical Method Validation
Sujan Banik, Ph.D.
Assistant Professor
Department of Pharmacy, NSTU
Validation
In recent years, the term validation has become familiar
in connection with pharmaceutical processes.
“Establishing documented evidence which provides a
high degree of assurance that a specific process will
consistently produce a product meeting its predetermined
specifications and quality characteristics”.
Purpose:
To ensure consistent product quality.
A new product or an old product manufactured using a
modified process or facility cannot be solid until the
‘process’ has been adequately validated.
Validation
The objective of any analytical measurement is to obtain
consistent, reliable and accurate data.
Validated analytical methods play a major role in
achieving this goal.
Validation of analytical procedures is the process of
determining the suitability of a given methodology for
providing useful analytical data.
Method validation is the process of demonstrating that
analytical procedures are suitable for their intended use
and that they support the identity, strength, quality, purity
and potency of the drug substances and drug products.
Method Life Cycle
Validation
Development Optimization
Today’s Validation Requirements
ICH/USP
GMPs
(legal) FDA
Parameters and Tests for Method Validation
“Validation of an analytical procedure is the process
by which it is established, by laboratory studies, that
the performance characteristics of the procedure
meet the requirements for the intended analytical
applications”.
Specificity/Selectivity
ICH defines specificity as
▪ “The ability to assess unequivocally the analyte in the
presence of components which may be expected to be
present.
▪ Ability of an analytical method to measure the analyte
free from interference due to other components.
▪ Specificity is the ability of a method to discriminate
between the intended analyte(s) and other components
in the sample.
Precision
The closeness of agreement (degree of scatter) between a
series of measurements obtained from multiple samplings of
the same homogeneous sample.
Precision may be considered at three levels: repeatability,
intermediate precision, and reproducibility.
Repeatability expresses the precision under the same
operating conditions over a short interval of time. Repeatability
is also termed intra-assay precision.
Intermediate precision expresses variations within
laboratories, such as different days, different analysts, different
equipment, and so forth.
Reproducibility expresses the precision between laboratories
(collaborative studies usually applied to standardization of
methodology).
Precision
A method’s intermediate precision may reflect
discrepancies in results obtained from:
different operators
inconsistent working practice
different instruments
standards and reagents from different suppliers
columns from different batches
a combination
Accuracy and Recovery
ICH defines the accuracy of an analytical procedure as
the closeness of agreement between the conventional
true value or an accepted reference value and the value
found.
Closeness of the test results obtained by the method
to the true value.
Accuracy and Precision
➢ The dictionary definition of both ‘accuracy’ and
‘precision’ are roughly the same, indicating that these
words may be used synonymously.
➢ However, in ‘Analytical Science’ they have two separate
meanings, the difference between them is best
illustrated by using target diagrams
Poor precision Good precision Good mean accuracy Good accuracy
poor accuracy poor accuracy poor precision good precision
Linearity and Calibration Curve
ICH defines linearity of an analytical procedure as its
ability (within a given range) to obtain test results that are
directly proportional to the concentration (amount) of
analyte in the sample.
Calibration graph
0.6
0.5
0.4
Signal
0.3
0.2
0.1
0
0 5 10 15 20 25
Concentration
Detection Limit (LOD)/Quantitation Limit (LOQ)
LOD
Lowest amount of analyte in a sample that can be
detected but not necessarily quantitated.
LOQ
Lowest amount of analyte in a sample that can be
quantified with suitable accuracy and precision.
Method validation -robustness
Robustness of an analytical method refers to it’s ability
to remain unaffected when subjected to small changes
in method parameters.
For example
In an hplc analysis the mobile phase is defined in terms of % organic
modifier, pH of the mobile phase, buffer composition, temperature
etc. A perfect mobile phase is one which allows small changes in the
composition without affecting the selectivity or the
quantitation of the method.
Alter all major parameters in order to ascertain when the method ceases
to function in accordance with specifications
Robustness
In the case of liquid chromatography, examples of
typical variations are:
• Influence of variations of pH in a mobile phase
• Influence of variations in mobile phase composition
• Different columns (different lots and/or suppliers)
• Temperature
• Flow rate
In the case of gas-chromatography, examples of typical
variations are:
• Different columns (different lots and/or suppliers)
• Temperature
• Flow rate