ANALYTICAL METHOD

VALIDATION

BY: P. VANAJA

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 INTRODUCTION
• Method validation is the process used to
confirm that the analytical procedure
employed for a specific test is suitable for its
intended use.

• Results from method validation can be used

to judge the quality, reliability and
consistency of analytical results.

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TYPES OF ANALYTICAL PROCEDURES TO BE VALIDATED

• Identification tests

• Quantitative tests for impurities content.

• Limit tests for the control of impurities.

• Quantitative tests of the active moiety in

samples of drug substance or drug product.
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 Analytical methods need to be validated or revalidated

 Before their introduction into routine use

 Whenever the conditions change for which

the method has been validated

 Whenever the method is changed

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 Laboratory Analytical Method Flow
During the Drug Development Process

Method Method Method

Development Validation Transfer

Approved
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 STEPS IN METHOD VALIDATION
• Develop a validation protocol or operating procedure
for the validation.
• Define the application, purpose, and scope of the
method.
• Define the performance parameters and acceptance
criteria.
• Define validation experiments.
• Verify relevant peformance characteristics of
equipment.
• Qualify materials
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• Define criteria for revalidation.
• Define type and frequency of system
suitability tests and/or analytical quality
control(AQC) checks.
• Document validation experiments and results
in the validation report.

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• Perform pre-validation experiments.
• Adjust method parameters or acceptance
criteria if necessary.
• Perform full internal (and external) validation
experiments.
• Develop SOPs for executing the method in
the routine.

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 PARAMETERS FOR METHOD
VALIDATION
• SPECIFICITY

• LINEARITY

• RANGE

• ACCURACY
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• ROBUSTNESS

• SYSTEM SUITABILITY TESTING

• PRECISION

• DETECTION LIMIT

• QUANTITATION LIMIT
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 SPECIFICITY

• The ability to assess unequivocally the

analyte in the presence of components which
may be expected to be present.

• An investigation of specificity should be

conducted during the validation of
identification tests, the determination of
impurities, and the assay.

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IDENTIFICATION

• Suitable identification tests should be able to

discriminate between compounds of closely
related structures which are likely to be
present.

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ASSAY AND IMPURITY TEST(S)
• For chromatographic procedures,
representative chromatograms should be used
to demonstrate specificity, and individual
components should be appropriately labeled.

• For critical separations, specificity can be

demonstrated by the resolution of the two
components which elute closest to each other.

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• In cases where a nonspecific assay is used,

 For ex: where a titration is adopted to assay

the drug substance for release, the
combination of the assay and a suitable test
for impurities can be used.

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 For the assay, the two results should be
compared.
 For the impurity tests, the impurity profiles
should be compared.

• Peak purity tests may be useful to show that

the analyte chromatographic peak is not
attributable to more than one component
(e.g., diode array, mass spectrometry).

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 LINEARITY

• Ability (within a given range) to obtain test

results which are directly proportional to the
concentration (amount) of analyte in the
sample.

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• Linearity should be evaluated by visual
inspection of a plot of signals as a function of
analyte concentration or content.

• To obtain linearity between assays and

sample concentrations, the test data may
have to be subjected to a mathematical
transformation prior to the regression
analysis.

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• Some analytical procedures, such as
immunoassays, do not demonstrate linearity
after any transformation.
• In this case, the analytical response should
be described by an appropriate function of
the concentration (amount) of an analyte in a
sample.

• For the establishment of linearity, a

minimum of five concentrations is
recommended.
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 RANGE

• The interval between the upper and lower

concentration of analyte in the sample.

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For the assay of a drug substance or a finished (drug)
product: Normally from 80 to 120 percent of the test
concentration;

For the determination of an impurity: From the reporting

level of an impurity 2 to 120 percent of the
specification;

Note: For validation of impurity test procedures carried

out during development, it may be necessary to
consider the range around a suggested (probable) limit.

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 ACCURACY

• The accuracy of an analytical procedure

expresses the closeness of agreement
between the value which is accepted either
as a conventional true value or an accepted
reference value and the value found.

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ASSAY
1. Drug substance:
(a)Application of an analytical procedure to an
analyte of known purity (e.g.,reference material);
(b)Comparison of the results of the proposed
analytical procedure with those of a second well-
characterized procedure, the accuracy of which is
stated and/or defined
(c)Accuracy may be inferred once precision,
linearity, and specificity have been established.

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2. Drug product:
(a) Application of the analytical procedure to
synthetic mixtures of the drug product
components to which known quantities of
the drug substance to be analyzed have been
added;

(b) Accuracy may be inferred once precision,

linearity, and specificity have been
established.
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IMPURITIES
• Accuracy should be assessed on samples
(drug substance/drug product) spiked with
known amounts of impurities.

• It should be clear how the individual or total

impurities are to be determined,
e.g. weight/weight or area percent, in all cases
with respect to the major analyte.

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RECOMMENDED DATA
• Accuracy should be assessed using a minimum
of 9 determinations over a minimum of 3
concentration levels covering the specified
range

• Reported as percent recovery by the assay of

known added amount of analyte in the sample
or as the difference between the mean and the
accepted true value together with the
confidence intervals.
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 ROBUSTNESS

• Measure of its capacity to remain unaffected

by small, but deliberate variations in method
parameters and provides an indication of its
reliability during normal usage.

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• It should show the reliability of an analysis
with respect to deliberate variations in
method parameters.

Examples of typical variations are:

• Stability of analytical solutions
• Extraction time

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In the case of liquid chromatography,
 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,
 Different columns
 Temperature
 Flow rate
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 PRECISION
• The precision of an analytical procedure
expresses the closeness of agreement
between a series of measurements obtained
from multiple sampling of the same
homogeneous sample under the prescribed
conditions.

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 Precision levels

Repeatability Intermediate precision Reproducibility

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 SYSTEM SUITABILITY TESTING

• 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
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 DETECTION LIMIT

• The detection limit of an individual analytical

procedure is the lowest amount of analyte in
a sample which can be detected but not
necessarily quantitated as an exact value.

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BASED ON VISUAL EVALUATION

• The detection limit is determined by the

analysis of samples with known
concentrations of analyte and by establishing
the minimum level at which the analyte can
be reliably detected.

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BASED ON SIGNAL-TO-NOISE
• Determination of the signal-to-noise ratio is
performed by comparing measured signals from
samples with known low concentrations of analyte
with those of blank samples and establishing the
minimum concentration at which the analyte can be
reliably detected.

• A signal-to-noise ratio between 3 or 2:1 is generally

considered acceptable for estimating the detection
limit.
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BASED ON THE STANDARD DEVIATION OF
THE RESPONSE AND THE SLOPE
• The detection limit (DL) may be expressed as:

3.3 σ
DL= ____________
S
σ = the standard deviation of the response
S = the slope of the calibration curve

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 QUANTITATION LIMIT
• The quantitation limit of an individual
analytical procedure is the lowest amount of
analyte in a sample which can be
quantitatively determined with suitable
precision and accuracy.

• The determination of impurities and/or

degradation products.

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BASED ON VISUAL EVALUATION
• The quantitation limit is generally
determined by the analysis of samples with
known concentrations of analyte and by
establishing the minimum level at which the
analyte can be quantified with acceptable
accuracy and precision.

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 BASED ON SIGNAL-TO-NOISE
• Determination of the signal-to-noise ratio is
performed by comparing measured signals
from samples with known low concentrations
of analyte with those of blank samples and by
establishing the minimum concentration at
which the analyte can be reliably quantified.

• A typical signal-to-noise ratio is 10:1.

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 BASED ON THE STANDARD DEVIATION OF
THE RESPONSE AND THE SLOPE
• The quantitation limit (QL) may be expressed
as:
10 σ
QL= _______________
S
• σ = the standard deviation of responses
• S = the slope of the calibration curve

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 REFERENCES
• www.ich.org
• Pharmaceutical process validation: an
international third addition
• http://www.fda.gov/cder/guidance/ichq2a.pdf
• http://www.nihs.go.jp/drug/validation/q2bw
ww.html

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THANQ

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G