1.
What is Validation?
• Validation is defined as establishing documented evidence
which provides a high degree of assurance that a specific process
will consistently produce a product meeting its pre-determined
specifications and quality attributes.
 Identification
 Determination of impurities
 Assay
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2.
Why 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 methods is also required by most
regulations.l.
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3.
Typical Analytical Procedures To
Be Validated
• Four most common 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
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4.
Revalidation
• Revalidation may be necessary in the following circumstances:
• Changes in the synthesis of the drug substance
• Changes in the composition of the finished product
• Changes in the analytical procedure
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5.
Parameters
1. Linearity and Range
2. Specificity
3. Precision
4. Accuracy
5. Limit of Detection
6. Limit of Quantitation
7. Robustness
8. System Suitability
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6.
Characteristic Identification Impurities Testing Assay
Quantitative Limit
Accuracy _ + _ +
Precision
a. Repeatability _ + _ +
b. Intermediate precision _ + _ +
Specificity + + + +
LOD _ _ + _
LOQ _ + _ _
Linearity _ + _ +
Range _ + _ +
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-signifies that this characteristic is not normally evaluated
+ signifies that this characteristic is normally evaluated

7.
1. Linearity and Range
LINEARITY
• Ability to obtain test results that are directly (or by a well-defined
mathematical transformation) proportional to the concentration
of analyte in samples within a given range. (y = mx + c)
• The following parameters should be determined:
 correlation coefficient
 y-intercept(c)
 slope of the regression line(m)
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8.
Determination of Linearity
• For establishment of linearity, minimum 5 concentrations are
recommended.
• Linearity results should be established by appropriate statistical
methods.
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y = 0.0868x + 0.019
R² = 0.9988
0
0.2
0.4
0.6
0.8
1
0 2 4 6 8 10 12

9.
• Transformations are also acceptable and may include log, square
root, or reciprocal (other transformations are acceptable)
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Conc.
(µg/ml)
Response
1 0.0625
2 0.25
3 0.562
4 0.922
5 1.562
y = 0.3671x - 0.4296
R² = 0.9515
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 2 4 6

10.
• If linearity is not attainable, a nonlinear model may be used. The
goal is to have a model (whether linear or nonlinear) that
describes closely the concentration-response relationship.
• Acceptance criteria: Linear regression r2 > 0.95
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Conc.
(µg/ml)
√Response
1 0.25
2 0.5
3 0.75
4 0.96
5 1.25
y = 0.246x + 0.004
R² = 0.9982
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 2 4 6

11.
RANGE
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• The range of an analytical procedure is the interval between the
upper and lower levels of analyte(including these levels) that
have been demonstrated with a suitable level of precision,
accuracy, and linearity.
• For assay tests, ICH requires the minimum specified range to be
80 to 120 percent of the test concentration. It also requires the
range for the determination of an impurity to extend from the
limit of quantitation or from 50 percent of the specification of
each impurity, whichever is greater, to 120 percent of the
specification

12.
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• Acceptable range having linearity, accuracy, precision.
• For Drug Substance & Drug product Assay
– 80 to 120% of test Concentration
• For Content Uniformity Assay
– 70 to 130% of test Concentration
• For Dissolution Test Method
– +/- 20% over entire Specification Range
• For Impurity Assays
– From Reporting Level to 120% of Impurity Specification for
Impurity Assays
– From Reporting Level to 120% of Assay Specification for
Impurity/Assay Methods

13.
2. Precision
• The precision of an analytical procedure expresses the closeness
of agreement (degree of scatter) between a series of
measurements obtained from multiple sampling of the same
homogeneous sample under the prescribed conditions.
• Sample prepared in six replicates on the same day and analysed
as per the method.
• The precision is reported in terms of %RSD
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14.
Concentration Absorbance
10 µg/ml
Mean
0.22
0.35
0.39
0.53
0.36
Less Variation More Variation
High Precision Low Precision
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Concentration Absorbance
10 µg/ml
Mean
0.28
0.31
0.29
0.30
0.29

15.
• Precision may be considered at three levels:
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Precision
Repeatability
Intermediate
Precision
Reproducibility

16.
1. Repeatability
• Repeatability expresses the precision under the same
operating conditions over a short interval of time.
• Repeatability should be assessed using a minimum of
9 determinations covering the specified range.
2. Intermediate Precision
• Intermediate precision expresses variations within
laboratories, such as different days, different analysts,
different equipment, and so forth
3. Reproducibility
• Reproducibility expresses the precision between
laboratories. It is assessed by means of an inter-
laboratory trial. (Defined as ruggedness in USP, ISO
17025)
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17.
• Following parameters should be reported:
a. Standard deviation
b. Relative standard deviation (coefficient of variation)
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Concentration
µg/ml
Absorbance SD & % RSD
8
0.337 0.00041,
1.223%
0.348
0.341
12
0.575 0.0106,
1.815%
0.583
0.596
20
0.967
0.0091,
0.933%
0.985
0.978
0
0.2
0.4
0.6
0.8
1
1.2
4 8 12 16 20 24
8
8
8
12
12
12
20
20
20

18.
3. Accuracy
• Closeness of agreement between the conventional true value /
an accepted reference value and the value found.
High Accuracy Less Accuracy
(Less Precision) (High Precision)
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19.
Determination of Accuracy
Drug Substance
a)application of analytical
procedure to a reference
material
b) to compare the results
c) accuracy may be inferred
once precision, linearity
and specificity have been
established.
Drug Product
a)application of the analytical
procedure to synthetic
mixtures to which known
quantities of the drug
substance have been added
b) to compare the results
c) accuracy may be inferred
once precision, linearity and
specificity have been
established.
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1. Assay

20.
2. Impurities (Quantitation)
 Assessed on samples (drug substance/drug product) spiked with
known amounts of impurities.
 Accuracy should be assessed using a minimum of 9 determinations
over a minimum of 3 concentration levels covering the specified
range (e.g., 3 concentrations/3 replicates each of the total
analytical procedure).
 Accuracy should be 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.
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21.
4. Limit of Detection & Limit of
Quantitation
LOD
 Lowest amount of analyte in a
sample that can be detected
but not necessarily
quantitated.
 Estimated by Signal to Noise
Ratio of 3:1.
LOQ
Lowest amount of analyte
in a sample that can be
quantified with suitable
accuracy and precision.
Estimated by Signal to
Noise Ratio of 10:1.
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22.
4. Limit of Detection & Limit of
Quantitation
• Limit of Detection:
• It is the lowest amount of analyte in a sample which can be
detected but not necessarily quantitated.
• Limit of Quantitation:
• It is the lowest amount of analyte in a sample which can be
quantitatively determined with suitable precision and accuracy.
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23.
Determination of LOD & LOQ
Limit of Detection
 Method
 Based on visual evaluation
 Based on standard deviation
of response and slope
LOD = 3.3 σ / Slope
 Signal to noise ratio 2:1 or 3:1
Limit of Quantitation
 Method
 Based on visual evaluation
 Based on standard deviation
of response and slope
LOD = 10 σ / Slope
 Signal to noise ratio 10:1
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24.
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25.
6. Specificity
• The ability to detect the analyte of interest in the presence of
interfering substances (typically impurities, degradants, matrix).
1. Identification
• Suitable identification tests should be able to discriminate
between compounds of closely related structures which are
likely to be present.
• The discrimination of a procedure may be confirmed by
obtaining positive results from samples containing the analyte,
coupled with negative results from samples which do not
contain the analyte.
• The identification test may be applied to materials structurally
similar to or closely related to the analyte to confirm that a
positive response is not obtained.
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26.
2. Assay and impurity test:
a. Impurities are available
• For the assay , this should involve demonstration of the
discrimination of the analyte in the presence of impurities
and/or excipients.
• This can be done by spiking pure substances with
appropriate levels of impurities and/or excipients and
demonstrating that the assay result is unaffected by the
presence of these materials.
• For the impurity test, the discrimination may be established
by spiking drug substance or drug product with appropriate
levels of impurities and demonstrating the separation of
these impurities individually and/or from other components
in the sample matrix.
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27.
b. Impurities are not available
• If impurity or degradation product standards are unavailable,
specificity may be demonstrated by comparing the test results
of samples containing impurities or degradation products to a
second well-characterized procedure e.g. pharmacopoeial
method or other validated analytical procedure.
• As appropriate, this should include samples stored under
relevant stress conditions: light, heat, humidity, acid/base
hydrolysis and oxidation.
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28.
7. Robustness
• The robustness of an analytical procedure is a 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.
• If measurements are susceptible to variations in analytical
conditions, the analytical conditions should be suitably
controlled or a precautionary statement should be included in
the procedure, such as:
• Use solution within 24 hours
• Maintain temperature below 25 degrees
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29.
• 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
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30.
8. System Suitability
• System suitability testing is an integral part of many 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.
• Determination: repeatability, tailing factor (T), capacity factor
(k’), resolution (R), and theoretical Plates (N)
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31.
System Suitability Requirements
Parameters Recommendations
K’ In general k’ ≥ 2.0
R
R >2, between the peak of interest and
the closest potential interferent (degradant,
internal STD, impurity, excipient, etc…)
T T ≤ 2
N In general N > 2000
Repeatability RSD ≤ 2.0% (n ≥ 5)
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32.
Conclusion
• When the method is properly validated consistent, reliable
and accurate results are obtained. Also, Validation of
analytical methods is also required by regulations. Hence it
is very important to validate any analytical method that has
been developed.
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33.
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