1.
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2.
 According FDA Validation is
“Established documented evidence which provides a high
degree of assurance that a specific process will continually
produce a product meeting its predetermined specifications
and quality attributes.”
 The Validated method should be stability indicating method,
which means the method that can detect the changes in
properties of drug substance or drug product with time
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3.
 Verification
 Compendial methods-Verification :
• These methods are listed in pharmacopoeia’s
• Use to evaluate defined characteristics of the drug
substance or drug product
• Suitability of Compendial methods must be verified
under actual conditions
• Should be partially validated
 Validation
 Non -Compendial methods-Validation
• Proposed by the applicant for use instead of Regulatory
Analytical Procedure
• Equivalency:
• Equivalency: required for an in-house method, when compendial standard is claimed
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4.
Validation
Development
Quality Control
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5.
ICH/USP
GMPs
FDA
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6.
Verification data is required.
 The compendial methods as published are typically validated based on an API or an
FPP originating from a specific manufacturer.
 Different sources of the same API or FPP may contain impurities and/or degradation
products that were not considered during the development of the monograph.
 The FPP may also contain excipients not present in the FPP(s) for which the
compendial method was developed.
 API
Assay: No validation generally required. Exception: specificity for any specified
impurities not in the monograph.
Purity: Full validation for specified impurities that are not included in the monograph
(specificity, linearity, accuracy, repeatability, intermediate precision,
LOD/LOQ).
 FPP
Assay: Specificity, accuracy and precision (repeatability). Eg. to rule out excipient
interference
Purity: Specificity, accuracy and precision (repeatability) for known imps.
 Full validation for specified impurities that are not included in the
monograph (specificity, linearity, accuracy, repeatability, intermediate
precision, LOD/LOQ)
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7.
 Full validation is required for an in-house purity/assay method.
 Equivalency must be demonstrated compared to the compendial
method
• Assay: same samples using the two methods;
• Related substances: same samples spiking with specified
impurities at the limits) using the two methods
 If equivalency is not demonstrated, it can not be claimed as
compendial standard
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8.
Equivalency is essentially two things:
 Ensure the in-house method is not inferior (in which case they
should adopt the compendial method)
 Ensure the compendial test can be met, if applied.
 Two samples are sufficient.
Note that a compendial method becomes an in-house method when
adjustments are made outside the allowable adjustments (Int.Ph,
USP, EP general chapter Chromatography)
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9.
 Specificity
 Precision
 Accuracy
 Linearity
 Range
 Quantitation Limit
 Detection Limit
 Robustness
 Stability in Analytical Solution
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10.
USP ICH
 Specificity
 Linearity and Range
 Accuracy
 Precision
 Limit of Detection
 Limit of Quantitation
 Ruggedness
 Robustness
 Specificity
 Linearity
 Range
 Accuracy
 Precision
 Repeatability
 Intermediate Precision
 Reproducibility
 Limit of Detection
 Limit of Quantitation
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11.
Type of
Analytical
Procedure
Identification
Impurity testing
Assay
Quantitative Limit Tests
Accuracy No Yes No Yes
Precision
Repeatability No Yes No Yes
Interm. Prec. No Yes No Yes
Specificity Yes Yes Yes Yes
LOD No No Yes No
LOQ No Yes No No
Linearity No Yes No Yes
Range No Yes No Yes
May be required, depending on the nature of the specific test
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12.
Testing a method to demonstrate it is suitable for its intended purpose and
the results obtained are reliable, accurate and reproducible.
Provides confidence that the method will perform properly under intended
conditions.
 Identification tests;
 Quantitative or limit tests for the control of impurities;
 Quantitative tests of the active moiety in samples of drug substance or drug
product or other selected component(s) in the drug product. Including assay,
Content Uniformity, dissolution, content of perseveratives.
Validation of analytical procedures requires:
 Qualified and calibrated instruments
 Documented methods
 Reliable reference standards
 Qualified analysts
 Sample integrity
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13.
 Ability of an analytical method to measure the analyte free
from interference due to other components.
 An investigation of specificity should be conducted during
the validation of identification tests, the determination of
impurities and assay
 Should be able to discriminate between compounds closely
related in structure.
 Confirmed by obtaining negative results for samples with
spiked related compounds and positive results for samples
with analyte.
 SELECTIVITY
“A measure of the discriminating power of a given analytical
procedure in differentiating between the analyte and other
components in the test sample.”
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14.
 Impurities/Degradants Available
 Spike with impurities/degradants
 Show resolution and a lack of interference
 Impurities/Degradants Not Available
 Stress Samples
 For assay, Stressed and Unstressed Samples should be compared.
 For impurity test, impurity profiles should be compared.
 Peak homogeneity tests should be performed
using PDA or mass spectrometry to show that the
analyte chromatographic peak is not attributable to
more than one component.
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15.
 SELECTIVITY & SPECIFICITY
15
Selectivity: Specificity:
If an analytical procedure is able to
separate and resolve the various
components of a mixture and detect
the analyte qualitatively
If the method determines or
measures quantitatively the
component of interest in the sample
matrix without separation
Restricted to qualitative detection of
the components of a sample
Restricted to quantitative
measurement of one or more analyte
Blank solution - to show no interference
Placebo - to demonstrate the lack of interference from excipients
Spiked samples - to show that all known related substances are resolved from each
other
Stressed sample of about 5% to 20% degradation is used to demonstrate the
resolution between degradants and the analyte of interest
Check peak purity of drug substance by photodiode array detector (PDA)
Representative chromatograms should be provided

16.
Temperature
 Humidity
 Acid Hydrolysis
 Base Hydrolysis
 Oxidation
 Light (UV/Vis/Fl)
Intent is to create 10 to 30 % Degradation
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17.
 The closeness of agreement (degree of scatter)
between a series of measurements obtained from
multiple samplings of the same homogeneous
sample.
Precision… Considered at 3 Levels
 Repeatability
 Intermediate Precision
 Reproducibility
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18.
 Repeatability
A minimum of nine determinations covering the specified
range for the procedure, e.g.
 three concentrations/three replicates each, or a
minimum of six determinations at 100% of the test
concentration
 Intermediate Precision
Within-laboratory variations
 usually on different days, different analysts and
different equipment.
 Reproducibility
Precision between laboratories
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19.
 Closeness of agreement between the value which is accepted either
as a conventional true value or an accepted reference value and the
value found
 Accuracy should be assessed using a
minimum of 9 determinations over a
minimum of 3 concentration levels covering
the specified range
 Accuracy is usually reported as percent
recovery by the assay (using the proposed
analytical procedure) 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. The
range for the accuracy limit should be within the linear range
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20.
 Drug Substance
For Assay
 Accuracy can be inferred once precision, linearity and
specificity have been established
For Related Substances/ Residual Solvents
 Accuracy should be assessed on samples spiked with
known amount of impurities
Drug Product
For Assay
 Known quantities of drug substance to be analysed have
been added to synthetic mixture of drug product
components.
For Related Substances/ Residual Solvents
 Accuracy should be assessed on samples spiked with
known amount of impurities
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21.
Accuracy range…..
Across the range of LOQ-150% of the target concentration
(shelf life limit), 3-5 concentrations, in triplicate each.
(LOQ, 50%, 100%, 150%)
Per cent recovery: in general, within 80-120%, depends on
the level of limit
Accurate &
precise
Accurate &
imprecise
Inaccurate &
precise
Inaccurate &
imprecise 21

22.
 Linearity of an analytical procedure is its ability to obtain test results
which are directly proportional to concentration of analyte in the
sample within a specified range.
 A series of samples should be prepared in which the analyte
concentrations span the claimed range of the procedure. If there is a
linear relationship, test results should be evaluated by appropriate
statistical methods
 Minimum of 5 concentration is recommended within the range
Range:
This is an expression of the lowest and highest levels of analyte that
have been demonstrated to be determinable for the product
The specified range is normally derived from linearity studies
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23.
 Assay : 80-120% of the theoretical content of active
 Content Uniformity: 70-130%
 Dissolution: ±20% of limits; eg if limits cover from
20% to 90% l.c. (controlled release),
linearity should cover 0-110% of l.c.
 Impurities: reporting level to 120%
of shelf life limit
 Assay/Purity by a single method:
reporting level of the impurities to
120% of assay limit
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24.
 Is the amount of analyte in a sample which can be
quantitatively determined with suitable precision and
accuracy
 Approaches (instrumental or non-instrumental):
 visual evaluation (Used for non-instrumental methods)
 signal to noise ratio (10:1 for Quantitation Limit)
 standard deviation of the response and the slope
Quantitation Limit = 10
S
 : SD of Response (The residual SD of regression line or SD of y
intercept of regression lines can be used as SD)
S : Slope of calibration curve
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25.
Signal
Noise
Peak A
LOD
Peak B
LOQ
Baseline
Ybl
LOD LOQ
Statistical estimate of
LOD & LOQ
LOD = 3.3 Sbl / b LOQ = 10 Sbl / b
Y = b X
+ a
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26.
 Is the amount of analyte in a sample which can be
detected but not necessarily quantitated as an exact
value
 Approaches (instrumental or non-instrumental):
 visual evaluation (Used for non-instrumental methods)
 signal to noise ratio (3:1 for Detection Limit)
 standard deviation of the response and the slope
Quantitation Limit = 3.3
S
 : SD of Response (The residual SD of regression line or SD of y
intercept of regression lines can be used as SD)
S : Slope of calibration curve
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27.
 Not required for assay/dissolution methods.
 Applicant should provide
 The method of determination
 The limits
 Chromatograms
 Visually identified
 Established by Repeatability
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28.
 Measure of its capacity to remain unaffected by small ,
but deliberate variations in method parameters and
provide an indication of its reliability during normal
usage
 If measurements are susceptible to variations in
analytical conditions, than it should be suitably
controlled or a precautionary statement should be
included in procedure
 System suitability parameters should be evaluated to
ensure that validity of analytical procedure is
maintained when ever used
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29.
 For Liquid Chromatography, typical variations are
 Influence of pH in a mobile phase ( ± 0.2 units)
 Influence of mobile phase composition (± 2.0% Organic phase)
 Different columns[ Lots/Suppliers]
 Temperature [Column Oven] (± 5° C)
 Flow rate ( ± 0.2ml/min)
 System Suitability
 Evaluate the System suitability parameters
 Validation of analytical method must be focused on critical separation
of components which can alter during change of conditions
 These critically separated components should be part of system
suitability
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30.
 Should be established
 By keeping the sample at RT or below 10°C and
injecting it at regular intervals
 No significant variations or trend should be observed
 Any significant variation in particular area / absorbance
should be compared or calculated against the initial value.
 In case of impurity the % of impurity present should be
verified against the initial .
 The outcome of the result should be mentioned in the
particular regular test procedure.
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31.
When Re- validation is required !!!
 Method parameters have been changed
 The scope of the method has been changed
 Synthetic methods have been changed
 Impurity profile has been changed (Due to change in
excipient).
 Changes in equipment or suppliers of critical supplies of
the API or final drug product will have the potential to
change their degradation profile and may require the
method to be redeveloped and revalidated.
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32.
 Integral part of many analytical procedures
 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 (in General): Repeatability, Tailing factor
(T), Capacity factor (k’), Resolution (R), and Theoretical
Plates (N)
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33.
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)
Assay: RSD ≤1% (API) or ≤ 2% (FPP), n ≥ 5
Impurities: in general, RSD ≤ 5% at the limit level,
up to 10% or higher at LOQ, n ≥ 6 33

34.
Allowed HPLC Adjustment
USP (Ref: General Chapter <621>) EP (Ref: General Chapter 2.2.46)
Column Length ±70% ±70%
Internal Diameter
Can be adjusted if linear velocity is
kept constant ±25%
Particle Size Reduction of 50%, no increase Reduction of 50%, no increase
Flow Rate
±50% or more as long as the linear
velocity is kept contant ±50%
Column Temperature ±10C ±10% Max 60C
Injection Volume
Change allowed as long as SST
criteria are met
May be decreased (if LOD and
repeatability ok)
PH ±0.2 Units
±0.2 Units (±1% for neutral
substances)
UV wavelength <±3 nm (in condition) <±3 nm ((in condition))
Conc. Salts in Buffer ±10% ±10%
Composition of mobile phase
Minor Components (<50%) ±30%
or ±10% absolute whichever is
smaller
Minor Components ±30% or ±2%
absolute whichever is larger
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35.
 Some 483 Observations
 Analytical methods are not adequately
validated
 There was no inadequate method validation
specificity data to demonstrate that each
method was capable of distinguishing the
active ingredient from its impurities and degradation products.
 Specificity studies did not include the minimum stress conditions of
acid and base hydrolysis, oxidation, thermal degradation and
photolysis, degradation schematic for the active ingredient that
identifies the major degradation products was not included for
each product.
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36.
 Some 483 Observations
 Stress studies conducted as part of method validation do
not target a minimum amount of degradation. … a
standard period of two hours as commonly used for stress
studies with no justification…
 Spreadsheets used to calculate linearity, percent recovery,
and final assay results for the cleaning validation of
…were not validated and the data transcribed from
chromatographs to the spreadsheets were not checked for
accuracy.
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37.
 FDA Waning Letter
 On addition to the example
of modifying both Compendial methods and
customer supplied methods, we also observed
the use of un-validated in-house methods as
well as un-validated modifications to in-house
methods.
 A statement indicating that the method has not been validated in the
particular formulation was included in the certificate of analysis
for…use of this statement does not absolve…from using valid,
accurate, and reproducible methods.
 OOS accuracy results reported by analyst 3 were never submitted in
the final report. Repeat analysis performed in a different system
passed specifications and these results were submitted in the report.
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38.
 FDA Warning Letter
 There is no assurance that qualification or maintenance of
the laboratory equipment can consistently produce valid
and accurate analytical results in that numerous examples
of test data were invalidated due to instrument
malfunction.
 Attempts to corroborate data in the validation report with
supporting raw data in the laboratory were difficult and
frustrating for the FDA personnel conducting the
inspection
 Raw data and calculations were not checked by a second
responsible individuals required by your procedures.
Inaccurate calculations were noted in the report 38

39.
 FDA Warning Letter
 The process validation samples were
assays using an HPLC method that had not been
validated. The method validation used for both products,
did not include a protocol that included specification and
acceptance criteria. … The method validation was not
reviewed and approved until during the current inspection.
Lots of both products were released for distribution prior
to completion of the method validation.
 Method validation for the product one product is
inadequate in that the data does not assess all variables,
such as different mobile phase concentrations and
analytes, to demonstrate that the method can sustain
variance.
39

40.
 Data for acceptance, release, stability will only be trustworthy
if the methods used are reliable
 More efficient use of resources in the R&D process, as a
consequence
 Validation should be Protocol driven
 Document each and every information with regard to
std/sample/reagent/impurities/process/ preparation etc.
 Document the details of each experiment with suitable way.
 Review each experiment after its completion and relate its
outcome with other experiments
 Identification and elimination of the need to duplicate studies
to meet different regulatory requirements
40

41.
 From My Side……
 Why is analytical monitoring necessary?
 What is the purpose of analytical validation?
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42.
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