1. The document provides an overview of method validation requirements from various regulatory bodies and guidelines. It discusses key validation parameters such as specificity, linearity, range, accuracy, precision, detection limit, and quantitation limit. 2. Validation is required to demonstrate that analytical methods are suitable for their intended purposes. It identifies potential sources of error and quantifies errors in the method. Validation includes parameters like linearity, range, accuracy, and precision. 3. The document provides details on establishing various validation parameters according to regulatory guidelines from ICH, FDA, and USP. It also discusses considerations for validating methods like instrument qualification and defines method life cycles.

1. 1
2016
Method Validation
Prepared by :
Santram Rajput
(Technical Manager)
Sigma Test & Research Centre

2. European and International regulatory bodies and their
guidelines on different aspects of QA
Body Full name Guidance on
Eurachem Focus for Analytical Chemistry in Europe Method validation
CITAC Cooperation of International Traceability in
Analytical Chemistry
Proficiency testing
Quality Assurance
EA European Cooperation for Accreditation Accreditation
CEN European Committee for Normalization Standardization
IUPAC International Union of Pure & Applied Chem. Method validation
ISO International Standardization Organisation Standardisation
AOAC
ILAC
Association of Official Analytical Chemists
International Laboratory Accreditation Cooperat.
Internal qual. Control
Proficiency testing
Accreditation
FDA US Food and Drug Administration Method validation
USP United States Pharmacopoeia Method validation
ICH International Conference on Harmonization Method validation
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3. Method Validation
 Validation of analytical procedures is the process of determining the
suitability of a given methodology for providing useful
analytical data.
J. Guerra, Pharm. Tech. March 1986
 Validation is the formal and systematic proof that a method compiles
w i t h t h e r e q u i r e m e n t s f o r t e s t i n g a p r o d u c t w h e n
observing a defined procedures.
G. Maldener, Chromatographia, July 1989
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4.  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
substances in products.
 Method validation is primarily concerned with:
identification of the sources of potential errors
quantification of the potential errors in the method
 An method validation describes in mathematical and quantifiable
t e r m s t h e p e r f o r m a n c e c h a r a c t e r i s t i c s o f a n a s s a y
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5. Examples of Methods That Require
Validation Documentation
 Chromatographic Methods - HPLC, GC, TLC, GC/MS, etc.
Pharmaceutical Analysis - In support of CMC.
Bioanalytical Analysis - In support of PK/PD/Clinical Studies.
 Spectrophotometric Methods – UV/VIS, IR, AAS, XRD, ICP-MS,
AAS, XRF, etc
 Particle Size Analysis Methods - Laser, Microscopic, Sieving, SEC, etc.
 Automated Analytical Methods - Robots, Automated Analysis.
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6. Considerations Prior to
Method Validation
Suitability of Instrument
 Status of Qualification and Calibration
Suitability of Materials
 Status of Reference Standards, Reagents, Placebo Lots
Suitability of Analyst
 Status of Training and Qualification Records
Suitability of Documentation
 Written analytical procedure and proper approved protocol
with pre-established acceptance criteria
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7. Validation Step
 Define the application, purpose and scope of the method.
 Analytes? Concentration? Sample matrices?
 Develop a analytical method.
 Develop a validation protocol.
 Qualification of instrument.
 Qualify/train operator
 Qualification of material.
 Perform pre-validation experiments.
 Adjust method parameters and/or acceptance criteria if necessary.
 Perform full validation experiments.
 Develop SOP for executing the method in routine analysis.
 Document validation experiments and results in the validation report.
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8. Purpose of Method Validation
 Identification of Sources and Quantitation of Potential errors
 Determination if Method is Acceptable for Intended Use
 Establish Proof that a Method Can be Used for Decision Making
 Satisfy Requirements
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9. What is not Analytical Method Validation?
 Calibration
The Process of Performing Tests on Individual System
Components to Ensure Proper function
For example) HPLC Detector calibration
 Wavelength Accuracy/ Linear Range/ Noise Level/ Drift
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10.  System Suitability
Test to verify the proper functioning of the operating system,
i.e., the electronics, the equipment, the specimens and the
analytical operations.
 Minimum Resolution of 3.0 between the analyte peak and
internal standard peaks
 Relative Standard Deviation of replicate standard injections
of not more than 10.0%
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11. 11
System Suitability
Sample
Validation
MethodAnalyst
Calibration
Pump
Detector
Injector
Data System
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12. Method Life Cycle
12
Validation
Development Optimization
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13. Verification vs. Validation
 Compendial vs. Non-compendial Methods
 Compendial methods-Verification
 Non-compendial methods-Validation requirement
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14. Published Validation Guidelines
 1978 Current Good Manufacturing Practices (cGMPs)
 1987 FDA Validation Guideline
 1989 Supplement 9 to USP XXI
 1994 CDER Reviewer Guidance:
Validation of Chromatographic Method
 1995 ICH Validation Definitions:
Q2A, Text on Validation of Analytical procedures
 1997 ICH Validation Methodology:
Q2B, Validation of Analytical Procedures: Methodology
 1999 Supplement 10 to USP 23 <1225>: Validation of Compendial Methods
 1999 CDER “Bioanalytical Method Validation for Human Studies”
 2000 CDER Draft “Analytical Procedures and Method Validation”
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15.  The objective of validation of an analytical
procedure is to demonstrate that it is suitable
for its intended purpose
15
ICH Guideline for
Industry
Q2A, Text on
Validation of
Analytical Procedures
March 1995
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16.  In practice, it is usually possible to design the experimental
work such that the appropriate validation characteristics
can be considered simultaneously to provide a sound,
overall knowledge of the capabilities of the analytical
procedure, for instance: Specificity, Linearity, Range,
Accuracy, and
Precision.
16
ICH Guideline for Industry
Q2B, Validation of Analytical
Procedures: Methodology
2016

17. Today’s Validation Requirements
17
ICH/USP
GMPs
(legal) FDA
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18. ICH/USP Validation Requirements &
Parameters
 Specificity
 Linearity
 Range
 Accuracy
 Precision
 Repeatability
 Intermediate Precision
 Reproducibility
 Limit of Detection
 Limit of Quantitation
18
ICH
 Specificity
 Linearity and Range
 Accuracy
 Precision
 Limit of Detection
 Limit of Quantitation
 Ruggedness
 Robustness
USP
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19. USP Data Elements Required
For Assay Validation
19
Analytical
Performance
Parameter
Assay
Category 1
Assay Category 2
Assay
Category 3Quantitative Limit Tests
Accuracy Yes Yes * *
Precision Yes Yes No Yes
Specificity Yes Yes Yes *
LOD No No Yes *
LOQ No Yes No *
Linearity Yes Yes No *
Range Yes Yes * *
Ruggedness Yes Yes Yes Yes
* May be required, depending on the nature of the specific test.
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20. ICH Validation Characteristics vs.
Type of Analytical Procedure
20
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
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21. Specificity/Selectivity
 Ability of an analytical method to measure the analyte free from
interference due to other components.
Specificity is the ability to assess unequivocally the analyte in the presence of components
which may be expected to be present. Typically these might include impurities, degradants,
matrix, etc.
Purity Tests: to ensure that all the analytical procedures performed allow an accurate
statement of the content of impurities of an analyte, i.e. related substances test, heavy
metals, residual solvents content, etc.
Assay (content or potency): to provide an exact result which allows an accurate statement
on the content or potency of the analyte in a sample.
 Selectivity describes the ability of an analytical method to differentiate
various substances in a sample
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22. Specificity: Impurities Assay
 Chromatographic Methods
 Demonstrate Resolution
 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.
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23. Forced Degradation Studies
 Temperature (50-60℃)
 Humidity (70-80%)
 Acid Hydrolysis (0.1 N HCl)
 Base Hydrolysis (0.1 N NaOH)
 Oxidation (3-30%)
 Light (UV/Vis/Fl)
Intent is to create 10 to 30 % Degradation
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24. Linearity
 Ability of an assay to
elicit a direct and
proportional response
to changes in analyte
concentration.
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25. Linearity Should be Evaluated
 By Visual Inspection of plot of signals vs. analyte
concentration
 By Appropriate statistical methods
 Linear Regression (y = mx + b)
 Correlation Coefficient, y-intercept (b), slope (m)
 Acceptance criteria: Linear regression r2 > 0.95
Requires a minimum of 5 concentration levels
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26. Range
 The specified range is normally derived from linearity studies and depends on the intended application of
the procedure. It is established by confirming that the analytical procedure provides an acceptable degree of
linearity, accuracy and precision when applied to samples containing amounts of analyte within or at the
extremes of the specified range of the analytical procedure.
 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
 From MDL to 100% of Impurity Specification Limit
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27. Accuracy
 Closeness of the test
results obtained by the
method to the true value.
27

28. Accuracy
 Should be established across specified range of
analytical procedure.
 Should be assessed using a minimum of 3 concentration
levels, each in triplicate (total of 9 determinations)
 Should be reported as:
 Percent recovery of known amount added or
 The difference between the mean assay result and the accepted
value
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29. Accuracy Data Set (1 of 3)
29
Amount
Added (mg)
Amount
Found (mg)
Percent
Recovery
0.0 0.0 ---
50.2 50.4 100.5
79.6 80.1 100.6
99.9 100.7 100.8
120.2 119.8 99.7
150.4 149.7 99.5
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30. Precision
 The closeness of agreement (degree of
scatter) between a series of
measurements obtained from
multiple samplings of the same
homogeneous sample.
Should be investigated using
homogeneous, authentic samples.
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31. Precision… Considered at 3 Levels
 Repeatability
 Intermediate Precision
 Reproducibility
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32. Repeatability
 Express the precision
under the same
operating conditions
over a short interval of
time.
 Also referred to as
Intra-assay precision
32
Should be assessed
using minimum of 9
determinations
(3 concentrations/ 3
replicates) or
Minimum of 6
determinations at the
100% level.
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33. Intermediate Precision
33
Express within-laboratory
variations.
Expressed in terms of
standard deviation,
relative standard deviation
(coefficient of variation)
and confidence interval.
Depends on the
circumstances under which
the procedure is intended
to be used.
Studies should include
varying days, analysts,
equipment, etc.
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34. Repeatability & Intermediate Precision
Day 1 Day 2
100.6 99.5
100.8 99.9
100.1 98.9
100.3 99.2
100.5 99.7
100.4 99.6
34
Grand
Mean = 100.0
RSD = 0.59%
Mean = 100.5
RSD = 0.24%
Mean = 99.5
RSD = 0.36%
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35. Reproducibility
 Definition: Ability reproduce data
within the predefined precision
 Determination: SD, RSD and
confidence interval
 Repeatability test at two different
labs.
Note: Data not required for BLA/NDA
Lab 1 Lab 2 Lab 3
Day
1
Day
2
Day
1
Day
2
Day
1
Day
2
Man
1
Man
2
Man
1
Man 2 Man
1
Man
2
3
Prep
3
Prep
3
Prep
3
Prep
3
Prep
3
Prep
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36. Detection Limit (LOD)/
Quantitation Limit (LOQ)
 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.
36
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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37. 1. Based in Visual Evaluations
- Used for non-instrumental methods
2. Based on Signal-to Noise-Ratio
- 3:1 for Detection Limit
- 10:1 for Quantitation Limit
3. Based on Standard Deviation of the Response and
the Slope
37
LOD and LOQ Estimated by
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38.  S = slope of calibration curve
 s = standard deviation of blank readings or
standard deviation of regression line
Validated by assaying samples at DL or QL
38
DL =
3.3s
QL =
10s
S S
LOD and LOQ Estimated by
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39. 39
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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40.  Definition: Capacity to remain unaffected by small but deliberate
variations in method parameters
 Determination: Comparison results under differing conditions
with precision under normal conditions
 Examples of typical variations in LC
 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
40
Robustness
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41. Ruggedness
 Degree of reproducibility of test results
under a variety of conditions
 Different Laboratories
 Different Analysts
 Different Instruments
 Different Reagents
 Different Days
 Etc.
 Expressed as %RSD
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42. 422016
Reference Sites
 www.fda.gov
 www.fda.gov/cder/
 www.waters.com
 www.usp.org
 www.ich.org
 www.aoac.org
 www.pharmweb.net

43. Thankyou
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