PHARM 462
2009
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Body
European and International regulatory bodies and
their guidelines on different aspects of QA
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
Association of Official Analytical Chemists
ILAC
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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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
with the requirements for testing a product when
observing a defined procedures.
G. Maldener, Chromatographia, July 1989
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 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 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
terms the performance characteristics of an assay
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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, NIR, AA, NMR,
XRD,MS

Capillary Electrophoresis Methods - Zone, Isoelectric Focusing

Particle Size Analysis Methods - Laser, Microscopic, Sieving, SEC, etc.

Automated Analytical Methods - Robots, Automated Analysis.
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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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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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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 FDA Requirements
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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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 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 2.0%
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System Suitability
Validation
Calibration
Pump
Injector
Detector
Data System
Analyst
Method
Sample
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Method Life Cycle
Validation
Development
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Optimization
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Verification vs. Validation
 Compendial vs. Non-compendial Methods

Compendial methods-Verification

Non-compendial methods-Validation requirement
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Compendial Analytical Procedures

The Analytical procedures in the USP 25/NF 20 are legally recognized under
section 501(b) of the Federal Food, Drug and Cosmetic Act as the regulatory
analytical procedures for the compendial items. The suitability of these
procedures must be verified under actual conditions of use. When using USP
25/NF 20 analytical procedures, the guidance recommends that information be
p r o v i d e d
f o r
t h e
f o l l o w i n g
characteristics:
 Specificity of the procedure
 Stability of the sample solution
 Intermediate precision
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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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Regulatory and Compliance
Requirements Review
 Validation of an analytical method is the
process by which it is established, by
laboratory studies, that the performance
characteristics of the method meet the
requirements for the intended analytical
applications
USP 23 General
Information <1225>
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 The accuracy, sensitivity, specificity, and
reproducibility of test methods employed by the firm
shall be established and documented. Such validation
and doc ume ntation m ay be accom plished i n
accordance
with
211.194(a)(2).
21 CFR PART 211 - CURRENT GOOD MANUFACTURING
PRACTICE FOR FINISHED PHARMACEUTICALS
Subpart I-Laboratory Controls
211.165 Testing and release for distribution (e)
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 The objective of validation of an analytical
procedure is to demonstrate that it is suitable
for its intended purpose
ICH Guideline for
Industry
Q2A, Text on
Validation of
Analytical Procedures
March 1995
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 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.
ICH Guideline for Industry
Q2B, Validation of Analytical
Procedures: Methodology
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Today’s Validation Requirements
ICH/USP
GMPs
(legal)
2009
FDA
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ICH/USP Validation Requirements &
Parameters
USP

Specificity

Linearity and Range

Accuracy

Precision

Limit of Detection

Limit of Quantitation

Ruggedness

Robustness
ICH
 Specificity
 Linearity
 Range
 Accuracy
 Precision

Repeatability

Intermediate Precision

Reproducibility
 Limit of Detection
 Limit of Quantitation
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USP Data Elements Required
For Assay Validation
Analytical
Performance
Parameter
Assay Category 2
Assay
Category 1
Quantitative
Limit Tests
Assay
Category 3
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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USP Categories
 Category 1: Quantitation of major components or
active ingredients
 Category 2: Determination of impurities or
degradation products
 Category 3: Determination of performance
characteristics
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ICH Validation Characteristics vs.
Type of Analytical Procedure
Type of
Analytical
Procedure
Impurity testing
Identification
Assay
Quantitative
Limit Tests
No
Yes
No
Yes
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
Accuracy
Precision
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Specificity/Selectivity
 Ability of an analytical method to measure the analyte free from
interference due to other components.
 Selectivity describes the ability of an analytical method to differentiate
various substances in a sample
Original term used in USP
Also Preferred by IUPAC and AOAC
Also used to characterize chromatographic columns
 Degree of Bias (Used in USP)
The difference in assay results between the two groups
-
the sample containing added impurities, degradation products, related chemical
compounds, placebo ingredients
-
the sample without added substances
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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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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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Linearity
 Ability of an assay to
elicit a direct and
proportional response
to changes in analyte
concentration.
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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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Range
 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
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Accuracy
 Closeness of the test
results obtained by the
method to the true value.
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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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Accuracy Data Set (1 of 3)
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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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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Precision… Considered at 3 Levels
 Repeatability
 Intermediate Precision
 Reproducibility
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Repeatability
 Express the precision
Should be assessed
under the same
using minimum of 9
operating conditions
determinations
over a short interval of
(3 concentrations/ 3
time.
replicates) or
 Also referred to as
Intra-assay precision
2009
Minimum of 6
determinations at the
100% level.
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Intermediate Precision
Express within-laboratory
Depends on the
variations.
circumstances under which
Expressed in terms of
the procedure is intended
standard deviation,
to be used.
relative standard deviation
Studies should include
(coefficient of variation)
varying days, analysts,
and confidence interval.
equipment, etc.
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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
Mean = 100.5
RSD = 0.24%
Mean = 99.5
RSD = 0.36%
Grand
Mean = 100.0
RSD = 0.59%
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Reproducibility
 Definition: Ability reproduce data
Lab 1
Lab 2
Lab 3
within the predefined precision
 Determination: SD, RSD and
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
confidence interval

Repeatability test at two different
labs.
Note: Data not required for BLA/NDA
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Detection Limit (LOD)/
Quantitation Limit (LOQ)
 LOD
LOQ
Lowest amount of analyte in a
Lowest amount of analyte
sample that can be detected
in a sample that can be
but not necessarily
quantified with suitable
quantitated.
accuracy and precision.
Estimated by Signal to Noise
Estimated by Signal to
Ratio of 3:1.
Noise Ratio of 10:1.
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LOD and LOQ Estimated by
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
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LOD and LOQ Estimated by
3.3s
DL =
S
QL =
10s
S
 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
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Y=bX+a
Statistical estimate of LOD & LOQ
LOD = 3.3 Sbl / b LOQ = 10 Sbl / b
Ybl
LOD
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LOQ
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Robustness
 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
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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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ICH/USP System Suitability
 ICH

Definition: evaluation of equipment, electronic,
analytical operations and samples as a whole

Determination: repeatability, tailing factor (T), capacity
factor (k’), resolution (R), and theoretical Plates (N)
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 USP 23 <621>

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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Re-validation
 When




Method parameters have been changed
The scope of the method has been changed
Synthetic methods have been changed
Impurity profile has been changed
 What

Preferably everything. Exceptions should be
scientifically justified
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How do we Know the expectations of
the FDA?
 FDA Form 483
 FDA Warning Letters
 Personal Experiences
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483 Observations
 There was 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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FDA Waning Letter
On addition to the example of modifying both compendial 
methods and customer supplied methods, we also observed
the use of unvalidated in-house methods as well as
u
n
v
a
l
i
d
a
t
e
d
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. (June 2000)
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FDA Systems Based Inspection:
Laboratory System
Feb – July 2002: 212 Inspections (US)
Method
Validation
13%
Training/Qual.
4%
Controls. General
35%
Stability Program
21%
Inadequate
Records
27%
* Reference: Albinus D’ Sa, FDA, CDER Office of Compliance, from AAPS, Nov. 2002 presentation.
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ICH Update:
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A Unique Approach
 International Conference on Harmonisation
(ICH) was created in 1990
 Agreement between the EU, Japan and the
USA to harmonize different regional
requirements for registration of pharmaceutical
drug products
 Unique because joint effort by regulators and
associated pharmaceutical industry trade
associations
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ICH Objectives
 Identification and elimination of the need to duplicate
studies to meet different regulatory requirements
 More efficient use of resources in the R&D process,
as a consequence
 Quicker access for patients to safe and effective new
medicines
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Working Groups
SAFETY
QUALITY
EFFICACY
MULTIDISCIPLINARY
STEERING COMMITTEE
Endorses topics, guidelines and monitors progress
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Related Site
 www.fda.gov
 www.fda.gov/cder/
 www.waters.com
 www.usp.org
 www.ich.org
 www.aoac.org
 www.pharmweb.net
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