PHARM 462

1 2009
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
CI TAC Cooperation of I nternational Traceability in
Analytical Chemistry
Proficiency testing
Quality Assurance
EA European Cooperation for Accreditation Accreditation
CEN European Committee for Normalization Standardization
I UPAC I nternational Union of Pure & Applied Chem. Method validation
I SO I nternational Standardization Organisation Standardisation
AOAC

I LAC
Association of Official Analytical Chemists

I nternational Laboratory Accreditation Cooperat.
I nternal qual. Control
Proficiency testing
Accreditation
FDA US Food and Drug Administration Method validation
USP United States Pharmacopoeia Method validation
I CH I nternational Conference on Harmonization Method validation
2 2009
Method Validation
Validation of analytical procedures is the process of determining the
sui t abi l i t y of a gi ven met hodol ogy f or provi di ng usef ul
analytical data.
J . Guerra, Pharm. Tech. March 1986

Validation is the formal and systematic proof that a method compiles
wi t h t h e r e q u i r e me n t s f o r t e s t i n g a p r o d u c t wh e n
observing a defined procedures.
G. Maldener, Chromatographia, J uly 1989
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2009
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
t e r ms t he pe r f o r ma nc e c ha 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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2009
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
Sample
Validation
Method Analyst
Calibration
Pump
Detector
Injector
Data System
2009
Method Life Cycle
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Validation
Development Optimization
2009
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 i t is establ ished, by
laboratory studies, that the performance
characteristics of the method meet the
requirements for the intended analytical
applications
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USP 23 General
Information <1225>
2009
The accuracy, s ens i t i vi t y, s peci f i ci t y, and
reproducibility of test methods employed by the firm
shall be established and documented. Such validation
and documentati on may be accompl i shed i n
a c c o r d a n c e w i t h 2 1 1 . 1 9 4 ( a ) ( 2 ) .
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21 CFR PART 211 - CURRENT GOOD MANUFACTURING
PRACTICE FOR FINISHED PHARMACEUTICALS
Subpart I-Laboratory Controls
211.165 Testing and release for distribution (e)
2009
The objective of validation of an analytical
procedure is to demonstrate that it is suitable
for its intended purpose
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ICH Guideline for
Industry
Q2A, Text on
Validation of
Analytical Procedures
March 1995
2009
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.

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ICH Guideline for Industry
Q2B, Validation of Analytical
Procedures: Methodology
2009
Todays Validation Requirements
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ICH/USP
GMPs
(legal)
FDA
2009
I CH/USP Validation Requirements &
Parameters
Specificity
Linearity
Range
Accuracy
Precision
Repeatability
Intermediate Precision
Reproducibility
Limit of Detection
Limit of Quantitation
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ICH
Specificity
Linearity and Range
Accuracy
Precision
Limit of Detection
Limit of Quantitation
Ruggedness
Robustness
USP
2009
USP Data Elements Required
For Assay Validation
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Analytical
Performance
Parameter
Assay
Category 1
Assay Category 2
Assay
Category 3
Quantitative 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.
2009
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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2009
I CH Validation Characteristics vs.
Type of Analytical Procedure
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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
2009
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: I mpurities 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 r
2
> 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)
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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
under the same
operating conditions
over a short interval of
time.
Also referred to as
Intra-assay precision
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Should be assessed
using minimum of 9
determinations
(3 concentrations/ 3
replicates) or
Minimum of 6
determinations at the
100% level.
2009
I ntermediate Precision
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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.
2009
Repeatability & I ntermediate 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
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Grand
Mean = 100.0
RSD = 0.59%
Mean = 100.5
RSD = 0.24%
Mean = 99.5
RSD = 0.36%
2009
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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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.
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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.
2009
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
2009
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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DL =
3.3s
QL =
10s
S S
LOD and LOQ Estimated by
2009
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Ybl
LOD LOQ
Statistical estimate of LOD & LOQ

LOD = 3.3 Sbl / b LOQ = 10 Sbl / b
Y = b X + a
2009
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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Robustness
2009
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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2009
I CH/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
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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)
2009
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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2009
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 foruse of this statement does not
a b s o l v e f r o m u s i n g v a l i d , a c c u r a t e , a n d
reproducible methods. (June 2000)
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FDA Systems Based I nspection:
Laboratory System
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Method
Validation
13%
Training/Qual.
4%
Stability Program
21%
Inadequate
Records
27%
Controls. General
35%
Feb July 2002: 212 Inspections (US)
* Reference: Albinus D Sa, FDA, CDER Office of Compliance, from AAPS, Nov. 2002 presentation.
2009
ICH Update:
2009 53
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

2009
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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
2009
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Working Groups
SAFETY EFFICACY


QUALITY MULTIDISCIPLINARY
STEERING COMMITTEE
Endorses topics, guidelines and monitors progress

2009
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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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