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12016
Method Validation Prepared by : Santram Ra-jput (Technical Man-ager)
Sigma Test & Research Centre
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 testingQuality 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. ControlProficiency testingAccreditation
FDA US Food and Drug Administration Method validation
USP United States Pharmacopoeia Method validation
ICH International Conference on Harmonization Method validation
22016
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
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
terms the performance characteristics of an assay
42016
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.
52016
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.
72016
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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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 10.0%
102016
11
System Suitability
Sample
Validation
MethodAnalyst
CalibrationPumpDetector
InjectorData System
2016
Method Life Cycle
12
Validation
Development Optimization
2016
Verification vs. Validation
Compendial vs. Non-compendial Methods
Compendial methods-Verification
Non-compendial methods-Validation requirement
132016
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”
142016
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 Vali-dation of Analyti-cal ProceduresMarch 1995
2016
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 In-dustry Q2B, Validation of Ana-lytical Procedures: Methodology
2016
Today’s Validation Requirements
17
ICH/USP
GMPs(legal) FDA
2016
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
2016
USP Data Elements Required For Assay Validation
19
Analytical PerformanceParameter
Assay Category
1
Assay Category 2Assay
Category 3
Quantita-tive Limit Tests
Accuracy Yes Yes * *Precision Yes Yes No YesSpecificity 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.
2016
ICH Validation Characteristics vs. Type of Analytical Procedure
20
Type of Ana-lytical
ProcedureIdentifica-
tion
Impurity testing
AssayQuantita-tive Limit Tests
Accuracy No Yes No YesPrecision Repeata-bility No Yes No Yes
Interm. Prec. No Yes No Yes
Specificity Yes Yes Yes YesLOD No No Yes NoLOQ No Yes No NoLinearity No Yes No YesRange No Yes No Yes
2016
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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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.
222016
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
232016
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 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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Accuracy
Closeness of the test
results obtained by the
method to the true value.
27
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)
29
Amount Added (mg)
AmountFound (mg)
Percent Re-covery
0.0 0.0 ---50.2 50.4 100.579.6 80.1 100.699.9 100.7 100.8120.2 119.8 99.7150.4 149.7 99.5
2016
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.302016
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
32
Should be as-sessed using min-imum of 9 deter-minations
(3 concentra-tions/ 3 repli-cates) or Minimum of 6 de-terminations at the 100% level.2016
Intermediate Precision
33
Express within-labo-
ratory variations.
Expressed in terms of
standard deviation,
relative standard de-
viation (coefficient of
variation) and confi-
dence interval.
Depends on the cir-
cumstances under
which the procedure
is intended to be
used.
Studies should in-
clude varying days,
analysts, equipment,
etc.2016
Repeatability & Intermediate Precision
Day 1 Day 2100.6 99.5100.8 99.9100.1 98.9100.3 99.2100.5 99.7100.4 99.6
34
GrandMean = 100.0RSD = 0.59%
Mean = 100.5RSD = 0.24%
Mean = 99.5RSD = 0.36%
2016
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
35
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
LOQLowest amount of an-
alyte in a sample that
can be quantified with
suitable accuracy and
precision.
Estimated by Signal to
Noise Ratio of 10:1. 2016
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
2016
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 =
3.3sQL =
10s
S S
LOD and LOQ Estimated by
2016
39
Ybl
LOD LOQ
Statistical estimate of LOD & LOQ
LOD = 3.3 Sbl / b LOQ = 10 Sbl / b
Y = b X + a
2016
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
Robust-ness
2016
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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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
Thankyou
432016