analytical method validation raaj gprac [compatibility mode]
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Analytical Method Validation 1
Analytical Method Validation Analytical Method Validation
RajashriRajashri OjhaOjha
Director, Director, RaajRaaj GPRACGPRAC
Analytical Method Validation 2
Why Method Validation is Important?
1. The purpose of analytical measurement is to get consistent,
reliable and accurate data.
Incorrect measurement results can lead to tremendous costs.
2. Equal importance for those working in a regulated and in an
accredited environment.
U.S. FDA, EMEA, EPA, AOAC, ISO
Analytical Method Validation 3
Background
NDA and ANDA must include the analytical procedures necessary
to ensure:
Identity, Strength, Quality, Purity, and Potency of the Drug
Substances and Drug product [21CFR 314.50(d)(l) and
314.94(a)(9)(i)]
Data to establish and reliability [21CFR 211.169(e) and
211.194(a)(2)]
Analytical Method Validation 4
Validation is an Old Concept But There are Many Problems
Lack of documented procedures and documented validation results
Sampling or Sample preparation step contribute to overall error.
Accessories and materials used for equipment qualification are not
qualified.
Limits for Operational Qualification
Lack of software validation and computer system validation
Qualification and validation are done at just one particular point in time.
Adaptation of acceptance criteria for qualification of new system
Analytical Method Validation 5
Validation Activity Including the Complete Analytical Procedure
Sampling
Sample Preparation
Analysis
Data Evaluation Reporting
Analytical Method Validation 6
Optimization of Validation
Additional value and Cost VS. Completeness of validation
Analytical Method Validation 7
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
Analytical Method Validation 8
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
Analytical Method Validation 9
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.
Validation Step
Analytical Method Validation 10
System Suitability
Sample
Validation
MethodAnalyst
Calibration
Pump
Detector
Injector
Data System
Analytical Method Validation 11
Verification vs. Validation
Compendial vs. Non-compendial Methods
Compendial methods-Verification
Regulatory analytical procedure in USP/NF
Non-compendial methods-Validation
Alternative analytical procedure proposed by the applicant for
use instead of the regulatory analytical procedure
Analytical Method Validation 12
Regulations and Guidelines of
Validation
US FDA 21 CFR (Code of Federal Regulations) Part 210 and 211
Part 210: cGMP in Manufacturing, Processing, Packaging, or Holding of
Drugs; General
Part 211: cGMP for Manufacturing Practice for Finished Pharmaceuticals
ICH Guidelines
Q2A, Text on Validation of Analytical procedures
(March 1995)
Q2B, Validation of Analytical Procedures: Methodology (May 1997)
USP Chapter <1225>
Validation of Compendial Methods
Analytical Method Validation 13
The accuracy, sensitivity, specificity, and reproducibility of test methods
employed by the firm shall be established and documented. Such
validation and documentation may be accomplished in 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)
Methods validation means establishing, through documented evidence,
a high degree of assurance that an analytical method will consistently
yield results that accurately reflect the quality characteristics of the
product tested.21 CFR PART 210 - CURRENT GOOD MANUFACTURING
PRACTICE IN MANUFACTURING, PROCESSING, PACKING,
OR HOLDING OF DRUGS
210.3 Definitions (b) (25)
Analytical Method Validation 14
The objective of validation of an analytical procedure is to
demonstrate that it is suitable for its intended purpose
ICH Guideline for Industry Q2A
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
Analytical Method Validation 15
ICH/USP Validation Requirements
Precision
Repeatability
Intermediate Precision
Limit of Detection
Limit of Quantitation
Robustness
Specificity
Linearity
Range
Accuracy
Analytical Method Validation 16
USP Data Elements Required For Assay Validation
Analytical
Performance
Parameter
Assay Category 1Assay 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.
Category 1: Quantitation of major components or active ingredients
Category 2: Determination of impurities or degradation products
Category 3: Determination of performance characteristics
Analytical Method Validation 17
ICH Validation Characteristics vs.
Type of Analytical Procedure
Type of Analytical
ProcedureIdentification
Impurity testingAssay
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
Analytical Method Validation 18
Specificity
Ability of an
analytical method
to measure the
analyte free from
interference due to
other components.Selectivity
Bias
Analytical Method Validation 19
Specificity: ICH/USP
An investigation of specificity should be conducted
during the validation of an identification test, an
impurities assay, and a potency assay.
Procedures used will depend on the intended
objective of the analytical procedure.
If a method can not completely discriminate, two
of more procedures are recommended.
Analytical Method Validation 20
Specificity: Identification
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.
Choice of potential interfering substances should be
based on sensible scientific judgment considering
substances that could likely occur.
Analytical Method Validation 21
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.
Analytical Method Validation 22
Pure and Impure HPLC peaks
Peak purity tests can also be evaluated with
The spectra of Photodiode array detectors
Mass spectrometry
Analytical Method Validation 23
Specificity: Potential Interference
Placebo
Drug Substance Degradants
Drug Product Degradants
Related Substances
Packaging Extractables
Analytical Method Validation 24
Forced Degradation Studies
Heat
Humidity
Acid Hydrolysis
Base Hydrolysis
Oxidation
Light
High Temperature (50 to 60 oC)
Humidity (70 to 80%)
Acid Hydrolysis (0.1 N)
Base Hydrolysis (0.1 N)
Peroxide Oxidation (3 to 30%)
Intense UV/Visible Light
Intent is to create 10 to 30 % Degradation
Analytical Method Validation 25
Specificity Study
ConditionDEG
#1
DEG
#2
DEG
#3
DEG
#4
Active ingredients
2 N HCl 0 0 17.23 4.71 95.17%
0.1 N
NaOH0 0 0 0 100%
30%
H2O2
1.12 1.41 1.65 1.2 99.98%
UV/Vis 0 0 4.68 0 94.67%
Analytical Method Validation 26
Linearity
Ability of an assay
to elicit a direct and
proportional
response to
changes in analyte
concentration.
Analytical Method Validation 27
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),
residual sum of squares
Requires a minimum of 5 concentration levels
Analytical Method Validation 28
Linearity Example
R square = 0.999
Slope = 0.97
y-intercept = 0.233
Line Eq.: Y = 0.97X + 0.233
Std. Error = 1.319
Std. Deviation of Slopes = 0.0079
Analytical Method Validation 29
Range
The interval between the
upper and lower
concentrations of analyte
in the sample that have
been demonstrate to have
a suitable level of
precision, accuracy, and
linearity.
Analytical Method Validation 30
Range
Normally derived from Linearity studies.
Established by confirming that the method provides
acceptable degree of linearity, accuracy, and
precision.
Specific range dependent upon intended
application of the procedure.
Analytical Method Validation 31
Minimum Specified Range:
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
Analytical Method Validation 32
Accuracy
Closeness of the test
results obtained by the
method to the true value.
Analytical Method Validation 33
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 (reference material) or
The difference between the mean assay result and the accepted
value
Analytical Method Validation 34
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
% Recovery % Recovery
99.2 98.9
99.3 99.3
99.4 99.7
Mean 99.3 99.3
Std.dev. 0.1 0.4
95%C.I 99.3±0.25 99.3±0.99
Analytical Method Validation 35
Analyte recovery at different concentration
AOAC manual for the Peer-Verified Methods program
Analyte Ingred. (%) Analyte ratio Unit Mean recovery (%)
100 1 100 % 98-102≥ 10 10-1 10 % 98-102≥ 1 10-2 1% 97-103
≥ 0.1 10-3 0.1% 95-1050.01 10-4 100 ppm 90-107
0.001 10-5 10 ppm 80-1100.0001 10-6 1 ppm 80-110
0.00001 10-7 100 ppb 80-1100.000001 10-8 10 ppb 60-115
0.0000001 10-9 1 ppb 40-120
Analytical Method Validation 36
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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Analytical Method Validation 37
Precision… Considered at 3 Levels
Repeatability
Intermediate Precision
Reproducibility
Analytical Method Validation 38
Repeatability
Express the precision
under the same
operating conditions
over a short interval
of time.
Also referred to as
Intra-assay precision
Should be assessed
using minimum of 9
determinations
(3 concentrations/ 3
replicates) or or
Minimum of 6
determinations at the
100% level.
Analytical Method Validation 39
Intermediate Precision
Express within-laboratory
variations.
Expressed in terms of standard
deviation, relative standard
deviation (coefficient of variation)
and confidence interval.
Known as part of Ruggedness in
USP
Depends on the
circumstances under which
the procedure is intended
to be used.
Studies should include
varying days, analysts,
equipment, etc.
Analytical Method Validation 40
GrandMean = 100.0RSD = 0.59%
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 = 100.5RSD = 0.24%CI = 100.5 ± 0.24
Mean = 99.5RSD = 0.36%CI = 99.5 ± 0.36
Analytical Method Validation 41
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
Reproducibility
Analytical Method Validation 42
Reproducibility Study
Lab 1 Lab 2 Lab 3
Day 1 Day 2 Day 1 Day 2 Day 1 Day 2
Analyst1
Analyst
2
Analyst
1
Analyst
2
Analyst
1
Analyst
2
3 Preps 3 Preps 3 Preps 3 Preps 3 Preps 3 Preps
Analytical Method Validation 43
Analyte concentration versus precision
AOAC manual for the Peer-Verified Methods program
Analyte % Analyte ratio Unit RSD (%)
100 1 100 % 1.3≥ 10 10-1 10 % 2.7≥ 1 10-2 1 % 2.8
≥ 0.1 10-3 0.1% 3.70.01 10-4 100 ppm 5.3
0.001 10-5 10 ppm 7.30.0001 10-6 1 ppm 11
0.00001 10-7 100 ppb 150.000001 10-8 10 ppb 21
0.0000001 10-9 1 ppb 30
Analytical Method Validation 44
Lowest amount of analyte in a
sample that can be detected
but not necessarily quantitated.
Estimated by Signal to Noise
Ratio of 3:1.
Detection Limit (DL)
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.
Quantitation Limit (QL)
Analytical Method Validation 45
Detection Limit (DL) and Quantitation
Limit (QL) 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
Analytical Method Validation 46
Based on Signal-to Noise-Ratio
Analytical Method Validation 47
Detection Limit (DL) and
Quantitation Limit (QL)
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
DL =3.3s
QL =10s
S S
Analytical Method Validation 48
Definition: Capacity to remain unaffected by small
but deliberate variations in method parameters
Determination: Comparison results under differing
conditions with precision under normal conditions
Variations may include: stability of analytical solution,
variation of pH in a mobile phase, different column
(lot/supplier), temperature, flow rate.
Robustness
Analytical Method Validation 49
Confuse of Precision Terms
Repeatability
Intermediate
Precision
Reproducibility
Ruggedness
Robustness
Analytical Method Validation 50
Precision Terms
Instrument Precision
Repeatability
Intermediate Precision
Reproducibility
Ruggedness
Robustness
- 10 Std. Injections
- One Analysis (6 preps)
- Two Analyses
- Two Lab.
- Many Variables
- Intentional Changes
Analytical Method Validation 51
Robustness Variations
All Assays
HPLC Assays
GC Assays
-Sample Prep Manipulation
-Extraction Time
-Mobile Phase Composition
-Different Columns
-Temperature
-Flow Rate
-Different Columns
-Temperature
-Flow Rate
Analytical Method Validation 52
Robustness-Mobile Phase Change
MeOH/
Water
Retention
Time 1
Retention
Time 2Resolution
75:25 11.94 16.41 7.39
80:20 8.47 11.17 6.17
85:15 7.81 10.18 5.93
Analytical Method Validation 53
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)
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)
Analytical Method Validation 54
Guidance on Re-Validation
“When sponsors make changes in the analytical
procedure, drug substance, drug product, the
changes, may necessitate revalidation of the
analytical procedures.”
“The degree of revalidation depends on the nature
of the change.”
“FDA intends to provide guidance in the future on
post-approval changes in analytical procedures.”
Analytical Method Validation 55
References
‘Analytical Methods Validation for FDA Compliance’ 교육교재 The Center for
Professional Advancement 2003. 3.12-14.
Guideline for submitting samples and analytical data for kethods validation
(Feb. 1987)
ICH Q2A
ICH Q2B
21 Code of Federal Registrations Part 210 and 211
Michael E. Swatrz and Ira S. Krull, Analytical method development and
validation. Mrcel Dekker, Inc. New York, 1997.
USP 23 <1225>
http://www.waters.com
Ludwig Huber, Validation and Qualification in Analytical Laboratories,
Interpharm Press Inc. Buffalo Grove, Illinois, 1999.