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 2
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 3
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 4
5. Validation Activity Including
the Complete Analytical Procedure
Sampling
Sample Preparation
Analysis
Data Evaluation Reporting
Analytical Method Validation 5
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 7
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 8
9. Validation Step
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.
Analytical Method Validation 9
10. System Suitability
Validation
Calibration
Pump Injector
Detector Data System
Analyst Method
Sample
Analytical Method Validation 10
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 11
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 12
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 13
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 14
15. ICH/USP Validation Requirements
Precision
Specificity Repeatability
Linearity Intermediate Precision
Range Limit of Detection
Accuracy Limit of Quantitation
Robustness
Analytical Method Validation 15
16. USP Data Elements Required For Assay Validation
Analytical Assay Category 2
Assay
Performance Assay Category 1
Quantitative Limit Tests Category 3
Parameter
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 16
17. ICH Validation Characteristics vs.
Type of Analytical Procedure
Type of Analytical Impurity testing
Identification Assay
Procedure 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 17
18. Specificity
Ability of an
analytical method
to measure the
analyte free from
interference due to
Selectivity
other components. Bias
Analytical Method Validation 18
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 19
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 20
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 21
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 22
23. Specificity: Potential Interference
Placebo
Drug Substance Degradants
Drug Product Degradants
Related Substances
Packaging Extractables
Analytical Method Validation 23
24. Forced Degradation Studies
Heat High Temperature (50 to 60 oC)
Humidity Humidity (70 to 80%)
Acid Hydrolysis Acid Hydrolysis (0.1 N)
Base Hydrolysis Base Hydrolysis (0.1 N)
Oxidation Peroxide Oxidation (3 to 30%)
Light Intense UV/Visible Light
Intent is to create 10 to 30 % Degradation
Analytical Method Validation 24
26. Linearity
Ability of an assay
to elicit a direct and
proportional
response to
changes in analyte
concentration.
Analytical Method Validation 26
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 27
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 28
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 29
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 30
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 31
32. Accuracy
Closeness of the test
results obtained by the
method to the true value.
Analytical Method Validation 32
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 33
34. Accuracy Data Set (1 of 3)
% Recovery % Recovery
Amount
Amount Percent 99.2 98.9
Found Recovery
Added (mg)
(mg) 99.3 99.3
0.0 0.0 ---
99.4 99.7
50.2 50.4 100.5
Mean 99.3 99.3
79.6 80.1 100.6
Std.dev. 0.1 0.4
99.9 100.7 100.8
95%C.I 99.3±0.25 99.3±0.99
Analytical Method Validation 34
35. Analyte recovery at different
concentration
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% 95-105
0.01 10-4 100 ppm 90-107
0.001 10-5 10 ppm 80-110
0.0001 10-6 1 ppm 80-110
0.00001 10-7 100 ppb 80-110
0.000001 10-8 10 ppb 60-115
0.0000001 10-9 1 ppb 40-120
AOAC manual for the Peer-Verified Methods program
Analytical Method Validation 35
36. Precision
Ball Ball Strike Strike
Ball Strike
Ball Ball Ball StrikeStrike
The closeness of agreement Ball Ball Strike
(degree of scatter) between a
series of measurements obtained
from multiple samplings of the
same homogeneous sample.
Should be investigated using
homogeneous, authentic samples.
Analytical Method Validation 36
37. Precision… Considered at 3 Levels
Repeatability
Intermediate Precision
Reproducibility
Analytical Method Validation 37
38. Repeatability
Express the precision Should be assessed
under the same using minimum of 9
operating conditions determinations
over a short interval (3 concentrations/ 3
of time. replicates) or
Also referred to as Minimum of 6
determinations at the
Intra-assay precision
100% level.
Analytical Method Validation 38
39. Intermediate Precision
Express within-laboratory Depends on the
variations. circumstances under which
Expressed in terms of standard the procedure is intended
deviation, relative standard to be used.
deviation (coefficient of variation) Studies should include
and confidence interval. varying days, analysts,
Known as part of Ruggedness in equipment, etc.
USP
Analytical Method Validation 39
40. 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 Mean = 99.5
RSD = 0.24% RSD = 0.36%
CI = 100.5 ± 0.24 CI = 99.5 ± 0.36
Grand
Mean = 100.0
RSD = 0.59%
Analytical Method Validation 40
41. 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
Analytical Method Validation 41
42. Reproducibility Study
Lab 1 Lab 2 Lab 3
Day 1 Day 2 Day 1 Day 2 Day 1 Day 2
Analyst Analyst Analyst Analyst Analyst Analyst
1 2 1 2 1 2
3 Preps 3 Preps 3 Preps 3 Preps 3 Preps 3 Preps
Analytical Method Validation 42
44. Detection Limit (DL) Quantitation Limit (QL)
Lowest amount of analyte in a Lowest amount of analyte in a
sample that can be detected sample that can be quantified
but not necessarily quantitated. with suitable accuracy and
precision.
Estimated by Signal to Noise
Estimated by Signal to Noise
Ratio of 3:1.
Ratio of 10:1.
Analytical Method Validation 44
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 45
47. Detection Limit (DL) and
Quantitation Limit (QL)
3.3s 10s
DL = QL =
S 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
Analytical Method Validation 47
48. 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
Variations may include: stability of analytical solution,
variation of pH in a mobile phase, different column
(lot/supplier), temperature, flow rate.
Analytical Method Validation 48
52. Robustness-Mobile Phase Change
MeOH/ Retention Retention
Resolution
Water Time 1 Time 2
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 52
53. ICH/USP System Suitability
ICH USP 23 <621>
System Suitability Requirements
Definition: evaluation of
equipment, electronic, Parameters Recommendations
analytical operations and
K’ In general k’ ≥ 2.0
samples as a whole
R > 2, between the peak of
Determination: repeatability, interest and the closest
R potential interferent
tailing factor (T), capacity (degradant, internal STD,
impurity, excipient, etc…..)
factor (k’), resolution (R), and
T T≤2
theoretical Plates (N)
N In general N > 2000
Repeatability RSD ≤ 2.0% (n ≥ 5)
Analytical Method Validation 53
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 54
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.
Analytical Method Validation 55
