Analytical Method Validation

Analytical Method Validation
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

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)]
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
Validation Activity Including

the Complete Analytical Procedure
Sampling
Sample Preparation
Analysis
Data Evaluation Reporting

Optimization of Validation
Additional value and Cost VS. Completeness of validation

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
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.
System Suitability
Validation
Calibration
Pump
Injector
Detector
Data System
Analyst
Method
Sample
10
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
11
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
12
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)
13
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

14
ICH/USP Validation Requirements
Precision
Specificity
Linearity
Range
Accuracy
Repeatability
Intermediate Precision
Limit of Detection
Limit of Quantitation
Robustness
15
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.

Category 1: Quantitation of major components or active ingredients
Category 2: Determination of impurities or degradation products
Category 3: Determination of performance characteristics
16
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
17
Specificity
Ability of an
analytical method to
measure the analyte
free from
interference due to
other components.
Selectivity
Bias
18
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.
19
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.
20
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.
21
Pure and Impure HPLC peaks
Peak purity tests can also be evaluated with

The spectra of Photodiode array detectors
Mass spectrometry
22
Specificity: Potential Interference
Placebo
Drug Substance Degradants
Drug Product Degradants
Related Substances
Packaging Extractables
23
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

24
Specificity Study
Condition
DEG
#1
DEG
#2
DEG
#3
DEG
#4
Active
ingredients
2 N HCl
17.23
4.71
95.17%
0.1 N
NaOH
100%
30%
H2O2
1.12
1.41
1.65
1.2
99.98%
UV/Vis
4.68
94.67%
25
Linearity
Ability of an assay
to elicit a direct and
proportional
response to
changes in analyte
concentration.
26
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

27
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
28
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.
29
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.
30
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
31
Accuracy
Closeness of the test

results obtained by the
method to the true
value.
32
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
33
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
99.3
99.3
Std.dev.
0.1
0.4
95%C.I
99.30.25
99.30.99
Mean
34
Analyte recovery at different

concentration
Analyte Ingred. (%)
Analyte ratio
Unit
Mean recovery (%)
100
100 %
98-102
10
10-1
10 %
98-102
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

35
Precision
The closeness of agreement
Ball Ball
Ball
Ball Ball Ball
Ball Ball
Strik Strike
Strike
e Strike
Strike
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.
36
Precision Considered at 3 Levels
Repeatability
Reproducibility
37
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
Intra-assay precision
determinations at the
100% level.
38
Express within-laboratory variations.
Depends on the
Expressed in terms of standard
circumstances under which
deviation, relative standard
the procedure is intended
deviation (coefficient of variation)
to be used.
and confidence interval.
Studies should include
Known as part of Ruggedness in
varying days, analysts,
USP
equipment, etc.
39
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
Mean = 100.5
RSD = 0.24%
CI = 100.5 0.24
99.6
Mean = 99.5
RSD = 0.36%
CI = 99.5 0.36
Grand
Mean = 100.0
RSD = 0.59%
40
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
41
Reproducibility Study
Lab 1
Lab 2
Lab 3
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
Analyst
1
Analyst
2
Analyst
1
Analyst
2
Analyst
1
Analyst
2
3 Preps
3 Preps
3 Preps
3 Preps
3 Preps
3 Preps
42
Analyte concentration versus precision

Analyte %
Analyte ratio
Unit
RSD (%)
100
100 %
1.3
10
10-1
10 %
2.7
10-2
1%
2.8
0.1
10-3
0.1%
3.7
0.01
10-4
100 ppm
5.3
0.001
10-5
10 ppm
7.3
0.0001
10-6
1 ppm
11
0.00001
10-7
100 ppb
15
0.000001
10-8
10 ppb
21
0.0000001
10-9
1 ppb
30
AOAC manual for the Peer-Verified Methods program

43
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
Estimated by Signal to Noise

Ratio of 3:1.
precision.
Estimated by Signal to Noise
Ratio of 10:1.
44
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
45
Based on Signal-to Noise-Ratio
46
Detection Limit (DL) and

Quantitation Limit (QL)
DL =
3.3s
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
47
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.
48
Confuse of Precision Terms
Repeatability
Reproducibility
Ruggedness
Intermediate
Precision
Robustness
49
Precision Terms
Instrument Precision
- 10 Std. Injections
Repeatability
- One Analysis (6 preps)
- Two Analyses
Reproducibility
- Two Lab.
Ruggedness
- Many Variables
Robustness
- Intentional Changes
50
Robustness Variations
All Assays
-Sample Prep Manipulation

-Extraction Time
HPLC Assays
-Mobile Phase Composition

-Different Columns
-Temperature
-Flow Rate
GC Assays
-Different Columns
-Temperature
-Flow Rate
51
Robustness-Mobile Phase Change
MeOH/
Water
Retention
Time 1
Retention
Time 2
Resolution
75:25
11.94
16.41
7.39
80:20
8.47
11.17
6.17
85:15
7.81
10.18
5.93
52
ICH/USP System Suitability

ICH
USP 23 <621>
Definition: evaluation of
equipment, electronic,
analytical operations and
samples as a whole
Determination: repeatability,
tailing factor (T), capacity
System Suitability Requirements

Parameters
Recommendations
In general k 2.0
R > 2, between the peak of

interest and the closest
potential interferent
(degradant, internal STD,
impurity, excipient, etc..)
factor (k), resolution (R), and

theoretical Plates (N)
T2
In general N > 2000
Repeatability
RSD 2.0% (n 5)
53
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.
54
References
Analytical Methods Validation for FDA Compliance 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.
55

Analytical Method Validation

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