Analytical Method Validation

Analytical Method Validation 1

 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

각국의 규제 및 규정들

Analytical Method Validation 2

 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

 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

 Validation Activity Including
the Complete Analytical Procedure

Sampling

Sample Preparation

Analysis

Data Evaluation Reporting

Analytical Method Validation 5

 Optimization of Validation

Additional value and Cost VS. Completeness of validation

Analytical Method Validation 6
 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

 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

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

 System Suitability

Validation

Calibration
Pump Injector
Detector Data System

Analyst Method

Sample
Analytical Method Validation 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

Analytical Method Validation 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

Analytical Method Validation 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)
Analytical Method Validation 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

Analytical Method Validation 14

 ICH/USP Validation Requirements

Precision

Specificity Repeatability

Linearity Intermediate Precision

Range Limit of Detection

Accuracy Limit of Quantitation

Robustness

Analytical Method Validation 15

USP Data Elements Required For Assay Validation

Assay Category 2
Analytical
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
 ICH Validation Characteristics vs.
Type of Analytical Procedure

Impurity testing
Type of
Analytical 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

 Specificity

Ability of an analytical
method to measure the
analyte free from
interference due to

other components.
Selectivity
Bias

Analytical Method Validation 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.

Analytical Method Validation 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.

Analytical Method Validation 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.

Analytical Method Validation 21

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
Specificity: Potential Interference

Placebo
Drug Substance Degradants
Drug Product Degradants
Related Substances
Packaging Extractables

Analytical Method Validation 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

Analytical Method Validation 24
 Specificity Study

DEG DEG DEG DEG Active

Condition
#1 #2 #3 #4 ingredients

2 N HCl 0 0 17.23 4.71 95.17%

0.1 N
0 0 0 0 100%
NaOH
30%
1.12 1.41 1.65 1.2 99.98%
H2O2

UV/Vis 0 0 4.68 0 94.67%

Analytical Method Validation 25

 Linearity

Ability of an assay
to elicit a direct
and proportional
response to
changes in analyte
concentration.

Analytical Method Validation 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

Analytical Method Validation 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

Analytical Method Validation 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.

Analytical Method Validation 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.

Analytical Method Validation 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

Analytical Method Validation 31

 Accuracy

Closeness of the test

results obtained by the
method to the true
value.

Analytical Method Validation 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

Analytical Method Validation 33

 Accuracy Data Set (1 of 3)

% Recovery % Recovery
Amount Amount
Percent 99.2 98.9
Added Found Recovery
(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

 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
 Ball Ball
Ball Ball
Precision Ball Ball
Ball Ball

Strike Strike
Strike
The closeness of agreement StrikeStrike
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

Precision… Considered at 3 Levels

Repeatability

Intermediate Precision

Reproducibility

Analytical Method Validation 37

 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
Minimum of 6
determinations at the
100% level.
Analytical Method Validation 38
 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 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 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
 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

 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

 Analyte concentration versus precision

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% 3.7
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

Analytical Method Validation 43

 Quantitation Limit (Q
Detection Limit (DL)
L)

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 with suitable accuracy and

quantitated. precision.

Estimated by Signal to Noise

Estimated by Signal to Noise
Ratio of 10:1.
Ratio of 3:1.

Analytical Method Validation 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

Analytical Method Validation 45

Based on Signal-to Noise-Ratio

Analytical Method Validation 46

 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

 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

 Confuse of Precision Terms

Repeatability Reproducibility

Ruggedness

Intermediate
Precision Robustness

Analytical Method Validation 49

 Precision Terms

Instrument Precision - 10 Std. Injections

Repeatability - One Analysis (6 preps)
Intermediate Precision - Two Analyses
Reproducibility - Two Lab.
Ruggedness - Many Variables
Robustness - Intentional Changes

Analytical Method Validation 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

Analytical Method Validation 51

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

 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 > 2, between the peak of
interest and the closest pot
R ential interferent (degradan
t, internal STD, impurity, ex
cipient, etc…..)
T T≤2
N In general N > 2000
Repeatability RSD ≤ 2.0% (n ≥ 5)
Analytical Method Validation 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.”

Analytical Method Validation 54

 References

‘Analytical Methods Validation for FDA Compliance’ 교육교재 The Center for Professi
onal Advancement 2003. 3.12-14.
Guideline for submitting samples and analytical data for kethods validation (Feb. 198
7)
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. Mrc
el Dekker, Inc. New York, 1997.
USP 23 <1225>
http://www.waters.com
Ludwig Huber, Validation and Qualification in Analytical Laboratories, Interpharm Pres
s Inc. Buffalo Grove, Illinois, 1999.

Analytical Method Validation 55