Method Validation

Prepared by :
Santram Rajput
(Technical Manager)
Sigma Test & Research Centre

2016
1
 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 Proficiency testing

Analytical Chemistry Quality 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 Association of Official Analytical Chemists Internal qual. Control
Proficiency testing
ILAC International Laboratory Accreditation Cooperat. Accreditation

FDA US Food and Drug Administration Method validation

USP United States Pharmacopoeia Method validation
ICH International Conference on Harmonization Method validation
2016 2
 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

2016 3
 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

2016 4
 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.

2016 5
 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

2016 6
 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.

2016 7
 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

2016 8
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

2016 9
 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%

2016 10
 System Suitability

Validation

Calibration
Pump Injector
Detector Data System

Analyst Method

Sample
2016 11
 Method Life Cycle

Validation

Development Optimization

2016 12
 Verification vs. Validation

 Compendial vs. Non-compendial Methods

 Compendial methods-Verification

 Non-compendial methods-Validation requirement

2016 13
 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”
2016 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, Text on
Validation of
Analytical Procedures
March 1995

2016 15
 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, Validation of Analytical
Procedures: Methodology

2016 16
Today’s Validation Requirements

ICH/USP

GMPs
(legal) FDA

2016 17
ICH/USP Validation Requirements &
Parameters

USP ICH
 Specificity
 Specificity
 Linearity
 Linearity and Range
 Range
 Accuracy
 Accuracy
 Precision
 Precision
 Limit of Detection
 Repeatability
 Limit of Quantitation
 Intermediate Precision
 Ruggedness
 Reproducibility
 Robustness
 Limit of Detection
 Limit of Quantitation
2016 18
 USP Data Elements Required
For Assay Validation
Analytical Assay Category 2
Assay Assay
Performance
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.

2016 19
 ICH Validation Characteristics vs.
Type of Analytical Procedure

Type of Impurity testing

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
2016 20
 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

2016 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.
2016 22
 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

2016 23
 Linearity
 Ability of an assay to
elicit a direct and
proportional response
to changes in analyte
concentration.

2016 24
 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

2016 25
 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

2016 26
 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

2016 28
 Accuracy Data Set (1 of 3)
Amount Amount Percent
Added (mg) Found (mg) Recovery
0.0 0.0 ---
50.2 50.4 100.5
79.6 80.1 100.6
99.9 100.7 100.8
120.2 119.8 99.7
150.4 149.7 99.5

2016 29
 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.

2016 30
Precision… Considered at 3 Levels

 Repeatability

 Intermediate Precision

 Reproducibility

2016 31
 Repeatability

 Express the precision Should be assessed

under the same using minimum of 9
operating conditions determinations

over a short interval of (3 concentrations/ 3

time. replicates) or

 Also referred to as Minimum of 6

determinations at the
Intra-assay precision
100% level.

2016 32
 Intermediate Precision

Express within-laboratory Depends on the

variations. circumstances under which

Expressed in terms of the procedure is intended

standard deviation, to be used.

relative standard deviation Studies should include

(coefficient of variation) varying days, analysts,

and confidence interval. equipment, etc.

2016 33
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%
Grand
Mean = 100.0
RSD = 0.59%
2016 34
 Reproducibility

 Definition: Ability reproduce data

Lab 1 Lab 2 Lab 3
within the predefined precision
Day Day Day Day Day Day
 Determination: SD, RSD and
1 2 1 2 1 2
confidence interval
Man Man Man Man 2 Man Man
 Repeatability test at two different 1 2 1 1 2
labs.
3 3 3 3 3 3
Prep Prep Prep Prep Prep Prep
Note: Data not required for BLA/NDA

35
 Detection Limit (LOD)/
Quantitation Limit (LOQ)

 LOD LOQ
Lowest amount of analyte in a Lowest amount of analyte

sample that can be detected in a sample that can be

but not necessarily quantified with suitable

quantitated. accuracy and precision.

Estimated by Signal to Noise Estimated by Signal to

Ratio of 3:1. Noise Ratio of 10:1.

2016 36
 LOD and LOQ 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

2016 37
 LOD and LOQ Estimated by

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

2016 38
 Y=bX+a

Statistical estimate of LOD & LOQ

LOD = 3.3 Sbl / b LOQ = 10 Sbl / b

Ybl

LOD LOQ

2016 39
 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

 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
2016 40
 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
2016 41
 Reference Sites
 www.fda.gov
 www.fda.gov/cder/
 www.waters.com
 www.usp.org
 www.ich.org
 www.aoac.org
 www.pharmweb.net

2016 42
 Thankyou

2016 43