VALIDATION OF ANALYTICAL

METHODS
Kelompok 10
1. Hani Hazarani (1311012022)
2. Novi Bakri (1311012025)
3. Hafsa Rahmi Latifa (1311012027)
4. Tri Ayuningtias (1311012029)
5. Poppy Agustin (1311012045)
 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)

2009 2
• Method validation is the process of demonstrating that analytical
procedures are suitable for their intended use and that they support the
i d e n t i t y, s t r e n g t h , q u a l i t y, p u r i t y a n d p o t e n c y o f t h e

drug substances and drug 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

2009 3
 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, NIR, AA, NMR, XRD,MS

• Capillary Electrophoresis Methods - Zone, Isoelectric Focusing
• Particle Size Analysis Methods - Laser, Microscopic, Sieving, SEC, etc.
• Automated Analytical Methods - Robots, Automated Analysis.

2009 4
 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
2009 5
 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.

2009 6
 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 FDA Requirements

2009 7
 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

2009 8
 • 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 2.0%

2009 9
 System Suitability

Validation

Calibration
Pump Injector
Detector Data System

Analyst Method

Sample
2009 10
 Method Life Cycle

Validation

Development Optimization

2009 11
 Verification vs. Validation

• Compendial vs. Non-compendial Methods

– Compendial methods-Verification

– Non-compendial methods-Validation requirement

2009 12
 Compendial Analytical Procedures

• The Analytical procedures in the USP 25/NF 20 are legally recognized under section

501(b) of the Federal Food, Drug and Cosmetic Act as the regulatory analytical

procedures for the compendial items. The suitability of these procedures must be

verified under actual conditions of use. When using USP 25/NF 20 analytical

procedures, the guidance recommends that information be provided for the

f o l l o w i n g

characteristics:

– Specificity of the procedure

– Stability of the sample solution

– Intermediate precision
2009 13
 Regulatory and Compliance
Requirements Review

• Validation of an analytical method is the process

by which it is established, by laboratory studies,
that the performance characteristics of the
method meet the requirements for the intended
analytical applications

USP 23 General
Information <1225>

2009 14
 • 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 .

21 CFR PART 211 - CURRENT GOOD MANUFACTURING PRACTICE FOR

FINISHED PHARMACEUTICALS

2009 15
• 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

2009 16
• 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

2009 17
 Today’s Validation Requirements

ICH/USP

GMPs
(legal) FDA

2009 18
 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
2009 19
 Specificity/Selectivity
• Ability of an analytical method to measure the analyte free from
interference due to other components.

• Selectivity describes the ability of an analytical method to differentiate

various substances in a sample
Original term used in USP
Also Preferred by IUPAC and AOAC
Also used to characterize chromatographic columns

• Degree of Bias (Used in USP)

The difference in assay results between the two groups
- the sample containing added impurities, degradation products, related chemical
compounds, placebo ingredients
- the sample without added substances

2009 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.

2009 21
 Linearity

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

2009 22
 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

2009 23
 Range
• 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 Assays
– From Reporting Level to 120% of Impurity Specification for Impurity Assays
– From Reporting Level to 120% of Assay Specification for Impurity/Assay
Methods

2009 24
 Accuracy

• Closeness of the test

results obtained by the
method to the true value.

25
 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

2009 26
 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.

2009 27
 Precision… Considered at 3 Levels

• Repeatability

• Intermediate Precision

• Reproducibility

2009 28
 Repeatability

• Express the precision Should be assessed using

under the same 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.

2009 29
 Intermediate Precision

Express within-laboratory Depends on the circumstances

variations. under which the procedure is

Expressed in terms of intended to be used.

standard deviation, relative Studies should include varying

standard deviation days, analysts, equipment, etc.

(coefficient of variation) and

confidence interval.

2009 30
 Reproducibility

• Definition: Ability reproduce data within the

predefined precision

• Determination: SD, RSD and confidence interval

– Repeatability test at two different labs.

31
 Detection Limit (LOD)/ Quantitation
Limit (LOQ)

• LOD LOQ
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 precision.

Ratio of 3:1. Estimated by Signal to Noise

Ratio of 10:1.

2009 32
 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

2009 33
 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

2009 34
 Y=bX+a

Statistical estimate of LOD & LOQ

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

Ybl

LOD LOQ

2009 35
 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

2009 36
 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

2009 37
 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)

2009 38
 Re-validation

• When
– Method parameters have been changed
– The scope of the method has been changed
– Synthetic methods have been changed
– Impurity profile has been changed

• What
– Preferably everything. Exceptions should be
scientifically justified
2009 39
 References
• USP 23 General Information
• ICH Guideline for Industry Q2B, Validation of
Analytical Procedures: Methodology
• ICH Guideline for Industry Q2A, Text on Validation
of Analytical Procedures March 1995
• 21 CFR PART 211 - CURRENT GOOD
MANUFACTURING PRACTICE FOR FINISHED
PHARMACEUTICALS
THANK YOU 