Razi

Vaccine & Serum

Research Institute

Analytical Method
Validation
A. Es-haghi
Ph.D.
Dept. of Physico chemistry
Razi vaccine and serum research institute

a.eshaghi@rvsri.ir

http://www.rvsri.ir/

Introduction

Razi
Vaccine & Serum
Research Institute

Test procedures for assessment of the quality levels of

pharmaceutical products are subject to various requirements
The CGMP regulations [21 CFR 211.194(a)] require that test
methods, which are used for assessing compliance of
pharmaceutical products with established specifications, must meet
proper standards of accuracy and reliability
according to these regulations [21 CFR 211.194(a)(2)], users of
analytical methods described in the USP and the NF are not required
to validate accuracy and reliability of these methods, but merely
verify their suitability under actual conditions of use
Recognizing the legal status of USP and NF standards, it is
essential, therefore, that proposals for adoption of new or revised
compendial analytical methods be supported by sufficient laboratory
data to document their validity

http://www.rvsri.ir/

What is Validation?

Razi
Vaccine & Serum
Research Institute

USP 30-NF 25, General Chapter <1225> Validation of

Compendial Methods

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.

ICH Guideline Q2A Text on Validation of Analytical

Procedures

The objective of validation of an analytical procedure is to

demonstrate that it is suitable for its intended purpose.

FDA draft guidance Analytical Procedures and Method

Validation

Methods validation is the process of demonstrating that

analytical procedures are suitable for their intended use.

http://www.rvsri.ir/

Analytical Method Validation

Razi
Vaccine & Serum
Research Institute

In actual practice, analytical methods validation involves

Established analytical method

Data from controlled samples
Statistical evaluation
Assessment of performance characteristics and applicability

http://www.rvsri.ir/

USP Performance Characteristics

Razi
Vaccine & Serum
Research Institute

Performance Characteristics are discrete attributes, derived

by actual execution of an analytical method using controlled
samples, which demonstrate the suitability and proper use of
that method for a specific application

http://www.rvsri.ir/

USP Performance Characteristics

Razi
Vaccine & Serum
Research Institute

SPECIFICITY
LINEARITY
RANGE
DETECTION LIMIT
QUANTITATION LIMIT
ACCURACY
PRECISION
RUGEDNESS
ROBUSTNESS
SYSYEM SUITABILITY

http://www.rvsri.ir/

Specificity / Selectivity

Razi
Vaccine & Serum
Research Institute

ICH Q2A and USP

the ability to assess unequivocally the analyte in the presence of
components that may be expected to be present, such as
impurities, degradation products, and matrix components

Lack of specificity of an individual analytical procedure may be

compensated by other supporting analytical procedure(s)
Other international authorities (IUPAC, AOAC) have preferred
the term Selectivity reserving Specificity for those
procedures that are completely selective

http://www.rvsri.ir/

Specificity

Razi
Vaccine & Serum
Research Institute

Identification
to ensure the identity of the analyte

Purity Test
accurate statement of the content of impurities of an analyte
(related substances, heavy metals, residual solvents, etc.)

Assay
an exact result which allows an accurate statement on the content
of potency of the analyte in a sample

http://www.rvsri.ir/

Specificity

Razi
Vaccine & Serum
Research Institute

Some analytical procedures are not sufficiently specific for the

intended purpose
Assay by titration
Assay of enantiomer by achiral method
Identification by UV absorbance

A combination of two or more analytical procedures is

recommended to achieve sufficient specificity

http://www.rvsri.ir/

Specificity

Razi
Vaccine & Serum
Research Institute

Should be one of the first validation items

When the criteria are not met, this indicates that the method is
not sufficiently developed
Poor specificity can impact accuracy, precision and linearity

http://www.rvsri.ir/

Linearity

Razi
Vaccine & Serum
Research Institute

The ability to generate responses which are directly

proportional to the concentration of an analyte in a
sample

http://www.rvsri.ir/

Linearity

Razi
Vaccine & Serum
Research Institute

A linear relationship requires fewer standards to define the

response curve than a non-linear one
justifies the use of a single point standard to quantitate across
the range

http://www.rvsri.ir/

Linearity

Razi
Vaccine & Serum
Research Institute

USP <1225>
Evaluated across the RANGE
Visual examination of a plot of signals as a function of
concentration
Statistical evaluation of correlation coefficient, y-intercept, slope,
and residual sum of squares of regression line or after
mathematical transformation

ICH Guidance
Performed directly on the drug substance (dilution of standard
stock solution)
Separate weighing of synthetic mixtures of the drug components
Minimum of five concentration is recommended

http://www.rvsri.ir/

Linearity

Razi
Vaccine & Serum
Research Institute

Required for
Assays (Category I)
Impurity tests (Quantitative Category II)
May be for Performance Tests (Category III-Dissolution, etc.)

Not required for

Limit tests (Category II- Impurity Test)
Identification

http://www.rvsri.ir/

Range

Razi
Vaccine & Serum
Research Institute

UPS <1225>
The interval between the upper and lower quantitation levels
of analyte (including these levels) demonstrated by suitable
precision, accuracy, and linearity

ICH Guidance
The following minimum specified ranges should be
considered
Assay of drug substance or finished drug product
from 80% to 120% of the test concentration
Content Uniformity
minimum of 70% to 130% of the test concentration,
unless otherwise justified

http://www.rvsri.ir/

Detection Limit/ Quantitation Limit

Razi
Vaccine & Serum
Research Institute

Characteristic for low level impurity assays

DL (LOD)
the lowest amount of any analyte which can be detected but not
necessarily quantitated

QL (LOQ)
the lowest amount of analyte that can be determined with
acceptable precision and accuracy

http://www.rvsri.ir/

Determination of Detection Limit

Razi
Vaccine & Serum
Research Institute

Non-instrumental Methods
analysis of samples with known concentrations of analyte to
establish the minimum concentration at which the analyte can be
reliably (the ability to reproducibly obtain a signal for the analyte)
detected
Examples
TLC, titrations, color compositions

Instrumental
Same as for non-instrumental methods, plus estimation of the DL
can be made at a value at which the signal to noise ratio (S/N) is
2:1 or 3:1

http://www.rvsri.ir/

Estimation of DL (ICH)

Razi
Vaccine & Serum
Research Institute

DL = 3.3 /S
Where: = Standard deviation (noise) of response
S = slope of the calibration curve for linearity

DL can be based on any of the following

of the blank
of the lowest level linearity point
the calibration plot (regression)

http://www.rvsri.ir/

Quantitation Limit
Definition

Razi
Vaccine & Serum
Research Institute

USP <1225>
the lowest amount of analyte in a sample that can be determined
with acceptable precision and accuracy under the stated
experimental conditions

Essentially identical to ICH Q2A

Quantitation Limit (QL) = Limit of Quantitation (LOQ)

http://www.rvsri.ir/

Determination of QL

Razi
Vaccine & Serum
Research Institute

Non-Instrumental
from analysis of samples with appropriate minimum level of
analyte quantified with acceptable accuracy and precision

Instrumental
same as non-instrumental, plus estimation of the QL can be made
at a value at which the signals to noise is 10:1

http://www.rvsri.ir/

Estimation of QL (ICH)

Razi
Vaccine & Serum
Research Institute

QL = 10 /S
where = standard deviation of response
S = slope of the calibration curve for linearity
can be based standard deviation of the blank or of the lowest
level linearity point

http://www.rvsri.ir/

Case Study

Razi
Vaccine & Serum
Research Institute

http://www.rvsri.ir/

Accuracy

Razi
Vaccine & Serum
Research Institute

Definition
USP
The closeness of the result obtained by the method to the true
value.
ICH
The closeness of the result obtained by the method to a value
that is accepted as conventionally true value or as a reference
value.

http://www.rvsri.ir/

Accuracy

Razi
Vaccine & Serum
Research Institute

The accuracy of an analytical method should be

established across its range

ICH recommends a minimum of 9 determinations over a

minimum of 3 concentrations covering the stated
quantitation range (3 test at 3 conc.)
Accuracy is calculated as
the percentage of recovery by the assay of the known added
amount of analyte in the sample
the difference between the mean and the accepted true
value, together with confidence intervals

http://www.rvsri.ir/

Accuracy

Razi
Vaccine & Serum
Research Institute

5 ways to determine the Accuracy

Testing of a Reference Standard

Mixture with Excipients (Spiked placebo)
Standard addition (Spiked sample)
Inferred from linearity and specificity data
Comparison with a method known to be accurate (reference
method)

http://www.rvsri.ir/

Accuracy
assay of a drug substance

Razi
Vaccine & Serum
Research Institute

Testing of a Reference Standard

Comparison with a reference standard method

http://www.rvsri.ir/

Recovery of Drug F Ref Std at different levels

Razi
Vaccine & Serum
Research Institute

http://www.rvsri.ir/

Comparison with Reference Method

Razi
Vaccine & Serum
Research Institute

There is no difference with p < 0.05

http://www.rvsri.ir/

Precision

Razi
Vaccine & Serum
Research Institute

The degree of agreement among results from individual

samplings of a homogeneous sample

http://www.rvsri.ir/

Precision

Razi
Vaccine & Serum
Research Institute

Precision is expressed as RSD

Precision should be evaluated across the specified
Quantitation range of the method
Determination
Assay individual samples of a homogeneous preparation
Calculate Standard Deviation or Relative Standard Deviation

Method precision should include all sources of variation from

sample preparation to rounding the final test result

http://www.rvsri.ir/

Precision

Razi
Vaccine & Serum
Research Institute

ICH recommends a minimum of 9 determinations over a

minimum of 3 concentrations covering the stated quantitation
range (3 test at 3 conc.)
OR

Using a minimum of 6 determinations at 100% of the test

concentration

http://www.rvsri.ir/

ICH-USP Level of Precision

Razi
Vaccine & Serum
Research Institute

Repeatability
agreement within a short period or the same analyst and
instrumentation

Intermediate precision
agreement in results intra-laboratory but from different days,
analysts and equipment (as appropriate)

Reproducibility
agreement in results between laboratories (as in a collaboration
study)

http://www.rvsri.ir/

Precision vs. Ruggedness

Razi
Vaccine & Serum
Research Institute

In USP, Reproducibility and intermediate precision are

also considered together with Ruggedness

http://www.rvsri.ir/

Precision
choice of acceptance criteria

Razi
Vaccine & Serum
Research Institute

Practical Considerations
Ability to meet criteria
may be instrument dependent
Maximum acceptance criteria for precision should be linked to
acceptance criteria for claim or specification to be able to obtain a
reliable result (reduce Out-Of Specifications)

http://www.rvsri.ir/

Precision

Razi
Vaccine & Serum
Research Institute

Repeatability (5 replicate injections)

Assay Limit (%)
99.0 101.0
98.5 101.5
98.0 102.0
95.0 105.0
90.0 110.0
Impurities (depending on level)

Max, % RSD
0.37
0.55
0.73
1.9
3.9
5 to 25

http://www.rvsri.ir/

Precision
choice of acceptance criteria

Razi
Vaccine & Serum
Research Institute

Intermediate Precision
FDA recommends a minimum of 2 different days with different
analysts
Acceptance criteria
Perform F-test
results between labs should not be statistically different
Typically about 2x repeatability (2 x RSD)
However, a statistical difference may still not be
significant (practical consideration). Need to evaluate
suitability for methods intended use

http://www.rvsri.ir/

Reproducibility Precision

Razi
Vaccine & Serum
Research Institute

FDA
at least 2 laboratories within the company

FDA
for multi-laboratory follow ASTM 691
Standard practice for conducting an laboratory study to
determine the precision of a test method

AOAC Protocol
8 samples, 8 laboratories

http://www.rvsri.ir/

Ruggedness

Razi
Vaccine & Serum
Research Institute

Reproducibility with the same samples using different

laboratories, analysts, days, reagent lots (same brand), and
environmental conditions
Precision should be evaluated across the specified
Quantitation range of the method

http://www.rvsri.ir/

Ruggedness vs. Robustness

Razi
Vaccine & Serum
Research Institute

Reproducibility of the test results under conditions normally

expected from laboratory to laboratory and from analyst to
analyst
Method parameters are kept constant (difference with
robustness)

http://www.rvsri.ir/

Ruggedness

Razi
Vaccine & Serum
Research Institute

Determination
Analysis of an homogeneous sample in different laboratories, by
different analysts, under prevalent environmental conditions using
the specified parameters
The degree of reproducibility of test results is determined as
function of the variables

http://www.rvsri.ir/

Ruggedness

Razi
Vaccine & Serum
Research Institute

study normal variables

Analyst
Day
Lab
Column

A, B, C
1, 2, 3
L, M N
X, Y, Z

Selected comparisons are evaluated since evaluating all

possible variations would require 81 experiments (3 x 3 x 3 x
3)
The influence of each variable may be evaluated using a multifactor experimental design (Plackett- Burman, Hendrix, etc.)

http://www.rvsri.ir/

Ruggedness

Razi
Vaccine & Serum
Research Institute

Degree of reproducibility of test results as a function of

the variables

http://www.rvsri.ir/

Robustness

Razi
Vaccine & Serum
Research Institute

The ability to remain unaffected by small but deliberate

variations in method parameters- evaluates reliability
Provides an indication of the reliability of the method
during normal usage

http://www.rvsri.ir/

Robustness

Razi
Vaccine & Serum
Research Institute

Evaluated by varying method parameters and determining the

effect (if any) on the results of the method
It should be considered early in the development of a method
If the results are susceptible to parameter variations, these
parameters should be adequately controlled and a
precautionary statement included in the method

http://www.rvsri.ir/

Robustness

Razi
Vaccine & Serum
Research Institute

Two approaches
Uni-variable
Systematically varying each parameter sequentially
Typically, each parameter may be varied by 5 10% above
and below the value set in the method
Multi-variable
Use of the statistical design
Central Composites
Fractional factorials,
Two-factor Plackett-Burman statistical approach
Hendrix

http://www.rvsri.ir/

System Suitability

Razi
Vaccine & Serum
Research Institute

Ensures that both methodology and instrumentation are

performing within expectation prior to the analysis of the
test samples

USP General Chapter <1225>

System suitability tests are based on the concept that the
equipment, electronics, analytical operations, and samples to be
analyzed constitute an integral system that can be evaluated as
such. System suitability test parameters to be established for a
particular procedure depend on the type of procedure being
evaluated. They are especially important in the case of
chromatographic procedures.

http://www.rvsri.ir/

System Suitability

Razi
Vaccine & Serum
Research Institute

Provides assurances that the system is working at the

time of analysis
Ensures that both methodology and instrumentation are
performing within expectations prior to the analysis of the
test samples
Should be monitored during run time to verify that criteria
remain realistic and achievable

http://www.rvsri.ir/

System Suitability

Razi
Vaccine & Serum
Research Institute

Parameters for chromatographic method

Resolution specificity
Column efficiency specificity
Relative standard deviation precision
Tailing factor accuracy and precision
QL sensitivity
Capacity factor specificity
Reference Standard Check - analyte

http://www.rvsri.ir/

USP Method Categories

Razi
Vaccine & Serum
Research Institute

Category I
Quantitative test for major active ingredients (chromatographic
and titration)

Category II
Impurities or degradation compounds; quantitative or limit tests

Category III
performance tests (dissolution, drug release, tapped density,
particle size)

Category IV
Identification tests (TLC, IR, UV, Chemical ID <191>

http://www.rvsri.ir/

USP <1225>
Data Elements for Validation

Razi
Vaccine & Serum
Research Institute

http://www.rvsri.ir/

ICH Q2B
Data Elements for Validation

Razi
Vaccine & Serum
Research Institute

May

be required, depending on the nature of the specific test

Is not needed if reproducibility has been performed

http://www.rvsri.ir/

Choice of Performance Characteristics

Razi
Vaccine & Serum
Research Institute

Not all Performance Characteristics are needed to validate

an analytical method or validated in the same way
Determine the expected use of the test procedure (e.g., assay,
dissolution, limit test, ID test)
Take into account the type of analytical technique used
(chromatographic, spectrophotometric, titration, etc.)

Establish the requirements of the analytical application

Degree of precision and accuracy needed
The concentration range of the analyte
The level of tolerable interference, if any

http://www.rvsri.ir/

Protocol for Final Validation Studies

Razi
Vaccine & Serum
Research Institute

Initial analytical methods are often developed in R&D

areas with some preliminary validation data
These procedures can be slightly modified when
transferred to QC areas for complete validation including
multi-site collaborative studies

http://www.rvsri.ir/

Validation Protocols

Razi
Vaccine & Serum
Research Institute

The validation protocol document contains

Description of the analytical method and its intended use
(purpose)
List of applicable performance characteristics
Appropriate acceptance criteria
Review and approval signatures

http://www.rvsri.ir/

Pre-validation Assessment

Razi
Vaccine & Serum
Research Institute

Equipment
Suitable for expected accuracy?

Reference Materials
Suitability characterized?

Analytical Method
Is procedure finalized?

Validation Protocol
Management / QA approved?

http://www.rvsri.ir/

Pre-validation Assessment

Razi
Vaccine & Serum
Research Institute

Equipment
Demonstrate that equipment used in validation studies is suitable
for use and is comparable to equipment used for routine analysis
Calibrated (as applicable)
Qualification should have been performed
Installation Qualification
Operation Qualification
Performance Qualification
Routine maintenance performed- proper working order

http://www.rvsri.ir/

Pre-validation Assessment

Razi
Vaccine & Serum
Research Institute

Reference Materials
evaluate suitability of use
FDA requires no additional evaluation of USP Reference
Standards
Standardization of reagents vs. Primary standards when available
(i.e., use of reagent grade chemical need qualification)
Principal analyte (purity and homogeneity)
Impurity Standards
chemical purity is very critical if used to determine
relative response
Solution stability of standard preparations

http://www.rvsri.ir/

Pre-validation Assessment

Razi
Vaccine & Serum
Research Institute

Analytical Method
must be final before validation
Individual procedure steps may impact overall method accuracy
or precision
Solution preparations
concentration of analyte in solution for analysis (suitable
range)
System suitability
necessary parameters and acceptance criteria must be
specified
Changes may negatively impact validation studies
previously performed
Later changes in tailing factor or resolution criteria from
robustness evaluation may have a significant impact on
integration in accuracy evaluations

http://www.rvsri.ir/

Standard and Sample Solution Preparation

Razi
Vaccine & Serum
Research Institute

Overall method precision and accuracy are also impacted by

sampling, weighing, and quantitative dilutions
No instrumental technology can overcome these errors
The precision and accuracy of a final analytical method can be
better than the precision and accuracy of the preparation of the
test solutions

http://www.rvsri.ir/

Revalidation

Razi
Vaccine & Serum
Research Institute

Revalidation may be necessary in the following cases

a submission to the USP of a revised analytical method
use of an established general method with a new product or
raw material

The ICH documents give guidance on the necessity for

revalidation in the following circumstances
changes in the synthesis of the drug substance
changes in the composition of the drug product
changes in the analytical procedure

http://www.rvsri.ir/

RVSRI deffects in Analytical Method Validation

Razi
Vaccine & Serum
Research Institute

Equipment
Personnel
Reference Materials
Analytical Methods
Validation Protocol
Change control
Weak links between different chain to final product be
completely assured

http://www.rvsri.ir/

Quick Quiz
Cumulative errors in serial dilution

Razi
Vaccine & Serum
Research Institute

To prepare 100 fold dilution of a stock solution, what is the

most precise procedure when considering the necessary
individual Class A pipette and volumetric flask tolerances?
A. 1.0 mL/10 mL; 1.0 mL/10 mL
B. 5.0 mL/50 mL; 5.0 mL/50 mL
C. 10.0 mL/100 mL; 10.0 mL/100 mL
D. 1.0 mL/100 mL
E. 2.0 mL/200 mL
F. 5.0 mL/500 mL

http://www.rvsri.ir/

Tolerance of Class A Glassware

(see USP <31> Volumetric apparatus)

Razi
Vaccine & Serum
Research Institute

http://www.rvsri.ir/

Quick Quiz
Cumulative errors in serial dilution

Razi
Vaccine & Serum
Research Institute

Sum of step errors

A. 1.0/10; 1.0/10L
B. 5.0/50; 5.0/50
C. 10.0/100; 10.0/100
D. 1.0/100
E. 2.0/200
F. 5.0/500

0.6 + 0.2 + 0.6 + 0.2 = 1.6%

0.2 + 0.1 + 0.2 + 0.1 = 0.6%
0.2 + 0.08 + 0.2 + 0.08 = 0.43%
0.6 + 0.08 = 0.68%
0.3 + 0.05 = 0.35%
0.2 + 0.03 = 0.23%

http://www.rvsri.ir/