Analytical Method

Validation
Muhammad Saqlain Tahir Ph.D.
Validation

A documented program that provides

a high degree of assurance that a
specific process, method, or system
will consistently produce a result
meeting predetermined acceptance
criteria.
The process to confirm that the
analytical procedure employed for a
specific test is suitable for intended
use and that they support the
identity, quality, purity and potency
of the drug substances and drug
products.
Why Validate?

 To trust the method

 Regulatory requirement
Pre-requisites

 A well-designed experimental
matrix (Written protocol)
 Step-by-step methodology (STP)
 Test samples of good quality
(should meet the quality as per
defined specifications)
 Peak purity (preferably >99%
purity)
 Equipments & analytical
instruments should be in
calibrated state.
Scope

The scope of the method and its

validation criteria should be defined
early in the process. These include
the following questions:
 What analytes should be
detected?
 What are the sample matrices?
 Are there interfering substances
expected, and, if so, should they
be detected and quantified?
Scope

 Are there any specific legislative

or regulatory requirements?
 Should information be qualitative
or quantitative?
 What are the required detection
and quantitation limits?
 What is the expected
concentration range?
 What precision and accuracy is
expected?
 How robust should the method
be?
Parameters to be evaluated

 Specificity  Accuracy (Recovery)

 Forced degradation study  Linearity
 System suitability  Range
 LOD & LOQ  Solution Stability
 Precision  Robustness
 Repeatability  System Suitability
 System
 Method
 Intermediate Precision
 Method Reproducibility
 Transfer of technology
 Compendial methods
What Methods to be Validated?

 Category I:- Analytical procedures for

quantitation of major components of
bulk drug substances or active
ingredients (including preservatives) in
finished pharmaceutical products.
 Category II:- Analytical procedures for
determination of impurities in bulk drug
substances or degradation compounds in
finished pharmaceutical products. These
procedures include quantitative assays
and limit tests.
 Category III:- Analytical procedures for
determination of performance
characteristics (e.g., dissolution,
deposition of emitted dose, and others).
 Category IV:- Identification tests
 Data Elements Required for Validation

Analytical Category II
Performance Category Category Category
Characteristics I Quantitative Limit Tests III IV
Accuracy Yes Yes a a No
Precision Yes Yes No Yes No
Specificity Yes Yes Yes a Yes
DL No No Yes a No
QL No Yes No a No
Linearity Yes Yes No a No
Range Yes Yes a a No
a May be required, depending on the nature of the specific test
Methodology

➢ No exact methodology given for

each parameter
➢ Only ICH guidelines are provided
but not to the extent of 100%
➢ Good understanding of each
performance characteristics most
important. This understanding
must be beyond the basic
definition of each parameter.
Precision

Definition: Types:
Closeness of agreement (degree of  Measurement / Injection
scatter) between a series of Repeatability (System Precision)
measurements obtained from multiple
 Method Repeatability (Method
sampling of the same homogeneous Precision)
sample under the prescribed
conditions Intermediate Precision - precision
under different laboratory conditions
Discussion:
(within laboratory variation, as on
Repeatability:- precision under same different days, or with different
operating conditions (within laboratory analysts, or equipments within the
over a short interval of time using the same laboratory).
same analyst with same equipment). Reproducibility – precision between
Repeatability is also termed intra-assay
laboratories
precision.
Precision

Procedure: Six replicate analysis of the samples

through the complete analytical
Six replicate measurements/
procedure from sample preparation
injections of standard preparation
to final result by two different
(System Precision)
analysts, columns, instruments,
Six replicate analysis of the samples different days (Intermediate
through the complete analytical Precision - Ruggedness)
procedure from sample preparation
to final result (Method Precision)
Precision - Repeatability

System Repeatability
Injection No.
Peak Area
1 1121533
2 1121262
3 1120831
4 1121177
5 1115016
6 1120936
Mean 1120125.8
SD 2515.6
% RSD 0.22
Precision - Repeatability

Method Repeatability
Sample No.
Assay (%)
1 98.82
2 99.20
3 99.41
4 98.51
5 98.75
6 98.68
Mean 98.89
SD 0.34
% RSD 0.34
Precision - Intermediate

Operator 1 Day 1 HPLC 1 Operator 2 Day 2 HPLC 2

Sample No.
Assay (%) Assay (%)
1 98.82 98.96
2 99.20 99.34
3 99.41 99.53
4 98.51 99.66
5 98.75 99.16
6 98.68 98.30
Mean 98.89 99.16
% RSD 0.34 0.49
Cumulative RSD (%) 0.42
Precision

Acceptance Criteria:

% RSD
Procedure System Precision Method Precision IMP
API DP API DP API DP
Assay 1.0/2.0 1.0/2.0* 1.0 2.0 1.0 3.0
Dissolution NA 1.0/2.0* NA 6.0 NA 5.0
Impurities 5.0* 5.0* 10.0 10.0 10.0 15.0

NA – Not Applicable DP – Drug Product IMP – Intermediate Precision

API – Active Pharmaceutical Ingredient * - As given in STP
Accuracy

Definition: Impurities:- drug substance/drug product

spiked with known amounts of impurities-
The accuracy of an analytical procedure minimum three levels
expresses the closeness of agreement
between the value which is accepted either ✓ LOQ level to 200% of specification and
as a conventional true value or an accepted each level in triplicate
reference value and the value found. This is
sometimes termed trueness. Evaluation:
Procedure: ✓ Recovery from amount added and
amount found
Assay/Dissolution:- Known amount of drug
substance spiked with synthetic mixtures of ✓ Precision (% RSD) at each level (for
drug product components (excipients) – three replicate preparations)
minimum of three levels
✓ 80%, 100% & 120% of test
concentration-assay;
✓ ± 20% of expected release-dissolution,
each level in triplicate
Accuracy

Acceptance Criteria:
Assay:-
Recovery should be between 98% to 102% (Depends upon the strength)
Dissolution:-
95% to 105%
Impurities:-
if, Specification is ≤ 0.2% : 85% to 115%
if, Specification is > 0.2% : 90% to 110%
At LOQ level : 80% to 120%
A simple logic behind this performance characteristic is whether the procedure is
capable of estimating a true value or not.
Linearity

Definition:
Linearity:- The Linearity of an analytical procedure is its ability (within a
given range) to obtain test results that are directly proportional to the
concentration of analyte in the sample.
Range:- The interval between the upper and lower level that have been
demonstrated to be determined with precision, accuracy and linearity using
this method as written.
Discussion:
In order to determine the quantity of any analyte present in unknown sample,
some kind of relation ship (mathematical/empirical) between concentration
and response is essential.
Linearity

Procedure:
Prepare a series of solutions (not less than five is recommended) with standard / reference
samples in the specified concentration range and analyze them as per method
Assay:-
80% to 120% of test concentration
CU:-
70% to 130% of test concentration
Dissolution:-
± 20% of expected release (Q) for immediate release
0 to 120% (for extended release)
Impurities:-
LOQ to 200% of specification
Linearity

Evaluation:
Slope:- indicates sensitivity of the method
Intercept:- indicates response for no analyte (interference)
Correlation Coefficient:- indicates the relation ship chosen is correct

Acceptance Criteria:
Correlation Coefficient should be not less than 0.999 for assay, CU,
dissolution test methods and 0.99 for impurities test method
Linearity

Area (y) Concentration ppm (x) Linest (y = mx + b)

885744 80 887331
1002727 90 999878
1111959 100 1112425
1223705 110 1224972
1337990 120 1337519
Correlation co-efficient 0.9999
Y- Intercept (b) -13045.0
% Y- Intercept -1.17
Slope (m) 11254.7
Linest equation y = 11254.7x - 13045.0
Range

Parameter Results
% RSD (Area) 0.22
% RSD (Contents) 0.49
% RSD (Cumulative) 0.42
Individual Recovery 98.63 – 100.19%
Average Recovery 99.21 – 99.59%
Correlation coefficient 0.9999
y - Intercept -13045.0
Slope of regression line 11254.7
Linest y = 11254.7x – 13045.0
LOD & LOQ

Limit of Detection:
Lowest amount of analyte in a sample which can be detected but not
necessarily quantitated, under the stated experimental conditions (LOD)

Limit of Quantitation:
LOQ: Lowest amount of analyte in a sample which can be quantitatively
determined with suitable precision and accuracy (LOQ)
LOD & LOQ

Approaches: Procedure:
Different approaches suggested by ICH, Derived from standard preparations
USP & EP. prepared for linearity studies (five
levels)
 Derived from the residual standard
deviation of regression line (Sy/x) Evaluation:
 Derived from the residual standard  Detection limit (DL) co-efficient 3.3
deviation of y-intercept of
 Quantitation limit (QL) co-efficient
regression lines (Sy)
10
 Signal to noise
Acceptance Criteria:
RSD of six replicate injections is ≤ 10%
for LOQ and between ˃ 10% ≤ 33.0%
for LOD
Solution Stability

Discussion: Procedure:
 It is often essential that solution Prepare test sample as per procedure and
(standards, test samples) be stable analyze at initial and at different time
enough to allow for delays covering intervals by keeping the sample at room
instrument breakdowns / overnight temperature (25°C) / refrigerator condition
analysis. (2 – 8°C)
 A minimum of 12 Hrs, 18 Hrs or 24 Hrs is Evaluation:
routinely recommended for
chromatographic methods for which % difference from initial response to
vialed solutions may remain on an specified interval for analyte / each
autosampler at ambient temperatures impurity.
due to various delays Acceptance Criteria:
A simple logic behind this study is to % difference is not more than
determine the period of time, a solution can
be held before analysis without Assay/CU: 2.0
compromising accuracy.
Dissolution 3.0
Impurities 10.0
Robustness
Definition:
Measure of its capacity to remain unaffected
by small, but deliberate variations in
method parameters and provides indication
of its reliability during normal usage.
Discussion:
Varying method parameters within a
realistic range and the quantitative
influence of the variables is determined,
and, if the influence of the parameter is
within a previously specified tolerance,
then, the parameter is said to be within the
method’s robustness range.
According to ICH Guidelines, robustness
should be considered early in the
development stage of a method.
Typical Variations Include:
 Flow rate (+ 10%)
 Wavelength (+ 2nm)
 Mobile phase composition, generally,
organic composition (+ 5%)
 Temperature (+ 2°C)
 pH of the mobile phase (+ 0.2units)
Procedure:
Analysis of resolution by proposed analytical
methodology and the method operated at
variable conditions
Robustness

Evaluation: Acceptance Criteria:

✓ System suitability parameters at
all variable conditions
✓ %age assay of sample at all
variable conditions
✓ Relative retention times at all
variable conditions (monitor the
separation at each variable
condition)
✓ System suitability criteria should
meet at each variable condition
✓ Cumulative RSD for % assay
results obtained at STP condition
and variable condition for each
variability.
Specificity

Definition: Selective refers to a method which

provides responses for a number of
Specificity of an analytical method is
chemical entities that may / may not
its ability to measure accurately an
be distinguished from each other. If
analyte in the presence of
each response is distinguished from
interferences, such as synthetic
all other responses, then the method
precursors, excipients, enantiomers
is said to be selective.
and known (or likely) degradation
product that may be expected to be Use of the term Selectivity is
present in the sample matrix. appropriate for the methods based
on techniques such as HPLC, GC.
Discussion:
Specific generally refers to a method
that produces a response from a
single analyte only
Specificity
Evaluation:
 Identification Tests:- Response for
compound of interest only
 Assay:- Peak purity of analyte peak (un-
spiked sample)
 Impurities:- Resolution between the
impurity(s) and / or degradants from
analyte – peak purity of analyte and
impurity(s) peaks (for spiked sample)
Acceptance Criteria:
➢ Identification Tests:- Positive response
for compound of interest only
➢ Assay:- No peak should be found at the
retention time of analyte peak and peak
purity of analyte peak should pass.
➢ Impurities:- Should pass peak purity of -
main analyte and impurity peaks
No peak should be found at the
retention time of analyte/impurity
➢ Dissolution:- No peak should be found at
the retention time of analyte. In case of
UV methodology, % difference should be
not more than 2.0
Specificity
Forced Degradation Studies
Introduction: Why perform Forced Degradation
Studies ?
 Forced degradation or stress
testing is undertaken to ➢ Address the stability of the
demonstrate specificity when compound
developing stability-indicating
methods ➢ Establish the degradation
pathway
 A stability-indicating method is
➢ Identify the degradation products
one that accurately quantitates
the active ingredients without ➢ Validate the stability indicating
interference from degradation power of the analytical
products, process impurities, procedures used
excipients or other potential
impurities
Specificity
Forced Degradation Studies
Procedure: When you conduct a Forced
Degradation Study ?
Perform analysis for each stressed
(acid / base / peroxide / thermal / ➢ Initiated at an early stage of
photolytic / humidity) sample as per development
methodology
➢ Repeated as methods, processes or
Normal initial stressed conditions to be formulations change, so it is an
applied ongoing effort.
✓ 1M HCl ➢ Evaluate the each unique
formulation before formal stability
✓ 1M NaOH begins
✓ 10% H2O2
✓ 105°C / at least 72 Hours
✓ 12000 Lux / at least 72 Hours
✓ 92% RH / 25°C / at least 72 Hours
Specificity
Forced Degradation Studies
Evaluation:
Assay:- % Difference of assay for Control
(Un-stressed) and each Stressed samples
Peak purity of analyte peak for Control and
Stressed sample
Impurities:- Peak purity of analyte peak for
Control and each Stressed sample
Acceptance Criteria:
Assay:- Peak purity of analyte peak in
Control and each Stressed sample should
pass
Impurities:- Peak purity of analyte peak in
Control and each Stressed sample should
pass
Points to remember:
If the degradation media degrades the drug
substance/drug product to too great extent
or do not degrade the drug substance/drug
product at all, then alternative action
should be taken (e.g., change the strength
of the degradation medium or exposure
time or apply heat over a period of time to
achieve minimum level of degradation)
Different numerical values were proposed
for the extent of degradation in recent
literature
* Minimum 5%
Specificity
System Suitability
 % RSD  Separation factor ()
 Area  The relative retention calculated
for two adjacent peaks
 Height
 No. of Theoretical plates
 Asymmetry
 A measure of column efficiency
 Also known as tailing factor
 S/N Ratio
 Resolution
 Separation of two components
 Similarity Factor

 Capacity (Retention) factor

 Ratio of time spent by analyte in
stationary phase to its time in
mobile phase
Time Management in Validation

There are no official guidelines on the sequence of If stability and robustness data is not available with
validation experiments and the optimal sequence method development data
can be depending on the method itself. Based on
experience, for HPLC method the following ✓ Stability
sequence has been proven to be useful for time
management. ✓ Robustness

If the method is proved as stable and robust under ✓ Specificity

method development (Pre-validation Programme) ✓ Linearity
✓ Specificity ✓ LOD & LOQ (if applicable)
✓ Linearity ✓ Precision
✓ LOD & LOQ (if applicable) ✓ Accuracy
✓ Precision ✓ Range
✓ Accuracy
✓ Range
✓ Stability
✓ Robustness
Validation Protocol

 Method principle  Test procedures to evaluate each

validation parameter and
 objective
proposed acceptance criteria
 Listing of responsibilities
 Plan or procedure when
 Laboratories involved and their acceptance criteria are not met
role in the validation
 Requirements for the final report
 Method categorization
 The validation process cannot
 List of reagents (including test proceed until the protocol and all
lots) and standards parties involved approve the
acceptance criteria
 Training
 Attachments
 Master test sheet
 Qualification/Calibration
Validation Report

Generally method validation report should have  Summary of report (overall view of
validation exercise, any critical issues,
 Objective and scope of the method recommendations etc., for the application
 Molecule details: IUPAC Name, CAS No. of method)
Molecular Formula, Molecular Weight and  Instrument out puts, which should
Molecular Structure etc. represent critical method parameters
 Detailed list of chemicals, reagents,  Specificity and LOD – for Identification Test
reference standards (Generally, photographs)
 Listing of equipment and its functional and  Selectivity / Specificity data (discriminating
performance requirements chromatogram, peak purity data, blank and
 Methodology followed placebo chromatograms and stressed
samples chromatograms)
 Validation data: parameter wise –  Linearity graphs
procedure, results, conclusion etc.
 Summary of data (results in brief –  Resolution and related system suitability
parameter wise) chromatograms
 Any out puts which are significant
Revalidation

Change Parameters to be validated

Synthetic route All parameters
Analytical procedure All parameters
Addition of new impurity Specificity, solution stability, linearity,
accuracy, range, LOQ & LOQ (for new impurity
only)
Composition of drug product Specificity, solution stability, precision &
accuracy
Change in specification Linearity, accuracy & range
Verification

USP <1225> states:

According to these regulations [21 CFR 211.194(a)(2)], users of analytical
methods described in USP-NF are not required to validate the accuracy and
reliability of these methods, but merely verify their suitability under actual
conditions of use.
GMP Guidance/Reference

 ICH Q2 R1 Validation of Analytical Procedures

 USP <1225> Validation of Compendial Procedures
Q&A

Thank you