METHOD VALIDATION

K.K. Nayak
Senior Chemist

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 Definitions
Validation is the process of demonstrating or
confirming the performance characteristics
of a method of analysis.
confirmation, through the provision of objective
evidence, that the requirements for a specific
intended use or application have been fulfilled
confirmation by examination and provision of
objective evidence that the particular
requirements for a specific intended use are
fulfilled
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 Why Method Validation?
 To minimize analytical and instrumental errors
 To give reliable and reproducible results in
accordance with the given specifications of the
test method
 To ensure the quality of the test results
 To meet accreditation requirement
 Objective evidence for defense against challenges
 To be assured of the correctness of results

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 When should methods be validated?
A method should be validated when it is necessary to verify
that its performance parameters are adequate for use for a
particular analytical problem. For example:

non-standard methods;
laboratory-designed/developed methods;
standard methods used outside their intended
scope;
amplifications and modifications of standard
methods.

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 When Method Validation Not
Required?

• Standard methods on condition that

– used within their scope of applicability (e.g.
matrices, ranges, etc)
– without modifications

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 Who carries out method validation?

The laboratory using a method is responsible for ensuring that it

is adequately validated, and if necessary for carrying out
further work to supplement existing data. For example, where
a method has been validated by a standards approving
organisation, such as AOAC International, the user will
normally need only to establish performance data for their
own use of the method.

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 EXTENT OF VALIDATION STUDIES

Suggestions as to the extent of validation and verification

measures for different circumstances are:
The laboratory is to use a “fully” validated method
The laboratory is to use a fully validated method,
but a new matrix is to be used
The laboratory is to use a well-established, but not
collaboratively studied, method.
 laboratory should verify and undertake precision
studies, bias studies and possibly linearity studies,

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 EXTENT OF VALIDATION STUDIES
 The method has been published in the scientific
literature together with some analytical characteristics.
 The laboratory should undertake precision
studies, bias studies, ruggedness, and linearity
studies.

Validation is always a balance between costs,

risks and technical possibilities.

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 EXTENT OF VALIDATION STUDIES

Extent of validation work for four types of analytical

applications. Example from the pharmaceutical sector

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 Validation Plan& Report
validation plan and validation report may consist
of the following sections:
Title
Planning
Performance Characteristics
Summary

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• Title:

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 Deciding what degree of validation is
required
• The laboratory has to decide which method performance
parameters need to be characterised in order to validate the
method.

Validation requirements may be specified in guidelines within

a particular sector of measurement relevant to the method
and it is recommended that where these are available they
are followed. For example validation of a method for food
analysis should be consistent with the validation strategy
used by AOAC International.

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 Ask yourself
• What analytes should be detected?
• What are the expected concentration levels?
• What are the sample matrices?
• Are there interfering substances expected, and, if so, should they be detected
and quantified?
• 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?
• Which type of equipment should be used? Is the method for one specific
instrument, or should it be used by all instruments of the same type?
• Will the method be used in one specific laboratory or should it be applicable in
all laboratories at one side or around the globe?
• What skills do the anticipated users of the method have?

07/20/2021 Before start to validate a method

K.K. Nayak (B. Pharm.) 13
 VAM Principles
There are six VAM principles:

Analytical measurements should be made

to satisfy an agreed requirement

Analytical measurements should be made

using methods and equipment which have been
tested to ensure they are fit for their purpose Analytical measurements made
in one location should be
consistent with those elsewhere

Staff making analytical measurements

should be both qualified and competent
to undertake the task Organisations making
analytical measurements
should have well defined
There should be a regular independent assessment quality control and quality
of the technical performance of the laboratory assurance procedures
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 Method development
and validation
‘Never attempt to re-invent the wheel!’
Before embarking on the development of a new method, always
research the chemical literature to see if a suitable one already
exists. If a suitable one is found, it will still be necessary however to
perform some method validation to prove that the method can be
successfully adapted to your laboratory, equipment and personnel.
More extensive validation is required for a brand new method.
Methods in any field of analysis may be defined in terms ‘Method
performance characteristics’ and it is these parameters plus a few
others, that are quantified during a method validation exercise.

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 Method validation
Validation, is the proof needed to ensure that an analytical
method can produce results which are reliable and reproducible
and which are fit for the purpose intended. The parameters that need to be
demonstrated are those associated with the ‘Performance characteristics’ together
with robustness, repeatability and reproducibility.
Many analytical methods appearing in the literature have not been
through a thorough validation exercise and thus should be treated with
caution until full validation has been carried out. Validation of a new
method (new to your laboratory), is a costly and time-consuming exercise,
however the result of not carrying out method validation could result in
litigation, failure to get product approval, costly repeat analysis and
loss of business and prestige.

You can now consider in more detail how validation is

carried out
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 Method performance
characteristics

A method’s performance is defined by a number of important

individual characteristics. There are:

Sensitivity Precision

Accuracy Limit of Detection (LoD)

Limit of Determination Bias

Selectivity Linearity

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 Accuracy and precision
The dictionary definition of both ‘accuracy’ and ‘precision’ are roughly
the same, indicating that these words may be used synonymously.
However in ‘Analytical Science’ they have two separate meanings, the
difference between them is best illustrated by using target diagrams

Poor precision Good precision Good mean accuracy Good accuracy

poor accuracy poor accuracy poor precision good precision
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 Accuracy and precision (2)

You saw from the previous slide, a set of results can be

either accurate and/or precise or can be neither accurate
nor precise. Thus accuracy may be defined as:
The closeness of the mean value from a replicate set of results to the
true or accepted value

Precision may be defined as:

The spread of results from a replicate set of measurements

The difference between the true value and the mean

measured value is termed bias. The spread of replicate data is
measured in terms of standard deviation (s) or variance (s2)
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 Random and systematic
errors
There are 2 types of error for which allowance may be made:
Random error Systematic error
Random error arises from variations Systematic error remains constant or
in parameters which are outside the may vary in a predictable way over a
control of the analyst, but which series of measurements and cannot be
influence the value of the reduced by making replicate
measurement being made. Because measurements. In theory, if known,
these errors are statistically random, this error can be allowed for.
the mean error should be zero if Eg: subtraction of blank values
sufficient measurements are made.

mean mean
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 Bias and variance
Cu by AAS

10.4
A solution containing copper was

Cu in ppm
10.2
analysed 10 times using atomic 10
9.8
absorption spectroscopy. 9.6
The results obtained in ppm were: 0 2 4 6 8 10 12
10.08, 9.80, 10.10, 10.21, 10.14, Replicate sample
9.88, 10.02, 10.12, 10.11, 10.09

We can now calculate the precision

Bias = Mean value - true value
of the data as standard deviation = 10.06 - 10.00
If the true value is known to be = 0.06 ppm
10.00 ppm, we can also calculate Standard deviation (SD) = 0.12(4)
the bias
Relative SD = 100 X SD/10.00 = 1.2%

Conclusion - the method gives both good accuracy (low bias) and
acceptable precision (RSD of 1.2%)
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 Sensitivity and selectivity
Assessment of method sensitivity

1200
Sensitivity is the change in measured 1000
signal for unit change in concentration 800

Signal
600
and can be obtained from the dy
400
calibration graph 200
0
dx
Sensitivity = dy/dx 0 10 20 30
Concentration

Selectivity is the ability of a method

to discriminate between the target
analyte and other constituents of the
sample. In many instances selectivity
is achieved by high performance
separation using chromatographic or
electrophoretic techniques.
07/20/2021 K.K. Nayak (B. Pharm.) Hplc chromatogram 22
Limits of detection (LoD)
and determination
Example: In an analysis of trace
Cd by plasma emission
These values refer to the statistical limits spectrometry the following data
below which results of detection or accurate were obtained:
quantitative measurements (determination) • mean blank (Bl) signal 4
should not be reported. The levels of both • SD of blank signal 12
• 500 ppb Cd 2000
are dependent upon the variability of the
signal when a blank containing none of the
LoD = Bl signal + 3(SD of Bl)
analyte is being measured. The signal = 4 + 3(12)
generated under these conditions is mostly = 40
signal noise and is assumed to exhibit a This equates to: [40/2000] X 500 ppb
normal distribution pattern. Both the blank = 10 ppb Cd
signal and the standard deviation of the The limit of determination uses a
blank signal need to be measured. From this similar formula, replacing the
data we can calculate both limits. 3 SD’s by 10. This gives the limit of
determination as 31 ppb Cd
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 Method validation -
linearity Calibration graph

0.6
0.5
Check linearity between 0.4

Signal
50 - 150% of the expected 0.3
0.2

analyte concentration 0.1

0
0 5 10 15 20 25
Concentration

Linearity
Most analytical methods are of a comparative type and thus require
calibration against accurately known standards to generate quantitative
data. Where possible calibration data should show a linear relationship
between analyte concentration and measured signal, however it is
acceptable under some circumstances, to use a non-linear relationship
up to the limit of the dynamic range.
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 Method validation -
specificity
Loss of specificity can be due to interferences and matrix
effects.
All likely interferences should be investigated and their effects on analyte
response determined over a range of concentrations. Measures can then be
put into place to mask, eliminate or separate them from the analyte.

Standard addition
procedures can be used
to identify matrix effects

no yes
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 Normal distribution curve

100
80

Frequency of
Method validation -

occurrence
60
40
20

precision 0
0 50 100 150
Data points
You have seen already that,
precision is measured in
terms of standard deviation (SD). Estimation of true mean
Assuming that the variability of
SD = [ Σ(xi - x)2/(n - 1)]1/2
the measurements is totally
random (obeys a normal where:
distribution curve) then xi = individual data point
a formula derived from this x = mean value of the data
n = the total number of data points
distribution may be used Σ = the sum of
to calculate standard deviation.
In practice around 8 - 10 data points are used normally to calculate the
SD, although statisticians would recommend 50
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 Method Validation -
repeatability & reproducibility
Methods need to be shown to be both repeatable and
reproducible. A replicate set of data produced at a particular
time point by an operator working with a particular set of
equipment in a given laboratory will verify repeatability. To show
reproducibility, the method must produce similar results when any
of these parameters are changed. The most likely changes are to
time and operator.
Two different operators
analysing milk
using different pieces of
equipment at different
times. The laboratory is
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the same.
K.K. Nayak (B. Pharm.) 27
 Method validation -
reliability
The reliability of a method can be Selection of reference
tested in a number of ways materials
from LGC

Test results from the new

method against an existing The best way of demonstrating
method which is known to accuracy is to analyse a reference
be accurate material or certified reference
material (CRM) if one is available

Add a known quantity of pure analyte

(spike) to a real sample or real sample
matrix and check that all of the added
substance can be measured
(recovered)
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 Method validation -
detection & quantitation limits
A method is not acceptable for accurate detection or
quantitation if the analyte level is likely be fall beneath
the limit(s) calculated based upon the blank signal and
its standard deviation (Refer to the formula given in slide 14,
in this part of the presentation). Analyte pre-concentration
then becomes necessary.
Mean sample signal
must be sufficiently
Variation in larger than the blank
sample signal
Variation in
so that positive
blank signal Mean sample detection or accurate
signal quantitation is
Mean blank
possible
signal
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 Method validation -
robustness
Robustness of an analytical method refers to it’s ability
to remain unaffected when subjected to small changes
in method parameters.
For example
In an hplc analysis the mobile phase is defined in terms of % organic
modifier, pH of the mobile phase, buffer composition, temperature
etc. A perfect mobile phase is one which allows small changes in the
composition without affecting the selectivity or the
quantitation of the method.

Alter all major parameters in order to ascertain when the method ceases
to function in accordance with specifications
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 Documentation of validated
methods
Once the validation process is complete it is important to
document the procedures so that the method can be
clearly and unambiguously implemented.

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Documentation of validated methods may
Include:
TITLE
SCOPE
WARNING & SAFETY PRECAUTIONS
DEFINITIONS
PRINCIPLE
REAGENTS & MATERIALS
APPARATUS & EQUIPMENT
SAMPLING & SAMPLES
CALIBRATION
QUALITY CONTROL
PROCEDURE
CALCULATION

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 Equipment validation:

• Demonstrate that equipment used in validation studies

is suitable for use and is comparable to equipment used
for routine analysis Calibrated (as applicable)

Qualifications should have been performed

Installation Qualification

Operation Qualification

Performance Qualification

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 Calibration of HPLC
Various Calibration parameters are:
Flow rate accuracy
 Injector accuracy
 System Precision
 Wavelength accuracy
 Detector linearity
 Injector linearity
 Gradient Performance Check
 Column oven temperature accuracy
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