Method Validation

By

Muhammad Imran Shaukat

April 2015

Muhammad Imran Shaukat

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Method Validation what is it

Specific intended use why are you analysing the samples and
what are the resulting data used for Objective evidence set of
results from validation studies Confirmation compare
information obtained with customers requirements

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Method Validation when is it require????

Non-standard methods
Laboratory-developed methods

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5.4

Test and calibration methods and validation

5.4.1 General
5.4.2 selection of method
5.4.3 Laboratory Developed Method
5.4.4 Non Standard Method
5.4.5 Validation of Method
5.4.5.1 Validation What is it
5.4.5.2 Validation of Non Standard Method
5.4.5.3 Range and Accuracy of the Values obtained
5.4.6 Estimation of Uncertainty
5.4.6.1 Apply Procedure for Estimation Uncertainty
5.4.6.2 Apply Rigorous Metrological and Statistical Calculation
5.4.6.3 All Uncertainty Components must be Taken into Methods of
accreditation

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What is a Method?
A method is a procedure for the analysis of a specific analyte

( for example determination of Mn in water)/Procedure for

calibration or measurement of a specific parameter

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Definition
Validation is the confirmation by examination
and
provision of objective evidence that the particular
requirements for a specific intended use are fulfilled
(ISO/IEC 17025 clause 5.4.5.1)
Verification confirmation, through the
provision of
objective evidence, that specified requirements have
been fulfilled (ISO 9000: 2005)
Note: intended use vs specified requirements
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5.4.1 GENERAL

Standard Method should be used and labs

should verify their own ability to achieve
satisfactory performance against documented
performance

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The laboratory shall do estimation of the measurement

uncertainty (where applicable )as well as statistical
techniques for analysis of test and/or calibration data.

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5.4.2 SELECTION OF

METHODS

Lab must be able to select / identify appropriate method

to be used. The other qualification of the lab such as
training records / test demonstration can also add to the
labs ability.

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The laboratory shall use test and/or calibration methods, including

methods for sampling, which meet the needs of the customer and
which are appropriate for the tests and/or calibrations it undertakes.
Methods published in international, regional or national standards
shall preferably be used

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 The laboratory shall ensure that it uses the latest valid edition of a

standard unless it is not appropriate or possible to do so. When

necessary, the standard shall be supplemented with additional details
to ensure consistent application.
When the customer does not specify the method to be used, the

laboratory shall select appropriate methods that have been published

either in international, regional or national standards, or by
reputable technical organizations, or in relevant scientific texts or
journals, or as specified by the manufacturer of the equipment

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5.4.3 LABORATORY DEVELOPED METHOD

In house development method must be validated and
authorized before use this simple statement has
significant impact on laboratory. To achieve this certified
reference material (CRM) should be used to determine
any systematic bias if not available /possible results
must be compared with the other technique preferably
based on different principle of analysis.

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5.4.4 NON STANDARD METHOD

The client must be told total agreement of use of non

standard methods.
The method must be appropriately validated, the method
such developed must include the following information.
Appropriate
Revision of document

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Use of Standard Method beyond applicable range

Validation is also required when Standard

Method is used outside their normal practice

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Non standard method

When it is necessary to use methods not covered by

standard methods, these shall be subject to
agreement with the customer and shall include a clear
specification of the customers requirements and the
purpose of the test and/or calibration. The method developed
shall have been validated appropriately before use.

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Non standard method

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procedures should contain at least the following information:

appropriate identification
scope;
description of the type of item to be tested or calibrated;
parameters or quantities and ranges to be determined;
apparatus and equipment, including technical performance
requirements;
reference standards and reference materials required

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5.4.5 VALIDATION OF METHOD

Validation is the confirmation by examination and

provision of objective evidence that the particular
requirement for a specific test / intended use are fulfilled

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VERIFICATION OF A METHOD

Verification is the confirmation by examination and

provision of objective evidence that the particular
requirements have been fulfilled

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Validation

Should be performed

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IF ?

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New Equipment
When a lab inducts new equipment validation
must be performed to prove that the lab is
capable of getting a valid result before
performing rest for the client

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Critical component of Equipment is replaced

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When Internal Control Shows that analytical

result is out of statistical control

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When participation in ILC shows that method

exceeds the statistical limits

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New test

Validation is required when a lab performs a test for the

first time

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PARAMETERS FOR METHOD VALIDATION

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How to determine performance of a method

The following techniques "as it is" and in combination are
used
Calibration using reference material
Comparison of results achieved with other methods
Inter-laboratory Comparison
Systematic assessment of the factors influencing the
results
Assessment of uncertainty based on scientific/statistical
principles
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PARAMETERS FOR METHOD VALIDATION

Accuracy
Sensitivity
The range
Linearity
recovery
PRECISION//repeatability and/or reproducibility
detection limit
Selectivity /Specificity of the method
Robustness against external influences and/or cross-sensitivity

against interference from the matrix of the sample/test object), as

assessed for the intended use, shall be relevant to the customers'
needs.

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ACCURACY

Accuracy
Accuracy is the closeness of the analytical value
to the true value (Eurachem )

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Accuracy
Accuracy is the measure of exactness of
an analytical method, or the closeness of
agreement between the measured value
and the value that is accepted as a
conventional true value or an accepted
reference value.

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Sensitivity
This is the change in the analytical response
divided by the corresponding change in the
concentration of a standard (calibration) curve,
i.e. the slope of the analytical calibration curve.
A method is said to be sensitive if a small
change in concentration of the analyte
causes a large change in the analytical
measurement.
Gradient of the calibration curve !!!
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Specificity/Selectivity

The Specificity of a method defines the ability of the

method to measure the analyte of interest to the
exclusion of other relevant components.

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Selectivity

Selectivity describes the ability of an analytical

method to differentiate various substances in a
sample.

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Specificity
Specify those substances which might be expected to

give rise to an interfering signal.

A check for random interferences should be performed

by analysis of a set of representative blank samples.

indicate the extent to which the method can distinguish

between the analyte of interest and interfering

substances

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PRECISION
The precision of procedure expresses the closeness of
agreement (degree of scatter) between a series of
measurements obtained from multiple sampling of the
homogenous sample under the prescribed conditions.
Precision may be considered
at three levels:
repeatability, intermediate precision and reproducibility.
Precision should be investigated using homogenous,
authentic (full scale) samples. However if it is not
possible to obtained a full scale sample it may be
investigated or bench-top scale sample solution.

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Following terms express precision:

a. Repeatability: Repeatability expresses the precision
under the same operating condition under a short interval
of time.
Repeatability is also termed intra- assay
precision.

b.

Reproducibility: Reproducibility expresses the

precision between laboratories (collaborative studies
usually applied to standardization of methodology)
c.
Intermediate Precision: Intermediate Precision
expresses within- laboratories variations: different days,
different analysts, different equipment, etc

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Nov 2014

2.8= t value x 2=2 x 1.4

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Limits of Detection
The Limit of Detection (LOD) of a method may
be defined as the concentration of analyte which
gives rise to a signal that is significantly
different from the control or blank.

The LOD is the lowest concentration of analyte

that can be distinguished from background
The results obtained at the Limit of Detection
are not necessarily Precise or Accurate

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Limit of detection (LoD) is based on the standard

deviation of the response and the slope. The detection
limit (LoD) may be expressed as:

DL = 3.3 /S
Where = the standard deviation of the
response (peak height, peak area etc.)
S = the slope of the calibration curve
For this purpose the blank + 3S
approach is
adequate
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Limits of Quantitation

The limits of quantitation (LOQ) are the

lowest and the highest concentrations of
analyte in a sample or specimen that can be
measured with an acceptable level of
accuracy and precision.

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Limit of quantification (LoQ) based on the standard deviation of

the response and the slope. The quantitation limit (QL) may be
expressed as:
QL = 10 /S
Where = the standard deviation of the response (peak height,
peak area etc.)
S = the slope of the calibration curve
The slope S may be estimated from the calibration curve of the
analyte.
For this purpose the blank + 10 approach is applicable
= standard deviation of sample blank or fortified sample blank
value.

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LOD/LOQ/LINEARITY

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ANOVA

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40
35

VALIDATION ANOVA/F TEST

C2

30
25

10

15 18 20

20

12 15 2
0
22 27 3
0

2
2
3
2

2
8
3
4

15
10
0

15
20
25
30
C1
Pearson correlation of C1 and C2 = 0.977.GOOD
ORRELATION
ANNOVA F=5.86 against 3.44 from F table

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Linearity and Range

The linearity of a method is its ability to elicit results that are

directly, or by a well defined mathematical transformation,

proportional to the concentration of analyte in the sample.

The range of the method is the area between the lower and the

upper limits of quantitation that is also linear.

Within the range of the method, results are accurate, precise

and linear

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Recovery expressed as a percentage :

R = {(CF-CU)/CA} x 100

CF = concentration of analyte measured in the fortified sample

CU = concentration of analyte measured in the un-fortified
sample

CA =

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concentration of analyte added in the fortified sample

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Recovery

The failure to determine all of the analyte

present in a sample may reflect an inherent
problem in the method. This could be due to
either problems in detecting the analyte or
problems with extraction efficiency. The fraction
or percentage of an analyte added to a test
sample that can be recovered in the analysis
is called recovery (Eurachem, 1998).
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ROBUSTNESS
The robustness of an analytical procedure is a
measure of its capacity to remain unaffected by
small, but deliberate, variation in method parameters
and provides an indication of its reliability during
normal usage.

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Ruggedness
Ruggedness is the reproducibility of the

test results obtained for identical

samples under normal (but variable) test
conditions./The variance in the analysis of
homogenous samples between analysts and
Laboratories.

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Correlation Coefficient,r:
The quantityr, called thelinear correlation
coefficient, measures the strength and
the direction of a linear relationship between two
variables.The linear correlation coefficient is
sometimes referred to as thePearson product
moment correlation coefficientin
honor of its developer Karl Pearson.
The mathematicalformulafor computingris:

wherenis the number of pairs of data.

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The quantityr, called thelinear correlation

coefficient, measures the strength and the

direction of a linear relationship between two
variables.

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The mathematicalformulafor
computingris:

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Correlation !!!
Positive correlation: Ifxandyhave a strong positive linear

correlation,ris close to +1. Anrvalue of exactly +1 indicates

a perfect positive fit.
Positive values

indicate a relationship betweenxandyvariables such that as values

forxincreases, values foryalso increase.
Negative correlation: Ifxandyhave a strong negative linear
correlation,ris close to -1. Anrvalue of exactly -1 indicates
a perfect negative fit.Negative values indicate a relationship
betweenxandysuch that as values forxincrease, values
forydecrease.
No correlation:If there is no linear correlation or a weak linear
correlation,ris close to 0.A value near zero means that there is
a random, nonlinear relationship between the two variables

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Coefficient of Determination,r2 orR2:

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Thecoefficient of determination,r2,is useful because it

gives the proportion of the variance (fluctuation) of one variable
that is predictable from the other variable.
It is a measure that allows us to determine how certain one can
be in making predictions from a certain model/graph.
Thecoefficient of determinationis the ratio of the
explained variation to the total variation .
Thecoefficient of determinationis such that 0<r2<1,
and denotes the strength of the linear association
betweenxandy.

Thecoefficient of determinationrepresents the percent

of the data that is the closestto the line of best fit.
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example,
ifr= 0.922, thenr2= 0.850, which means that
85% of the total variation inycan be explained by
the linear relationship betweenx andy(as
described by the regression equation). The other
15% of the total variation inyremains unexplained.

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SLOPE of the line???

think the most useful form of straight-line equations is the "slope-

intercept" form:
y = mx + b
This is called the slope-intercept form because "m" is theslopeand "b"
gives they-intercept
SLOPE DETERMINES ????

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