1

SELECTION,
VERIFICATION AND
VALIDATION OF METHOD
08 JUL 2016
IRRI Grain Quality & Nutrition Services Laboratory

Selection
2

ISO/IEC 17025:2005
Clause 5.4.2
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

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Verification
3

The provision of objective evidence that a

given item fulfills specified requirements.
[VIM 2.44]

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Validation
4

Verification, where the specified

requirements are adequate for intended
use. [VIM 7]

Confirmation by examination and

provision of objective evidence that the
particular requirements for a specific
intended use are fulfilled. (ISO/IEC 17025)

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Selection
Type of Analytical Application

Performance
Characteristic
Selectivity

Identificati
on Test

Quantitati
ve test for
impurity

Limit
test for
impurity

Quantificati
on of main
component

Limit of Detection

Limit of
Quantitation

Working range

Trueness (bias)

Precision
(repeatability and
intermediate
precision)

x signifies normally validated performance characteristics for four

types ofGuide. The Fitness for Purpose of Analytical Methods: A
EURACHEM
Laboratory
Guideapplications
to Method Validation and Related Topics, 2nd ed.
analytical
2014

Definition
6

Reagent Blank - reagents used during the

analytical process (including solvents used for
extraction or digestion) are analyzed in order
to determine if they contribute to
measurement signal. The signal arising from
the analyte can then be corrected accordingly.

Method Blank reagent blanks that are

taken through the whole measurement
procedure or method process

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Definition
7

Sample Blank these are essentially

sample matrices with no analyte
present. These are difficult to obtain but
such materials are necessary to give a
realistic estimate of interferences that
would be encountered in the analysis of
test samples.

EURACHEM Guide.The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Performance
Characteristics

Method Performance Characteristics

9

Selectivity
Limit of Detection (LOD) and Limit of
Quantitation (LOQ)
Working Range
Trueness (bias, recovery)
Precision (repeatability, intermediate
precision, reproducibility)
Ruggedness (robustness)

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Method Performance Characteristics

10

***Measurement Uncertainty is not a

performance characteristic but a property
of the results obtained using that method.

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Selectivity
11

The ability of a method to determine

accurately and specifically the analyte of
interest in the presence of other
components in a sample matrix under the
stated conditions of the test. [EURACHEM
Guide]

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Selectivity
12

What to do

How many times

Analyze test sample

containing various
suspected
interferences in the
presence of the
analytes of interest

What to calculate
from the data
Examine
interference effect.

EURACHEM Guide - Selectivity

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Comment
If detection or
quantification is
inhibited by the
interferences, further
method development
will be required.

Concepts on Low Concentration

Measurements
13

1. Establish a value that an analyte level is

significantly different from zero (critical value
or decision limit)
2. Know the lowest concentration of the analyte
that can be detected by the method at a
specified level of confidence (limit of detection,
minimum detectable value or detection limit)
3. Establish the lowest level at which the
performance is acceptable for a typical
application (limit of quantitation)
EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A
Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Limit of Detection
14

The lowest amount of analyte in a sample

which can be detected but not necessarily
quantitated as an exact value. [ICH Q2A
and ICH Q2B, ASEAN Guide]

The lowest content that can be measured

with reasonable statistical certainty.
[AOAC-PVMC]

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

S0
(standard deviation
from results of
measurement)
15

Lim
it of
Det
ecti
on

Results
blank
corrected
during
routine?

YES

NO

s0 '

s0
n

Use the calculated s0 for

computing the value of
LOD and LOQ
EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A
Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

s 0 ' s0

1 1

n nb

Limit of Detection
16

Instrument Detection Limit

is based on sample analysis, often a reagent
blank presented directly to the instrument
(omitting sample preparation steps). When
the reagent blanks do not go through the
whole measurement procedure, the
calculated data will give the Instrument
LOD.

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

EURACHEM Guide LOD (Instrument Detection Limit)

How
What to calculate
What to do
many
Comment
from the data
times
Replicate
10
Calculate standard
for non-blank corrected
measurements of
deviation of the
results
s
reagent blanks
results, s0.
s ' 0
0

or

Calculate s0.

Replicate
measurements of
reagent blanks
spiked with low
concentrations of
analyte

Calculate LOD

LOD 3 s0 '

This approach is
acceptable when
blank samples or
test samples at low
concentrations
cannot be obtained
EURACHEM Guide.The Fitness for Purpose of Analytical Methods: A
Laboratory Guide
to Method Validation and Related Topics, 2nd ed. 2014

For blank-corrected results

s0 ' s0

1
1

n
nb

S0 standard deviation of
result at or near zero
concentration
S0 standard deviation for
calculating LOD and LOQ
n number of replicate
observations averaged
when reporting results
nb number of blank
observations averaged
when calculating
blank
17
correction

Limit of Detection
18

Method Detection Limit

LOD is based on the analysis of samples that
have been taken through the whole
measurement procedure using results calculated
with the same equation as the test sample.
Samples that are suitable for estimation of LOD
or LOQ are: blank samples (matrices with no
detectable analyte), test samples with
concentrations of analyte close to or below the
expected LOD.
EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A
Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

EURACHEM Guide LOD (Method Detection Limit)

How
What to calculate
What to do
many
Comment
from the data
times
Replicate
10
Calculate standard
for non-blank corrected
19
measurements of
deviation of the
results
s
blank samples
results, s0.
s0 ' 0
or
Replicate
measurements of
test samples with
low analyte
concentration

Calculate s0.
Calculate LOD

LOD 3 s0 '

or
Replicate
measurement of
reagent blanks

For blank-corrected results

s0 ' s0

1
1

n
nb

S0 standard deviation of
result at or near zero
concentration
S0 standard deviation for
calculating LOD and LOQ

Or

n number of replicate
observations averaged
when reporting results

Replicate
measurements
EURACHEM Guide.of
The Fitness for Purpose of Analytical Methods: A
reagent
blanks
Laboratory
Guide to Method Validation and Related Topics, 2nd ed.
2014 with low
spiked

nb number of blank
observations averaged
when calculating blank
correction

Limit of Detection
20

LOD based on standard deviation of QC

results at low concentration levels under
intermediate precision conditions.
1.
2.

The standard deviation s0 is equal to s0

Since blank correction is no longer
required, LOD can be computed directly
by using s0
multiplied by 3.

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Limit of Quantitation
21

The lowest level of analyte that can be

determined with acceptable performance.

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

EURACHEM Guide LOQ

How
What to do
many
times
Replicate
10
22
measurements of
blank samples
or
Replicate
measurements of
test samples with
low analyte
concentration
or

What to calculate
from the data

Comment

Calculate standard
deviation of the
results, s0.

for non-blank corrected

results

Calculate s0.

For blank-corrected results

Calculate LOQ
IUPAC default kQ
value is usually 10

LOQ kQ s0 '
LOQ 10 s0 '

Replicate
measurements of
reagent blanks

s0 '

s0

s0 ' s0

n
1
1

n
nb

S0 standard deviation of
result at or near zero
concentration
S0 standard deviation for
calculating LOD and LOQ

or

n number of replicate
observations averaged
when reporting results

Replicate
measurements
EURACHEM Guide.of
The Fitness for Purpose of Analytical Methods: A
reagent
blanks
Laboratory
Guide to Method Validation and Related Topics, 2nd ed.
2014 with low
spiked

nb number of blank
observations averaged
when calculating blank
correction

Limit of Quantitation
23

LOQ based on the standard deviation of

quality control results at low concentration
levels under intermediate precision
conditions.
1. The standard deviation s0 is equal to s0
2. Since blank correction is no longer
required, LOQ can be computed directly
by using s0 multiplied by 10.
EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A
Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Working Range
24

The interval over which the method

provides results with an acceptable
uncertainty.
The lower end is bounded by the limit of
quantification and the upper end of the
working range is defined by
concentrations at which significant
anomalies in analytical sensitivity are
observed.

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Working Range
25

Instrument Working Range

In method validation, the instrument
working range is assessed to: confirm the
relationship (e.g. linear, curvilinear, etc) of
the instrument response between the LOQ
and upper end of the instrument working
range;

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Working Range
26

Method Working Range

Assessing method working range requires
use of sample blanks and samples with
known concentrations. These samples shall
undergo the entire measurement procedure
(method) and preferably cover the whole
working range. Results are calculated based
on the method.

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

EURACHEM Guide Working and linear range

How
What to calculate from
What to do
many
the data
times
1.Measure blank and
1
Plot
27
calibration standards
Response y-axis
at 6-10
Concentration x-axis
concentrations
Visually identify
(evenly spaced
approximate linear range
across the range of
and upper and lower
interest)
working range boundaries.
Proceed to step 2.
2.Measure blank plus
1
Plot
6-10 calibration
Response y-axis
standards, 2 to 3
Concentration x-axis
times. (calibration
Calculate appropriate
standard
regression statistics.
concentrations
Calculate and plot residuals
evenly spaced across
(difference between
linear range.
observed y-value and
calculated y-value predicted
by the straight line, for each
x value). Linearity is
confirmed when there is
random distribution of
trends
EURACHEM Guide. The Fitness for Purposeresiduals.
of Analytical Systematic
Methods: A
indicate
non-linearity
or
Laboratory Guide to Method Validation and
Related Topics,
2nd ed.
2014
change in variance with

Comment
Visual confirmation
if working range is
linear or not

If the standard
deviation is
proportional to
concentration,
consider using
weighted regression
rather than simple
non-weighted linear
regression.
In certain
circumstances, it
may be better to try
to fit a non-linear
curve to the data.
Functions higher

EURACHEM Guide Working and linear range

How
What to calculate from the
What to do
many
data
times
3.Calibrate
1
Plot measured concentration y28
according to the
axis against test sample
proposed
concentration x-axis.
calibration
Visually identify approximate
procedure.
linear range and upper and lower
Measure according
working range boundaries.
to the method,
Calculate appropriate regression
blank plus RMs or
statistics. Calculate and plot
spiked sample
residuals (difference between
blanks 2 to 3 times
observed y-value and calculated
at 6-10 different
y-value predicted by the straight
concentrations
line, for each x value). Linearity is
evenly spaced
confirmed when there is random
across the range of
distribution of residuals.
interest.
Systematic trends indicate nonlinearity

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Comment
This is to assess
whether
instrument range
and calibration
procedure are fit
for purpose.

Accuracy
29

The closeness of agreement between a

measured quantity value and a true
quantity value of a measurand [VIM 2.13]

Therefore, it is normally studied as two

components, trueness and precision. In
addition, an increasingly common
expression of accuracy is measurement
uncertainty.

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Trueness (bias)
30

The closeness of agreement between the

average of an infinite number of replicate
measured quantity values and a reference
quantity value. [VIM 2.14]

Trueness is inversely related to systematic

measurement error which may be
estimated as bias.

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

EURACHEM Guide Trueness

How many What to calculate from the
What to do
times
data
Measure RM
10
Calculate
31
using candidate
bias,
method

b x xref

Comment
bias taking into
account effect of
method and
laboratory bias

%relative bias,

b (%)

x xref

100

xref
% relative recovery
(apparent
recovery)
R (%)
Measure matrix
blanks/test
samples
unspiked and
spiked with the
analyte of
interest over a
range of
concentrations

10

x
xref

100

Calculate
relative spike recovery,

R ' (%)

x 'spiked x
xspike

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Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

100

assess net
recovery of
added spike

EURACHEM Guide Trueness

How many
What to do
times
10
32 Measure RM/test
sample using
candidate
method and
alternative
method

What to calculate from the

data
Calculate
bias,

b x xref
%relative bias,

b (%)

x xref
xref

100

% relative recovery (apparent

recovery)

R (%)

x
xref

100

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Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Comment
alternative
method may be a
reference method

Estimated Recovery
33

Analyte
Concentration,
%

Analyte
Ratio

100

100%

98 102

> 10

10-1

10%

98 102

>1

10-2

1%

97 103

> 0.1

10-3

0.10%

95 105

0.01

10-4

100 ppm

90 107

0.001

10-5

10 ppm

80 110

0.0001

10-6

1 ppm

80 110

0.00001

10-7

100 ppb

80 110

0.000001

10-8

10 ppb

60 115

0.0000001

10-9

1 ppb

40 120

AOAC Manual Peer Verified Methods

Unit

Mean
% Recovery

Precision
34

The closeness of agreement between

indications or measured quantity values
obtained by replicate measurements on
the same or similar objects under specified
conditions. [VIM 2.15]

Measurement precision is related to

random measurement error [VIM 2.19] and
is a measure of how close results are to
one another.

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Precision
35

Repeatability
is a measure of the variability in results when a
measurement is performed by a single analyst using
the same equipment over a short timescale.
Intermediate Precision
gives an estimate of the variation in results when
measurements are made in a single laboratory but
under conditions that are more variable than
repeatability conditions (different analysts,
extended timescale, different pieces of
equipment, etc).
EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A
Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Precision
36

Reproducibility
is a measure of the variability in results
between laboratories using the same
method or using different methods but
intending to measure the same quantity.

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

EURACHEM Guide Precision

How many
What to calculate
What to do
times
from the data
a. same
6 to 15
Calculate standard
analyst and
replicates
deviation of results for
37
equipment,
for each
each material
same
material
laboratory,
short
timescale
b. different
6 to 15
Calculate standard
analysts and
replicates
deviation of results for
equipment,
for each
each material
same
material
laboratory,
extended
timescale
c. different
6 to 15
Calculate repeatability
analysts and
groups of
standard deviation
equipment,
duplicate
from ANOVA results
same
measureme for each material.
laboratory,
nts obtained
extended
under
Calculate betweentimescale
repeatability group standard
conditions
deviation from ANOVA
and ofcombine
EURACHEM Guide. on
The different
Fitness for Purpose
Analytical with
Methods: A
days/
standard
Laboratory Guide to
Method Validationrepeatability
and Related Topics,
2nd ed.
2014
equipment
deviation for each

Comment
Estimates Repeatability
standard deviation sr for
each material

Estimates Intermediate
Precision standard
deviation sI for each
material

Estimates Repeatability
standard deviation sr for
each material
Estimates Intermediate
Precision standard
deviation sI for each
material

EURACHEM Guide Precision

How many
What to calculate
What to do
times
from the data
d. different
6 to 15
Calculate repeatability
analysts and
groups of
standard deviation
38
equipment,
duplicate
from ANOVA results
different
measureme for each material.
laboratory,
nts obtained
extended
under
Calculate betweentimescale
repeatability laboratory standard
conditions in deviation from ANOVA
different
results and combine
laboratories with repeatability
for each
standard deviation for
material
each material

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Comment
Estimates Repeatability
standard deviation sr for
each material
Estimates Reproducibility
standard deviation sR for
each material. This
requires a special interlaboratory comparison
(collaborative trial)

Estimated Precision
39

Analyte
Concentration,
%

Unit

%RSD

100

100%

1.3

10

10%

2.3

1%

2.7

0.1

0.10%

3.7

0.01

100 ppm

5.3

0.001

10 ppm

7.3

0.0001

1 ppm

11

0.00001

100 ppb

15

0.000001

10 ppb

21

0.0000001

1 ppb

30

AOAC Manual Peer Verified Methods

Ruggedness
40

The ruggedness of an analytical procedure

is a measure of its capacity to remain
unaffected by small, but deliberate
variations in method parameters and
provides an indication of its reliability
during normal usage. [ICH Q2A and ICH
Q2B, ASEAN Guide]

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

EURACHEM Guide Ruggedness

How many
What to do
times
Identify variables
Most
which
effectively
41
could have a
evaluated
significant
using
effect on method
experimental
performance.
designs.
e.g. 7
Set up experiments parameters
(analysing RMs or
can be studied
test
in 8
samples) to monitor experiments
the
using a
effect on
Plackettmeasurement
Burman
results of
experimental
systematically
design.
changing the
variables.

What to calculate from

the data
Determine the effect of
each
change of condition on
the
measurement results.
Rank the variables in
order of
the greatest effect on
method
performance.
Carry out significance
tests to
determine whether
observed
effects are statistically
significant.

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Comment
Design quality
control or modify
the method in
order
to control the
critical variables,
e.g. by stating
suitable tolerance
limits in the
standard
operating
procedure

Analytical Sensitivity
42

Analytical sensitivity is the change in

instrument response which corresponds to
a change in the measured quantity

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

Analytical Sensitivity
43

Useful Applications:
1. Theoretical analytical sensitivity is
sometimes known. (e.g. ion-selective
electrode show a Nernstian behavior.)
2. In spectrophotometric analysis,
absorbance can be predicted from BeerLamberts law.

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

*Measurement Uncertainty
44

Interval associated with the measurement

result which expresses the range of values
that can reasonably be attributed to the
quantity being measured.
It is not a performance characteristic but a
property of the results obtained using the
method.

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, 2nd ed.
2014

45

Exercise

Exercise
46

Method Validation for Determination of Fe in food

sample was done using a AAS. The expected Fe
content of the sample is around 100g/g.
a. What are the performance characteristics that
should be included in the study?
b. The method for routine requires duplicate
analysis for the blank and test sample.
AAS Reading,
Results were calculated
as : g/mL 50mL
Fe, g/g
Weight Sample, g
Calculate for LOD and LOQ using the data
obtained from the validation study.

47