1

SELECTION,
VERIFICATION AND
VALIDATION OF METHOD
08 JUL 2016

masih
 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
5 Type of Analytical Application
Performance Quantitative Limit test Quantification
Characteristic Identification
test for for of main
Test
impurity impurity component
Selectivity x x x x
Limit of Detection x
Limit of Quantitation x
Working range x x
Trueness (bias) x x
Precision (repeatability x x
and intermediate
precision)
x signifies normally validated performance characteristics for four types of
analytical applications

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A Laboratory Guide
to Method Validation and Related Topics, 2nd ed. 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
8 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
Analyze test sample
containing various
suspected
interferences in the
presence of the
analytes of interest
What to calculate
How many times Comment
from the data
1 Examine If detection or
interference effect. quantification is
inhibited by the
interferences, further
method development
will be required.

EURACHEM Guide - Selectivity

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A Laboratory Guide
to Method Validation and Related Topics, 2nd ed. 2014
 Concepts on Low Concentration
13
Measurements
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

Results blank
corrected YES
Limit of Detection

during
routine?

NO

s0 1 1
s0 ' s 0 ' s0
n n nb

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

1 1
Replicate measurements Calculate LOD s0 ' s0
of reagent blanks spiked n nb
with low concentrations LOD 3 s0 '
of analyte S0 standard deviation of result
at or near zero concentration

This approach is S0 standard deviation for

acceptable when blank calculating LOD and LOQ
samples or test samples n number of replicate
at low concentrations observations averaged when
cannot be obtained reporting results
nb number of blank observations
averaged when calculating blank
correction
EURACHEM Guide.The Fitness for Purpose of Analytical Methods: A Laboratory Guide
to Method Validation and Related Topics, 2nd ed. 2014 17
 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 many What to calculate from the
What to do Comment
times data
19Replicate measurements 10 Calculate standard for non-blank corrected results
of blank samples deviation of the results, s0. s
s0 ' 0

or n
Calculate s0. For blank-corrected results
Replicate measurements
1 1
of test samples with low Calculate LOD s0 ' s0
analyte concentration n nb
LOD 3 s0 '
or S0 standard deviation of result
at or near zero concentration
Replicate measurement
S0 standard deviation for
of reagent blanks
calculating LOD and LOQ
Or n number of replicate
observations averaged when
Replicate measurements reporting results
of reagent blanks spiked nb number of blank
with low analyte observations averaged when
concentration calculating blank correction
EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A Laboratory Guide
to Method Validation and Related Topics, 2nd ed. 2014
 Limit of Detection
20

LOD based on standard deviation of QC 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, 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 many What to calculate from the
What to do Comment
times data
22Replicate measurements 10 Calculate standard for non-blank corrected results
of blank samples deviation of the results, s0. s
s0 ' 0

or n
Calculate s0. For blank-corrected results
Replicate measurements
1 1
of test samples with low Calculate LOQ s0 ' s0
analyte concentration n nb
IUPAC default kQ value is
usually 10
or S0 standard deviation of result
LOQ kQ s0 ' at or near zero concentration
Replicate measurements
LOQ 10 s0 ' S0 standard deviation for
of reagent blanks
calculating LOD and LOQ
or n number of replicate
observations averaged when
Replicate measurements reporting results
of reagent blanks spiked nb number of blank
with low concentrations observations averaged when
of analyte calculating blank correction
EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A Laboratory Guide
to Method Validation and Related Topics, 2nd ed. 2014
 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 many
What to do What to calculate from the data Comment
times
1.Measure blank and 1 Plot Visual confirmation if
27calibration standards at 6- Response y-axis working range is linear
10 concentrations (evenly Concentration x-axis or not
spaced across the range of Visually identify approximate
interest) linear range and upper and lower
working range boundaries. Proceed
to step 2.
2.Measure blank plus 6-10 1 Plot If the standard deviation
calibration standards, 2 to Response y-axis is proportional to
3 times. (calibration Concentration x-axis concentration, consider
standard concentrations Calculate appropriate regression using weighted
evenly spaced across statistics. Calculate and plot regression rather than
linear range. residuals (difference between simple non-weighted
observed y-value and calculated y- linear regression.
value predicted by the straight line, In certain circumstances, it
for each x value). Linearity is may be better to try to
confirmed when there is random fit a non-linear curve to
distribution of residuals. Systematic the data. Functions higher
trends indicate non-linearity or than quadratic are
change in variance with level generally not advised.

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

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A Laboratory Guide
to Method Validation and Related Topics, 2nd ed. 2014
 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 do What to calculate from the data Comment
times
31Measure RM using 10 Calculate bias taking into
candidate method bias, account effect of
method and
b x xref laboratory bias
%relative bias,
x xref
b (%) 100
xref
% relative recovery (apparent
recovery)
x
R (%) 100
xref
Measure matrix 10 Calculate assess net recovery of
blanks/test samples relative spike recovery, added spike
unspiked and spiked x 'spiked x
with the analyte of R ' (%) 100
interest over a range xspike
of concentrations

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 do What to calculate from the data Comment
times
32Measure RM/test 10 Calculate alternative method
sample using bias, may be a reference
candidate method method
and alternative
b x xref
method %relative bias,
x xref
b (%) 100
xref
% relative recovery (apparent
recovery)
x
R (%) 100
xref

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A Laboratory Guide
to Method Validation and Related Topics, 2nd ed. 2014
 Estimated Recovery
33
Analyte Analyte Mean
Unit
Concentration, % Ratio % Recovery
100 1 100% 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

 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 from the
What to do Comment
times data
a. same analyst 6 to 15 Calculate standard Estimates Repeatability standard
37and equipment, replicates for deviation of results for each deviation sr for each material
same laboratory, each material material
short timescale
b. different 6 to 15 Calculate standard Estimates Intermediate Precision
analysts and replicates for deviation of results for each standard deviation sI for each
equipment, same each material material material
laboratory,
extended
timescale
c. different 6 to 15 groups Calculate repeatability Estimates Repeatability standard
analysts and of duplicate standard deviation from deviation sr for each material
equipment, same measurements ANOVA results for each
laboratory, obtained under material. Estimates Intermediate Precision
extended repeatability standard deviation sI for each
timescale conditions on Calculate between-group material
different days/ standard deviation from
equipment for ANOVA and combine with
each material repeatability standard
deviation for each material

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 from the
What to do Comment
times data
d. different 6 to 15 groups Calculate repeatability Estimates Repeatability standard
38analysts and of duplicate standard deviation from deviation sr for each material
equipment, measurements ANOVA results for each
different obtained under material. Estimates Reproducibility standard
laboratory, repeatability deviation sR for each material. This
extended conditions in Calculate between- requires a special inter-laboratory
timescale different laboratory standard comparison (collaborative trial)
laboratories for deviation from ANOVA
each material results and combine with
repeatability standard
deviation for each material

EURACHEM Guide. The Fitness for Purpose of Analytical Methods: A Laboratory Guide
to Method Validation and Related Topics, 2nd ed. 2014
 Estimated Precision
39
Analyte
Unit %RSD
Concentration, %
100 100% 1.3
10 10% 2.3
1 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
What to do How many times What to calculate from the data Comment
Identify variables which Most effectively Determine the effect of each Design quality
41could have a significant evaluated using change of condition on the control or modify
effect on method experimental measurement results. the method in order
performance. designs. to control the
e.g. 7 parameters Rank the variables in order of critical variables,
Set up experiments can be studied in the greatest effect on method e.g. by stating
(analysing RMs or test 8 experiments performance. suitable tolerance
samples) to monitor the using a limits in the
effect on measurement Plackett-Burman Carry out significance tests to standard operating
results of systematically experimental determine whether observed procedure
changing the variables. design. 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
 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 Beer-Lamberts 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. Results were
g/mL 50mL
calculated as : Fe, g/g AAS Reading,
Weight Sample, g
Calculate for LOD and LOQ using the data
obtained from the validation study.
47

Trial Wt (g) Method Blank Sample Blank

Reading (g/mL) Reading (g/mL)
1 1.0003 0.0691 0.2070
2 1.0003 0.0604 0.2017
3 1.0003 0.0702 0.2051
4 1.0001 0.0756 0.1998
5 1.0003 0.0740 0.2097
6 1.0006 0.0631 0.2615
7 1.0000 0.0633 0.1908
8 1.0000 0.0482 0.2042
9 1.0004 0.0561 0.1907
10 1.0004 0.0509 0.2179