Method Validation in a Single Laboratory

Method validation in A Single
Laboratory
Dr. Maha El-Bidaoui

Analytical measurements ?
 ..Made to satisfy an agreed requirement = a clear

defined objective
 ..Made using methods & equipment tested earlier to
ensure fitness for purpose
 .. Made in one location must be consistent with tests
made elsewhere
 ..Should be made in the frame of well defined QA/QC
procedures
 Staff: should be qualified & competent to undertake the

task (demonstrate a satisfactory performance).
 There should be a regular independent assessment of
the technical performance of a laboratory
Method Val_M. El-Bidaoui 2

Analytical methods & Validation
 ISO 17025 requirements for carrying out t/c, including
sampling using standard methods, non-standard methods, and
laboratory-developed methods.
 The laboratory shall ensure that it uses the latest valid edition
of a standard, unless it is not appropriate or possible to do so.
 Validation includes: Sampling, Transportation & Handling.
 It should also measure the influence of instrumental, human &

environmental factors on the uncertainty of the results.
 ISO definition: Validation

5.4.5.1. “Confirmation by examination and the provision of
objective evidence that the particular requirements for a
specific intended use are fulfilled.”
 La Validation d’une méthode est la
procédure par laquelle on démontre,
preuves expérimentales à l’appui, que les
performances de cette méthode
permettent de répondre aux exigences de
l’usage auquel elle est destinée.

Standard & official methods
Source: standard-setting organizations e.g. ISO, AOAC,

ASTM, etc.
 Standard Methods thoroughly investigated & fit-for-the-
purpose. Are less prone to criticism; contributes to an
increase in the laboratory's reputation & respect.
 Standard methods accepted by regulatory bodies such as
EPA, FDA, governments, become ‘Official’ methods.
They are thoroughly validated before their acceptance,
widely recognised & their levels of accuracy, precision,
& interferences are well known.
 Drawback: absence of flexibility in operations. Their
development towards full acceptance takes so much time
that latest developments are not incorporated.
Standard & official methods
 New analytical methods in international journals are well

described & thoroughly peer reviewed.
 E.g. Analytical Chemistry, Analytical Chimica Acta, Fresenius
J. of Anal. Chemistry, J.AOAC; etc.
 ‘Copying' a method from a journal needs always

adaptation as seldom the application is identical to the
conditions published (different substrate, physical or
chemical form of the analyte, instruments).

Submitting Laboratory
In-house Method Validation Study Protocol Development
Method Track Review AOAC I.

Staff
AOAC method validation
Proprietary Methods Non-proprietary Methods

Application Fees, Legal Agreements
Protocol, Package Insert, & QA review
Performance Tested Official Methods of Peer-Verified Methods

Methods Evaluation Analysis Evaluation Evaluation
In-house Method Validation Data Review In-house Method Validation &Peer Laboratory
AOAC RI Staff, General Referee and 2 Expert data Review
Reviewers (General Referee, Technical Referee, and 2-3
Technical Reviewers
Independent Laboratory In-House Method Validation Data

Review
Evaluation
Data Review to Verify Claims, Insert Collaborative Study Protocol Review (General
Accuracy, QA Certification Referee, Safety & Statistical Advisors, &
(AOAC RI Staff, General Referee and 2 Expert Method Committee)
Reviewers
Performance tested Method Collaborative Study Peer Verified Method

Certification
Collaborative Study Data review (General Referee, Safety &

Statistical Advisors, & Method Committee and Official Methods
Board)
First Action Official Method of Analysis
Membership Use, Feedback & Vote

Final Action Official Method of Analysis
Analytical methods
Methods are often described as:

 „quantitative‟,
 „semi-quantitative‟ or
 „qualitative‟
depending on the accuracy & the precision
achievable.

Stages of the application, validation &
use of analytical methods
Related
Stages Generally used terms
expressions
1 Development or adaptation of analytical procedure Pre-validation
Establishment of acceptable performance (validation) in

2 Validation proper
the authors laboratory
Demonstration of acceptable performance in 2nd or 3rd

3a Validation study
laboratory
Demonstration of acceptable performance in full inter-

3b Full study
laboratory collaborative study
4 Regular use of the method – performance verification

Validation of
Sampling
(a) For the representativiness of the sample,
(b) To minimize questions on sample authenticity.
These aspects should be included in SOPs for the receipt & acceptance of
samples.
Use of incurred analyte samples.
Determination in replicates.
Variance associated with sub-sampling is to be combined with Variance of

the Recovery.
For testing if the comminuted lab sample is statistically well mixed,

Wallace/Kratochvil’s procedure is recommended.
The uncertainty (U) of the method should be the combined U of sample prep.
& analysis.
Validation of
Sampling & Measurement
Method Validation-parameters :
- Accuracy (trueness, bias)
- precision
- reproducibility, - repeatability
- specificity
- selectivity
- limit of detection, - limit of determination
- linearity
- range
- sensitivity
- Ruggedness & Robustness

But before..!!

Data Variation
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- -
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inaccurate, imprecise accurate, imprecise
+ +
+ +
- -
- -
inaccurate, precise accurate, precise
Data Variation
+ +
+ +
- -
- -
+ +
+ +
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- -
Data Variation
+ +
+ +
- -
- -
+ +
+ +
- -
- -
Data Variation
+ +
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- -
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+ +
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Method Validation-parameters
Sample validation:
For the representativiness of the sample
- Accuracy (trueness, bias)

Measure of exactness of an analytical method,
Closeness of agreement between the accepted, the conventional value, or
the true value or an accepted reference value & the value found.
Measured or reported as the % of analyte recovered by spiking samples in a

blind study.
For the quantitation of impurities, accuracy is determined by analysing
samples spiked with known amounts of impurities.
A minimum number of determinations (from 5 onwards) should cover a

minimum of 3 concentration levels considering the specified range.

Trueness or Bias (systematic error)
 Use of (certified) reference materials when

available;
 Use of fortified or spiked samples provided
blind to the analyst;
 Comparison of results with those obtained with
a recognised reference method, if one is
available;
 Documented through the use of control charts.

Bias - Trueness
 Usually, one corrects for bias
x  CRM
Bias (%) 
CRM
Bias
Reference Average
-
Value Test result

- Precision
Measure of the degree of repeatability of a test method under similar

conditions.
Expressed as the % RSD for a statistically significant number of samples.
Precision is best determined at 3 different levels:
repeatability,
intermediate precision,
&
Precision
reproducibility
True
Method Val_M. El-Bidaoui value 20
- Precision
Repeatability is the results of the method operating over a short time

interval under the same conditions.
Ex. It is determined from a minimum of 9 determinations covering the
specified range of the procedure (e.g., 3 levels, 3 repetitions each) or from a
minimum of 6 determinations at 100% of the test or target concentration.
Intermediate precision is the results from within lab variations due to random
events such as different days, analysts, equipment, etc.
Reproducibility refers to the results of collaborative studies between

laboratories.
Documentation in support of precision studies should include the SD, RSD, CV,
& the confidence interval.

Precision
•Repeatability:
•represents the within laboratory variation (Sr)
• r = t x √2 x Sr is the repeatability limit.
•Reproducibility:
•Represents both variation within laboratory and
between laboratories (SR)
•R = t x √2 x SR

Repeatability
• Definition (ISO 5725-1:1994(E))

• Precision under repeatable conditions
• Repeatable conditions – where independent
test results are obtained with:
• the same method on
• identical items in
• the same laboratory by
• the same operator using
• the same equipment within short periods of time
Repeatability
 Minimum 4 independent experiments, on different days, using for

each matrix of interest, 6 blank samples; 6 samples fortified at 0.5 x
the level of interest; 6 samples fortified at the level of interest; 6
samples fortified at 2 x the level of interest.
 Samples may be fortified by the analyst.

 The analyst should report results for at least 1 set of fortified
samples, provided blind, containing a minimum of 3 duplicates in the
range 0.5 to 2 x the level of interest, plus another set consisting of a
minimum of 3 different samples in duplicate prepared from incurred
samples or fortified samples.
 These experiments can also be used to assess Trueness or Bias &

Recovery.
Reproducibility
•Definition (ISO 5725-1:1994(E))

•Precision under reproducible conditions
•Reproducible conditions – where test
results are obtained with:
 the same method on
 identical items in
 different laboratories with
 different operators using
 different equipment
Repeatability versus
Reproducibility
Parameter Repeatability Reproducibility

Method The same The same
Test items Identical Identical
Laboratory The same Different
Operator The same Different
Equipment The same Different
Time Short intervals Not defined

The AOAC manual for the Peer Verified Methods program
includes estimated precision data as a function of analyte
concentration within or between days
Analyte % Analyte ratio Unit RSD (%)

100 1 100% 1.3
10 10-1 10% 2.8
1 10-2 1% 2.7
0.1 10-3 0.1 % 3.7
0.01 10-4 100 ppm 5.3
0.001 10-5 10 ppm 7.3
0.0001 10-6 1 ppm 11
0.00001 10-7 100 ppb 15
0.000001 10-8 10 ppb 21
0.0000001 10-9 1 ppb 30
Horwitz’ curve

Critical Factors Affecting
Accuracy
1. Sampling
2. Sample processing / homogeneity
3. Stability
4. Extraction efficiency

Accuracy
Sample processing
 should be proven to have no significant effect on the measured
analyte levels
 For testing if the comminuted laboratory sample is statistically well
mixed, the Wallace/Kratochvil‟s procedure is recommended.
 The uncertainty (U) of this stage should be included in the combined

U.
Statistical significance
 If due to processing we recover >80% or we obtain an insignificant
difference (p = 0.05), the result should be considered acceptable to
validate quantitative & semi-quantitative procedures

Trueness or Bias
Sample, extracts, standard solution storage
 stability of analyte(s) in solutions & sample extracts may
vary greatly, according to the physico-chemical
properties of the analyte(s) & the nature of extracts;
 stability must be investigated during method validation;
 Analyte levels in extracts & calibration solutions must not

decline significantly (p = 0.05) at work temperature
during the time required to analyse a batch of samples;
 Storage of extracts is validated, up to the temperature &

elapsed time studied, if there is no statistically significant
difference (p = 0.05) in levels before & after storage.
The AOAC manual for the Peer Verified Methods program
includes estimated recovery data as a function of analyte
concentration
Active Ingredient
Analyte ratio Unit Mean recovery [%]
[%]
100 1 100% 98-102
>=10 10-1 10% 98-102
>=1 10-2 1% 97-103
>=0.1 10-3 0.1 % 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 -9
10Method 1 ppb 40-120
Val_M. El-Bidaoui 32
Extraction efficiency
 Bound analytes do not usually form a major proportion.
Nevertheless, validation of extraction efficiency remains
important.
 Rigorous validation of extraction efficiency of organic
analytes to be performed with samples containing
incurred analyte(s).
 Suitable certified reference materials containing incurred
analytes are very useful.
 Validation may require identification & quantification of
radio-labelled analyte(s), metabolites & all other
degradation products (beyond most laboratories‟
capabilities).

Extraction efficiency Alternative
approaches
1. Comparison with extraction of samples using a
procedure which has previously been validated
rigorously;
2. Comparison with extraction of samples by very
different extraction techniques; e.g., using a different
solvent (especially if coupled with a more effective
sample disintegration technique);
3. Analysis of a proficiency test material containing
incurred analyte, where the consensus analyte level
has been determined by a number of laboratories.

Recovery
Hill Recovery should normally be determined at two levels:

1. at or about the lowest calibrated level
2. at or about the action level.
 Quantitative Methods: the mean R should be within 70-110% & the RSD
within ±10%.
 Semi-quantitative method: mean R 50-120%, RSD ±25%, or 20-150%, RSD
±10%.
 The results should not be corrected for recovery! (against the IUPAC
recommendation)
NMKL perform recovery study with minimum 5 blank & 5 fortified samples;
 The lower the concentration of the analyte, the more determinations should
be carried out as the random error is increasing;
 apply t-test to demonstrate whether or not the obtained recoveries are
significantly different from 100 %.

Specificity
 The detection system response is wholly

attributable to the analyte (MS or MS/MS).
 Demonstration that a non-specific detector

response increases with analyte quantity may not
be sufficient, as the increasing response could be
produced by an impurity or unsuspected
degradation product.

Specificity
 Where the detected species is a degradation

product (or a metabolite of the analyte) but is not
included in the definition of the accepted limit, the
specificity, & perhaps the accuracy of the method,
cannot be validated.
 if the method itself does not provide adequate
proof of analyte identity, a quantitative
confirmatory method should be applied.

- Specificity
Ability to measure accurately & specifically the analyte of interest in the

presence of other components that could be present in the sample matrix.
A measure of the degree of interference from other active ingredients,

excipients, impurities, & degradation products, ensuring that a response is
due to a single component only. i.e. that no co-elutions exist.
Specificity is measured & documented in a separation by the resolution, plate

count (efficiency), & tailing factor.
Specificity can be evaluated with modern photodiode array detectors

that compare spectra collected across a peak mathematically as an indication
of peak homogeneity.
The term specificity can be divided into 2 categories:

identification, and assay/impurity tests.
- Specificity
For identification purposes:

Specificity is demonstrated by the:
• Ability to discriminate between compounds of

closely related structures;
• Comparison to known reference materials.

- Specificity
For assay and impurity tests:
Specificity is demonstrated by the:
Resolution of the 2 closest eluting compounds (usually

the analyte of interest & an impurity).
If impurities are available, it must be demonstrated that
the assay is unaffected by the presence of spiked
materials (impurities &/or excipients).
If the impurities are not available, the test results are

compared to a 2nd well-characterised procedure.

Examples of pure & impure HPLC
peaks (Agilent)
 Chromatography
does not indicate any
impurity in either
peak. However,
spectral evaluation
identifies the peak on
the left as impure

Linearity & Range
 Linearity: the ability of the method to elicit test results that

are directly proportional to analyte concentration within a
given range.
 Range is the interval between the upper & lower levels of
analyte (inclusive) that have been demonstrated to be
determined with precision, accuracy & linearity.
 For assay, the minimum specified range is from 80-120%
of the target concentration.
 For an impurity test, the minimum range is from the
reporting level of each impurity, to 120% of the
specification.
 For content uniformity testing, the minimum range is 70-
130% of the test or target concentration, & for dissolution
testing, +/- 20% over the specified range of the test.
Linearity & Range

Calibration function
Response vs. concentration in solvent
 Determination based on a curve consisting of minimum of

5 concentrations (excluding zero).
 Linearity: 0x, 1/2x, 1x, 2x and 3x with external analytical

standards; x = legal limit or expected concentration;
 NMKL: 6 points with blank or reference samples covering

the working range, repeated at least once,
r> 0.999;
 EURACHEM 6+ points;

Calibration function: Hill
 If the levels to be calibrated approach the limits of the

dynamic range of the detection system, increased # of low
&/or high level calibration points must be incorporated at
these levels.
 At method validation, calibration curves should be derived

from 2 replicate determinations each at 3 points.
 For detection systems with inherently non-linear response

(e.g. S-specific FPD), it is essential to determine the
response curve at both the lower & higher ends of the scale
& a minimum of 3 replicates at each of the points.

Calibration function: Hill
 Where detection system response is linear with analyte

concentration or mass (i.e. y = a + bx), a significant
deviation from a value of 1 for “b” should be investigated.
 Stability of detection system response, & the required

frequency of calibration, should be investigated.
 For chromatographic analyses, the validation should define

whether peak H or A integration is to be used.
 Exact procedure for calibration with mixed isomer (or

similar) standards should also be clearly defined at method
validation.
Analytical function
 Response vs. concentration in matrix, including blank;

 Same as calibration function but using blank matrix
material. More than 1 source of blank matrix material
should be used.
 The linearity of analytical function should be checked at
0.5X, X & 2 X, if X > LOD
 The effects, if any, of co-extractives from the samples on
the detection system calibration should be assessed at
method validation.
 These "matrix effects" may be observed as increased or
decreased responses, compared with those produced by
simple solvent solutions of the analyte.
 The effects should be checked during validation of
performance.
Limit of detection
 The lowest content of the analyte in the

matrix that can be detected with reasonable
statistical certainty (not quantified!).
 For analytical methods in which baseline
noise may be measured, the limit of
detection is usually established using a 3:1
signal-to-noise ratio.
 Blank measurement.
Limit of detection
 AOAC PVMP: n 20 blank determinations

 EURACHEM: 10 blank samples to determine SD of blank
response LD = 3 + 4.65xSD.
 NMKL: instrumental noise is enlarged to measure SD. Extracts
of more concentrated blank samples than normal are used to
demonstrate that there is no interference from any component.
 IUPAC: y = yB + 3SB
 Using results from 10-20 blank extracts is not practical for Multi-
analytes‟ Methods or multi-matrix methods.
 Bravenboer‟s practical solution: Make a calibration curve in the
relevant matrix. Determine slope (b) & the residual standard
deviation of the curve (Sres). The limit of detection is defined
as: Cdet = 3 x Sres/b

Lloyd Curie
 A visiting professor at NIST once pointed out that
measurement professionals are given a difficult task
by some of customers.
 Analysts are asked to perform measurements with
tools and techniques of finite precision and in the
end to produce digital answers, preferably binary:
 yes or no,
 safe or unsafe,
 above or below the regulatory limit.

Limit of detection Hill
 Adoption of a lowest calibrated level.

 Limits of detection or quantification always vary with time,
equipment, etc., which tends to make them irreproducible.
 Method validation of these parameters may be of little value.
 LCL should be decided based on the purpose of the method.
 The important is to demonstrate that the LCL can be detected
consistently & that the response is consistent.
 Internal reproducibility will be determined by performance

validation.
 If the analyte response is superimposed on a „background‟
signal, great caution is required to determine the true LCL &
should normally be at least 3 times the maximum background
level.

Limit of quantitation (determination)
 The lowest concentration of an analyte in a sample

determined with acceptable precision & accuracy.
 The lowest concentration point on the calibration
curve.
 Usually measured as recovery too.
 When the determination is made with respect to the
background noise, a 10:1 signal-to-noise ratio is
typically used.
 EURACHEM: 10 blank samples to determine SD of
blank response LoD = 10SD
 IUPAC: y = yB + 10SB
 Bravenboer: Cquan = 2 x Cdet or
Cquan = 6 x Sres/b
Limit of detection & limit of quantitation
 The figure shows how

efficiency & peak shape can
affect the S/N.
 Sharper peaks result in a
higher S/N, resulting in lower
LOQs & LDs.
 Chromatographic
determination of LOQ/LOD
should take into account both
the type & age of the column,
which is usually determined
over the course of time as
experience with the method
grows.

LOD / LOQ
Peak B
LOQ
Peak A
LOD
Baseline noise

Limit of identification
 The lowest concentration at which a

positive confirmation of the analyte may be
made.
Example:
 For GC/MS confirmations, the EU has
established criteria based on the ratios of 4
characteristic fragment ions.

Ruggedness
 Ruggedness is the degree of reproducibility of

the results obtained under a variety of
conditions, expressed as %RSD.
 The conditions include different labs, analysts,

instruments, reagents, days, etc.
 Some guidelines chose to cover the topic of

ruggedness as part of precision
(reproducibility).
Robustness
 The capacity of a method to remain unaffected by

small deliberate variations in method parameters.
 It is evaluated by varying method parameters
such as percent organic, pH, ionic strength,
temperature, etc., & determining the effect (if any)
on the results of the method.
 Robustness should be considered early in the
development of a method.
 If the results of a method are susceptible to
variations in method parameters, these
parameters should be adequately controlled.

Recommended Validation Characteristics of the Various Types of Tests
Type of Tests / Identification Testing for Impurities Assay Specific

Characteristics Quantitative Limit Dissolution (Measurement Tests
Only), Content/Potency
Accuracy - + - + +4
Precision- - + - + +4
Repeatability
Precision- - +1 - +1 +4
Intermediate
Precision
Specificity +2 + + +5 +4
DL - -3 + - -
QL - + - - -
Linearity - + - + -
Range - + - + -
Robustness - + -3 + +4
NOTE:
- Signifies that this characteristic is not normally evaluated.
+ Signifies that this characteristic is normally evaluated.
1 In cases where reproducibility has been performed, intermediate precision is not needed.
2 Lack of specificity for an analytical procedure may be compensated for by the addition of a second analytical procedure.
3 May be needed in some cases.
4 May not be needed in some cases.
5 Lack of specificity for an assay for release may be compensated for by impurities testing.
Important information
 Contaminants, additives, any components expected to be present;

non-specific effect of matrices; effects of transformation products or
metabolic products.
 E.g. Formulations of a product contain minor components which may
be found in samples.
 Minor quantities of metabolites present in samples from treated
animals. Parent compound maybe the major residue & quantitation is
based on its measurement.
 Drugs from the same group (list drugs) were tested & did not have
retention times which interfere in the analysis. Other drugs commonly
used in combination with the target compound which were tested (list
drugs) did not interfere in the analysis.
 Any differences observed in the background for samples of the same
matrix from different sources, or for the different matrices to which the
analyses may be applied should be noted.
Detection Records
Include clearly in the validation report labelled copies of:

 chromatograms,
 Spectra,
 other relevant analytical outputs for standards, controls
and samples.
 Information provided should include sample
identification, analyst, date & analytical conditions.
 The information should be provided in printed (hard
copy) format.

Acceptable within laboratory
performance Characteristics
Concentration Repeatability Reproducibility Trueness
mg/kg CVA% CVL% CVA% CVL% Range of mean

% recovery
< or = 0.001 35 36 53 54 50120
> 0.001 - < or = 0.01 30 32 45 46 60120
> 0.01- < or = 0.1 20 22 32 34 70120
> 0.1 < or = 1 15 18 23 25 70110
>1 10 14 16 19 70110

Representative samples
Group Common properties Commodity group Representative species
Plant products
I. High water and Leafy vegetables spinach or lettuce
chlorophyll Brassica leafy vegetables broccoli, cabbage, kale, green
content Legume vegetables beans,
green peas
II. High water and low or Pome fruits apple, pear
no chlorophyll Stone fruits peach, cherry
content Berries strawberry
Small fruits grape,
Fruiting vegetables tomato, bell pepper, melon
Root vegetables mushroom
potato, carrot, parsley
III. High acid content Citrus fruits orange, lemon
IV. High sugar content raisins, dates
V. High oil or fat Oil seeds avocado, sunflower seed,

Nuts walnut, pecan nut, pistachios
VI. Dry materials Cereals wheat, rice or maize grains
Cereal products wheat bran, wheat floor
Commodities e.g. garlic, hops, tea, spices,
requiring cranberry
individual test
Situation Degree of validation
New method developed for particular problem F
Existing method tested for applicability to particular problem F
new matrix F or P
Established method revised to incorporate improvements P or F
Established method extended or adapted to a new problem (analyte, ) P or F
When quality control indicates an established method is changing with time P or F
Established method in a different laboratory P or E
Established method with different instrumentation P
Established method with different operator P,
New reagent batches P
The laboratory takes an already validated and verified method into use after it P
has been out of use for long time.
Changes in laboratory premises which may influence the results P
New collaboratively studied methods P
New collaboratively studied methods intended to be used on new matrix or with P
new instrument(s)
Validated but collaboratively not tested P+ E
Method is published in scientific literature with performance characteristics P+ E
Method is published in scientific literature without performance characteristics F3

The Validation Process

References
EURACHEM/CITAC Guide:
The fitness for purpose of Analytical Methods: A
Laboratory Guide to Method validation and Related
Topics (http://www.eurachem.ul.pt/)
International Union of Pure and Applied Chemistry:

Harmonized guidelines for single laboratory
validation of methods of analysis
Pure Appl. Chem., Vol. 74, No. 5, pp. 835–855, 2002

Method Validation in a Single Laboratory

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Method validation in A Single

Dr. Maha El-Bidaoui

 ..Made to satisfy an agreed requirement = a clear

 Staff: should be qualified & competent to undertake the

Method Val_M. El-Bidaoui 2

 Validation includes: Sampling, Transportation & Handling.

 It should also measure the influence of instrumental, human &

 ISO definition: Validation

Method Val_M. El-Bidaoui 4

Source: standard-setting organizations e.g. ISO, AOAC,

 New analytical methods in international journals are well

 ‘Copying' a method from a journal needs always

Method Val_M. El-Bidaoui 6

Method Track Review AOAC I.

Proprietary Methods Non-proprietary Methods

Performance Tested Official Methods of Peer-Verified Methods

Independent Laboratory In-House Method Validation Data

Performance tested Method Collaborative Study Peer Verified Method

Collaborative Study Data review (General Referee, Safety &

First Action Official Method of Analysis

Membership Use, Feedback & Vote

Method Val_M. El-Bidaoui 7

Methods are often described as:

Method Val_M. El-Bidaoui 8

Establishment of acceptable performance (validation) in

Demonstration of acceptable performance in 2nd or 3rd

Demonstration of acceptable performance in full inter-

4 Regular use of the method – performance verification

Method Val_M. El-Bidaoui 9

Use of incurred analyte samples.

Variance associated with sub-sampling is to be combined with Variance of

For testing if the comminuted lab sample is statistically well mixed,

Method Val_M. El-Bidaoui 11

Method Val_M. El-Bidaoui 12

- Accuracy (trueness, bias)

Measured or reported as the % of analyte recovered by spiking samples in a

A minimum number of determinations (from 5 onwards) should cover a

Method Val_M. El-Bidaoui 17

 Use of (certified) reference materials when

Method Val_M. El-Bidaoui 18

Method Val_M. El-Bidaoui 19

Measure of the degree of repeatability of a test method under similar

Precision is best determined at 3 different levels:

Repeatability is the results of the method operating over a short time

Reproducibility refers to the results of collaborative studies between

Method Val_M. El-Bidaoui 21

Method Val_M. El-Bidaoui 22

• Definition (ISO 5725-1:1994(E))

 Minimum 4 independent experiments, on different days, using for

 Samples may be fortified by the analyst.

 These experiments can also be used to assess Trueness or Bias &

•Definition (ISO 5725-1:1994(E))

Parameter Repeatability Reproducibility

Test items Identical Identical

Laboratory The same Different

Operator The same Different

Equipment The same Different

Time Short intervals Not defined

Method Val_M. El-Bidaoui 26

Analyte % Analyte ratio Unit RSD (%)

Method Val_M. El-Bidaoui 28

Method Val_M. El-Bidaoui 29

 The uncertainty (U) of this stage should be included in the combined