2002D0657 — EN — 15.03.2003 — 001.001 — 1
This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents
►B COMMISSIONDECISION
of►C1 14August2002 ◄
implementing Council Directive 96/23/EC concerning the performance of analytical methods and
theinterpretationofresults
(notified under document number C(2002) 3044)
(TextwithEEArelevance)
(2002/657/EC)
(OJ L 221, 17.8.2002, p. 8)
Amended by:
Official Journal
No page date
►M1Commission Decision 2003/181/EC of 13 March 2003 L 71 17 15.3.2003
Corrected by:
►C1Corrigendum, OJ L 239, 6.9.2002, p. 66 (2002/657/EC)
▼B
COMMISSIONDECISION
of►C1 14August2002 ◄
implementing Council Directive 96/23/EC concerning the perfor-
manceofanalyticalmethodsandtheinterpretation ofresults
(notified under document number C(2002) 3044)
(TextwithEEArelevance)
(2002/657/EC)
THE COMMISSION OF THE EUROPEAN COMMUNITIES,
Having regard to the Treaty establishing the European Community,
Having regard to Council Directive 96/23/EC of 29 April 1996 on
measures to monitor certain substances and residues thereof in live
animalsand animal productsand repealing Directives85/358/EEC and86/469/EEC and Decisions 89/187/EEC and 91/664/EEC (
1), and in
particular the second subparagraph of Article 15(1) thereof,
Whereas:
(1)The presence of residues in products of animal origin is a matter
of concern for public health.
(2)Commission Decision 98/179/EC of 23 February 1998 layingdown detailed ruleson official s ampling for the monitoring of
certain substances and residues thereof in live animals and
animal products(
2) provides that the analysis of samples is to
be carried out exclusively by laboratories approved for officialresidue control by the competent national authority.
(3)It is necessary to ensure the quality and comparability of theanalytical results generated by laboratories approved for officialresidue control. This should be achieved by using quality assur-ance systems and specifically by applying of methods validatedaccording to common proceduresand performance criteria and
by ensuring traceability to common standards or standards
commonly agreed upon.
(4)Council Directive 93/99/EEC of 29 October 1993 on the subjectof additional measures concerning the official control of food-
stuffs and Decision 98/179/EC (
3) require official control
laboratoriesto be accredited according to ISO 17025 (1) fromJanuary 2002 onwards. Pursuant to Decision 98/179/EC, partici-pation in an internationally recognised external quality controlassessment and accreditation scheme is required for approvedlaboratories. Moreover, approved laboratories must prove their
competence by regular and successful participation in adequate
proficiency testing schemes recognised or organised by thenational or Community reference laboratories.
(5)A network of Community reference laboratories, national refer-
ence laboratoriesand national control laboratoriesoperates
under Directive 96/23/EC to enhance coordination.
(6)As a result of advances in analytical chemistry since the adop-tion of Directive 96/23/EC the concept of routine methodsand
reference methods has been superseded by criteria approach, in
which performance criteria and proceduresfor the validation ofscreening and confirmatory methods are established.
(7)It is necessary to determine common criteria for the interpreta-
tion of test results of official control laboratories in order to
ensure a harmonised implementation of Directive 96/23/EC.
(8)It is necessary to provide for the progressive establishment ofminimum required performance limits(MRPL) of analytical2002D0657 — EN — 15.03.2003 — 001.001 — 2
(1) OJ L 125, 23.5.1996, p. 10.
(2) OJ L 65, 5.3.1998, p. 31.
(3) OJ L 290, 24.11.1993, p. 14.
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method for substances for which no permitted limit has been
established and in particular for those substances whose use isnot authorised, or is specifically prohibited in the Community,
in order to ensure harmonised implementation of Directive 96/
23/EC.
(9)Commission Decision 90/515/EEC of 26 September 1990 layingdown the reference methodsfor detecting res iduesof heavy
metalsand ars enic (
1), Commission Decision 93/256/EEC of 14
May 1993 laying down the methodsto be us ed for detectingresidues of substances having a hormonal or a thyrostaticaction (
2), and of Commission Decision 93/257/EEC of 15 April
1993 laying down the reference methodsand the lis t of the
national reference laboratoriesfor detecting res idues (3), aslas t
amended by Decision 98/536/EC (4) have been re-examined
before in order to take account of developmentsin s cientificand technical knowledge, have been found outdated in theirscope and provisions and should accordingly be repealed withthisDecis ion.
(10)In order to allow methods for the analysis of official samples tobe adapted to the provisions of this Decision, a transitionalperiod should be laid down.
(11)The measures provided for in this Decision are in accordancewith the opinion of the Standing Committee on the Food Chainand Animal Health,
HAS ADOPTED THIS DECISION:
Article 1
Subjectmatter andscope
This Decision provides rules for the analytical methods to be used in
the testing of official samples taken pursuant to Article 15(1), second
sentence, of Directive 96/23/EC and specifies common criteria for theinterpretation of analytical results of official control laboratories forsuch samples.
This Decision shall not apply to substances for which more specific
ruleshave been laid down in other Community legis lation.
Article 2
Definitions
For the purpose of this Decision the definitions in Directive 96/23/EC
and in the ►
M1Annex I ◄to this decision shall apply.
Article 3
Analyticalmethods
The Member States shall ensure that official samples taken pursuant to
Directive 96/23/EC are analysed using methods that:
(a) are documented in test instructions, preferably according to ISO
78-2 (6);
(b) comply with Part 2 of the ►M1Annex I ◄to thisDecis ion;
(c) have been validated according to the proceduresdes cribed in Part 3
of the►M1Annex I ◄;
(d) comply with the relevant minimum required performance limits
(MRPL) to be established in accordance with Article 4.2002D0657 — EN — 15.03.2003 — 001.001 — 3
(1) OJ L 286, 18.10.1990, p. 33.
(2) OJ L 118, 14.5.1993, p. 64.
(3) OJ L 118, 14.5.1993, p. 75.
(4) OJ L 251, 11.9.1998, p. 39.
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Article 4
Minimumrequiredperformance limits
Member States shall ensure that the analytical methods used for
detecting the following substances meet the minimum required perfor-
mance limits (MRPLs) set out in Annex II, against the matrixesreferred to in that Annex:
(a) chloramphenicol;
(b) nitrofuran metabolites;
(c) medroxyprogesterone.
Article 5
Qualitycontrol
The Member States shall ensure the quality of the results of the
analysis of samples taken pursuant to Directive 96/23/EC, in particularby monitoring tests and/or calibration results according to Chapter 5.9of ISO 17025 (1).
Article 6
Interpretationofresults
1. The result of an analysis shall be considered non-compliant if the
decision limit of the confirmatory method for the analyte is exceeded.
2. If a permitted limit has been established for a substance, the deci-
sion limit is the concentration above which it can be decided with a
statistical certainty of 1 – αthat the permitted limit hasbeen truly
exceeded.
3. If no permitted limit has been established for a substance, the
decision limit is the lowest concentration level at which a method candiscriminate with a statistical certainty of 1 – αthat the particular
analyte ispres ent.
4. For substances listed in Group A of Annex I to Directive 96/23/
EC, the αerror shall be 1 % or lower. For all other substances, the α
error shall be 5 % or lower.
Article 7
Repeal
Decisions 90/515/EEC, 93/256/EEC and 93/257/EEC are repealed.
Article 8
Transitionalprovisions
The methods for the analysis of official samples of substances listed in
Group A of Annex I to Directive 96/23/EC, which satisfy the criteriaset out in Decisions 90/515/EEC, 93/256/EEC and 93/257/EEC maybe used for up to two years after this Decision enters into force.Methods currently applied for substances listed in Group B of AnnexI to Directive 96/23/EC shall comply with this Decision at the latest
five yearsafter the date of application of thisDecis ion.
Article 9
Dateofapplication
This Decision shall apply from 1 September 2002.2002D0657 — EN — 15.03.2003 — 001.001 — 4
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Article 10
Addressees
This Decision is addressed to the Member States.2002D0657 — EN — 15.03.2003 — 001.001 — 5
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ANNEXI
PERFORMANCE CRITERIA, OTHER REQUIREMENTS ANDPROCE-
DURESFORANALYTICALMETHODS
1.DEFINITIONS
1.1. Accuracy means the closeness of agreement between a test result and
the accepted reference value (2). It isdetermined by determining true-
ness and precision.
1.2. Alpha ( α)error means the probability that the tested sample is
compliant, even though a non-compliant measurement has been
obtained (false non-compliant decision).
1.3. Analyte means the substance that has to be detected, identified and/or
quantified and derivatives emerging during its analysis.
1.4. Beta ( β)error means the probability that the tested sample is truly non-
compliant, even though a compliant measurement has been obtained
(false compliant decision).
1.5. Bias means the difference between the expectation of the test result and
an accepted reference value (2).
1.6. Calibration standard means a device for measurements that represents
the quantity of substance of interest in a way that ties its value to a
reference base.
1.7. Certified reference material (CRM) meansa material that hashad a
specified analyte content assigned to it.
1.8. Co-chromatography meansa procedure in which the extract prior to the
chromatographic step(s) is divided into two parts. Part one is chromato-
graphed as such. Part two is mixed with the standard analyte that is to
be measured. Then this mixture is also chromatographed. The amount
of added standard analyte has to be similar to the estimated amount of
the analyte in the extract. Thismethod isdes igned to improve the iden-
tification of an analyte when chromatographic methodsare us ed,
especially when no suitable internal standard can be used.
1.9. Collaborative study means analysing the same sample by the same
method to determine the performance characteristics of the method.
The study covers random measurement error and laboratory bias.
1.10. Confirmatory method meansmethodsthat provide full or complemen-
tary information enabling the substance to be unequivocally identified
and if necessary quantified at the level of interest.
1.11. Decision limit (CC α)meansthe limit at and above which it can be
concluded with an error probability of αthat a sample is non-compliant.
1.12. Detection capability (CC β)means the smallest content of the substance
that may be detected, identified and/or quantified in a sample with an
error probability of β.In the case of substances for which no permitted
limit has been established, the detection capability is the lowest concen-
tration at which a method isable to detect truly contaminated s amples
with a statistical certainty of 1 – β.In the case of substances with an
established permitted limit, this means that the detection capability is
the concentration at which the method isable to detect permitted limit
concentrations with a statistical certainty of 1 – β.
1.13. Fortified sample material means a sample enriched with a known
amount of the analyte to be detected.
1.14. Interlaboratory study (comparison) means organisation, performance
and evaluation of tests on the same sample by two or more laboratories
in accordance with predetermined conditionsto determine tes ting
performance. According to the purpose the study can be classified as
collaborative study or proficiency study.
1.15. Internal Standard (IS) means a substance not contained in the sample
with physical-chemical properties as similar as possible to those of the
analyte that hasto be identified and which isadded to each s ample as
well asto each calibration s tandard.
1.16. Laboratory sample means a sample prepared for sending to a laboratory
and intended for inspection or testing.
1.17. Level of interest means the concentration of substance or analyte in a
sample that is significant to determine its compliance with legislation.2002D0657 — EN — 15.03.2003 — 001.001 — 6
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1.18. Minimum required performance limit (MRPL) meansminimum content
of an analyte in a sample, which at least has to be detected and
confirmed. It isintended to harmonis e the analytical performance of
methods for substances for which no permitted limited has been estab-
lished.
1.19. Performance characteristic means functional quality that can be attrib-
uted to an analytical method. This may be for instance specificity,
accuracy, trueness, precision, repeatability, reproducibility, recovery,
detection capability and ruggedness.
1.20. Performance criteria meansrequirementsfor a performance character-
istic according to which it can be judged that the analytical method is
fit for the purpose and generates reliable results.
1.21. Permitted limit meansmaximum res idue limit, maximum level or other
maximum tolerance for substances established elsewhere in Community
legislation.
1.22. Precision means the closeness of agreement between independent test
results obtained under stipulated (predetermined) conditions. The
measure of precision usually is expressed in terms of imprecision and
computed as standard deviation of the test result. Less precision is
determined by a larger standard deviation (2).
1.23. Proficiency study means analysing the same sample allowing labora-
tories to choose their own methods, provided these methods are used
under routine conditions. The study has to be performed according to
ISO guide 43-1 (3) and 43-2 (4) and can be used to assess the reprodu-
cibility of methods.
1.24. Qualitative method meansan analytical method which identifiesa
substance on the basis of its chemical, biological or physical properties.
1.25. Quantitative method meansan analytical method which determinesthe
amount or mass fraction of a substance so that it may be expressed as a
numerical value of appropriate units.
1.26. Reagent blank determination meansthe complete analytical procedure
applied without the test portion or using an equivalent amount of
suitable solvent in place of the test portion.
1.27. Recovery means the percentage of the true concentration of a substance
recovered during the analytical procedure. It isdetermined during vali-
dation, if no certified reference material isavailable.
1.28. Reference material meansa material of which one or s everal properties
have been confirmed by a validated method, so that it can be used to
calibrate an apparatusor to verify a method of meas urement.
1.29. Repeatability meansprecis ion under repeatability conditions(2).1.30. Repeatability conditionsmeansconditionswhere independent tes t
results are obtained with the same method on identical test items in
the same laboratory by the same operator using the same equipment (2).
1.31. Reproducibility meansprecis ion under reproducibility conditions(2)(4).1.32. Reproducibility conditions means conditions where test results are
obtained with the same method on identical test items in different
laboratorieswith different operatorsus ing different equipment (2)(4).
1.33. Ruggedness means the susceptibility of an analytical method to changes
in experimental conditions which can be expressed as a list of the
sample materials, analytes, storage conditions, environmental and/or
sample preparation conditions under which the method can be applied
as presented or with specified minor modifications. For all experimental
conditions which could in practice be subject to fluctuation (e.g. stabi-
lity of reagents, composition of the sample, pH, temperature) any
variations which could affect the analytical result should be indicated.
1.34. Sample blank determination meansthe complete analytical procedure
applied to a test portion taken from a sample from which the analyte
isabs ent.
1.35. Screening method means methods that are used to detect the presence
of a substance or class of substances at the level of interest. These
methods have the capability for a high sample throughput and are used
to sift large numbers of samples for potential non-compliant results.
They are specifically designed to avoid false compliant results.
1.36. Single laboratory study (in-house validation) means an analytical study
involving a single laboratory using one method to analyse the same or2002D0657 — EN — 15.03.2003 — 001.001 — 7
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different test materials under different conditions over justified long
time intervals.
1.37. Specificity means the ability of a method to distinguish between the
analyte being measured and other substances. This characteristic is
predominantly a function of the measuring technique described, but
can vary according to class of compound or matrix.
1.38. Standard addition means a procedure in which the test sample is
divided in two (or more) test portions. One portion is analysed as such
and known amounts of the standard analyte are added to the other test
portions before analysis. The amount of the standard analyte added has
to be between two and five timesthe es timated amount of the analyte in
the sample. This procedure is designed to determine the content of an
analyte in a sample, taking account of the recovery of the analytical
procedure.
1.39. Standard analyte meansan analyte of known and certified content and
purity to be used as a reference in the analysis.
1.40. Substance means matter of particular or definite chemical constitution
and itsmetabolites .
1.41. Test portion means the quantity of material drawn from the test sample
on which the test or observation is carried out.
1.42. Test sample means a sample prepared from a laboratory sample and
from which test portions will be taken.
1.43. Trueness means the closeness of agreement between the average value
obtained from a large series of test results and an accepted reference
value. Trueness is usually expressed as bias (2).
1.44. Units means those units described in ISO 31 (20) and Directive 71/354/
EC (19).
1.45. Validation meansthe confirmation by examination and the provis ion of
effective evidence that the particular requirementsof a s pecific intended
use are fulfilled (1).
1.46. Within-laboratory reproducibility means precision obtained in the same
laboratory under stipulated (predetermined) conditions (concerning e.g.
method, test materials, operators, environment) over justified long time
intervals.
2.PERFORMANCE CRITERIA ANDOTHER REQUIREMENTS FOR
ANALYTICALMETHODS
Analytical methods or combinations of methods other than those described
below may only be used for screening or confirmatory purposes if it can be
proven that they fulfil the relevant requirements established in this Decision.
2.1. GENERAL REQUIREMENTS
2.1.1.Handlingofsamples
Samples shall be obtained, handled and processed in such a way that
there is a maximum chance of detecting the substance. Sample handling
procedures shall prevent the possibility of accidental contamination or
loss of analytes.
2.1.2.Performance oftests
2.1.2.1.Recovery
During the analysis of samples the recovery shall be determined in each
batch of samples, if a fixed recovery correction factor is used. If the
recovery is within limits, the fixed correction factor may then be used.
Otherwise the recovery factor obtained for that specific batch shall be
used, unless the specific recovery factor of the analyte in the sample is
to be applied in which case the standard addition procedure (see 3.5) or
an internal standard shall be used for the quantitative determination of
an analyte in a sample.
2.1.2.2.Specificity
A method shall be able to distinguish between the analyte and the other
substances under the experimental conditions. An estimate to which
extent this is possible has to be provided. Strategies to overcome any
foreseeable interference with substances when the described measuring
technique is used, e.g. homologues, analogues, metabolic products of
the residue of interest have to be employed. It is of prime importance2002D0657 — EN — 15.03.2003 — 001.001 — 8
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that interference, which might arise from matrix components, is investi-
gated.
2.2. SCREENING METHODS
Only those analytical techniques, for which it can be demonstrated in a docu-
mented traceable manner that they are validated and have a false compliant
rate of < 5 % ( β-error) at the level of interest shall be used for screening
purposes in conformity with Directive 96/23/EC. In the case of a suspected
non-compliant result, this result shall be confirmed by a confirmatory method.
2.3. CONFIRMATORY METHODS FOR ORGANIC RESIDUES AND
CONTAMINANTS
Confirmatory methods for organic residues or contaminants shall provide infor-
mation on the chemical structure of the analyte. Consequently methods based
only on chromatographic analysis without the use of spectrometric detection
are not suitable on their own for use as confirmatory methods. However, if a
single technique lacks sufficient specificity, the desired specificity shall be
achieved by analytical procedures consisting of suitable combinations of clean-
up, chromatographic separation(s) and spectrometric detection.
The following methods or method combinations are considered suitable for the
identification of organic residues or contaminants for the substance groups indi-
cated:
Table1
Suitableconfirmatorymethodsfororganicresiduesorcontaminants
Measuring techniqueSubstances Annex 1 96/
23/ECLimitations
LC or GC with mass-spec-
trometric detectionGroupsA and B Only if following either an on-line or an off-
line chromatographic separation
Only if full scan techniques are used or using
at least 3 (group B) or 4 (group A) identifica-
tion pointsfor techniquesthat do not record
the full mass spectra
LC or GC with IR spectro-
metric detectionGroupsA and B Specific requirements for abs orption in IR
spectrometry have to be met
LC-full-scan DAD Group B Specific requirements for absorption in UV
spectrometry have to be met
LC -fluorescence Group B Only for molecules that exhibit native fluores-
cence and to moleculesthat exhibit fluores -
cence after either transformation or
derivatisation
2-D TLC - full-scan UV/
VISGroup B Two-dimensional HPTLC and co-chromato-
graphy are mandatory
GC-Elektron capture
detectionGroup B Only if two columnsof different polarity are
used
LC-immunogram Group B Only if at least two different chromatographic
systems or a second, independent detection
method are used
LC-UV/VIS (single wave-
length)Group B Only if at least two different chromatographic
systems or second, independent detection
method are used.
2.3.1.Commonperformancecriteria andrequirements
Confirmatory methods shall provide information on the chemical struc-
ture of the analyte. When more than one compound givesthe s ame
response, then the method cannot discriminate between these
compounds. Methods based only on chromatographic analysis without
the use of spectrometric detection are not suitable on their own for use
asconfirmatory methods .
Where used in the method, a suitable internal standard shall be added to
the test portion at the beginning of the extraction procedure. Depending
on availability, either stable isotope-labelled forms of the analyte,
which are particularly suited for mass-spectrometric detection, or
compounds that are structurally related to the analyte shall be used.2002D0657 — EN — 15.03.2003 — 001.001 — 9
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When no suitable internal standard can be used, the identification of the
analyte shall be confirmed by co-chromatography. In this case only one
peak shall be obtained, the enhanced peak height (or area) being
equivalent to the amount of added analyte. With gaschromatography
(GC) or liquid chromatography (LC), the peak width at half-maximum
height shall be within the 90-110 % range of the original width, and the
retention timess hall be identical within a margin of 5 %. For thin layer
chromatography (TLC) methods, only the spot presumed to be due to
the analyte shall be intensified; a new spot shall not appear and the
visual appearance shall not change.
Reference or fortified material containing known amountsof analyte, at
or near either the permitted limit or the decision limit (non-compliant
control s ample) aswell ascompliant control materialsand reagent
blanks should preferably be carried through the entire procedure simul-
taneously with each batch of test samples analysed. The order for
injecting the extracts into the analytical instrument is as follows:
reagent blank, compliant control sample, sample(s) to be confirmed,
compliant control sample again and finally non-compliant control
sample. Any variation from this sequence shall be justified.
2.3.2.Additionalperformancecriteriaandotherrequirementsforquanti-
tativemethodsofanalysis
2.3.2.1.Trueness of quantitative methods
In the case of repeated analyses of a certified reference material, the
guideline rangesfor the deviation of the experimentally determined
recovery corrected mean mass fraction from the certified value are as
follows:
Table2
Minimumtruenessofquantitativemethods
Mass fraction Range
≤1µg/kg – 50 % to + 20 %
>1 µg/kg to 10 µg/kg – 30 % to + 10 %
≥10µg/kg – 20 % to + 10 %
When no such CRMs are available, it is acceptable that trueness of
measurements is assessed through recovery of additions of knownamountsof the analyte(s ) to a blank matrix. Data corrected with the
mean recovery are only acceptable when they fall within the ranges
shown in Table 2.
2.3.2.2.Precision of quantitative methods
The inter-laboratory coefficient of variation (CV) for the repeated
analysis of a reference or fortified material, under reproducibility condi-
tions, shall not exceed the level calculated by the Horwitz Equation.
The equation is:
CV = 2
(1 - 0,5 log C)
where C is the mass fraction expressed as a power (exponent) of 10
(e.g. 1 mg/g = 10-3). Examplesare s hown in the table 3.
Table3
Examples for reproducibility CVs for quantitative methods at a range of analyte mass
fractions
Mass fraction Reproducibility CV(%)
1µg/kg (*)
10µg/kg (*)
100 µg/kg 23
1 000 µg/kg (1 mg/kg) 16
(*) For mass fractions lower than 100 µg/kg the application of the Horwitz Equation givesunacceptable
high values. Therefore, the CVs for concentrations lower than 100 µg/kg shall be as low as possible.2002D0657 — EN — 15.03.2003 — 001.001 — 10
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For analyses carried out under repeatability conditions, the intra-labora-
tory CV would typically be between one half and two thirdsof the
above values. For analyses carried out under within-laboratory reprodu-
cibility conditions, the within-laboratory CV shall not be greater than
the reproducibility CV.
In the case of substances with an established permitted limit, the
method shall achieve within-laboratory reproducibility not greater than
the corresponding reproducibility CV at a concentration of 0,5 × the
permitted limit.
2.3.3.Performance criteria and other requirements for mass spectro-
metric detection
Mass spectrometric methods are suitable for consideration as confirma-
tory methodsonly following either an on-line or an off-line
chromatographic separation.
2.3.3.1.Chromatographic separation
For GC-MS procedures, the gas chromatographic separation shall be
carried out using capillary columns. For LC-MS procedures, the chro-
matographic separation shall be carried out using suitable LC columns.
In any case, the minimum acceptable retention time for the analyte
under examination istwice the retention time corres ponding to the
void volume of the column. The retention time (or relative retention
time) of the analyte in the test portion shall match that of the calibra-
tion standard within a specified retention time window. The retention
time window shall be commensurate with the resolving power of the
chromatographic system. The ratio of the chromatographic retention
time of the analyte to that of the internal standard, i.e. the relative
retention time of the analyte, shall correspond to that of the calibration
solution at a tolerance of ± 0,5 % for GC and ± 2,5 % for LC.
2.3.3.2.Mass spectrometric detection
Mass-spectrometric detection shall be carried out by employing MS-
techniques such as recording of full mass spectra (full scans) or selected
ion monitoring (SIM), aswell asMS-MSntechniquess uch asSelected
Reaction Monitoring (SRM), or other suitable MS or MS-MSntechni-
ques in combination with appropriate ionisation modes. In high-
resolution mass spectrometry (HRMS), the resolution shall typically be
greater than 10 000 for the entire mass range at 10 % valley.
Full scan: When mass spectrometric determination is performed by
the recording of full scan spectra, the presence of all measured diag-
nostic ions (the molecular ion, characteristic adducts of the molecular
ion, characteristic fragment ions and isotope ions) with a relative inten-
sity of more than 10 % in the reference spectrum of the calibration
standard is obligatory.
SIM: When mass spectrometric determination is performed by frag-
mentography, the molecular ion shall preferably be one of the selected
diagnostic ions (the molecular ion, characteristic adducts of the mole-
cular ion, characteristic fragment ions and all their isotope ions). The
selected diagnostic ions should not exclusively originate from the
same part of the molecule. The signal-to-noise ratio for each diagnostic
ion shall be ≥3:1.
Full scan and SIM: The relative intensities of the detected ions,
expressed as a percentage of the intensity of the most intense ion or
transition, shall correspond to those of the calibration standard, either
from calibration standard solutions or from spiked samples, at compar-
able concentrations, measured under the same conditions, within the
following tolerances:
Table4
Maximumpermittedtolerancesforrelativeionintensitiesusingarangeofmassspectro-
metric techniques
Relative intensity
(% of base peak)EI-GC-MS
(relative)CI-GC-MS, GC-MSn
LC-MS, LC-MSn
(relative)
>5 0% ±1 0% ±2 0%
> 20 % to 50 % ± 15 % ± 25 %
> 10 % to 20 % ± 20 % ± 30 %
≤10 % ± 50 % ± 50 %2002D0657 — EN — 15.03.2003 — 001.001 — 11
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Interpretation of mass spectral data: The relative intensities of
the diagnostic ions and/or precursor/product ion pairs have to be identi-
fied by comparing spectra or by integrating the signals of the single
mass traces. Whenever background correction is applied, this shall be
applied uniformly throughout the batch (see 2.3.1, paragraph 4) and
shall be clearly indicated.
Full scan: When full scan spectra are recorded in single mass spectro-
metry, a minimum of four ions shall be present with a relative intensity
of≥10 % of the base peak. The molecular ion shall be included if it is
present in the reference spectrum with a relative intensity of ≥10 %.
At least four ions shall lie within the maximum permitted tolerances for
relative ion intensities (Table 5). Computer-aided library searching may
be used. In this case, the comparison of mass spectral data in the test
samples to that of the calibration solution has to exceed a critical match
factor. This factor shall be determined during the validation process for
every analyte on the basis of spectra for which the criteria described
below are fulfilled. Variability in the spectra caused by the sample
matrix and the detector performance shall be checked.
SIM: When mass fragments are measured using other than full-scan
techniques, a system of identification points shall be used to interpret
the data. For the confirmation of substances listed in Group A of Annex
I of Directive 96/23/EC, a minimum of 4 identification pointss hall be
required. For the confirmation of substances listed in Group B of Annex
I of Directive 96/23/EC, a minimum of 3 identification pointsare
required. The table below shows the number of identification points
that each of the basic mass spectrometric techniques can earn.However, in order to qualify for the identification pointsrequired for
confirmation and the sum of identification points to be calculated:
(a) a minimum of at least one ion ratio shall be measured, and
(b) all relevant measured ion ratios shall meet the criteria described
above, and
(c) a maximum of three separate techniques can be combined to
achieve the minimum number of identification points.
Table5
The relationship between a range of classes of mass fragment and identification points
earned
MS technique Identification pointsearned per ion
Low resolution mass spectrometry (LR) 1,0
LR-MSnprecursor ion 1,0
LR-MSntransition products 1,5
HRMS 2,0
HR- MSnprecursor ion 2,0
HR-MSntransition products 2,5
Footnotes:
(1) Each ion may only be counted once.
(2) GC-MS using electron impact ionisation is regarded as being a different technique to GC-MS using
chemical ionisation.
(3) Different analytes can be used to increase the number of identification points only if the derivatives
employ different reaction chemistries.
(4) For substances in Group A of Annex 1 to Directive 96/23/EC, if one of the following techniques are
used in the analytical procedure: HPLC coupled with full-scan diode array spectrophotometry (DAD);HPLC coupled with fluorescence detection; HPLC coupled to an immunogram; two-dimensional TLCcoupled to spectrometric detection; a maximum of one identification point may be contributed,
providing that the relevant criteria for these techniques are fulfilled.
(5) Transition products include both daughter and granddaughter products.
Table6
Examples of the number of identification points earned for a range of techniques and
combinationsthereof(n=aninteger)
Technique(s) Number of ions Identification points
GC-MS (EI or CI) N n
GC-MS (EI and CI) 2 (EI) + 2 (CI) 42002D0657 — EN — 15.03.2003 — 001.001 — 12
▼B
Technique(s) Number of ions Identification points
GC-MS (EI or CI) 2 deriva-
tives2 (Derivative A) + 2 (Deri-
vative B)4
LC-MS N n
GC-MS-MS 1 precursor and 2 daugh-
ters4
LC-MS-MS 1 precursor and 2 daugh-
ters4
GC-MS-MS 2 precursor ions, each with
1 daughter5
LC-MS-MS 2 precursor ions, each with
1 daughter5
LC-MS-MS-MS 1 precursor, 1 daughter and
2 granddaughters5,5
HRMS N 2 n
GC-MS and LC-MS 2 + 2 4
GC-MS and HRMS 2 + 1 4
2.3.4.Performance criteria and other requirements for chromatography
coupledtoinfrareddetection
Adequate peaks: Adequate peaks are absorption maxima in the
infrared spectrum of a calibration standard fulfilling the following
requirements.
2.3.4.1.Infra-red detection
Absorption maximum: This shall be in the wavenumber range
4 000-500 cm-1.
Intensity of absorption: This shall not be less than either:
(a) a specific molar absorbance of 40 with respect to peak base line; or
(b) a relative absorbance of 12,5 % of the absorbance of the most
intense peak in the region 4 000-500 cm-1
when both are measured with respect to zero absorbance, and 5 % ofthe absorbance of the most intense peak in the region 4 000-500 cm
-1
when both are measured with respect to their peak base line.
Note: Although adequate peaksaccording to (a) may be preferred from
a theoretical point of view, those according to (b) are easier to deter-
mine in practice.
The number of peaks in the infrared spectrum of the analyte whose
frequencies correspond with an adequate peak in the spectrum of the
calibration standard, within a margin of ± 1 cm-1isdetermined.
2.3.4.2.Interpretation of infra-red spectral data
Absorption shall be present in all regions of the analyte spectrum which
correspond with an adequate peak in the reference spectrum of the cali-
bration standard. A minimum of six adequate peaks is required in the
infrared spectrum of the calibration standard. If there are less than six
adequate peaks (7), the spectrum at issue cannot be used as a reference
spectrum. The ‘score’, i.e. the percentage of the adequate peaks found
in the infrared spectrum of the analyte, shall be at least 50. Where there
isno exact match for an adequate peak, the relevant region of the
analyte spectrum shall be consistent with the presence of a matching
peak. The procedure isonly applicable to abs orption peaksin the
sample spectrum with an intensity of a least three times the peak to
peak noise.
2.3.5.Performance criteria andotherrequirementsforthedetermination
ofananalyteusingLCwithotherdetectiontechniques
2.3.5.1.Chromatographic separation
An internal standard shall be used if a material suitable for this purpose
is available. It shall preferably be a related standard with a retention
time close to that of the analyte. The analyte shall elute at the retention
time that is typical for the corresponding calibration standard under the2002D0657 — EN — 15.03.2003 — 001.001 — 13
▼B
same experimental conditions. The minimum acceptable retention time
for an analyte shall be two times the retention time corresponding to the
void volume of the column. The ratio of the retention time of the
analyte to that of the internal standard, i.e. the relative retention time
of the analyte, shall be the same as that of the calibration standard in
the appropriate matrix, within a margin of ± 2,5 %.
2.3.5.2.Full-scan UV/VIS detection
The performance criteria for LC methodshave to be fulfilled.
The absorption maxima in the spectrum of the analyte shall be at the
same wavelengths as those of the calibration standard within a margin
determined by the resolution of the detection system. For diode array
detection, thisistypically within ± 2 nm. The s pectrum of the analyte
above 220 nm shall, for those parts of the two spectra with a relative
absorbance ≥10 %, not be visibly different from the spectrum of the
calibration standard. This criterion is met when firstly the same maxima
are present and secondly when the difference between the two spectra is
at no point observed greater than 10 % of the absorbance of the calibra-
tion standard. In the case computer-aided library searching and
matching are used, the comparison of the spectral data in the test
samples to that of the calibration solution has to exceed a critical match
factor. This factor shall be determined during the validation process for
every analyte on the basis of spectra for which the criteria described
above are fulfilled. Variability in the spectra caused by the sample
matrix and the detector performance shall be checked.
2.3.5.3.Performance criteria for fluorimetric detection
The performance criteria for LC methodshave to be fulfilled.Thisappliesto moleculesthat exhibit native fluores cence and to mole-
cules that exhibit fluorescence after either transformation or
derivatisation. The selection of the excitation and emission wavelengths
in combination with the chromatographic conditionss hall be done in
such a way to minimise the occurrence of interfering components in
blank sample extracts.
The nearest peak maximum in the chromatogram shall be separated
from the designated analyte peak by at least one full peak width at
10 % of the maximum height of the analyte peak.
2.3.5.4.Performance criteria for the determination of an analyte by an LC-
immunogram
A LC immunogram is not suitable on its own for use as a confirmatory
method.
Relevant criteria for LC methodshave to be fulfilled.
The pre-defined quality control parameters, e.g. non-specific binding,
the relative binding of the control samples, the absorbance value of
the blank have to be within the limitsobtained during validation of
the assay.
The immunogram has to be constructed of at least five fractions.
Each fraction shall be less than half of the peak width.
The fraction with the maximum content of the analyte hasto be the
same for the suspect sample, the non-compliant control sample and the
standard.
2.3.5.5.Determination of an analyte using LC with UV/VIS detection (single
wavelength)
LC with UV/VIS detection (single wavelength) is not suitable on its
own for use as a confirmatory method.
The nearest peak maximum in the chromatogram shall be separated
from the designated analyte peak by at least one full peak width at
10 % of the maximum height of the analyte peak.
2.3.6.Performance criteria andotherrequirementsforthedetermination
of an analyte by 2-DTLC coupled to full-scan UV/VIS spectro-
metric detection
Two-dimensional HPTLC and co-chromatography are mandatory.The RF valuesof the analyte s hall agree with the RF valuesof the s tan-
dardswithin ±5 %.2002D0657 — EN — 15.03.2003 — 001.001 — 14
▼B
The visual appearance of the analyte shall be indistinguishable from
that of the standard.
For spots of the same colour the centre of the nearest spot should be
shall separated from the centre of the spot of the analyte by at least
half the sum of the spot diameters.
The spectrum of the analyte shall not be visually different from the
spectrum of the standard, as described for full-scan UV/VIS detection.
In the case computer-aided library searching and matching are used, the
comparison of the spectral data in the test samples to that of the calibra-
tion solution has to exceed a critical match factor. This factor shall be
determined during the validation process for every analyte on the basis
of spectra for which the criteria described above are fulfilled. Varia-
bility in the spectra caused by the sample matrix and the detector
performance shall be checked.
2.3.7.Performance criteria andrequirements forthedetermination ofan
analyte by GC in combination with electron capture detection
(ECD)
An internal standard shall be used if a material suitable for this purpose
is available. It shall preferably be a related substance with a retention
time close to that of the analyte. The analyte shall elute at a retention
time which is typical for the corresponding calibration standard under
the same experimental conditions. The minimum acceptable retention
time for an analyte shall be two times the retention time corresponding
to the void volume of the column. The ratio of the retention time of the
analyte to that of the internal standard, i.e. the relative retention time of
the analyte, shall be the same as that of the calibration standard in the
appropriate matrix, within a margin of ± 0,5 %. The nearest peak
maximum in the chromatogram shall be separated from the designated
analyte peak by at least one full peak width at 10 % of the maximum
height of the analyte peak. For additional information, co-chromato-
graphy may be used.
2.4. CONFIRMATORY METHODS FOR ELEMENTS
Confirmatory analyses for chemical elements shall be based on the concept of
unequivocal identification and accurate aswell asprecis e quantification by
meansof phys ical-chemical propertiesunique to the chemical element at hand
(e.g. element characteristic wavelength of emitted or absorbed radiation, atomic
mass) at the level of interest.
The following methodsor combinationsof methodsare cons idered s uitable for
the identification of chemical elements:
Table7
Suitableconfirmatory methodsforchemical elements
Technique Measured parameter
Differential pulse anodic stripping voltametry Electric signal
Atomic absorption spectrometry
Flame Absorption wavelengthHydride generation Absorption wavelength
Cold vapour Absorption wavelength
Electrothermal atomisation (graphite furnace) Absorption wavelength
Atomic emission spectrometry
Inductively coupled plasma Emission wavelength
Mass spectrometry
Inductively coupled plasma Mass-to-charge-ratio
2.4.1.Common performance criteria and other requirements for confir-
matorymethods
Reference or fortified material containing known amountsof analyte, at
or near either the maximum permitted limit or the decision limit (non-
compliant control sample) as well as compliant control materials and
reagent blankss hould preferably be carried through the entire procedure
simultaneously with each batch of test samples analysed. The recom-2002D0657 — EN — 15.03.2003 — 001.001 — 15
▼B
mended order for injecting the extractsinto the analytical ins trument is
as follows: reagent blank, compliant control sample, sample to be
confirmed, compliant control sample and finally non-compliant control
sample. Any variation from this shall be justified.
In general, most analytical techniques require complete digestion of the
organic matrix to obtain solutions prior to determination of the analyte.
This can be achieved by using microwave mineralisation procedures,
which minimise the risk of loss and/or contamination of the analytes
of interest. Decontaminated Teflon vessels of good quality shall be
used. If other wet or dry digestion methods are resorted to, documented
evidence shall be available to exclude potential loss or contamination
phenomena. As an alternative to digestion, separation procedures (e.g.
extraction) may under certain circumstances be chosen to separate
analytesfrom matrix componentsand/or to concentrate analytesin
order to introduce them into the analytical equipment.
As regards calibration, be it external or based on the standard addition
method, care shall be taken not to exceed the working range established
for the analysis. In the case of external calibration, it is mandatory that
calibration standards are prepared in a solution that matches as closely
as possible the composition of the sample solution. Background correc-
tion shall be also applied if required by specific analytical
circumstances.
2.4.2.Additionalperformancecriteriaandotherrequirementsforquanti-
tativemethodsofanalysis
2.4.2.1.Trueness of quantitative methods
In the case of repeated analyses of a certified reference material for
elements, the deviation of the experimentally determined mean content
from the certified value shall not lie outside the limit ± 10 %. When no
such CRMs are available, it is acceptable that trueness of measurements
is assessed through recovery of additions of known amounts of the
element to the unknown samples. Attention is drawn to the fact that,
unlike the analyte, the added element isnot chemically bound in thereal matrix and that therefore results obtained by this approach have
lesser validity than those achieved through the use of CRMs. Recovery
data are only acceptable when they are within ± 10 % of the target
value.
2.4.2.2.Precision of quantitative methods
In the case of repeated analysis of a sample carried out under within-
laboratory reproducibility conditions, the intra-laboratory coefficient of
variation (CV) of the mean shall not exceed the following values:
Table8
CVsforquantitativemethodsatarangeofelementmassfractions
Mass fraction CV (%)
≥10µg/kg to 100 µg/kg 20
> 100 µg/kg to 1 000 µg/kg 15
≥1 000 µg/kg 10
2.4.3.Specific requirements for differential pulse anodic stripping volta-
metry(DPASV)
Complete destruction of organic matter in samples prior to DPASV
determinations is of the greatest importance. No broad signals due to
the presence of organic materials shall be seen in the voltamograms.
Inorganic matrix constituents may influence peak heights in DPASV.
Therefore, quantification hasto be done by the method of s tandard
additions. Specimens of typical voltamograms of a sample solution
shall be supplied with the method.
2.4.4.Specificrequirementsforatomicabsorptionspectrometry(AAS)
Thistechnique isbas ically mono-elemental and requirestherefore opti-
misation of the experimental settings depending on the particularelement to be quantified. Wherever possible, results shall be checked
qualitatively and quantitatively by resorting to alternative absorption
lines (ideally, two different lines shall be selected). Calibration stan-
dards shall be prepared in a solution matrix that matches as closely as
possible that of the sample measurement solution (e.g. acid concentra-2002D0657 — EN — 15.03.2003 — 001.001 — 16
▼B
tion or modifier composition). To minimise blank values, all reagents
shall be of the highest available purity. Depending on the mode chosen
to vaporise and/or atomise the sample, various types of AAS can be
distinguished.
2.4.4.1.Specific requirements for flame AAS
The instrument settings shall be optimised for each element. Especially
the gascompos ition and flow rateshave to be checked. A continuum
source corrector shall be used to avoid interferences caused by back-
ground absorption. In the case of unknown matrices, a check shall be
made asto whether or not background correction isrequired.
2.4.4.2.Specific requirements for graphite furnace AAS
Contamination in the laboratory often affectsaccuracy when working at
ultra-trace levelsin the graphite furnace. Therefore high purity reagents ,
deionised water and inert plastic ware for sample and standard handling
should be used. The instrument settings for each element shall be opti-
mised. Especially the pre-treatment- and atomisation-conditions
(temperature, time) and the matrix modification have to be checked.
Working under isothermal atomisation conditions (e.g. transverse
0heated graphite tube with integrated Lvov platform (8) will reduce
the influence of the matrix concerning the atomisation of the analyte.
In combination with matrix modification and Zeeman-background
correction (9), quantification by meansof a calibration curve bas ed
upon measuring of aqueous standard solutions will be allowed.
2.4.5.Specific requirements for hydride generation atomic absorption
spectrometry
Organic compounds containing elements such as arsenic, bismuth,
germanium, lead, antimony, selenium, tin and tellurium can be very
stable and require oxidative decomposition to obtain correct results for
total element content. Therefore, microwave digestion or high-pressure
ashing under strong oxidative conditions is recommended. The greatest
care shall be devoted to the complete and reproducible conversion of
the elements into their corresponding hydrides.
The formation of arsenic hydride in hydrochloric acid solution with
NaBH4depends on the oxidation state of arsenic (As III: fast formation,
As V: longer formation period). To avoid a loss of sensitivity for the
determination of AsV with flow injection technique, caus ed by the
short reaction time in this system, As V has to be reduced to As III
after the oxidative decomposition. Potassium iodide/ascorbic acid or
cysteine are suitable for this purpose. Blanks, calibration solutions and
sample solutions shall be treated in the same way. Working with a
batch system allows determining both arsenic species without affecting
accuracy. Due to the delayed formation of AsV-hydride, calibration
shall be performed by peak area integration. The instrument settings
shall be optimised. The gas flow, which transfers the hydride to the
atomisator, is especially important and shall be checked.
2.4.6.Specific requirements for cold vapour atomic absorption spectro-
metry
Cold vapour is used only in the case of mercury. Due to volatilisation
and adsorption losses of elemental mercury, special care is necessary
during the whole analysis. Contamination by reagents or the environ-
ment hasto be avoided carefully.
Organic compoundscontaining mercury require oxidative decompos i-
tion to obtain correct results for total mercury content. For
decomposition, sealed systems with microwave digestion or high pres-
sure asher are to be used. Special care is required for cleaning the
equipment that had contact with mercury.
Working with the flow injection technique isadvantageous . For lower
decision limits, adsorption of elemental mercury on gold/platinum
adsorber followed by thermal desorption is recommended. Contact ofthe adsorber or the cell with moisture will disturb the measurement
and shall be avoided.
2.4.7.Specific requirements for inductively coupled plasma atomic emis-
sionspectrometry(ICP-AES)
Inductively coupled plasma atomic emission spectrometry (10) is a
multi-element method, which allows a simultaneous measurement of
various elements. To use the ICP-AES, the samples first have to be
digested to decompose organic matrices. Sealed systems with micro-
wave digestion or high pressure ashing shall be used. For a2002D0657 — EN — 15.03.2003 — 001.001 — 17
▼B
meaningful ICP-AES analysis, the instrument calibration and element or
wavelength selection play an essential role. For instrument calibration,
in case of linear calibration curves, it is usually necessary to measure
calibration solutions of only four concentrations, because ICP-AES cali-
bration curvesare generally linear over four to s ix ordersof magnitude
of concentration. Calibration of the ICP-AES system should normally
be performed with a multi-element standard, which shall be prepared
in a solution that contains the same acid concentration as the measure-
ment solution. For the linear curve, the element concentrations shall be
checked.
The selection of wavelengths for measurement of the emission from the
analytesisappropriate for the concentrationsof the elementsto be
determined. When the analyte concentration fallsouts ide of the working
range of an emission line, a different emission line shall be used. At
first, the most sensitive emission line (not interfered) shall be chosen,
then a less sensitive line. When working at or near the detection limit,
the most sensitive line for the respective analyte is usually the best
choice. Spectral and background interferencesare caus ing the major
difficulties in ICP-AES. Possible interferences are e.g. simple back-
ground shift, sloping background shift, direct spectral overlap and
complex background shift. Each of these interferences has its own
causes and remedies. Depending on the matrices, interference correc-
tions and optimisation of operating parameters shall be applied. Some
interferencescan be avoided by dilution or by adaptation of the
matrices. With each batch of test samples analysed, reference and forti-
fied material containing known amountsof the analyte(s ) aswell as
blank material shall be treated in the same way as the test samples.
For testing for a drift, the standard shall be checked e.g. after 10
samples. All reagents and the plasma gas shall be of the highest avail-
able purity.
2.4.8.Specific requirements for inductively coupled mass spectrometry
(ICP-MS)(11))
The determination of trace elements of average atomic mass, such as
chromium, copper and nickel may be subject to strong interference
from other isobaric and polyatomic ions. This can be circumvented
only when a resolution power of at least 7 000-8 000 is available. Diffi-
culties associated with the MS techniques include instrumental drift,
matrix effectsand molecular ion interference (m/z < 80). Multiple
internal standardisation covering the same mass range as the elements
to be determined isrequired to correct ins trumental drift and matrix
effects.
Complete decomposition of organic matter in samples prior to ICP-MS
measurements is required. As in the AAS, after digestion in sealed
vessels, volatile elements e.g. iodine are to be transferred to a stable
oxidation state. Most severe interference results from molecular ion
combinations of argon (plasma gas), hydrogen, carbon, nitrogen and
oxygen (dissolution acids, plasma gas impurities and entrained atmo-
spheric gases) and the sample matrix. Complete digestion, background
measurements, appropriate choice of analytical masses sometimes asso-
ciated with a lower abundance (poorer detection limit) and of
dissolution acids, e.g. nitric acid, are required to avoid interferences.
For the elementsto be determined, interferencesare to be excluded by
the appropriate choice of specific analytical masses including confirma-
tion of isotope ratios. Instrument response considering Fano-factors
shall be checked for each measurement by the use of internal standards.
3.VALIDATION
Validation shall demonstrate that the analytical method complies with the
criteria applicable for the relevant performance characteristics.
Different control purposes require different categories of methods. The
following table determines which performance characteristic shall be verified
for which type of method.2002D0657 — EN — 15.03.2003 — 001.001 — 18
▼B
Table9
Classification of analytical methods by the performance characteristics that have to be deter-
mined
Detection
limit CCßDecision
limit CC αTrueness/
recoveryPrecisionSelectivity/
specificityApplic-
ability/
ruggedness/
stability
Qualitative
methodsS+–––++
C++––++
Quantita-
tive
methodsS+––+++
C++++++
S = screening methods; C = confirmatory methods; + = determination is mandatory.
3.1. VALIDATION PROCEDURES
Thischapter providesexamplesand/or referencesfor validation proceduresof
analytical methods. Other approaches to demonstrate that the analytical method
complieswith performance criteria for the performance characteris ticsmay be
used, provided that they achieve the same level and quality of information.
Validation can also be performed by conducting an interlaboratory study such as
established by Codex Alimentarius, ISO or the IUPAC (12), or according toalternative methods such as single laboratory studies or in-house validation
(13)(14). This part focuses on single laboratory studies (on in-house validation)
using a modular approach. This approach consists of:
1. a set of common performance characteristics independent of the validation
model used and
2. more specific model-dependent procedures as described in Table 10.
Table10
Model-independentandmodel-dependentperformance parameters
Validation
Model-independent performance
parametersModel-dependent performance parameters
Common performance charac-
teristics (3.1.1)Conventional validation
approach (3.1.2)In-house validation approach
(3.1.3)
Specificity Recovery Recovery
Trueness Repeatability Repeatability
Ruggedness: minor changes Within-laboratory reproduci-
bilityWithin-laboratory reproduci-
bility
Stability Reproducibility Reproducibility
Decision limit (CC α) Decision limit (CC α)
Detection capability (CC β) Detection capability (CC β)
Calibration curvesCalibration curve
Ruggedness: major changes Ruggedness
3.1.1.Model-independentperformancecharacteristics
Irrespective of the validation approach chosen, the following perfor-
mance characteristics have to be determined. To minimise the
workload, a carefully designed and statistically sound approach can be
used to combine experiments performed to determine different para-
meters.
3.1.1.1.Specificity
For analytical methods, the power of discrimination between the analyte
and closely related substances (isomers, metabolites, degradation2002D0657 — EN — 15.03.2003 — 001.001 — 19
▼B
products, endogenous substances, matrix constituents, etc) is important.
Two approaches are necessary to check for interferences.
Therefore, potentially interfering substances shall be chosen and rele-
vant blank samples shall be analysed to detect the presence of possible
interferences and to estimate the effect of the interferences:
— select a range of chemically related compounds (metabolites, deri-
vatives, etc.) or other substances likely to be encountered with the
compound of interest that may be present in the samples;
— analyse an appropriate number of representative blank samples (n
≥20) and check for any interferences (signals, peaks, ion traces)
in the region of interest where the target analyte is expected to
elute;
— additionally, representative blank samples shall be fortified at a rele-
vant concentration with substances that are likely to interfere with
the identification and/or quantification of the analyte;
— after analysis, investigate whether:
— the presence may lead to a false identification,
— the identification of the target analyte ishindered by the
presence of one or more of the interferences, or
— the quantification isinfluenced notably.
3.1.1.2.Trueness
In thisparagraph, the determination of truenes s(one component of
accuracy) is described. Trueness can only be established by means of
certified reference material (CRM). A CRM be used whenever avail-
able. The procedure isdes cribed in detail in ISO 5725-4 (5). An
example isgiven below:
— analyse six replicates of the CRM in accordance with the test
instructions for the method,
— determine the concentration of the analyte present in each sample of
the replicates,
— calculate the mean, the standard deviation and the coefficient of
variation (%) for these concentrations,
— calculate the trueness by dividing the detected mean concentration
by the certified value (measured as concentration) and multiply by
100, to express the result as a percentage.
Trueness ( %) = mean recovery-corrected concentration detected × 100/
certified value.
If no CRM is available, instead of trueness, the recovery can be deter-
mined asdes cribed under 4.1.2.1 below.
3.1.1.3.Applicability/ruggedness (minor changes)
Such studies use the deliberate introduction of minor reasonable varia-
tions by the laboratory and the observation of their consequences.
The pre-investigative studies have to be carried out by selecting factors
of the sample pre-treatment, clean up and analysis, which may influ-
ence the measurement results. Such factors may include the analyst,
the source and the age of reagents, solvents, standards and sample
extracts, the rate of heating, the temperature, the pH-value as well as
many other factorsthat may occur in the laboratory. Thes e factors
should be modified in an order of magnitude that matches the devia-
tions usually encountered among laboratories.
— Identify possible factors that could influence the results.
— Vary each factor slightly.— Conduct a ruggedness test using the approach of Youden (15)(16).
(Other approved methodsmay be us ed at thispoint. The Youden
approach, however, keepsthe required time and effort to a
minimum). The Youden approach isa fractional factorial des ign.
Interactionsbetween the different factorscannot be detected.
— Where a factor is found to influence the measurement results signif-
icantly, conduct further experimentsto decide on the acceptability
limitsof thisfactor.
— Factors that significantly influence the results should be identified
clearly in the method protocol.
The basic idea is not to study one alteration at a time but to introduce
several variations at once. As an example, let A, B, C, D, E, F, G
denote the nominal valuesfor s even different factorsthat could influ-
ence the results, if their nominal values are changed slightly. Let their
alternative values be denoted by the corresponding lower case letters a,2002D0657 — EN — 15.03.2003 — 001.001 — 20
▼B
b, c, d, e, f and g. Thisres ultsin 27or 128 different possible combina-
tions.
It is possible to choose a subset of eight of these combinations that
have a balance between capital and small letters (Table 11). Eight
determinationshave to be made, which will us e a combination of the
chosen factors (A-G). The results of the determinations are shown in
Table 11 below asS-Z.
Table11
Experimentdesignforruggednessstudies(minorchanges)
Factor value
FCombination of determinationsnumber
12345678
A / a AAAAa a a a
B / b BBbbBBb b
C / c CcCcCcCc
D / d D DddddD D
E/e E e E e e E e E
F / f FffFFffF
G/g G g g G g G G g
Observed
result RSTUV W XYZ
For calculations see examples for ruggedness testing in 3.3.
3.1.1.4.Stability
It has been observed that insufficient stability of the analyte or matrix
constituents in the sample during storage or analysis may give rise to
significant deviations in the outcome of the result of analysis. Further-
more, the stability of the calibration standard in solution should be
checked. Usually analyte stability is well characterised under various
storage conditions. Monitoring of the storage condition will form part
of the normal laboratory accreditation system. When this is not known,
examplesare given below on how the s tability can be determined.
Stability of the analyte in solution:— Prepare fresh stock solutions of the analyte(s) and dilute as specified
in the test instructions to yield sufficient aliquots (e.g. 40) of each
selected concentration (around the minimum required performance
limit for substances for which no permitted limit has been estab-
lished or around the permitted limit for other substances. Prepare
both solutions of the analyte used for fortification and used in the
final analysis solution, and any other solution that is of interest
(e.g. derivatised standards).
— Measure the analyte content in the freshly prepared solution
according to the test instructions.
— Dispense appropriate volumes into suitable containers, label and
store according to the scheme:
Table12
Schemeforthedeterminationofanalytestabilityinsolution
–2 0º C +4º C +2 0º C
Dark 10 aliquots10 aliquots10 aliquots
Light 10 aliquots
— The storing time could be selected as one, two, three and four
weeks or longer if necessary, e.g. until the first degradation
phenomena are observable during identification and/or quantifica-
tion. The maximum storing time and the optimum storing
conditionshave to be recorded.2002D0657 — EN — 15.03.2003 — 001.001 — 21
▼B
— The calculation of the concentration of the analyte(s) in each aliquot
should be carried out by using the solution of the analyte freshly
prepared at the time of analysis as 100 %.
Analyte Remaining (%) = Ci× 100/Cfresh
Ci= concentration at time point
Cfresh= concentration of fresh solution
Stability of analyte(s) in matrix
— Whenever possible, incurred samples should be used. When no
incurred material isavailable, matrix fortified with the analyte
should be used.
— When incurred material isavailable, the concentration in the mate-
rial should be determined while the material is still fresh. Further
aliquotsof material could be taken after one, two, four and 20
weeks and the concentrations should be determined. The tissue
should be stored at least minus 20 ºC or lower if required.
— If no incurred material isavailable, take s ome blank material and
homogenise it. Divide the material into five aliquots. Fortify each
aliquot with the analyte, which should preferably be prepared in a
small quantity of aqueous solution. Analyse one aliquot immedi-
ately. Store the remaining aliquotsat leas t minus20 ºC or lower if
required and analyse them after one, two, four and 20 weeks.
3.1.1.5.Calibration curves
When calibration curvesare us ed for quantification:
— at least five levels (including zero) should be used in the construc-
tion of the curve,
— the working range of the curve should be described,
— the mathematical formula of the curve and the goodness-of-fit of the
data to the curve should be described,
— acceptability rangesfor the parametersof the curve s hould be
described.
When serial calibration based on a standard solution is necessary,
acceptable rangess hall be indicated for the parametersof the calibra-
tion curve, which may vary from series to series.
3.1.2.Conventionalvalidationprocedures
The calculation of the parametersin accordance with conventional
methods requires the performance of several individual experiments.
Each performance characteristic has to be determined for each major
change (see under applicability/ruggedness above). For multi-analyte
methods, several analytes can be analysed simultaneously, as long as
possibly relevant interferences are ruled out previously. Several perfor-
mance characteristics can be determined in a similar way. So, to
minimise workload, it is advised to combine experiments as much as
possible (e.g., repeatability and within-laboratory reproducibility with
specificity, analysis of blank samples to determine the decision limit
and testing for specificity).
3.1.2.1.Recovery
If there isno CRM available, the recovery hasto be determined by
experiments using fortified blank matrix using, for example, the
following scheme:
— select 18 aliquots of a blank material and fortify six aliquots at each
of 1, 1,5 and 2 timesthe minimum required performance limit or
0,5, 1 and 1,5 timesthe permitted limit,
— analyse the samples and calculate the concentration present in each
sample,
— using the equation below, calculate the recovery for each sample,
— calculate the mean recovery and CV from the six results at each
level,
— % Recovery = 100 × measured content/fortification level.2002D0657 — EN — 15.03.2003 — 001.001 — 22
▼B
Thisconventional method for the determination of recovery isa variant
of the standard addition method described in 3.5, when:
— the sample is considered as a blank sample instead of a sample to
be analysed,
— it iscons idered that yield (1) and recovery (2) are similar for the two
test portions,
— the test samples have the same masses and the test portion extracts
the same volumes,
— the amount of the calibration standard that is added to the second
(spiked) test portion is noted xADD.( xADD=ρA.VA),
— x1 is the measured value for the blank and x2 the measured value
for the second (spiked) test portion,
— then, % Recovery = 100 (x2–x1)/xADD.
When any of the above conditionsis(or iss uppos ed) not to be
achieved, then the complete procedure for determination of the recovery
by mean of the standard addition method as described in 3.5 has to be
performed.
3.1.2.2.Repeatability
— Prepare a set of samples of identical matrices, fortified with the
analyte to yield concentrationsequivalent to 1, 1,5 and 2 timesthe
minimum required performance limit or 0,5, 1 and 1,5 timesthe
permitted limit.
— At each level the analysis should be performed with at least six
replicates.
— Analyse the samples.— Calculate the concentration detected in each sample.— Find the mean concentration, standard deviation and the coefficient
of variation (%) of the fortified samples.
— Repeat these steps on at least two other occasions.
— Calculate the overall mean concentrationsand CVsfor the fortified
samples.
3.1.2.3.Within-laboratory reproducibility
— Prepare a set of samples of specified test material (identical or
different matrices), fortified with the analyte(s) to yield concentra-
tionsequivalent to 1, 1,5 and 2 timesthe minimum required
performance limit or 0,5, 1 and 1,5 timesthe permitted limit.
— At each level the analysis should be performed with at least six
replicates.
— Repeat these steps on at least two other occasions with different
operatorsand different environmental conditions , e.g. different
batches of reagents, solvents etc., different room temperatures,
different instruments, etc. if possible.
— Analyse the samples.
— Calculate the concentration detected in each sample.— Find the mean concentration, standard deviation and the coefficient
of variation ( %) of the fortified samples.
3.1.2.4.Reproducibility
When reproducibility hasto be verified, laboratoriess hould participate
in collaborative studies according to ISO 5725-2 (5).
3.1.2.5.Decision Limit (CC α)
The decision limit has to be established according to the requirements
for identification or identification plusquantification asdefined under
‘Performance criteria and other requirementsfor analytical methods ’
(part 2).
In the case of substances for which no permitted limit has been estab-
lished, CC αcan be established:
— either by the calibration curve procedure according to ISO 11843
(17) (here referred to ascritical value of the net s tate variable). In
this case blank material shall be used, which is fortified at and2002D0657 — EN — 15.03.2003 — 001.001 — 23
(1) Yield: that fraction of mass of the analyte contained in the sample, which is present in
the final extract.
(2) Recovery (here): that fraction of mass of the analyte added to the sample, which is
present in the final extract. Throughout the rest of the document it is assumed that yield
and recovery are equal and therefore only the term ‘recovery’ isus ed.
▼B
above the minimum required performance level in equidistant steps.
Analyse the samples. After identification, plot the signal against the
added concentration. The corresponding concentration at the y-inter-
cept plus2,33 timesthe s tandard deviation of the within-laboratory
reproducibility of the intercept equalsthe decis ion limit. Thisis
applicable to quantitative assays only ( α= 1 %),
— or by analysing at least 20 blank materials per matrix to be able to
calculate the signal to noise ratio at the time window in which the
analyte is expected. Three times the signal to noise ratio can be
used as decision limit. This is applicable to quantitative and qualita-
tive assays.
In the case of substances an with established permitted limit, CC αcan
be established:
— either by the calibration curve procedure according to ISO 11843
(17) (here referred to ascritical value of the net s tate variable). In
this case blank material shall be used, which is fortified around the
permitted limit in equidistant steps. Analyse the samples. After
identification, plot the signal against the added concentration. The
corresponding concentration at the permitted limit plus 1,64 times
the standard deviation of the within-laboratory reproducibility
equalsthe decis ion limit ( α= 5 %),
— or by analysing at least 20 blank materials per matrix fortified with
the analyte(s) at the permitted limit. The concentration at the
permitted limit plus 1,64 times the corresponding standard deviation
equal the decision limit ( α= 5 %).
See also Article 5 and point 3.2.
3.1.2.6.Detection capability (CC β)
The detection capability should be determined according to the require-
mentsfor s creening, identification or identification plusquantification
asdefined (s ee part 2).
In the case of substances for which no permitted limit has been estab-
lished, CC βcan be established by:
— the calibration curve procedure according to ISO 11843 (17) (here
referred to asminimum detectable value of the net s tate variable).
In this case representative blank material shall be used, which is
fortified at and below the minimum required performance level in
equidistant steps. Analyse the samples. After identification, plot the
signal against the added concentration. The corresponding concen-
tration at the decision limit plus 1,64 times the standard deviation
of the within-laboratory reproducibility of the mean measured
content at the decision limit equals the detection capability ( β= 5 %),
— analysing at least 20 blank materials per matrix fortified with the
analyte(s) at the decision limit. Analyse the samples and identify
the analytes. The value of the decision limit plus 1,64 times the
standard deviation of the within-laboratory reproducibility of the
measured content equals the detection capability ( β= 5 %),
— where no quantitative results are available, the detection capability
can be determined by the investigation of fortified blank material
at and above the decision limit. In this case the concentration level,
where only ≤5 % false compliant results remain, equals the detec-
tion capability of the method. Therefore, at least 20 investigations
for at least one concentration level have to be carried out in order
to ensure a reliable basis for this determination.
In the case of substances for which a permitted limit has been estab-
lished, CC βcan be established:
— either by the calibration curve procedure according to ISO 11843
(17) (here referred to asminimum detectable value of the net s tate
variable). In this case representative blank material shall be used,
which is fortified around the permitted limit in equidistant steps.
Analyse the samples and identify the analyte(s). Calculate the stan-dard deviation of the mean measured content at the decision limit.
The corresponding concentration at the value of the decision limit
plus1,64 timesthe s tandard deviation of the within-laboratory
reproducibility equalsthe detection capability ( β= 5 %),
— or by analysing at least 20 blank materials per matrix fortified with
the analyte(s) at the decision limit. The value of the decision limit
plus 1,64 times the corresponding standard deviation equals the
detection capability ( β= 5 %).
See also section 3.2.2002D0657 — EN — 15.03.2003 — 001.001 — 24
▼B
3.1.2.7.Ruggedness (major changes)
The analytical method should be tested under different experimental
conditions, which include for example, different species, different
matrices or different sampling conditions. The changes introduced
should be major. The importance of these changes can be evaluated,
for instance, using the Youden approach (15)(16). Each performance
characteristic should be determined for all major changes that have
been shown to have a significant effect on the performance of the
assay.
3.1.3.Validationaccordingtoalternative models
When alternative validation proceduresare applied, the underlying
model and strategy with the respective prerequisites, assumptions and
formulae shall be laid down in the validation protocol or at least refer-
encess hall be given to their availability. In the following an example
for an alternative approach isgiven. When applying e.g. the in-hous e
validation model, the performance characteristics are determined in a
manner that permitsvalidation for major changeswithin the s ame vali-
dation procedure. Thisrequiresdes ign of an experimental plan for
validation.
3.1.3.1.Experimental plan
An experimental plan hasto be des igned depending on the number of
different species and different factors under investigation. Hence, the
first step of the entire validation procedure shall consider the sample
populationsthat will be analys ed in the laboratory in the future in order
to select the most important species and those factors which may influ-
ence the measurement results. Subsequently, the concentration range
shall be chosen in a purpose-adapted way according to the level of
interest.
Example:
— several analytes can be investigated simultaneously with the analy-
tical method being validated,
— two variationsof the leading factor have been identified (A and B).
Leading factorsform the bas ison which the factor levelsare
combined. These leading factors may include factors such as species
or matrix. In thisexample the leading factor wasvaried on two
levels, i.e. two different species (species A and B) were considered.
In general, it is possible to vary the leading factors on more than
two levels, which only increase the number of analyses to be
performed,
— the selected factors are to be varied on two levels (indicated as
either + or –).
Table13
Examplesforfactorsconsideredimportantforavalidationprocedure
Gender of the animal (factor 1)
Breed (factor 2)
Transport conditions (factor 3)Storing conditions(factor 4)
Freshness of the sample (factor 5)
Fattening conditions(factor 6)Different operatorswith different experience (factor 7).
Table14
Possibleexperimentalplanfortheaboveexample
SpeciesFactor 1 Factor 2 Factor 3 Factor 4 Factor 5 Factor 6 Factor 7Sample
No
A ++++–+– 1
A+ + – – + – – 2A +–+–––+ 3
A +––++++ 42002D0657 — EN — 15.03.2003 — 001.001 — 25
▼B
SpeciesFactor 1 Factor 2 Factor 3 Factor 4 Factor 5 Factor 6 Factor 7Sample
No
A– + + – + + + 5
A –+–+––+ 6
A ––+++–– 7
A –––––+– 8
B +++++–+ 9B+ + – – – + + 1 0
B +–+–++–1 1
B+ – – + – – – 1 2
B –++–––– 1 3
B –+–+++–1 4
B– – + + – + + 1 5
B ––––+–+ 1 6
As each sample (each factor level combination) has to be spiked with
four different concentrationsaround the level of interes t, and one blank
sample shall be analysed for each level, 5 x 16 = 80 analyses have to be
performed for the entire validation experiment.
From these 80 measurement results it is possible to compute (13)(14).
Recovery
— repeatability per concentration level (s
ir),
— within-laboratory reproducibility per concentration level (sir),
— decision limit (CC α),
— detection capability (CC β),
— power curve ( β-error rate versus concentration (see 3.1.3.2),
— ruggedness of major changes; ruggedness to minor changes can be
determined according to paragraph 3.1.1.3,
— 16 sample-related calibration curves,
— one overall calibration curve,— prediction interval of the overall calibration curve,
— matrix-induced deviations(s
mat),
— run-induced deviations(srun),
— effect of the individual factors on the measurement results.
These performance characteristics allow the comprehensive evaluation
of the performance level of the method, since not only the influence
of the individual factors is investigated, but also the relevant combina-
tions of these factors. With the help of this experiment design it is
possible to decide if one or the other of the selected factors shall be
excluded from the overall calibration curve, because it significantly
deviates from the standard deviations of the other factors.
3.1.3.2.Power curve
The power curve providesinformation about the detection capability of
the method within the chosen concentration range. It refers to the β-
error risk when applying the investigated method. The power curve
allowsto calculate the detection capabilitiesfor the res pective cate-
gories(s creening, confirmation) or types(qualitative or quantitative) of
methodsfor a certain β-error (e.g. 5 %).2002D0657 — EN — 15.03.2003 — 001.001 — 26
▼B
Figure 1
Power curve
Figure 1 shows an example of the graphical establishment of detection
capability (CC β)of an analytical method. Thisparticular method hasa
remaining risk of taking a false decision of 5 % at a concentration of
0,50 µg/kg. At a concentration of 0,55 µg/kg the risk of taking a false
compliant decision decreases to 1 %.
3.1.3.3.Reproducibility
The determination of a method'sreproducibility by the s ingle laboratory
studies (in-house validation) concept requires repeated participation in
proficiency studies in accordance with ISO guide 43-1 (3) and 43-2
(4). The laboratories are allowed to choose their own methods, provided
these methods are used under routine conditions. The standard deviation
of the laboratory can be used to assess the reproducibility of the
method.2002D0657 — EN — 15.03.2003 — 001.001 — 27
▼B
3.2. GRAPHICAL REPRESENTATION OF THE DIFFERENT ANALYTICAL
LIMITS
Figure 2
Substances for which no permitted limit has been established
2002D0657 — EN — 15.03.2003 — 001.001 — 28
▼B
Figure 3
Substances with an established permitted limit
2002D0657 — EN — 15.03.2003 — 001.001 — 29
▼B
3.3. CALCULATION EXAMPLE FOR RUGGEDNESS TESTING OF MINOR
CHANGES ACCORDING TO THE YOUDEN APPROACH (16)
Comparisonofaverages(A)
AA=Σ(Ai)/4
AB=Σ(Bi)/4
AC=Σ(Ci)/4
AD=Σ(Di)/4
AE=Σ(Ei)/4
AF=Σ(Ei)/4
AG=Σ(Gi)/4
Aa=Σ(ai)/4
Ab=Σ(bi)/4
Ac=Σ(ci)/4
Ad=Σ(di)/4
Ae=Σ(ei)/4
Af=Σ(fi)/4
Ag=Σ(gi)/4Compare the averagesof the capital letters(AAto AG) with the averagesof their
corresponding small letters (Aato Ag). If a factor have an effect, the difference
will be significant larger than the differences of the other factors.
A robust method should not be affected by changes encountered almost
certainly between laboratories.
If there is no outstanding difference, the most realistic measure of the random
error is given by the seven differences.
Differences(Di) Squaresof differences(Di2)
Da=A–a= Σ(Ai)–Σ(ai)
Db=B–b= Σ(Bi)–Σ(bi)
Dc=C–c= Σ(Ci)–Σ(ci)
Dd=D–d= Σ(Di)–Σ(di)
De=E–e= Σ(Ei)–Σ(ei)
Df=F–f= Σ(Fi)–Σ(fi)
Dg=G–g= Σ(Gi)–Σ(gi)Da2= value a
Db2= value b
Dc2= value c
Dd2= value d
De2= value e
Df2= value f
Dg2= value g
Standard deviation of the differences Di(SDi):
When SDiis significantly larger than the standard deviation of the method
carried out under within-laboratory reproducibility conditionsaccording (s ee
above) it isa foregone conclus ion that all factorstogether have an effect on
the result even if every single factor does not show a significant influence and
that the method is not sufficiently robust against the chosen modifications.
3.4. CALCULATION EXAMPLES FOR THE IN-HOUSE VALIDATION
PROCEDURE
Examples and calculations for the in-house validation protocol as described
under validation according to alternative models(3.1.3) (13) (14).
3.5. EXAMPLES FOR THE STANDARD ADDITION METHOD
A test sample with a content T of the analyte is divided in two test portions 1
and 2 of respective masses m1and m2. The test portion 2 is spiked with a
volume VAof a solution of concentration ρAof the analyte. Two extractsof
the test portions of respective volumes V1and V2are obtained after extraction
and purification steps of the method. The recovery of the analyte is supposed
to be rc. Both extracts are assayed with a measurement method of sensitivity b
and give an analytical response of x1and x2respectively.
If assumed that rc and b are the same for the analyte in the native sample and in
the spiked sample, then the content T can be calculated as:
T=x1.V1.ρA.VA/(x2.V2.m1−x1.V1.m2)
The method will allow the determination of the recovery rc. Then, in addition
with the assay described above, part of the extract of the test portion 1 (volume
V3) iss piked with a known amount ρB.VBof the analyte and assayed. The analy-
tical response is x3and the recovery is:
rc = x2.V1.V2.ρB.VB/[x3.V1.V3(T.m2+ρA.VA)−x2.V2.T.m1(V3−VB)]2002D0657 — EN — 15.03.2003 — 001.001 — 30
▼B
Moreover, it is possible to calculate the sensitivity b, as:
b=x1.V1/rc.T.m1
All conditionsof application and detailshave been des cribed (18).
4.ABBREVIATIONSUSED
AAS Atomic absorption spectrometry
AES Atomic emission spectrometry
AOAC-I Association of Official Analytical Chemists INTERNATIONALB bound fraction (immunoassays)CI chemical ionisation
CRM Certified reference material
CV coefficient of variation2 D two dimensionalDAD diode array detectionDPASV differential pulse anodic stripping voltametry
ECD electron capture detection
EI electronic impact ionisationGC gaschromatographyHPLC high performance liquid chromatographyHPTLC high performance thin layer chromatography
HRMS high resolution (mass spectrometry)
ICP-AES inductively coupled plasma-atomic emission spectrometryICP-MS inductively coupled plasma-mass spectrometryIR infraredISO International Standard Organisation
LC liquid chromatography
LR(MS) low resolution (mass spectrometry)MRPL Minimum required performance limitMS mass spectrometry
m/z mass/charge ratio
RF relative migration to the solvent front (TLC)RSDL relative standard deviations of the laboratorySIM selected ion monitoringTLC thin layer chromatography
UV ultra violet light
VIS visible light
5.REFERENCES
(1) ISO 17025: 1999 General requirement for the competence of calibration
and testing laboratories.
(2) ISO 3534-1: 1993 Statistical Methods for quality control — Vol. 1
vocabulary and symbols.
(3) ISO Guide 43-1: 1997 Proficiency testing by interlaboratory compari-
sons — Part 1: Development and operation of proficiency testing
schemes.
(4) ISO Guide 43-2: 1997 Proficiency testing by interlaboratory compari-
sons — Part 2: Selection and use of proficiency testing schemes by
laboratory accreditation bodies.
(5) ISO 5725: 1994 Accuracy (trueness and precision) of measurement
methods and results — Part 1: General principles and definitions; ISO
5725-2 Part 2: Basic method for the determination of repeatability and
reproducibility of a standard measurement method; Part 4: Basic
methods for the determination of the trueness of a standard measure-
ment method.
(6) ISO 78-2: 1999 Chemistry — Layouts for standards — Part 2: Methods
of chemical analysis.2002D0657 — EN — 15.03.2003 — 001.001 — 31
▼B
(7) W.G de Ruig and J.M Weseman ‘A new approach to confirmation by
infrared spectrometry’ J. Chemometrics 4 (1990) 61-77.
(8) See e.g. May, T.W., Brumbaugh, W.G., 1982, Matrix modifier and
L'vov platform for elimination of matrix interferencesin the analys is
of fish tissues for lead by graphite furnace atomic absorption spectro-
metry: Analytical Chemistry 54(7): 1032-1037 (90353).
(9) Applicationsof Zeeman Graphite Furnace Atomic Abs orption Spectro-
metry in the Chemical Laboratory and in Toxicology, C. Minoia, S.
Caroli (Eds.), Pergamon Press (Oxford), 1992, pp. xxvi + 675.
(10) Inductively Coupled Plasmas in Analytical Atomic Spectrometry, A.
Montaser, D. W. Golighty (Eds.), VCH Publishers, Inc. (New York),
1992.
(11) Plasma Source Mass Spectrometry Developments and Applications, G.
Holland, S. D. Tanner (Eds.), The Royal Society of Chemistry, 1997, p.
329.
(12) IUPAC (1995), Protocol for the design, conduct and interpretation of
method-performance studies, Pure & Applied Chem, 67, 331.
(13) Jülicher, B., Gowik, P. and Uhlig, S. (1998) Assessment of detection
methods in trace analysis by means of a statistically based in-house
validation concept. Analyst, 120, 173.
(14) Gowik, P., Jülicher, B. and Uhlig, S. (1998) Multi-residue method for
non-steroidal anti-inflammatory drugs in plasma using high perfor-
mance liquid chromatography-photodiode-array detection. Method
description and comprehensive in-house validation. J. Chromatogr.,
716, 221.
(15) OAC-I Peer Verified Methods, Policies and Procedures, 1993, AOAC
International, 2200 Wilson Blvd., Suite 400, Arlington, Virginia
22201-3301, USA.
(16) W.J. Youden; Steiner, E.H.; ‘Statistical Manual of the AOAC–Associa-
tion of Official Analytical Chemists’, AOAC-I, Washington DC: 1975,
p. 35 ff.
(17) ISO 11843: 1997 Capability of detection — Part 1: Termsand defini-
tions, Part 2: Methodology in the linear calibration case Part 2:
Methodology in the linear calibration case.
(18) R.W. Stephany & L.A. van Ginkel: ‘Yield or recovery: a world of
difference’. ProceedingsEight Euro Food Chem, Vienna, Aus tria
September 18-20 (1995) Federation of European Chemical Societies,
Event 206. ISBN 3-900554-17X, page 2 to 9.
(19) Directive 71/354/EEC of 18 October 1971 on the approximation of the
lawsof the Member Statesrelating to unitsof meas urement, OJ L 243,
29.10.1971, p. 29).
(20) ISO 31-0: 1992 Quantitiesand units— Part 0: General principles2002D0657 — EN — 15.03.2003 — 001.001 — 32
▼M1
ANNEX II
Minimumrequiredperformancelimits
Substance and/or metabolite Matrixes MRPL
Chloramphenicol Meat
Eggs0,3 µg/kg
Milk
Urine
Aquaculture products
Honey
Medroxyprogesterone acetate Pig kidney fat 1 µg/kg
Nitrofuran metabolites:
— furazolidone Poultry meat 1 µg/kg for all
— furaltadone Aquaculture products
— nitrofurantoin
— nitrofurazone2002D0657 — EN — 15.03.2003 — 001.001 — 33