Foodstuffs - Guidelines for the calibration and quantitative determination of pesticide residues and organic contaminants using chromatographic methods

This Technical Specification gives guidelines for the execution of calibration and quantitative evaluation of chromatographic procedures for the determination of pesticides and organic contaminants in residue analysis. In addition, the essential requirements for calibration are outlined.
The calibration of analytical procedures and the evaluation of analytical results need to be conducted according to uniform principles in order to allow for a comparison of analytical results (even from different analytical procedures). They constitute the basis of any method validation and of the quality assurance within laboratories [1], [2], [3].
This Technical Specification does not consider issues of identification/qualification and extraction efficiency.

Lebensmittel - Anleitung zur Kalibrierung und quantitativen Bestimmung von Pestizidrückständen und organischen Kontaminanten mit chromatographischen Verfahren

Diese Technische Spezifikation enthält Leitlinien für die Durchführung der Kalibrierung und die quantitative Auswertung von Analysenergebnissen bei der Anwendung chromatographischer Methoden für die Bestimmung von Pflanzenschutzmittelrückständen und organischen Kontaminanten in der Rückstandsanalyse. Dabei werden auch die wesentlichen Anforderungen an die Kalibrierung erläutert.
Die Kalibrierung von Analysenverfahren und die Auswertung von Analysenergebnissen müssen nach einheitlichen Grundsätzen erfolgen, um Analysenergebnisse (auch aus verschiedenen Analysenverfahren) miteinander vergleichen zu können. Sie sind Grundlage sowohl der Validierung von Methoden als auch der laborinternen Qualitätssicherung [1], [2], [3].
Diese Technische Spezifikation berücksichtigt keine Fragen zur Identifizierung/Qualifizierung von Analyten und zur Extraktionseffizienz.

Produits alimentaires - Lignes directrices pour l’étalonnage et le dosage des résidus de pesticides et contaminants organiques par des méthodes chromatographiques

La présente Spécification technique fournit des lignes directrices concernant la mise en œuvre de l’étalonnage et de l’évaluation quantitative de procédures chromatographiques pour la recherche de pesticides et de contaminants organiques dans le cadre d’analyses de résidus. En outre, elle présente les exigences essentielles en matière d’étalonnage.
L’étalonnage des modes opératoires d’analyse et l’évaluation des résultats d’analyse doivent être réalisés conformément à des principes uniformes afin de pouvoir comparer les résultats d’analyse (même obtenus à partir de différentes procédures). Ils constituent la base de la validation d’une méthode ainsi que de l’assurance qualité au sein des laboratoires [1], [2], [3].
La présente Spécification technique ne s’intéresse pas aux problèmes d’identification/de qualification et d’efficacité de l’extraction.

Živila - Smernice za kalibracijo ter kvantitativno določanje ostankov pesticidov in organskih onesnaževal z uporabo kromatografskih metod

Ta tehnična specifikacija podaja smernice za izvedbo kalibracije in kvantitativnega vrednotenja kromatografskih postopkov za določevanje pesticidov in organskih onesnaževal pri analizi ostankov. Opisane so tudi bistvene zahteve za kalibracijo.
Kalibracijo analitskih postopkov in vrednotenje analitskih rezultatov je treba izvesti v skladu z enotnimi načeli, da se omogoči primerjava analitskih rezultatov (tudi iz različnih analitskih postopkov). Ti tvorijo podlago za vsako vrednotenje metod in zagotavljanje kakovosti v laboratorijih [1], [2], [3].
Ta tehnična specifikacija ne obravnava zadev v zvezi z identifikacijo/kvalifikacijo in učinkovitostjo ekstrakcije.

General Information

Status
Published
Publication Date
03-Sep-2019
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
04-Sep-2019
Completion Date
04-Sep-2019

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SLOVENSKI STANDARD
SIST-TS CEN/TS 17061:2019
01-november-2019
Nadomešča:
SIST-TS CEN/TS 17061:2017

Živila - Smernice za kalibracijo ter kvantitativno določanje ostankov pesticidov in

organskih onesnaževal z uporabo kromatografskih metod

Foodstuffs - Guidelines for the calibration and quantitative determination of pesticide

residues and organic contaminants using chromatographic methods
Lebensmittel - Anleitung zur Kalibrierung und quantitativer Bestimmung von
Pflanzenschutzmittelrückständen und organischen Kontaminanten mit
chromatographischen Verfahren

Produits alimentaires - Lignes directrices pour l’étalonnage et le dosage des résidus de

pesticides et contaminants organiques par des méthodes chromatographiques
Ta slovenski standard je istoveten z: CEN/TS 17061:2019
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST-TS CEN/TS 17061:2019 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST-TS CEN/TS 17061:2019
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SIST-TS CEN/TS 17061:2019
CEN/TS 17061
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
September 2019
TECHNISCHE SPEZIFIKATION
ICS 67.050 Supersedes CEN/TS 17061:2017
English Version
Foodstuffs - Guidelines for the calibration and quantitative
determination of pesticide residues and organic
contaminants using chromatographic methods

Produits alimentaires - Lignes directrices pour Lebensmittel - Anleitung zur Kalibrierung und

l'étalonnage et le dosage des résidus de pesticides et quantitativer Bestimmung von

contaminants organiques par des méthodes Pflanzenschutzmittelrückständen und organischen

chromatographiques Kontaminanten mit chromatographischen Verfahren

This Technical Specification (CEN/TS) was approved by CEN on 14 July 2019 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to

submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS

available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in

parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 17061:2019 E

worldwide for CEN national Members.
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CEN/TS 17061:2019 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

1 Scope .................................................................................................................................................................... 4

2 Normative references .................................................................................................................................... 4

3 Terms and definitions ................................................................................................................................... 4

4 Principle ............................................................................................................................................................. 4

5 General ................................................................................................................................................................ 4

6 Execution and calculation of calibrations .............................................................................................. 5

6.1 General/specifications .................................................................................................................................. 5

6.2 Calibration functions ..................................................................................................................................... 7

6.3 Test for matrix effects ................................................................................................................................... 9

6.4 Basic calibration and calibration by means of external standard................................................. 9

6.5 Calculation by means of internal standard ......................................................................................... 11

6.6 Calibration using standard addition procedures ............................................................................. 15

6.7 Procedural calibration ............................................................................................................................... 17

6.8 Calibration with chemical conversion of the standard .................................................................. 17

7 Quality assurance ......................................................................................................................................... 17

7.1 Qualification of the chromatographic system .................................................................................... 17

7.2 Examination of integration results ........................................................................................................ 18

7.3 Permissible blank values in relation to the lower limit of working range ............................. 18

7.4 Frequency of calibrations ......................................................................................................................... 19

7.5 Number of analytes to be calibrated for “multi-methods” ............................................................ 19

7.6 Handling of substances with multiple peaks ..................................................................................... 19

7.7 Quality control chart ................................................................................................................................... 19

8 Expression of results (units, number of significant figures) ........................................................ 22

9 Examples ......................................................................................................................................................... 22

9.1 Example 1 — Test for variance inhomogeneity ................................................................................ 22

9.2 Example 2 — Selection of appropriate calibration function ........................................................ 23

9.3 Example 3 — Test for matrix effects ..................................................................................................... 30

9.4 Example 4 — Calibration with external standard ............................................................................ 31

9.5 Example 5 — Calibration with internal standard ............................................................................. 33

9.6 Example 6 — Calculation with stable-isotope labelled standard ............................................... 34

9.7 Example 7 — Standard addition to final extract ............................................................................... 35

Annex A (informative) Abbreviations ................................................................................................................ 37

Bibliography ................................................................................................................................................................. 39

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European foreword

This document (CEN/TS 17061:2019) has been prepared by Technical Committee CEN/TC 275 “Food

analysis - Horizontal methods”, the secretariat of which is held by DIN.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

This document will supersede CEN/TS 17061:2017.
Compared to CEN/TS 17061:2017, the following changes have been made:
— Annex A (informative) containing a list of abbreviations was added;
— The document has been editorially revised.
— Annex A (informative) contains a list of abbreviations.

According to the CEN/CENELEC Internal Regulations, the national standards organisations of the

following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,

Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
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CEN/TS 17061:2019 (E)
1 Scope

This Technical Specification gives guidelines for the execution of calibration and quantitative evaluation

of chromatographic procedures for the determination of pesticides and organic contaminants in residue

analysis. In addition, the essential requirements for calibration are outlined.

The calibration of analytical procedures and the evaluation of analytical results need to be conducted

according to uniform principles in order to allow for a comparison of analytical results (even from

different analytical procedures). They constitute the basis of any method validation and of the quality

assurance within laboratories [1], [2], [3].

This Technical Specification does not consider issues of identification/qualification and extraction

efficiency.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
No terms and definitions are listed in this document.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
4 Principle

This document describes the approach for the calibration of chromatographic procedures. The

following types of calibration are discussed in more detail:
— external calibration with linear calibration function;
— external calibration with quadratic calibration function;
— calibration with internal standard and linear calibration function;
— calibration with internal standard and quadratic calibration function;

— calibration with standards labelled with stable isotopes (isotopic dilution analysis);

— standard addition to final extract;
— standard addition to sample.

For this purpose, the calibration function and the selection criteria are illustrated on the basis of

examples. The calculation formulae refer to the final extract ready for analysis (“test solution”).

The description is rounded off by essential items of quality assurance, e.g. the qualification of

chromatographic systems or the quality control chart.
5 General

Calibration of a system is understood as the determination of a functional relationship between a

measurable quantity and a concentration to be determined. The chosen type of calibration depends on

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the various analytical problems/tasks. It is performed in connection with the respective series of

measurements.

Basic calibration is regarded as the determination of the functional relationship when an analyte is to be

determined for the first time by means of a particular measurement system.

Depending on the problem and on the type of reference solution used, it is distinguished between:

— calibration with external standard;
— calibration with internal standard;
— calibration with standard addition;
— calibration of the entire procedure.

In case of the calibration with external standard, the calibration solutions can be prepared either with a

pure solvent (standards in solvent) or with sample extracts which evidently do not contain significant

amounts of the analyte(s) (matrix-matched standards).

The application of a simple linear-regression calculation requires a linear relationship between the

content of substance and the measured value. The linearity test can be performed visually and/or

mathematically. A mathematical check is performed, e.g. by means of the goodness-of-fit test according

to Mandel or by means of residual analysis. The residuals are the deviations of the measurement values

from the values predicted by the regression line (see 9.1, Example 1).
6 Execution and calculation of calibrations
6.1 General/specifications
6.1.1 Working range

The range of measurement represented by the lowest and the highest calibration point constitutes the

range of concentrations for which the determined calibration function applies (working range). Only

within this range, the measured values are reliable and, therefore, can be used for the calculation of

analyte contents. At the upper and lower end, the prediction interval becomes wider i.e. the

measurement error increases progressively. The highest precision is found in the middle of the working

range [4].

The detector response from the analytes in the sample extract has to lie within the working range.

Extracts containing residues above the calibrated range shall be diluted. If the calibration solutions are

matrix-matched the matrix concentration in the calibration standards should also be diluted, see [5].

The calibration range shall be adjusted to the respective residue concentrations in the test solution

(real-sample concentrations which often occur in practice) and should cover a maximum of two orders

of magnitudes. Where appropriate, several calibration functions shall be established by means of

calibration solutions.

The lower limit of the practical working range usually represents the lowest calibration level, see [5]. It

shall be equal to or lower than the Reporting Limit (RL). The RL may not be lower than the Limit of

Quantification (LOQ).
6.1.2 Number of calibration points
6.1.2.1 General

For the working range of calibration functions, calibration solutions with different concentrations of

pesticides or contaminants are prepared (depending on the requirement, three to five calibration

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CEN/TS 17061:2019 (E)

points), the concentrations of which are as equidistantly distributed over the working range as possible.

The concentrations shall start at the lower limit of the practical working range. If the working range has

to cover one order of magnitude, three calibration points are necessary, while five calibration points are

necessary for two orders of magnitude (depending on the covered concentration range, e.g. 1, 3, 10, 30

and 100 times the lowest calibrated concentration).
6.1.2.2 Acceptability of single-point calibrations

A single-point calibration is sufficient if the linearity of the calibration function has been checked over a

longer period of time and has been evaluated as stable and if the blank values as well as the intercept

are negligibly small. The concentration level should be in the upper fraction of the working range. The

analyte concentrations in the calibration and test solutions should be within the range proposed

DG-SANTE, if the test solution is compared to one calibration solution only (see [5] for more details).

However, a check of the basic calibration shall be made every working day, and the measurement of a

minimum number of representative analytes is indispensable, see [5].

6.1.3 Permissible quantities for determination of response (peak areas or peak heights and

peak ratio, respectively)

The analyte signal, thus the peak, produced by the detector can be quantitatively evaluated through

determination of the peak height or peak area. By principle, the height as well as the area of the peak

depends on the analyte concentration and mass, respectively. The peak height indicates the distance

from the baseline to the maximum of the peak. In case of well resolved peaks, the peak height is

proportional to the analyte concentration. The evaluation by means of peak height should only be

performed in case of reproducible peak shape and constant width at half-height (half-width). It leads to

difficulties if there are two peaks with a poorer resolution than approximately 1,25.

A resolution R can be defined by means of the distance ΔE and the width 4 × s of the peaks, see Figure 1

and Formula 1. The width 4 × s is determined through the intercept of both inflexional tangents on the

baseline or calculated from the standard deviation s.
R = (1)
4 × s

In case of well resolved peaks, the peak area is proportional to the analyte concentration. In contrast to

the peak height, the peak area usually provides accurate results, even for asymmetric peaks. The

prerequisite for peak-area determinations is always the precise definition of the baseline.

Key
t retention time
Y intensity
Figure 1 — Definition of the resolution of peaks
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CEN/TS 17061:2019 (E)

The calculation by means of the intensity ratios of peaks (peak ratio) is used for calibrations with

internal standard (ISTD), e.g. for EN 15662 (QuEChERS) and for calibrations with internal standard

labelled with stable isotopes (stable-isotope labelled standards). This procedure of calibration and

calculation requires to know at least the ratio of amounts of internal standard added to test samples (or

to extract aliquots) and to calibration standards.
6.1.4 Stability of calibration functions

The calibration standards should be injected at least at the start and end of a sample sequence

(bracketing calibration). Bracketed samples containing pesticide residues or organic contaminants

should be re-analysed if the drift between the two bracketing injections exceeds the limit given by

DG-SANTE. In general such bracketed samples have to be re-analysed if the calibration level

corresponding to the RL was not measurable throughout the batch, see [5] for more details.

6.2 Calibration functions
6.2.1 Selection of appropriate calibration function

Calibration functions can be linear, logarithmic, exponential as well as polynomial of 2 order.

Whenever possible, the simplest acceptable calibration function should be used. The use of linear

weighted regression (e.g. 1/x or 1/x weighting) is recommended.

A calibration function is a unique plotting of the set of all x-values (concentration or mass) against the

set of all y-values (peak area or height), i.e. exactly one y-value is assigned to each x-value.

Before determining the established type of calibration function and testing the linearity of a calibration

function, the homogeneity of variances should be checked first as it represents a basic prerequisite for

the applicability of statistical methods.

For this purpose, calibration solutions are prepared where between three and ten concentration levels

with two to six measurements per concentration are recommended or ten standard samples of the

lowest and the highest working-range concentration are separately analysed at a time. The variation

(scatter) of the measured values at the limits of the working range is tested for significant differences by

means of a simple F-test for variance inhomogeneity.

In case that the F-test indicates a significant difference of variances, there are three opportunities to

proceed:
— selection of a narrower working range;
— application of weighted regression;
— application of multiple curve fitting.

The non-consideration of a present inhomogeneity of variances results in a wider prediction interval so

that an analytical result which has been determined accordingly shows a higher uncertainty of

measurement.

The linearity of the calibration function can be mathematically checked by means of a linearity test. For

this purpose, the goodness-of-fit test according to Mandel or the residual analysis is appropriate. In

every case, a graphical representation (plot) of the calibration data are recommended (see 9.2,

Example 2).
6.2.2 Visual linearity test

In the simplest case, the determination of the type of calibration function is performed by means of

graphical representation of the calibration data including the calibration line and a subjective

assessment. If this indicates an obvious nonlinearity of the measured values (see Figure 2), a separate

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CEN/TS 17061:2019 (E)

statistic linearity test can be omitted. In cases of doubt, however, the linearity should be checked

mathematically [5].

Calibration curves (graphs) shall not be forced through the zero point (origin). Further examples can be

found in Clause 9 on Examples.
Key
X concentration
Y measured value
Figure 2 — Graphical representation of calibration data
6.2.3 Mathematical check of linearity

Within the laboratory, the mathematical check of linearity is of secondary importance. Regarding the

execution of linearity tests refer to the Examples 1 and 2 and to [6].

As an important criterion of linearity, the coefficient of determination, R , is well accepted. However,

the coefficient of determination does not allow a sufficient statement regarding the statistical

significance of the linear relationship. Calculation of the residuals is recommended to avoid

overreliance on coefficients of determination. If individual residuals exceed an acceptable level as

defined by DG-SANTE, an alternative calibration function shall be used according to [5].

6.2.4 Calibration with interpolation functions

Several methods of measurement show a basically nonlinear relationship between the measured

signals and the concentration and the amount of analyte, respectively. In cases where the linear

regression is inappropriate, a polynomial of 2 order (quadratic calibration function) is usually fitted to

the calibration data.

Like the linear calibration function, the polynomial of 2 order has a prediction interval. Nevertheless,

the functional equation has a higher degree of complexity which is why this procedure needs computer

support for evaluation.

In case of the nonlinear calibration function, the number of necessary calibration points depends on the

desired accuracy and on the reasonable effort. For a global interpolation function, a single mathematical

equation describes the entire calibration. However, this is only possible in exceptional cases. In most

cases, such a function does not show the expected properties between the calibration points (e.g.

polynomial “oscillation”). Therefore, local interpolation functions are employed where each interval

between two interpolation nodes is characterized by a separate interpolation function. The application

of the calibration function requires the selection of the corresponding local interpolation function for

each signal value.
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In case of interpolation with polynomials calculated from calibration points in proximity, the curvature

of the calibration graph between the interpolation nodes can also be taken into account. The

computational effort increases especially when determining the inverse function. Therefore, the

calibration function is often calculated once the x-values and y-values have been interchanged.

The calibration function piecewise defined by polynomials will show break (knee) points between the

interpolation nodes. With further increased computational effort, this can be prevented by means of

spline functions which are also piecewise defined. If derivations are needed, these, starting from a

certain differentiation level, will become discontinuous.
6.3 Test for matrix effects

The test for matrix effects is performed by means of a comparison of the calibration lines from the

calibration with standards in a pure solvent and with standards in a matrix (matrix-matched standards)

(see Figure 11). Significantly higher or lower signals in matrix calibration indicate matrix effects (see

9.3, Example 3).

Matrix-matched standards can be prepared in the same way as standards in solvents. Typically, the

solvent used to fill up is replaced by an extract of blank samples (residue-free material), that is

prepared according to the method used for the routine sample preparation (generally without use of an

internal standard). The matrix-matched standards prepared in this way shall contain ≥ 80 % (V/V) of

the test solution of control samples in order to obtain comparable matrix effects of samples and

standards during GC- or HPLC-MS(/MS) analysis. Blank samples of the same commodities as the test

sample (apple for apple samples etc.) are used to optimally compensate for matrix effects.

The stability of matrix-matched standards can be lower than that of standards in solvents and,

therefore, shall be controlled periodically.

If the quantitative evaluation of a peak is impaired by peaks of co-extracted accompanying substances

(e.g. peak overlaps), an extract solution should be injected for comparison which contains the

corresponding accompanying substance in the test solution of the same but residue-free test material.

6.4 Basic calibration and calibration by means of external standard
6.4.1 Basic calibration

The basic calibration serves as a qualification test for the measurement system used, i.e. no steps of

sample preparation (treatment) such as extraction or digestion are undertaken in the course of it, but

only standard solutions are analysed.

To perform the basic calibration, equal volumes of calibration solutions with appropriate

concentrations are injected and the peak areas/heights obtained are plotted against the concentrations

of the calibration solutions (see 9.4, Example 4). Evaluation is carried out with the most suitable

calibration function. It is important that a continuous and reproducible relationship between signal

intensity and analyte concentration is obtained.

A validated calibration function of the basic calibration can be also used for quantification with external

standards if it has been proven before that the matrix does not cause a significant increase or decrease

of the analyte signal.

6.4.2 Calculation by means of external standard in case of linear calibration function without

significant ordinate intercept

The quantitative evaluation is done by determination of the peak areas (or peak heights) and

comparison with the peak areas (or peak heights) of analyte solutions with known concentrations. In

the course of this, equal volumes of the test solutions and calibration solutions are injected into the

chromatographic column. If the signal of the analyte is within the linear range and if the straight line

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CEN/TS 17061:2019 (E)

intercepts the ordinate in the range of the origin (of coordinates), the analyte concentration in the test

solution can be calculated according to the following very simple Formula (2):
ρ = (2)
where
ρ is the mass concentration of the analyte in the test solution [e.g. µg/ml];

y is the peak area (or peak height) of the analyte measured in the test solution [e.g. counts];

b is the slope of the calibration function [e.g. counts⋅ml/µg].

6.4.3 Calculation by means of external standard using linear calibration function

If a significant ordinate intercept has been calculated for the calibration function but the analyte signal

is within the linear range, the analyte concentration in the test solution is calculated by rearranging the

established calibration function according to Formula (3):
yc−
( )
(3)
ρ =
where
ρ is the mass concentration of the analyte in the test solution [e.g. µg/ml];

y is the peak area (or peak height) of the analyte in the test solution [e.g. counts];

b is the slope of the calibration function [e.g. counts⋅ml/µg];
c is the ordinate intercept of the calibration function [e.g. counts].

6.4.4 Calculation by means of external standard using quadratic calibration function

A quadratic calibration function can be used if the distribution of residuals or other mathematical tests

indicate that a linear calibration function is inappropriate. For a calculation of the analyte concentration

in the test solution in case of quadratic calibration function, the rearrangement of the equation

theoretically yields two solutions of which only one makes analytical sense, see Formula (4):

−±b b − 4 ac − y
( )
(4)
ρ =
where
ρ is the concentration of the analyte in the test so
...

SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17061:2019
01-maj-2019

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Foodstuffs - Guidelines for the calibration and quantitative determination of pesticide

residues and organic contaminants using chromatographic methods
Lebensmittel - Anleitung zur Kalibrierung und quantitativer Bestimmung von
Pflanzenschutzmittelrückständen und organischen Kontaminanten mit
chromatographischen Verfahren

Produits alimentaires - Lignes directrices pour l’étalonnage et le dosage des résidus de

pesticides et contaminants organiques par des méthodes chromatographiques
Ta slovenski standard je istoveten z: FprCEN/TS 17061
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
kSIST-TS FprCEN/TS 17061:2019 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
kSIST-TS FprCEN/TS 17061:2019
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kSIST-TS FprCEN/TS 17061:2019
FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17061
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
March 2019
ICS 67.050 Will supersede CEN/TS 17061:2017
English Version
Foodstuffs - Guidelines for the calibration and quantitative
determination of pesticide residues and organic
contaminants using chromatographic methods

Produits alimentaires - Lignes directrices pour Lebensmittel - Anleitung zur Kalibrierung und

l'étalonnage et le dosage des résidus de pesticides et quantitativer Bestimmung von

contaminants organiques par des méthodes Pflanzenschutzmittelrückständen und organischen

chromatographiques Kontaminanten mit chromatographischen Verfahren

This draft Technical Specification is submitted to CEN members for Vote. It has been drawn up by the Technical Committee

CEN/TC 275.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

aware and to provide supporting documentation.

Warning : This document is not a Technical Specification. It is distributed for review and comments. It is subject to change

without notice and shall not be referred to as a Technical Specification.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprCEN/TS 17061:2019 E

worldwide for CEN national Members.
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Contents Page

European foreword ....................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

4 Principle ............................................................................................................................................................. 5

5 General ................................................................................................................................................................ 5

6 Execution and calculation of calibrations .............................................................................................. 6

6.1 General/specifications .................................................................................................................................. 6

6.1.1 Working range.................................................................................................................................................. 6

6.1.2 Number of calibration points ...................................................................................................................... 6

6.1.3 Permissible quantities for determination of response (peak areas or peak heights

and peak ratio, respectively) ...................................................................................................................... 7

6.1.4 Stability of calibration functions ............................................................................................................... 8

6.2 Calibration functions ..................................................................................................................................... 8

6.2.1 Selection of appropriate calibration function ...................................................................................... 8

6.2.2 Visual linearity test ........................................................................................................................................ 8

6.2.3 Mathematical check of linearity ................................................................................................................ 9

6.2.4 Calibration with interpolation functions................................................................................................ 9

6.3 Test for matrix effects ................................................................................................................................ 10

6.4 Basic calibration and calibration by means of external standard.............................................. 10

6.4.1 Basic calibration ........................................................................................................................................... 10

6.4.2 Calculation by means of external standard in case of linear calibration function

without significant ordinate intercept ................................................................................................. 10

6.4.3 Calculation by means of external standard using linear calibration function....................... 11

6.4.4 Calculation by means of external standard using quadratic calibration function ............... 11

6.4.5 Calculation by means of external standard using nonlinear calibration function or

weighted regression .................................................................................................................................... 12

6.5 Calculation by means of internal standard ......................................................................................... 12

6.5.1 General ............................................................................................................................................................. 12

6.5.2 Calculation with internal correction standard .................................................................................. 13

6.5.3 Calculation with stable-isotope labelled standard .......................................................................... 14

6.6 Calibration using standard addition procedures ............................................................................. 16

6.6.1 General ............................................................................................................................................................. 16

6.6.2 Standard addition to the final extract................................................................................................... 16

6.6.3 Standard addition to the sample before extraction ......................................................................... 17

6.7 Procedural calibration ............................................................................................................................... 18

6.8 Calibration with chemical conversion of the standard .................................................................. 18

7 Quality assurance ......................................................................................................................................... 18

7.1 Qualification of the chromatographic system .................................................................................... 18

7.1.1 Equipment qualification ............................................................................................................................ 18

7.1.2 Drift of a calibration system ..................................................................................................................... 19

7.1.3 Performance criteria for GC and HPLC measurements .................................................................. 19

7.2 Examination of integration results ........................................................................................................ 19

7.3 Permissible blank values in relation to the lower limit of working range ............................. 19

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7.4 Frequency of calibrations .......................................................................................................................... 20

7.5 Number of analytes to be calibrated for “multi-methods” ............................................................. 20

7.6 Handling of substances with multiple peaks ...................................................................................... 20

7.7 Quality control chart ................................................................................................................................... 20

8 Expression of results (units, number of significant figures)......................................................... 23

9 Examples .......................................................................................................................................................... 23

9.1 Example 1 — Test for variance inhomogeneity ................................................................................. 23

9.2 Example 2 — Selection of appropriate calibration function ......................................................... 24

9.3 Example 3 — Test for matrix effects ...................................................................................................... 31

9.4 Example 4 — Calibration with external standard ............................................................................ 32

9.5 Example 5 — Calibration with internal standard ............................................................................. 34

9.6 Example 6 — Calculation with stable-isotope labelled standard ................................................ 35

9.7 Example 7 — Standard addition to final extract ............................................................................... 36

Annex A (informative) Abbreviations ................................................................................................................ 38

Bibliography ................................................................................................................................................................. 40

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European foreword

This document (FprCEN/TS 17061:2019) has been prepared by Technical Committee CEN/TC 275

“Food analysis - Horizontal methods”, the secretariat of which is held by DIN.
This document is currently submitted to the Vote on TS.
This document will supersede CEN/TS 17061:2017.
Compared to CEN/TS 17061:2017, the following changes have been made:
— Annex A (informative) containing a list of abbreviations was added;
— The document has been editorially revised.
Annex A (informative) contains a list of abbreviations.
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1 Scope

This Technical Specification gives guidelines for the execution of calibration and quantitative evaluation

of chromatographic procedures for the determination of pesticides and organic contaminants in residue

analysis. In addition, the essential requirements for calibration are outlined.

The calibration of analytical procedures and the evaluation of analytical results need to be conducted

according to uniform principles in order to allow for a comparison of analytical results (even from

different analytical procedures). They constitute the basis of any method validation and of the quality

assurance within laboratories [1], [2], [3].

This Technical Specification does not consider issues of identification/qualification and extraction

efficiency.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
No terms and definitions are listed in this document.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp
4 Principle

This document describes the approach for the calibration of chromatographic procedures. The

following types of calibration are discussed in more detail:
— external calibration with linear calibration function;
— external calibration with quadratic calibration function;
— calibration with internal standard and linear calibration function;
— calibration with internal standard and quadratic calibration function;

— calibration with standards labelled with stable isotopes (isotopic dilution analysis);

— standard addition to final extract;
— standard addition to sample.

For this purpose, the calibration function and the selection criteria are illustrated on the basis of

examples. The calculation formulae refer to the final extract ready for analysis (“test solution”).

The description is rounded off by essential items of quality assurance, e.g. the qualification of

chromatographic systems or the quality control chart.
5 General

Calibration of a system is understood as the determination of a functional relationship between a

measurable quantity and a concentration to be determined. The chosen type of calibration depends on

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the various analytical problems/tasks. It is performed in connection with the respective series of

measurements.

Basic calibration is regarded as the determination of the functional relationship when an analyte is to be

determined for the first time by means of a particular measurement system.

Depending on the problem and on the type of reference solution used, it is distinguished between:

— calibration with external standard;
— calibration with internal standard;
— calibration with standard addition;
— calibration of the entire procedure.

In case of the calibration with external standard, the calibration solutions can be prepared either with a

pure solvent (standards in solvent) or with sample extracts which evidently do not contain significant

amounts of the analyte(s) (matrix-matched standards).

The application of a simple linear-regression calculation requires a linear relationship between the

content of substance and the measured value. The linearity test can be performed visually and/or

mathematically. A mathematical check is performed, e.g. by means of the goodness-of-fit test according

to Mandel or by means of residual analysis. The residuals are the deviations of the measurement values

from the values predicted by the regression line (see 9.1, Example 1).
6 Execution and calculation of calibrations
6.1 General/specifications
6.1.1 Working range

The range of measurement represented by the lowest and the highest calibration point constitutes the

range of concentrations for which the determined calibration function applies (working range). Only

within this range, the measured values are reliable and, therefore, can be used for the calculation of

analyte contents. At the upper and lower end, the prediction interval becomes wider i.e. the

measurement error increases progressively. The highest precision is found in the middle of the working

range [4].

The detector response from the analytes in the sample extract has to lie within the working range.

Extracts containing residues above the calibrated range shall be diluted. If the calibration solutions are

matrix-matched the matrix concentration in the calibration standards should also be diluted, see [5].

The calibration range shall be adjusted to the respective residue concentrations in the test solution

(real-sample concentrations which often occur in practice) and should cover a maximum of two orders

of magnitudes. Where appropriate, several calibration functions shall be established by means of

calibration solutions.

The lower limit of the practical working range usually represents the lowest calibration level, see [5]. It

shall be equal to or lower than the Reporting Limit (RL). The RL may not be lower than the Limit of

Quantification (LOQ).
6.1.2 Number of calibration points
6.1.2.1 General

For the working range of calibration functions, calibration solutions with different concentrations of

pesticides or contaminants are prepared (depending on the requirement, three to five calibration

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points), the concentrations of which are as equidistantly distributed over the working range as possible.

The concentrations shall start at the lower limit of the practical working range. If the working range has

to cover one order of magnitude, three calibration points are necessary, while five calibration points are

necessary for two orders of magnitude (depending on the covered concentration range, e.g. 1, 3, 10, 30

and 100 times the lowest calibrated concentration).
6.1.2.2 Acceptability of single-point calibrations

A single-point calibration is sufficient if the linearity of the calibration function has been checked over a

longer period of time and has been evaluated as stable and if the blank values as well as the intercept

are negligibly small. The concentration level should be in the upper fraction of the working range. The

analyte concentrations in the calibration and test solutions should be within the range proposed

DG-SANTE, if the test solution is compared to one calibration solution only (see [5] for more details).

However, a check of the basic calibration shall be made every working day, and the measurement of a

minimum number of representative analytes is indispensable, see [5].

6.1.3 Permissible quantities for determination of response (peak areas or peak heights and

peak ratio, respectively)

The analyte signal, thus the peak, produced by the detector can be quantitatively evaluated through

determination of the peak height or peak area. By principle, the height as well as the area of the peak

depends on the analyte concentration and mass, respectively. The peak height indicates the distance

from the baseline to the maximum of the peak. In case of well resolved peaks, the peak height is

proportional to the analyte concentration. The evaluation by means of peak height should only be

performed in case of reproducible peak shape and constant width at half-height (half-width). It leads to

difficulties if there are two peaks with a poorer resolution than approximately 1,25.

A resolution R can be defined by means of the distance ΔE and the width 4 × s of the peaks, see Figure 1

and Formula 1. The width 4 × s is determined through the intercept of both inflexional tangents on the

baseline or calculated from the standard deviation s.
R = (1)
4 × s

In case of well resolved peaks, the peak area is proportional to the analyte concentration. In contrast to

the peak height, the peak area usually provides accurate results, even for asymmetric peaks. The

prerequisite for peak-area determinations is always the precise definition of the baseline.

Key
t retention time
Y intensity
Figure 1 — Definition of the resolution of peaks
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The calculation by means of the intensity ratios of peaks (peak ratio) is used for calibrations with

internal standard (ISTD), e.g. for EN 15662 (QuEChERS) and for calibrations with internal standard

labelled with stable isotopes (stable-isotope labelled standards). This procedure of calibration and

calculation requires to know at least the ratio of amounts of internal standard added to test samples (or

to extract aliquots) and to calibration standards.
6.1.4 Stability of calibration functions

The calibration standards should be injected at least at the start and end of a sample sequence

(bracketing calibration). Bracketed samples containing pesticide residues or organic contaminants

should be re-analysed if the drift between the two bracketing injections exceeds the limit given by

DG-SANTE. In general such bracketed samples have to be re-analysed if the calibration level

corresponding to the RL was not measurable throughout the batch, see [5] for more details.

6.2 Calibration functions
6.2.1 Selection of appropriate calibration function

Calibration functions can be linear, logarithmic, exponential as well as polynomial of 2 order.

Whenever possible, the simplest acceptable calibration function should be used. The use of linear

weighted regression (e.g. 1/x or 1/x weighting) is recommended.

A calibration function is a unique plotting of the set of all x-values (concentration or mass) against the

set of all y-values (peak area or height), i.e. exactly one y-value is assigned to each x-value.

Before determining the established type of calibration function and testing the linearity of a calibration

function, the homogeneity of variances should be checked first as it represents a basic prerequisite for

the applicability of statistical methods.

For this purpose, calibration solutions are prepared where between three and ten concentration levels

with two to six measurements per concentration are recommended or ten standard samples of the

lowest and the highest working-range concentration are separately analysed at a time. The variation

(scatter) of the measured values at the limits of the working range is tested for significant differences by

means of a simple F-test for variance inhomogeneity.

In case that the F-test indicates a significant difference of variances, there are three opportunities to

proceed:
— selection of a narrower working range;
— application of weighted regression;
— application of multiple curve fitting.

The non-consideration of a present inhomogeneity of variances results in a wider prediction interval so

that an analytical result which has been determined accordingly shows a higher uncertainty of

measurement.

The linearity of the calibration function can be mathematically checked by means of a linearity test. For

this purpose, the goodness-of-fit test according to Mandel or the residual analysis is appropriate. In

every case, a graphical representation (plot) of the calibration data are recommended (see 7.2,

Example 2).
6.2.2 Visual linearity test

In the simplest case, the determination of the type of calibration function is performed by means of

graphical representation of the calibration data including the calibration line and a subjective

assessment. If this indicates an obvious nonlinearity of the measured values (see Figure 2), a separate

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statistic linearity test can be omitted. In cases of doubt, however, the linearity should be checked

mathematically [5].

Calibration curves (graphs) shall not be forced through the zero point (origin). Further examples can be

found in Clause 9 on Examples.
Key
X concentration
Y measured value
Figure 2 — Graphical representation of calibration data
6.2.3 Mathematical check of linearity

Within the laboratory, the mathematical check of linearity is of secondary importance. Regarding the

execution of linearity tests refer to the Examples 1 and 2 and to [6].

As an important criterion of linearity, the coefficient of determination, R , is well accepted. However,

the coefficient of determination does not allow a sufficient statement regarding the statistical

significance of the linear relationship. Calculation of the residuals is recommended to avoid

overreliance on coefficients of determination. If individual residuals exceed an acceptable level as

defined by DG-SANTE, an alternative calibration function shall be used according to [5].

6.2.4 Calibration with interpolation functions

Several methods of measurement show a basically nonlinear relationship between the measured

signals and the concentration and the amount of analyte, respectively. In cases where the linear

regression is inappropriate, a polynomial of 2 order (quadratic calibration function) is usually fitted

to the calibration data.

Like the linear calibration function, the polynomial of 2 order has a prediction interval. Nevertheless,

the functional equation has a higher degree of complexity which is why this procedure needs computer

support for evaluation.

In case of the nonlinear calibration function, the number of necessary calibration points depends on the

desired accuracy and on the reasonable effort. For a global interpolation function, a single mathematical

equation describes the entire calibration. However, this is only possible in exceptional cases. In most

cases, such a function does not show the expected properties between the calibration points (e.g.

polynomial “oscillation”). Therefore, local interpolation functions are employed where each interval

between two interpolation nodes is characterized by a separate interpolation function. The application

of the calibration function requires the selection of the corresponding local interpolation function for

each signal value.
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In case of interpolation with polynomials calculated from calibration points in proximity, the curvature

of the calibration graph between the interpolation nodes can also be taken into account. The

computational effort increases especially when determining the inverse function. Therefore, the

calibration function is often calculated once the x-values and y-values have been interchanged.

The calibration function piecewise defined by polynomials will show break (knee) points between the

interpolation nodes. With further increased computational effort, this can be prevented by means of

spline functions which are also piecewise defined. If derivations are needed, these, starting from a

certain differentiation level, will become discontinuous.
6.3 Test for matrix effects

The test for matrix effects is performed by means of a comparison of the calibration lines from the

calibration with standards in a pure solvent and with standards in a matrix (matrix-matched standards)

(see Figure 11). Significantly higher or lower signals in matrix calibration indicate matrix effects (see

9.3, Example 3).

Matrix-matched standards can be prepared in the same way as standards in solvents. Typically, the

solvent used to fill up is replaced by an extract of blank samples (residue-free material), that is

prepared according to the method used for the routine sample preparation (generally without use of an

internal standard). The matrix-matched standards prepared in this way shall contain ≥ 80 % (V/V) of

the test solution of control samples in order to obtain comparable matrix effects of samples and

standards during GC- or HPLC-MS(/MS) analysis. Blank samples of the same commodities as the test

sample (apple for apple samples etc.) are used to optimally compensate for matrix effects.

The stability of matrix-matched standards can be lower than that of standards in solvents and,

therefore, shall be controlled periodically.

If the quantitative evaluation of a peak is impaired by peaks of co-extracted accompanying substances

(e.g. peak overlaps), an extract solution should be injected for comparison which contains the

corresponding accompanying substance in the test solution of the same but residue-free test material.

6.4 Basic calibration and calibration by means of external standard
6.4.1 Basic calibration

The basic calibration serves as a qualification test for the measurement system used, i.e. no steps of

sample preparation (treatment) such as extraction or digestion are undertaken in the course of it, but

only standard solutions are analysed.

To perform the basic calibration, equal volumes of calibration solutions with appropriate

concentrations are injected and the peak areas/heights obtained are plotted against the concentrations

of the calibration sol
...

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