CEN/TS 17061:2019

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

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.

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

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.
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
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
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.
∆E
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
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
nd
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
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
nd
regression is inappropriate, a polynomial of 2 order (quadratic calibration function) is usually fitted to
the calibration data.
nd
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.
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
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):
y
A
ρ = (2)
A
b
where
ρ is the mass concentration of the analyte in the test solution [e.g. µg/ml];
A
y is the peak area (or peak height) of the analyte measured in the test solution [e.g. counts];
A
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−
( )
A
(3)
ρ =
A
b
where
ρ is the mass concentration of the analyte in the test solution [e.g. µg/ml];
A
y is the peak area (or peak height) of the analyte in the test solution [e.g. counts];
A
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
( )
A
(4)
ρ =
A
2a
where
ρ is the concentration of the analyte in the test solution [e.g. µg/ml];
A
y is the peak area (or peak height) of the analyte in the test solution [e.g. counts];
A
a is the slope of the calibration function in the quadratic term of the equation [e.g.
2 2
counts⋅ml /µg ];
b is the slope of the calibration function in the linear term of the equation [e.g. counts⋅ml/µg];
c is the ordinate intercept of the calibration function [e.g. counts].
6.4.5 Calculation by means of external standard using nonlinear calibration function or
weighted regression
If the execution of calibrations with nonlinear (e.g. exponential or logarithmic) calibration function
becomes necessary, one will usually rely on appropriate instrument software for calculating the analyte
concentration in the final extract. Before using such software, it shall be checked with suitable self-
calculated examples.
The same applies to the calculation of analyte content using a calibration function which has been
obtained by means of weighted regression.
6.5 Calculation by means of internal standard
6.5.1 General
The internal standard shall neither belong to the substance group to be analysed nor be present in the
sample. It shall have sufficient signal intensities and needs to be separately detectable.
For one analysis, several ISTDs can be used in order to detect and, if appropriate, compensate various
errors which occur during preparation (treatment) and measurement. The ISTD solution can be added
at different points of the analytical course and for various reasons [7]:
— before extraction and sample digestion, respectively, to compensate for all errors in the entire
procedure (correction standard for the calculation of analyte concentration, i.e. classic ISTD for
quantification);
— before extraction and sample digestion, respectively, only to control the recovery (single steps of
preparation) without making any correction (quality-assurance standard or surrogate standard);
— after the preparation has been completed and before the instrumental measurement, for the
compensation of sensitivity fluctuations of the instrument and variations of injection volumes
during the measurement sequence (injection standard) and for the calculation of the recovery
(rates) of correction standards, respectively.
Hereafter, only the application as correction standard for the entire procedure is considered. This
application is only reasonable if the ISTD, in comparison to the analyte, shows nearly the same
behaviour during preparation and detection. The use of stable-isotope labelled compounds as ISTD is
particularly advantageous.
The identification of the residues contained in the test solution is first carried out by means of their
relative retention times related to the retention time of the respective ISTD. But before that, the
calibration line needs to be determined using ISTD addition as well. For calibration and for testing the
linearity of the measurement signal, the ratio of the peak areas of analyte and ISTD is, separately for
each agent, plotted against the concentration ratio of analyte and ISTD in the respective calibration
solution (see 9.5, Example 5).
Possible errors:
If an internal standard is used for the calculation of residues, its signal intensity influences all quantified
contents and, consequently, the accuracy of analyses. Unwanted factors can change the intensity of the
internal standard and, thereby, lead to errors in the calculation of residue contents. Thus, losses of
internal standard during sample purification or the selective signal suppression (matrix effect) give rise
to overestimation of residue contents. To the contrary, the contents of all detected analytes are
underestimated as a consequence of signal enhancement of ISTD due to matrix effects. Such matrix
effects are caused by coeluting sample components and usually occur to an increasing degree only for
certain matrices.
In general, internal standards should be selected so that the sources of error described above are as low
as possible (losses, matrix-induced effects, etc.).
In order to avoid the errors mentioned above, the signal intensity of an internal standard within a
sequence shall be checked, which can involve the comparison to the intensity of a second ISTD, where
appropriate. In case of significant deviations of this signal intensity in individual samples, additional
measures of quality assurance are necessary. If an individual signal suppression of the ISTD occurs, the
conspicuous samples can be evaluated against a second ISTD or without any ISTD. It is also possible to
verify matrix effects in conspicuous samples by comparing the peak area of the ISTD in the test solution
with that of the calibration solution containing the same ISTD concentration.
6.5.2 Calculation with internal correction standard
Calibration solutions with ISTD are prepared by diluting a constant volume of the internal standard
cal
solution (V ) and a variable volume of the analyte solution with solvent to a defined volume (V ).
cal
ISTD
cal
The added volume of internal standard shall yield a concentration of the standard ( ρ ) which, as far
ISTD
as possible, represents the expected concentration of the ISTD in the test solution ( ρ ).
ISTD
The concentration and, thus, the absolute peak intensity of the internal standard in the final extract of
the sample should be 80 % to 120 % of the peak intensity of the internal standard in the calibration
standards, see Formula (5):
cal stock
V ⋅ ρ
cal ISTD ISTD
ρρ≈= (5)
ISTD ISTD
V
cal
where
cal
is the concentration of the internal standard in the calibration standard [e.g. µg/ml];

ρ
ISTD
is the expected concentration of the internal standard in the test solution [e.g. µg/ml];
ρ
ISTD
cal
is the volume of the ISTD solution which has been used for preparing the calibration
V
ISTD
standard for measurement [e.g. ml];
stock
is the concentration of the ISTD solution which has been used for preparing the
ρ
ISTD
calibration standard for measurement [e.g. µg/ml];
V is the volume of the calibration standard which is ready for measurement and contains
cal
the analyte and ISTD [e.g. ml].
The calibration solutions obtained are used to establish a calibration function by plotting the respective
cal cal
quotients (ratios) of the peak areas of analyte ( y ) and internal standard ( y ) against the
A ISTD
cal cal
corresponding concentration ratios of analyte ( ) to internal standard ( ) see Formula (6):
ρ ρ
A ISTD
cal cal cal cal
yy/ = f(/ρ ρ ) (6)
A ISTD A ISTD
Depending on the quality of regression, a linear or quadratic calibration function is used. The
calculation of the analyte concentration in the test solution (ρ ) is then performed analogously to the
A
equations given in 6.4.3 and 6.4.4, respectively. But in both equations, the peak area of the analyte (y )
A
has to be replaced by the quotient of analyte and internal standard peak areas (y / y ) which has to
A ISTD
be calculated here.
For linear calibration, the calculation is performed according Formula (7):
yy/ − c
( )
A ISTD
ρρ× (7)
A ISTD
b
The quadratic calibration function is given in Formula (8):
−±b b − 4 ac − y / y
( )
A ISTD
ρρ× (8)
A ISTD
2a
where
ρ is the concentration of the analyte in the test solution [e.g. µg/ml];
A
is the expected concentration of the internal standard in the test solution [e.g. µg/ml];
ρ
ISTD
y is the peak area (or peak height) of the analyte measured in the test solution [e.g. counts];
A
is the peak area (or peak height) of the internal standard measured in the test solution
y
ISTD
[e.g. counts];
a is the slope of the calibration function in the quadratic term of the equation
[dimensionless];
b is the slope of the calibration function in the linear term of the equation [dimensionless];
c is the ordinate intercept of the calibration function [dimensionless].
An example of calculation for a linear calibration function is given in Example 5 in 9.5.
The calibration can be simplified if an identical concentration of the internal standard in the calibration
cal
solutions and test solution is ensured ( ρ = ρ ). In this case, it is possible to directly plot the
ISTD ISTD
cal cal cal
dependence of the peak-area ratio y / y against the analyte concentration ( ρ ) in the
A ISTD A
calibration solutions as calibration line. This leads to a simplification of the calculation of analyte
concentration in the test solution because the multiplication with the concentration of internal standard
in the test solution can be omitted.
Thus, the calculation for the linear calibration is performed according to Formula (9):
yy/ − c
( )
A ISTD
ρ = (9)
A
b
The following applies for a quadratic calibration function in Formula (10):
−±b b − 4 ac − y / y
( )
A ISTD
(10)
ρ =
A
2a
6.5.3 Calculation with stable-isotope labelled standard
Extraction and treatment losses of the analyte and, if applicable, matrix effects are usually compensated
very well during measurement if internal standards are added before extraction which differ from the
2 1 13 12
analyte only in terms of the isotopes in the molecule (e.g. H instead of H or C instead of C). This
is due to the almost identical chemical and physical properties of analyte and standard and, thus, the
identical losses and effects.
=
=
If a known mass of a stable-isotope labelled standard is added to a test sample of known mass, the
execution of analysis and the calculation of analyte concentration become significantly easier and a
solvent calibration will be possible. This kind of quantification is also called isotope dilution analysis.
To be able to apply this technique:
— a stable-isotope labelled compound (often very expensive) of the analyte to be determined shall be
available;
— the proportion of the native analyte in the stable-isotope labelled standard shall be small so that the
quantification is not interfered;
— a mass-selective detector shall be used.
If these requirements are fulfilled, a calculation of the analyte concentration can be performed by
means of simplest calibration without exact knowledge of, for example, extraction volume, aliquot
quantities, dilution factors, matrix effects and other concentrations. In general, stable-isotope labelled
standards can be subject to isotope-exchange processes. The stable-isotope labelled standards shall be
selected so that these exchange processes are negligible. It still has to be ensured that the signals of
both standards do not significantly influence each other.
Calibration solutions with stable-isotope labelled standard are prepared by diluting a constant volume
cal
of the internal standard solution (V ) and a variable volume of the solution of native analyte with
ISTD
solvent to a defined volume (V ). The added volume of stable-isotope labelled internal standard shall
cal
cal
yield a concentration of the standard ( ρ ) which only nearly represents the expected concentration
ISTD
of the ISTD in the test solution ( ρ ) of the sample. It is not necessary to accurately know the
ISTD
concentration difference.
The concentration and, thus, the absolute peak intensity of the stable-isotope labelled standard in the
final extract of the sample should be 30 % to 300 % of the peak intensity of the stable-isotope labelled
standard in the calibration standards, see Formula (11):
cal cal
0,,30 × ρρ≤ ≤×3 0 ρ (11)
ISTD ISTD ISTD
where
cal
is the mass concentration of the stable-isotope labelled internal standard in the
ρ
ISTD
calibration standard [e.g. µg/ml];
is the expected mass concentration of the stable-isotope labelled internal standard in the
ρ
ISTD
test solution [e.g. µg/ml].
The calibration solutions obtained are used to establish a linear calibration function (see Formula 12)
cal
by plotting the respective quotients of the peak areas of analyte ( y ) and stable-isotope labelled
A
cal cal
internal standard ( y ) against the quotient of analyte mass ( m ) and mass of the stable-isotope
ISTD A
cal
labelled internal standard ( m ).
ISTD
cal cal cal cal
(12)
y //y = b×+m m c
A ISTD A ISTD
If the ratio of the analyte signal divided by the signal of the stable-isotope labelled internal standard is
within the calibrated range, the analyte mass in the sample is calculated by Formula (13):
yy/ − c
( )
A ISTD
m × m (13)
A ISTD
b
where
m is the mass of the (native) analyte in the test sample [e.g. µg];
A
y is the peak area (or peak height) of the analyte measured in the test solution [e.g. counts];
A
is the peak area (or peak height) of the stable-isotope labelled standard measured in the
y
ISTD
test solution [e.g. counts];
is the mass of the stable-isotope labelled standard added to the sample before extraction

m
ISTD
[e.g. µg];
b is the slope of the calibration function [dimensionless];
c is the ordinate intercept of the calibration function [dimensionless].
In this case, the mass fraction (w ) of the residue or analyte in the test sample is directly calculated by
A
the known mass of the test sample (m ) as in Formula (14):
sample
m
A
w = (14)
A
m
sample
Then, the calculation of the analyte concentration can be performed using the corresponding extraction
and aliquot volumes according to the sample treatment.
An example of calculation for a calibration with stable-isotope labelled standard is given in 9.6 (see
Example 6).
6.6 Calibration using standard addition procedures
6.6.1 General
In standard addition, the test sample or its extracts are always spiked with different amounts of the
analyte to be determined. The content of analyte in the sample is then calculated by comparing the
analyte signal of the unspiked sample with the analyte signals of the spiked samples or extracts.
The use of standard addition requires [7]:
— the knowledge of the approximate analyte content in the sample in order to appropriately adjust
the spiking levels;
— that the concentration of analyte in the spiked samples is within the working range of the linear
calibration function.
6.6.2 Standard addition to the final extract
aliquot
During this procedure, aliquots of the final extract are spiked with quantities ( m )of the analyte to
add
be analysed which are known before measurement and have graduated concentration (e.g. 1 : 2 : 3). All
spiked aliquots are brought to the same volume (V ) by adding solvent. Following this, the peak
End
areas of the analyte (y ) in
...