SIST EN 16923:2023

SLOVENSKI STANDARD
SIST EN 16923:2023
01-februar-2023
Nadomešča:
SIST EN 16923:2017
Živila - Določevanje toksinov T-2 in HT-2 v žitu in žitnih proizvodih za dojenčke in
majhne otroke s HPLC-MS/MS po čiščenju s SPE
Foodstuffs - Determination of T-2 toxin and HT-2 toxin in cereals and cereal products for
infants and young children by SPE clean up and HPLC-MS/MS
Lebensmittel - Bestimmung von T-2-Toxin und HT-2-Toxin in Getreide und Säuglings-
und Kleinkindernahrung auf Getreidebasis mit HPLC-MS/MS nach SPE-Reinigung
Produits alimentaires - Dosage des toxines T-2 et HT-2 dans les céréales et les produits
céréaliers pour nourrissons et enfants en bas âge par SPE et CLHP-SM/SM
Ta slovenski standard je istoveten z: EN 16923:2022
ICS:
67.060 Žita, stročnice in proizvodi iz Cereals, pulses and derived
njih products
67.230 Predpakirana in pripravljena Prepackaged and prepared
hrana foods
SIST EN 16923:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 16923:2023

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SIST EN 16923:2023


EN 16923
EUROPEAN STANDARD

NORME EUROPÉENNE

November 2022
EUROPÄISCHE NORM
ICS 67.060; 67.230 Supersedes EN 16923:2017
English Version

Foodstuffs - Determination of T-2 toxin and HT-2 toxin in
cereals and cereal products for infants and young children
by SPE clean up and HPLC-MS/MS
Produits alimentaires - Dosage des toxines T-2 et HT-2 Lebensmittel - Bestimmung von T 2 Toxin und HT 2
dans les céréales et les produits céréaliers pour Toxin in Getreide und Säuglings- und
nourrissons et enfants en bas âge par purification par Kleinkindernahrung auf Getreidebasis mit HPLC
SPE et CLHP-SM/SM MS/MS nach SPE-Reinigung
This European Standard was approved by CEN on 9 October 2022.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

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, Türkiye 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
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 16923:2022 E
worldwide for CEN national Members.

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SIST EN 16923:2023
EN 16923:2022 (E)
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 5
5 Reagents . 5
6 Apparatus and equipment . 8
7 Procedure. 9
7.1 Preparation of the test sample . 9
7.2 Preparation of the solid phase column . 9
7.3 Extraction of T-2 toxin and HT-2 toxin . 9
7.4 Clean-up by solid phase filtration . 10
7.5 LC-MS/MS-analysis . 10
7.6 Identification . 10
8 Calculation . 11
9 Precision . 12
9.1 General . 12
9.2 Repeatability . 12
9.3 Reproducibility . 12
10 Test report . 13
14
Annex A (informative) Example chromatograms (API 4000™) .
Annex B (informative) Example conditions for suitable LC-MS/MS systems . 18
B.1 System settings for SCIEX API 4000™ and SCIEX API 4000™ QTrap . 18
B.1.1 Settings for chromatography . 18
B.1.2 Detector parameters . 18
B.2 System settings for SCIEX API 2000 . 20
B.2.1 Settings for chromatography . 20
B.2.2 Detector parameters . 21
B.3 System settings for SCIEX API 3000™ . 22
B.3.1 Settings for chromatography . 22
B.3.2 Detector parameters . 22
B.4 System settings for Micromass Quattro LC . 23
B.4.1 Settings for chromatography . 23
B.4.2 Detector parameters . 24
Annex C (informative) Precision data . 25
Bibliography . 27
2

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SIST EN 16923:2023
EN 16923:2022 (E)
European foreword
This document (EN 16923:2022) has been prepared by Technical Committee CEN/TC 275 “Food
analysis - Horizontal methods”, the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by May 2023, and conflicting national standards shall be
withdrawn at the latest by May 2023.
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 supersedes EN 16923:2017.
In comparison with the previous edition, the following technical modifications have been made:
— the second elution step in the solid phase extraction in 7.4 is more clearly described.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: 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, Türkiye and the United
Kingdom.
3

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SIST EN 16923:2023
EN 16923:2022 (E)
Introduction
The mycotoxin T-2 toxin and its metabolite HT-2 toxin belong to the group of trichothecenes which are
produced by various Fusarium species. Cereals like maize, wheat, barley, oats and rye are most likely to
be affected.
WARNING 1 — Suitable precaution and protection measures need to be taken when carrying out working
steps with harmful chemicals. The latest version of the hazardous substances ordinance, Regulation (EC)
No 1907/2006 [3], should be taken into account as well as appropriate national statements, e.g. such as
in [4].
WARNING 2 — The use of this document can involve hazardous materials, operations and equipment.
This document does not purport to address all the safety problems associated with its use. It is the
responsibility of the user of this document to establish appropriate safety and health practices and
determine the applicability of regulatory limitations prior to use.
WARNING 3 — T-2 toxin and its metabolite HT-2 toxin are known to have carcinogenic effects.
4

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EN 16923:2022 (E)
1 Scope
This document describes a method for the determination of T-2 toxin and HT-2 toxin in cereals and
cereal-based products, e.g. oats, intended for nutrition of infants and young children by high performance
liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) after cleanup by solid
phase extraction (SPE) [5].
The method has been validated for HT-2 toxin in oat flour at levels of 9,3 µg/kg and 28,1 µg/kg, oat flakes
at levels of 16,5 µg/kg and 21,4 µg/kg, and breakfast cereals (containing oat flakes) at a level of 8,1 µg/kg
and for T-2 toxin in oat flour at levels of 4,4 µg/kg and 8,3 µg/kg, oat flakes at levels of 4,9 µg/kg and
6,6 µg/kg and breakfast cereals (containing oat flakes) at a level of 3,5 µg/kg.
Laboratory experiences [6] have shown that the method is also applicable to highly swelling materials
(dry cereal-based porridges and modified starches), but these were not examined in the method
validation study. Details are outlined in 7.3.
The method can also be applied to oat-by-products at higher levels of T-2- and HT-2 toxin. In this case,
the dilution steps need to be considered [6].
The method can also be applied to cereals and cereal products for infants and young children based on
e.g. wheat, barley and rice. In this case, the method needs to be in-house-validated for each material. At
the time of the interlaboratory study, planned range was 10 µg/kg to 100 µg/kg, and it is known from the
pre-study that the method works well in the whole range, although final validation was only done in the
range from 3,5 µg/kg to 28,1 µg/kg.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696)
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:
— ISO Online browsing platform: available at https://www.iso.org/obp/ui
— IEC Electropedia: available at https://www.electropedia.org/
4 Principle
T-2 toxin and HT-2 toxin are extracted with acetonitrile-water mixture and by shaking manually or with
a laboratory blender. A solid phase extraction column or a pass through column is used to clean up and
concentrate the filtered and diluted extract, see also [7]. The toxins are determined by HPLC coupled with
tandem mass spectrometry.
5 Reagents
Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696,
unless otherwise specified. Solvents shall be of quality for HPLC analysis, unless otherwise specified.
5

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EN 16923:2022 (E)
5.1 Acetonitrile, HPLC grade.
5.2 Methanol, HPLC grade.
5.3 Solvent mixture.
Mix 20 parts of acetonitrile (5.1) and 80 parts of water (20+80, v+v).
5.4 Extraction mixture.
Mix 84 parts of acetonitrile (5.1) and 16 parts of water (84+16, v+v).
5.5 Eluent for LC-MS/MS.
Examples of eluents suitable for LC-MS/MS systems are given in Annex B. Filter the solution through a
membrane filter (6.18).
5.6 Nitrogen, purity of at least 99,9 %.
5.7 Activated charcoal for column chromatography (particle size: 63 µm to 200 µm).
5.8 Aluminium oxide (neutral, for liquid chromatography).
1
5.9 Finely ground/pulverized diatomaceous earth (diatomite, kieselgur), e.g. Celite® 545 .
2
5.10 Siliconization reagent, e.g. Surfasil™ (optional).
5.11 Cyclohexane, analytical quality, (optional).
5.12 Preparation of the diluted siliconization reagent, (optional).
Add e.g. 50 ml of a siliconization reagent (5.10) to 950 ml cyclohexane (5.11).
5.13 Formic acid, HPLC quality.
5.14 Ammonia solution, substance concentration c(NH ) = 13,4 mol/l or mass concentration
3
ρ(NH ) = 250 g/l.
3
5.15 Ammonium acetate (CH CO NH ), LC-MS/MS quality.
3 2 4
5.16 Anti-clogging material, such as washed sea sand, glass beads, or polyethylene beads, (optional).
5.17 Stock solution of T-2 toxin, mass concentration ρ = 100 μg/ml, in acetonitrile.
T-2-toxin, e.g. crystalline or as a film, purity greater than 98 % mass fraction, or as certified standard
solution.
5.18 Stock solution of HT-2 toxin, ρ = 100 μg/ml, in acetonitrile.

1
Celite® 545 is a trade name of a product commercially available from various suppliers. This information is given for
the convenience of users of this document and does not constitute an endorsement by CEN of the products named.
Equivalent products may be used if they can be shown to lead to the same results.
2
Surfasil™ is a trade name of a product commercially available from various suppliers. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN of the products named. Equivalent
products may be used if they can be shown to lead to the same results.
6

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EN 16923:2022 (E)
HT-2 toxin, e.g. crystalline or as a film, purity greater than 98 % mass fraction, or as certified standard
solution.
13
5.19 Internal standard solution of [ C ]-T-2 toxin, ρ = 25 μg/ml, in acetonitrile.
24
Stock solution of T-2 toxin, mass concentration ρ = 25 μg/ml, in acetonitrile, taking into account the
certified purity and/or concentration.
13
Other suitable isotopic labelled standards of T-2 toxin than the [ C ]-T-2 toxin may be used.
24
13
5.20 Internal standard solution of [ C ]-HT-2 toxin, ρ = 25 μg/ml, in acetonitrile.
22
Stock solution of HT-2 toxin, ρ = 25 μg/ml, in acetonitrile, taking into account the certified purity and/or
concentration.
13
Other suitable isotopic labelled standards of HT-2 toxin than the [ C ]-HT-2 toxin may be used.
22
5.21 Mixed standard solution, ρ = 500 ng/ml.
Pipette 25 µl of each T-2 toxin and HT-2 toxin stock solution (5.17 and 5.18), respectively, into a 5 ml
volumetric flask, and dilute up to the mark with solvent mixture (5.3).
This solution can be stored at −18 °C for 12 months.
5.22 Mixed internal standard solution, ρ = 1000 ng/ml.
13
Dilute 200 µl of internal standard solution of [ C ]-T-2 toxin (5.19) and 200 µl of internal standard
24
13
solution of [ C ]-HT-2 toxin (5.20) with solvent mixture (5.3) in a 5 ml volumetric flask.
22
This solution can be stored at –18 °C for 6 months.
5.23 Calibration solutions.
For the calibration of the measuring system, prepare calibration solutions within a range from 5 ng/ml
to 100 ng/ml.
Prepare e.g. the following calibration solutions as outlined in Table 1:
Table 1 — Examples of suitable calibration solutions
Mixed
Mass Mixed
Mass internal Solvent
Calibration concentration standard
concentration standard mixture
solution per isotope solution
per analyte solution (5.3)
labelled analyte (5.21)
(5.22)
 ng/ml ng/ml µl µl µl
IS-Blank 0 50 – 50 950
1 5 50 10 50 940
2 10 50 20 50 930
3 20 50 40 50 910
4 40 50 80 50 870
5 60 50 120 50 830
6 80 50 160 50 790
7 100 50 200 50 750
7

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6 Apparatus and equipment
Usual laboratory apparatus and, in particular, the following.
6.1 Laboratory balance, accuracy of 0,01 g.
6.2 Analytical balance, accuracy of 0,1 mg.
6.3 Ultrasonic bath.
6.4 Laboratory shaker for test tubes.
6.5 Manual dispensers, microlitre syringes or microlitre pipettes for 10 µl to 5 ml.
6.6 Dispenser, suitable for 20 ml.
6.7 250 ml-Erlenmeyer flasks with stoppers, or 250 ml-centrifuge tubes.
6.8 Syringe filters (0,45 µm), or centrifugal filters (e.g. Durapore® PVDF (0,45 µm), or Millipore
3
Ultrafree-MC® 0,5 ml), fitting with centrifuge for reaction vessels, e.g. Eppendorf® vessels.
6.9 Folded filter, pore size 4 µm to 7 µm, diameter 100 mm.
6.10 Laboratory centrifuge.
6.11 Cartridges (6 ml), made from polypropylene (PP) and corresponding frits from polyethylene (PE),
1
, 6 ml, 500 mg.
or commercially available SPE columns, e.g. CHROMABOND® Carbon/Alox/Celite®
6.12 SPE vacuum/elution station.
6.13 Laboratory shaker, e.g. overhead shaker.
3
6.14 Laboratory blender, e.g. Ultra Turrax® .
6.15 Test tubes, suitable for a volume up to 10,0 ml.
6.16 Siliconized test tubes (optional).
After thorough cleaning of the test tubes (6.15), fill up to the top with the diluted siliconization reagent
(5.12) and allow them to stand for 1 min. Then, pouring out the reagent solution, make sure to collect it
for repeated usage. Afterwards rinse the tubes with cyclohexane (5.11) and acetonitrile (5.1) or methanol
(5.2) successively in this order. The rinsing solutions may be used again. Finally rinse the tubes twice
with double-distilled water and allow them to dry.
WARNING — Surfasil™, being a chloride silane solvent, readily reacts with water by forming hydrochloric
acid vapour. Therefore, never rinse tubes with water directly after derivatization.
Tubes that are not siliconized, such as those made from polypropylene, may be used, if formally proved
suitable.

3
Durapore® PVDF, Millipore Ultrafree-MC®, Ultra Turrax ®, TurboVap®LV Zymark and Surfasil™ are trade names of
products commercially available from various suppliers. Eppendorf® vessel is an example of a product commercially
available from Eppendorf, Chromabond® is the trade name of a product, commercially available from by Macherey-Nagel.
This information is given for the convenience of users of this document and does not constitute an endorsement by CEN
of the products named. Equivalent products may be used if they can be shown to lead to the same results.
8

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3
6.17 Concentration evaporator workstation, e.g. TurboVap® Zymark , or similar.
6.18 Membrane filters for aqueous solutions (pore size 0,45 µm).
6.19 LC-MS/MS system with the following components:
6.19.1 HPLC pump.
6.19.2 Injection system.
6.19.3 HPLC column, e.g. octadecylsilane (ODS), that ensures base line separation to distinguish peaks
of the T-2 toxin and HT-2 toxin from all other signals, 150 mm length, 2,00 mm inner diameter, particle
size 5 µm, suitable reversed-phase pre-column.
Columns of different dimensions may also be used.
6.19.4 Column thermostat.
6.19.5 Tandem mass spectrometer (MS/MS).
6.19.6 Data evaluation system.
7 Procedure
7.1 Preparation of the test sample
Grind and homogenize the sample to particle sizes less than 1 mm before analysis.
7.2 Preparation of the solid phase column
Mix 42 g of activated charcoal (5.7) with 30 g of neutral Al O (5.8) and 18 g of Celite 545 (5.9) in a glass
2 3
vessel (500 ml) and homogenize with a shaker (6.13) for 1 h (ratio 7:5:3 activated charcoal/neutral
Al O /Celite 545; m/m/m). Place the homogenized mixture, 0,5 g respectively, in empty 6 ml cartridges
2 3
provided with three PE frits (2 frits below, and one on top for covering).
Alternatively, commercially available SPE-columns may be used. For this reason, clean up procedure shall
be checked for recovery and shall be optimized if necessary [7].
7.3 Extraction of T-2 toxin and HT-2 toxin
Weigh 25,0 g of the homogenized and finely ground sample (7.1) with an accuracy of 0,1 g into a 250 ml
beaker/Erlenmeyer flask, or into a 250 ml centrifuge tube (6.7), add 100 ml of the extraction mixture
(5.4) and close the vessel. Shake the mixture with a shaker (6.13) for approximately 1 h at room
temperature.
Alternatively, use a laboratory blender (6.14) for extraction. In this case, homogenize the mixture for
3 min at a great speed.
After extraction, pass slightly more than 10 ml extract through a folded filter (6.9) into a glass vessel.
Centrifuge this portion at 2 500 × g at room temperature for 10 min. Remove 10 ml of the upper solution
of the centrifugate.
If highly swelling food matrices are analysed, increase the water content in the extraction medium up to
200 % or alternatively reduce the weight of the sample amount down to 50 % of the described amount.
To prevent clogging of the swelling material, add the same amount of e.g. sea sand (5.16) as the sample
weight.
Take volume and/or weight adjustments into account in the final calculation.
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7.4 Clean-up by solid phase filtration
Plug the prepared column containing 0,5 g of activated charcoal/Al O /Celite (7.2) on the SPE station
2 3
(6.12), and place a test tube (6.15) beneath to collect the eluate. Pass 5,0 ml of the extract (7.3) through
the SPE-column and collect the eluate. Apply a low vacuum in order to obtain an elution speed of 1 drop
to 2 drops per s. Rinse the cartridge two times with 5 ml of extraction mixture (5.4), and collect the
eluates also in the same test tube.
Add 25 µl of mixed internal standard solution (5.22) to the combined eluates, and evaporate to dryness
with nitrogen (5.6) using a concentration evaporator workstation (at 45 °C for 30 min, and 70 kPa gas
pressure).
Re-dissolve the residue in 500 µl of solvent mixture (5.3) by shaking turbulently (6.4) for 60 s, and, if
necessary, apply an ultrasonic bath (6.3) for 5 min at room temperature. The solution should be filtered
through syringe filters (6.8) or centrifugal filters (0,45 µm, 0,5 ml) at minimum 10 000 g in order to
discard any micro-particles to give the injection solution.
If necessary, the process of sample purification may be interrupted without analyte loss by storing the
sample extracts (evaporated to dryness) at 4 °C for several days, or at −18 °C for two weeks.
In case the residue level exceeds the calibration range, the residue should be dissolved in more than
500 µl of solvent mixture.
7.5 LC-MS/MS-analysis
Depending on the LC-MS/MS system, inject e.g. 10 µl to 25 µl of the injection solution derived from 7.4.
Set up a suitable measuring system by weighing in analytes and optimizing the separation and detection
parameters. Annex A lists some example chromatograms in Figure A.1 to Figure A.4, and Annex B lists
some suitable parameters.
The adopted measuring systems shall meet with the following requirements as in Table 2:
Table 2 — Prerequisites for precursor ions and product ions
Minimum-Signal-to-Noise Ratio Minimum-Signal-to-Noise Ratio
Analyte
for the Quantification Trace for the Confirmation Trace
a
T-2 toxin 50:1 at 250 pg 30:1 at 250 pg
HT-2 toxin 10:1 at 250 pg 5:1 at 250 pg
13
[ C ]-T-2 toxin 50:1 at 250 pg 30:1 at 250 pg
24
13
[ C ]-HT-2 toxin 10:1 at 250 pg 5:1 at 250 pg
22
a
250 pg is the calculated absolute mass amount of the analyte injected on the column. Signal is
peak height, noise is base line noise as observed in the extracted ion chromatogram.
7.6 Identification
Identify each mycotoxin by comparing the retention times of the calibration solutions with that of the
sample test solution. Identify the analyte on the basis of at least two mass transitions. In addition, the
retention times (peaks in both mass traces) and the area ratio of the two peaks shall match that of the
standard substance [8].
Identify the analyte on the basis of at least two mass transitions. In addition, the retention times (peaks
in both mass traces) and the area ratio of the two peaks shall match that of the standard substance.
10

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8 Calculation
For the quantification of T-2 toxin and HT-2 toxin, the corresponding isotope labelled standard is used as
internal standard. Carry out a multi-point calibration with a standard solution, for example according to
Table 1 in 5.23.
For the linear regression plot the peak area ratio from each analyte against the corresponding
concentration ratio. The mass concentration of each toxin in the injection solution is derived from linear
regression in ng/ml, see Formula (1).
A ρ
a a
ab+ (1)
Cal Cal
A ρ
IS IS
where
A is the peak area of the analyte in the calibration solution (5.23);
a
A is the peak area of the internal standard in the calibration solution (5.23);
IS
a is the slope of the calibration graph determined by the calibration solutions (5.23);
Cal
ρ is the mass concentrations of the analytes in the calibration solutions (5.23), in ng/ml;
a
ρ is the internal standard concentration in the calibration solution (5.23), in ng/ml, usually
IS
50 ng/ml;
b is the axis intercept of the calibration determined by the calibration solutions (5.23).
Cal
s
Calculate the mass concentration of the analyte in the sample test solution ρ , in ng/ml using
a
Formula (2):
s
A
a
−b
Cal
s
A
ss
IS
ρρ= (2)
a IS
a
Cal
where
s
is the response of the analyte in the sample test solution;
A
a

s
is the response of the internal standard in the sample test solution;
A
IS

s
is the mass concentration of the internal standard in the sample test solution of 50 ng/ml.
ρ
IS

Calculate the mass fraction of the analyte, w in µg/kg using Formula (3):
a
s
ρ ××VV
a ex im
(3)
w=
a
mV×
s Aliquot
where
is the volume of extraction solution, in ml, here 100 ml;
V
ex

is the volume of solvent mixture used to re-dissolve the evaporated sample, in ml, here
V
im

0,5 ml;
is the mass of the test portion, in g, here 25 g;
m
s

is the sample extract aliquot, in ml, here 5 ml.
V
Aliquot

11
=

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