SIST EN 17521:2021
(Main)Foodstuffs - Determination of Alternaria toxins in tomato, wheat and sunflower seeds by SPE clean-up and HPLC-MS/MS
Foodstuffs - Determination of Alternaria toxins in tomato, wheat and sunflower seeds by SPE clean-up and HPLC-MS/MS
This European Standard specifies a method for the determination of five Alternaria toxins in wheat, tomato juice and sunflower seed samples by liquid chromatography tandem mass spectrometry (LC-MS/MS). The method includes the analysis of Altenuene (ALT), Alternariol (AOH), Alternariol monomethyl ether (AME) in the range of 1 μg/kg to 100 μg/kg, and Tentoxin (TEN) in the range of 5 μg/kg to 500 μg/kg, and Tenuazonic acid (TEA) in the range of 10 μg/kg to 1000 μg/kg.
Lebensmittel - Bestimmung von Alternariatoxinen in Tomaten, Weizen und Sonnenblumenkernen mit Flüssigchromatographie und Tandem-Massenspektrometrie
Dieses Dokument legt ein Verfahren zur Bestimmung von fünf Alternaria-Toxinen an Weizen, Tomatenmark und Sonnenblumenkernen durch Hochleistungs-Flüssigkeitschromatographie (HPLC) in Verbindung mit Tandem-Massenspektrometrie (MS/MS) fest.
Das Verfahren wurde mit sowohl natürlich kontaminierten als auch dotierten Weizen-, Tomatenmark- und Sonnenblumenkernproben validiert.
Die Validierungswerte für Altenuen (ALT) lagen im Bereich von 2,18 μg/kg bis 13,8 μg/kg.
Die Validierungswerte für Alternariol (AOH) lagen im Bereich von 1,82 μg/kg bis 46,7 μg/kg.
Die Validierungswerte für Alternariolmonomethylether (AME) lagen im Bereich von 1,29 μg/kg bis 47,2 μg/kg.
Die Validierungswerte für Tentoxin (TEN) lagen im Bereich von 5,29 μg/kg bis 218 μg/kg.
Die Validierungswerte für Tenuazonsäure (TEA) lagen im Bereich von 41,8 μg/kg bis 1 618 μg/kg.
Mit diesem Verfahren sind Bestimmungsgrenzen von 1 μg/kg für ALT (ausgenommen bei Weizen und Sonnenblumenkernen), AOH und AME, von 5 μg/kg für TEN und von 10 μg/kg für TEA oder niedriger erreichbar.
Produits alimentaires - Détermination de la teneur en toxines d'Alternaria dans la tomate, le blé et les graines de tournesol purification par SPE et CLHP-SM/SM
Le présent document décrit un mode opératoire pour la détermination de la teneur en cinq toxines
d’Alternaria dans la purée de tomate, le blé et les graines de tournesol par chromatographie liquide
haute performance (CLHP) couplée à la spectrométrie de masse en tandem (SM/SM).
La méthode a été validée avec des échantillons aussi bien naturellement contaminés que dopés de blé,
de purée de tomate et de graines de tournesol.
Les teneurs de validation pour l’altenuène (ALT) étaient comprises entre 2,18 μg/kg et 13,8 μg/kg.
Les teneurs de validation pour l’alternariol (AOH) étaient comprises entre 1,82 μg/kg et 46,7 μg/kg.
Les teneurs de validation pour l’alternariol monométhyl éther (AME) étaient comprises entre
1,29 μg/kg et 47,2 μg/kg.
Les teneurs de validation pour la tentoxine (TEN) étaient comprises entre 5,29 μg/kg et 218 μg/kg.
Les teneurs de validation pour l’acide ténuazonique (TEA) étaient comprises entre 41,8 μg/kg et
1 618 μg/kg.
Il est possible d’atteindre des limites de quantification de 1 μg/kg pour l’ALT (sauf dans le cas du blé et
des graines de tournesol), l’AOH et l’AME, de 5 μg/kg pour la TEN et de 10 μg/kg pour le TEA, ou des
valeurs inférieures à l’aide de la présente méthode.
Živila - Določevanje Alternaria toksinov v paradižniku, pšenici in sončničnih semenih z SPE čiščenjem in HPLC-MS/MS
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN 17521:2021
01-december-2021
Živila - Določevanje Alternaria toksinov v paradižniku, pšenici in sončničnih
semenih z SPE čiščenjem in HPLC-MS/MS
Foodstuffs - Determination of Alternaria toxins in tomato, wheat and sunflower seeds by
SPE clean-up and HPLC-MS/MS
Lebensmittel - Bestimmung von Alternariatoxinen in Tomaten, Weizen und
Sonnenblumenkernen mit Flüssigchromatographie und Tandem-Massenspektrometrie
Produits alimentaires - Détermination de la teneur en toxines d'Alternaria dans la tomate,
le blé et les graines de tournesol purification par SPE et CLHP-SM/SM
Ta slovenski standard je istoveten z: EN 17521:2021
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST EN 17521:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN 17521:2021
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SIST EN 17521:2021
EN 17521
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2021
EUROPÄISCHE NORM
ICS 67.050
English Version
Foodstuffs - Determination of Alternaria toxins in tomato,
wheat and sunflower seeds by SPE clean-up and HPLC-
MS/MS
Produits alimentaires - Détermination de la teneur en Lebensmittel - Bestimmung von Alternariatoxinen in
toxines d'Alternaria dans la tomate, le blé et les graines Tomaten, Weizen und Sonnenblumenkernen mit
de tournesol purification par SPE et CLHP-SM/SM Flüssigchromatographie und Tandem-
Massenspektrometrie
This European Standard was approved by CEN on 21 June 2021.
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, 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
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17521:2021 E
worldwide for CEN national Members.
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SIST EN 17521:2021
EN 17521:2021 (E)
Contents Page
European foreword . 3
Introduction . 3
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 5
5 Reagents . 6
6 Apparatus and equipment . 9
7 Procedure . 11
8 Calculation . 14
9 Precision . 14
10 Test report . 17
Annex A (informative) Example chromatograms . 18
Annex B (informative) Example conditions for suitable HPLC-MS/MS systems . 21
Annex C (informative) Precision data . 26
Bibliography . 37
2
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SIST EN 17521:2021
EN 17521:2021 (E)
European foreword
This document (EN 17521:2021) 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 February 2022, and conflicting national standards shall
be withdrawn at the latest by February 2022.
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 has been prepared under a Standardization Request given to CEN by the European
Commission and the European Free Trade Association.
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, Turkey and the United
Kingdom.
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SIST EN 17521:2021
EN 17521:2021 (E)
Introduction
Alternaria species, the most prevalent being A. alternata, produce more than 70 secondary metabolites,
but only a few of them have been structurally identified and reported as mycotoxins [1]. Alternaria fungi
are common plant pests in cereals, oilseeds, fruits and vegetables, among other foods, and the presence
of Alternaria toxins in these commodities has been widely reported [1]. These toxins do not only
contaminate harvests but they can also spoil foods at refrigerator temperatures.
Among these Alternaria toxins altenuene (ALT), alternariol (AOH), alternariol monomethyl ether (AME),
tentoxin (TEN) and tenuazonic acid (TEA) are the ones of major concern. ALT, AOH, AME are dibenzo-α-
pyrones, TEN is a cyclic tetrapeptide and TEA is a tetramic acid derivative. ALT and TEA have shown high
acute toxicity in vitro and in animal experiments. AME and AOH are not very acutely toxic; however, they
have been described to induce genotoxic and mutagenic effects [1 - 3].
These toxins generally appear in tomato products, cereals and oilseeds (e.g. sunflower seeds), therefore
they are the focus of this document.
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 [4] should be taken into account as well as appropriate national statements.
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 compatibility with regulatory limitations prior to use.
WARNING 3 — Some Alternaria toxins exhibit genotoxic and mutagenic effects.
4
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SIST EN 17521:2021
EN 17521:2021 (E)
1 Scope
This document specifies a procedure for the determination of five Alternaria toxins in wheat, tomato
puree and sunflower seeds by high-performance liquid chromatography (HPLC) coupled with tandem
mass spectrometry (MS/MS).
The method has been validated with naturally contaminated and spiked samples of wheat, tomato puree
and sunflower seeds.
Validation levels for altenuene (ALT) ranged from 2,18 μg/kg to 13,8 μg/kg.
Validation levels for alternariol (AOH) ranged from 1,82 μg/kg to 46,7 μg/kg.
Validation levels for alternariol monomethyl ether (AME) ranged from 1,29 μg/kg to 47,2 μg/kg.
Validation levels for tentoxin (TEN) ranged from 5,29 μg/kg to 218 μg/kg.
Validation levels for tenuazonic acid (TEA) ranged from 41,8 μg/kg to 1 618 μg/kg.
Limits of quantification of 1 μg/kg for ALT (except in wheat and sunflower seeds – 1,4 μg/kg and
1,2 μg/kg, respectively), AOH and AME; 5 μg/kg for TEN and 10 μg/kg for TEA or lower are achievable
using this method.
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:
• IEC Electropedia: available at https://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
4 Principle
A test portion of the sample spiked with the isotopically-labelled internal standards is extracted with a
mixture of methanol, water and acetic acid. The sample/extraction solvent mixture is centrifuged and an
aliquot of the supernatant is collected. The extract is diluted with an equal volume of 1 % (φ) aqueous
acetic acid solution, and concentrated on a polymeric solid-phase extraction (SPE) adsorbent. The extract
is eluted from the SPE column with a methanol and ethyl acetate solution. The eluate is then evaporated,
reconstituted, filtered through a polytetrafluoroethylene (PTFE) syringe filter and subsequently analysed
by HPLC-MS/MS.
5
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SIST EN 17521:2021
EN 17521:2021 (E)
5 Reagents
Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696,
unless otherwise specified. Solutions shall be of quality for LC-MS analysis, unless otherwise specified.
Commercially available solutions with equivalent properties to those listed may be used.
5.1 Nitrogen compressed gas, purity equivalent to φ = 99,99 % or better.
5.2 Water (H O), HPLC grade.
2
5.3 Water (H O), LC-MS grade.
2
5.4 Methanol (CH OH), analytical grade.
3
5.5 Methanol (CH OH), LC-MS grade.
3
5.6 Ethyl acetate (CH COOC H ), analytical grade or higher.
3 2 5
5.7 Ammonium hydroxide (NH OH), LC-MS grade, mass fraction w(NH OH) = 25 %.
4 4
5.8 Ammonium hydroxide (NH OH), w(NH OH) = 2,3 %.
4 4
Add 1 ml of ammonium hydroxide 25 % (5.7) to a 10 ml volumetric flask containing approximately 5 ml
of water (5.3) and fill up to the mark with water (5.3).
5.9 Acetic acid (CH COOH), w ≥ 99,7 %.
3
5.10 Ammonium acetate (CH COONH ), LC-MS grade.
3 4
5.11 Ammonium acetate solution (CH COONH ), molar concentration c = 1 mol/l.
3 4
Dissolve 77,08 g of ammonium acetate (5.10) in 1 l of water (5.3).
5.12 Extraction mixture, methanol + water + acetic acid (85 + 14 + 1, V + V + V) mixture.
Mix 850 ml of methanol (5.4) with 140 ml of water (5.2) and 10 ml of acetic acid (5.9).
5.13 Aqueous acetic acid solution, acetic acid + water (1 + 99, V + V), φ = 1 %.
Mix 10 ml of acetic acid (5.9) with 990 ml of water (5.2) and homogenize well.
5.14 Elution solution, methanol + ethyl acetate (75 + 25, V + V).
Mix 750 ml of methanol (5.4) with 250 ml of ethyl acetate (5.6) and homogenize well.
5.15 HPLC mobile phase A, 5 mmol/l ammonium acetate buffer at pH approximately 8,0.
Mix 5 ml of ammonium acetate solution (5.11) and approximately 200 µl of ammonium hydroxide 2,3 %
(5.8) with 900 ml of water (5.3). Adjust the volume with water (5.3) to 1 l and homogenize well.
Check the pH of the mobile phase A. The pH shall be between 7,95 and 8,05. Adjust with ammonium
hydroxide 2,3 % (5.8) to be within that range.
5.16 HPLC mobile phase B, methanol (5.5).
5.17 Alternaria toxins’ standards, e.g. crystalline, as a film or as a reference material.
5.17.1 Altenuene, at least w = 96 % purity.
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5.17.2 Alternariol, at least w = 96 % purity.
5.17.3 Alternariol monomethyl ether, at least w = 96 % purity.
5.17.4 Tentoxin, at least w = 96 % purity.
5.17.5 Tenuazonic acid, at least w = 96 % purity.
Laboratories can seek for a higher purity, if available on the market.
5.18 Isotopically-labelled internal standards, e.g. crystalline or as a standard solution.
5.18.1 Altenuene isotopically-labelled internal standard (ALT-ISTD), e.g. ALT-(methoxy-d3,
) (ALT-d ).
methyl-d3 6
5.18.2 Alternariol isotopically-labelled internal standard (AOH-ISTD), e.g. AOH-(methyl-d )
3
(AOH-d ).
3
5.18.3 Alternariol monomethyl ether isotopically-labelled internal standard (AME-ISTD), e.g.
AME-(1-methyl-d ) (AME-d ).
3 3
5.18.4 Tentoxin isotopically-labelled internal standard (TEN-ISTD), e.g. TEN-d .
3
13
5.18.5 Tenuazonic acid isotopically-labelled internal standard (TEA-ISTD), e.g. TEA-(acetyl- C )
2
13
(TEA- C ), mixture of diastereomers in methanol.
2
Laboratories can seek for isotopically-labelled internal standards with a high degree of deuteration or
13
C-enrichment.
WARNING – Protective clothing, gloves and safety goggles should be worn at all times, and all standard
and sample preparation stages should be carried out in a fume hood.
5.19 Stock solutions of ALT, AOH, AME, TEN and TEA, e.g. at a mass concentration ρ = 100 µg/ml.
In case of crystalline powders, weigh in e.g. 5 mg of ALT (5.17.1), AOH (5.17.2), AME (5.17.3),
TEN (5.17.4) and TEA (5.17.5) to the nearest 0,1 mg into individual 50 ml volumetric flasks and fill up to
the mark with methanol (5.5). Homogenize vigorously.
In case of dried-down films, reconstitute the standard in the vial according to the certificate of each
individual standard.
The stock solutions are stable for at least six months if stored at ≤ −18 °C.
NOTE For the purpose of photometrically verifying the concentration of gravimetrically prepared standards,
molar extinction coefficients are available in the literature for AOH [5], AME [5], ALT [6], TEN [7] and TEA [8].
5.20 Standard solution 1.
Prepare a methanolic standard mixture that contains ALT (5.17.1), AOH (5.17.2), AME (5.17.3) in
500 ng/ml concentration, TEN (5.17.4) in 2 500 ng/ml concentration and TEA (5.17.5) in 5 000 ng/ml
concentration.
For that purpose, transfer e.g. 25 µl of the stock solutions of ALT, AOH and AME (5.19) into a 5 ml
volumetric flask. Transfer e.g. 125 µl of stock solution of TEN (5.19) and e.g. 250 µl of stock solution of
TEA (5.19) into the same 5 ml volumetric flask. Fill up to the mark with methanol (5.5). Homogenize
vigorously. The standard solution 1 is stable for at least six months if stored at ≤ −18 °C.
NOTE The exact volumes to be used to prepare the standard solution 1 (5.20) are derived from the exact
concentration of the stock solutions (5.19).
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5.21 Standard solution 2.
Prepare a methanolic standard mixture that contains ALT (5.17.1), AOH (5.17.2), AME (5.17.3) in
100 ng/ml concentration, TEN (5.17.4) in 500 ng/ml concentration and TEA (5.17.5) in 1 000 ng/ml
concentration. Standard solution 2 is prepared by diluting standard solution 1 (5.20).
Transfer 1 000 µl of the standard solution 1 (5.20) into a 5 ml volumetric flask. Fill up to the mark with
methanol (5.5). Homogenize vigorously. Standard solution 2 is stable for at least six months if stored
at ≤ −18 °C.
5.22 Stock solutions of ALT-ISTD, AOH-ISTD, AME-ISTD, TEN-ISTD and TEA-ISTD, e.g. at a mass
concentration ρ = 750 µg/ml.
Prepare the stock solutions, e.g. dissolve 1,12 mg of crystalline powders of ALT-ISTD (5.18.1), AOH-ISTD
(5.18.2), AME-ISTD (5.18.3), TEN-ISTD (5.18.4) and TEA-ISTD (5.18.5) in individual vials with 1 500 µl of
methanol (5.5). Homogenize vigorously. The stock solutions are stable for at least six months if stored
at ≤ −18 °C. TEA-ISTD might be supplied already as a methanolic solution with approximately the same
concentration.
5.23 Internal standard solution 1.
Prepare a methanolic standard mixture that contains AOH-ISTD (5.18.2), AME-ISTD (5.18.3) and TEN-
ISTD (5.18.4) in 5 μg/ml concentration, ALT-ISTD (5.18.1) in 10 μg/ml concentration and TEA-ISTD
(5.18.5) in 25 μg/ml concentration.
For that purpose, transfer e.g. 33 µl of the stock solutions of AOH-ISTD, AME-ISTD and TEN-ISTD (5.22)
into a 5 ml volumetric flask. Transfer e.g. 67 µl of stock solution of ALT-ISTD (5.22) and e.g. 167 µl of stock
solution of TEA-ISTD (5.22) into the same 5 ml volumetric flask. Fill up to the mark with methanol (5.5).
Homogenize vigorously. The internal standard solution 1 is stable for at least six months if stored
at ≤ −18 °C.
5.24 Internal standard solution 2.
Prepare a methanolic standard mixture that contains AOH-ISTD (5.18.2), AME-ISTD (5.18.3) and TEN-
ISTD (5.18.4) in 500 ng/ml concentration, ALT-ISTD (5.18.1) in 1 000 ng/ml concentration and TEA-ISTD
(5.18.5) in 2 500 ng/ml concentration. Internal standard solution 2 is prepared by diluting internal
standard solution 1 (5.23).
Transfer 1 000 µl of the internal standard solution 1 (5.23) into a 10 ml volumetric flask. Fill up to the
mark with methanol (5.5). Homogenize vigorously. The internal standard solution 2 is stable for at least
six months if stored at ≤ −18 °C.
1
5.25 Polysorbate 20 (C H O ) (Tween®20 ), analytical grade.
58 114 26
5.26 Polysorbate 20 (C H O ) solution, φ = 2 % in water.
58 114 26
Pipette 2 ml of polysorbate 20 (5.25) into a 100 ml volumetric flask and fill up to the mark with water
(5.2). Homogenize well. This solution can be used for three months if stored at approximately 4 °C.
1
Tween®20 is a trade name of a polysorbate 20-type nonionic surfactant available from different suppliers.
This information is given for the convenience of users of this document and does not constitute an endorsement
by CEN of this product. Equivalent products may be used if they can be shown to lead to the same results.
8
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5.27 Calibration solutions.
Add different volumes of standard solution (5.20 resp. 5.21) and the internal standard solution 2 (5.24)
to five HPLC vials (6.18) e.g. as listed in Table 1. Add the methanol (5.5) volumes specified in Table 1 and
mix gently. Add 600 µl of HPLC mobile phase A (5.15), cap the vial and mix it (6.15) for approximately
20 s.
Prepare a sixth vial (blank) without standard solutions and without internal standard solution and use it
as an instrumental blank.
Table 1 — Preparation of calibration solutions
ALT AOH AME TEN TEA
Calibration Standard Standard Internal Methanol Equivalent concentration
solution solution 1 solution 2 standard (5.5)
(5.20) (5.21) solution 2
(5.24)
µl µl µl µl µg/kg
1 – 10 50 340 1 1 1 5 10
2 – 50 50 300 5 5 5 25 50
3 – 100 50 250 10 10 10 50 100
4 50 – 50 300 25 25 25 125 250
5 200 – 50 150 100 100 100 500 1000
Blank – – – 400 – – – – –
6 Apparatus and equipment
Usual laboratory glassware and equipment and, in particular, the following.
6.1 pH meter.
6.2 Polypropylene (PP) centrifuge tube, (50 ml) with scale on it.
6.3 Laboratory balance, accuracy of 0,01 g.
6.4 Analytical balance, accuracy of 0,01 mg.
6.5 Adjustable mechanical vertical or horizontal shaker.
2
6.6 High speed blending device (e.g. Ultra-turrax® ).
6.7 Centrifuge, with temperature control and capable of generating a relative centrifugal force of
approximately 3 200 g.
6.8 Graduated volumetric pipettes, 10 ml capacity.
2
Ultra-turrax and Strata-XL are examples of suitable products available commercially. This information is given
for the convenience of users of this document and does not constitute an endorsement by CEN of these
products. Equivalent products may be used if they can be shown to lead to the same results.
9
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6.9 Displacement pipettes, e.g. 10 μl, 20 μl, 100 μl, 250 μl and 1 000 μl capacity, with appropriate tips.
6.10 Solid-phase extraction (SPE) column, with hydrophilic modified styrene polymer with 6 ml
reservoir capacity, 200 mg adsorbent mass and, 100 μm particle size or smaller.
2
NOTE Phenomenex Strata-XL , with 6 ml reservoir capacity, 200 mg adsorbent mass and 100 µm particle size
have shown to meet these specifications.
6.11 PP reservoirs (approximately 25 ml), fit to SPE columns (6.10).
6.12 Polytetrafluoroethylene (PTFE) syringe filter, 0,2 µm pore size and e.g. 13 mm or 15 mm of
diameter.
6.13 Syringe with needle, 1 ml.
6.14 Vacuum manifold, for SPE clean-up, with taps.
6.15 Mixer, with high shear rate.
6.16 Sample concentrator, with temperature control and gas supply.
6.17 Glass receiving tubes, for sample elution and evaporation.
6.18 Silanized glass HPLC vials, approximately 1,5 ml capacity and crimp caps or equivalent.
6.19 Beakers, 250 ml capacity.
6.20 Volumetric flasks, 5 ml, 10 ml, 50 ml, 100 ml and 1 l capacity.
6.21 HPLC-MS/MS system, with the following components.
6.21.1 HPLC pump, capable of maintaining a binary gradient at flow rates appropriate for the analytical
column in use with sufficient accuracy.
6.21.2 Degasser, optional, for degassing HPLC mobile phases.
6.21.3 Injection system, capable of injecting an appropriate volume of test solution with sufficient
accuracy.
6.21.4 HPLC reversed phase column.
A suitable column and appropriate HPLC conditions providing sufficient retention capacity of the first
eluting analyte. Acceptable chromatograms, as shown in Annex A, can be achieved using a column with a
minimum capacity factor of 3 (k′ ≥ 3,0) and a minimum plate number of 2 000 (N ≥ 2 000) for any of the
analytes.
Examples of suitable columns and analytical conditions are reported in Annex A and Annex B.
6.21.5 Pre-column, recommended, with the same stationary phase material as the analytical column
(6.21.4).
6.21.6 Column oven, capable of maintaining a constant temperature.
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6.21.7 Triple stage mass spectrometer.
Triple quadrupole, ion trap or quadrupole linear ion trap instrument, amongst others, equipped with an
electrospray ionization (ESI) interface and operated in multiple reaction monitoring (MRM) mode. Any
ionization mode (typically negative, or positive) giving sufficient yield might be employed.
6.21.8 Computer-based instrument control and data evaluation system.
7 Procedure
7.1 General
If not stated otherwise, all reagents, samples and equipment used in Clause 7.2 to 7.5 shall be at room
temperature before any kind of manipulation takes place.
7.2 Preparation of the test sample
Solid laboratory samples shall be finely ground and homogenized. Care shall be taken not to overheat the
sample during this process. Cryo-milling of fatty samples might be required.
Semi-solid samples shall be thoroughly homogenized by vigorous shaking for approximately 10 min.
NOTE When applying slurry mixing it is essential to end up in the prescribed extraction mixture. A lower
content of organic solvent can result in extractability limitations of less water soluble Alternaria toxins.
7.3 Extraction
Weigh 2,00 g to the nearest 0,01 g into a 50 ml centrifuge tube (6.2) for single analysis. Add 100 μl of
internal standard solution 2 (5.24).
For tomato puree: add 14,0 ml of extraction mixture (5.12). For wheat and sunflower seeds flour: add
15,0 ml of extraction mixture (5.12).
Cap the tube tightly and mix it for approximately 10 s to obtain a homogeneous suspension.
Sunflower seeds flour can get sticky and clog during manipulation. To allow for a complete extraction,
these samples need to be sufficiently dispersed in the extraction mixture using a high speed blender (6.6)
for approximately 30 s prior to the extraction.
Shake it vigorously for 45 min ± 1 min with a shaker (6.5).
A small amount of the sunflower seeds sample might stick to the wall of the tubes during extraction. In
such event, take the tubes out of the shaker and shake it by hand for 5 s to remove the material from the
walls. Bring the tubes back into the shaker and continue the extraction.
Centrifuge the sample at room temperature for at least 10 min at approximately 3 200 g (6.7) and
transfer 7,5 ml of the supernatant (equal to 1,0 g sample) into a new 50 ml centrifuge tube (6.2).
Dilute with an equal volume (7,5 ml) of 1 % aqueous acetic acid solution (5.13). Homogenize by mixing
for approximately 5 s.
After dilution of the extract, turbidity might form in the solution depending on the sample. Centrifugation
may be performed for 10 min at approximately 3 200 g and room temperature (6.7) to avoid clogging of
the SPE cartridge.
11
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7.4 Solid phase extraction clean up
Connect the SPE column (6.10) to the vacuum manifold (6.14).
Condition the column by passing through 7 ml of methanol (5.4), followed by 7 ml of water (5.2) and 4 ml
of 1 % aqueous acetic acid solution (5.13).
Close the tap under the column and pipette another 3 ml of 1 % aqueous acetic acid solution (5.13) into
the SPE column. Attach a reservoir (6.11) onto the SPE column.
Load the diluted sample into the reservoir. Wash the 50 ml centrifuge tube, which contained the diluted
sample, with 4 ml of 1 % aqueous acetic acid solution (5.13) and load it into the reservoir. Open the tap
and let the extract pass through at a flow rate of approximately 1 drop per second.
Remove the reservoir and wash the column with 4 ml of polysorbate 20 solution (5.26) followed by 4 ml
of 1 % aqueous acetic acid solution (5.13). Dry the column thoroughly with vacuum (approximately
10 min).
7.5 Preparation of the sample test solution
Place a glass receiving tube (6.17) in the vacuum manifold. Elute the extract with 7 ml of elution solution
(5.14) into the glass receiving tube. After the solution has passed through, dry the column for about 10 s
with vacuum and collect all eluate.
If the elution solution does not start dropping spontaneously, a gentle vacuum may be applied in order
to trigger the flow. After the first drops flow from the column, the vacuum is not needed.
Evaporate the eluate in the glass receiving tube (6.17) at 50 °C using the sample concentrator (6.16) and
a gentle stream of nitrogen (5.1).
Add 400 µl of methanol (5.5) to the dry extract and re-dissolve the residue on the tube walls by mixing
for at least 20 s.
Add 600 µl of HPLC mobile phase A (5.15) and mix for at least another 20 s.
Filter the extract through a PTFE syringe filter (6.12), with the aid of a 1 ml syringe (6.13), into an HPLC
vial (6.18).
If filtering does not result in a clear test solution, centrifugation of the test solution may be performed.
7.6 HPLC-MS/MS analysis
7.6.1 HPLC-MS/MS operating conditions
A combination of analytical column, mobile phase composition, gradient settings and injection volume
shall be such that it allows obtaining acceptable chromatographic separation of the analytes and reliable
results at the required levels, with sufficient selectivity.
Annex A illustrates an example chromatogram, and Annex B gives example conditions for suitable
HPLC-MS/MS systems.
7.6.2 Calibration curve
Before starting the injection sequence the HPLC-MS/MS instrument shall be equilibrated by injecting
blank solutions and matrix-fr
...
SLOVENSKI STANDARD
oSIST prEN 17521:2020
01-julij-2020
Živila - Določevanje toksinov "Alternaria" v paradižniku, pšenici in sončničnih
semenih s čiščenjem SPE in HPLC-MS/MS
Foodstuffs - Determination of Alternaria toxins in tomato, wheat and sunflower seeds by
SPE clean-up and HPLC-MS/MS
Lebensmittel - Bestimmung von Alternariatoxinen in Tomaten, Weizen und
Sonnenblumenkernen mit Flüssigchromatographie und Tandem-Massenspektrometrie
Produits alimentaires - Détermination de la teneur en toxines d'Alternaria dans la tomate,
le blé et les graines de tournesol purification par SPE et CLHP-SM/SM
Ta slovenski standard je istoveten z: prEN 17521
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
oSIST prEN 17521:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 17521:2020
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oSIST prEN 17521:2020
DRAFT
EUROPEAN STANDARD
prEN 17521
NORME EUROPÉENNE
EUROPÄISCHE NORM
May 2020
ICS 67.050
English Version
Foodstuffs - Determination of Alternaria toxins in tomato,
wheat and sunflower seeds by SPE clean-up and HPLC-
MS/MS
Produits alimentaires - Détermination de la teneur en Lebensmittel - Bestimmung von Alternariatoxinen in
toxines d'Alternaria dans la tomate, le blé et les graines Tomaten, Weizen und Sonnenblumenkernen mit
de tournesol par chromatographie liquide couplée à la Flüssigchromatographie und Tandem-
spectrométrie de masse en tandem Massenspektrometrie
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 275.
If this draft becomes a European Standard, 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.
This draft European Standard was established by CEN 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, 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 European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17521:2020 E
worldwide for CEN national Members.
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Contents Page
European foreword . 3
Introduction . 4
7.1 Preparation of the test sample . 11
7.2 Extraction . 11
7.3 Solid phase extraction clean up . 11
7.4 Preparation of the sample test solution . 12
7.5 LC-MS/MS analysis . 12
7.5.1 LC-MS/MS operating conditions . 12
7.5.2 Calibration curve . 12
7.5.3 Injection sequence . 12
7.6 Identification . 13
7.7 Calibration . 13
9.1 General. 14
9.2 Repeatability . 14
9.3 Reproducibility . 14
Annex A (informative) Example chromatograms . 17
Annex B (informative) Example conditions for suitable LC-MS/MS systems . 20
Annex C (informative) Precision data . 24
Bibliography . 37
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European foreword
This document (prEN 17521:2020) 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 CEN Enquiry.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
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Introduction
Alternaria species, the most prevalent being A. alternata, produce more than 70 secondary metabolites,
but only a few of them have been structurally identified and reported as mycotoxins [1]. Alternaria
fungi are common plant pests in cereals, oilseeds, fruits and vegetables, among other foods, and the
presence of Alternaria toxins in these commodities has been widely reported [1]. These toxins do not
only contaminate harvests but they can also spoil foods in the fridge-temperatures.
Among these Alternaria toxins altenuene (ALT), alternariol (AOH), alternariol monomethyl ether
(AME), tentoxin (TEN) and tenuazonic acid (TEA) are the ones of major concern. ALT, AOH, AME are
dibenzo-α-pyrones, TEN is a cyclic tetrapeptide and TEA is a tetramic acid derivative. ALT and TEA have
shown high acute toxicity in vitro and in animal experiments. AME and AOH are not very acutely toxic;
however, they have been described to induce genotoxic and mutagenic effects [1] to [3].
These toxins generally appear in tomato products, cereals and oilseeds (e.g. sunflower seeds), therefore
they are the focus of this document.
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 [4] should be taken into account as well as
appropriate national statements.
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 compatibility with regulatory limitations prior to use.
WARNING 3 — Some Alternaria toxins exhibit genotoxic and mutagenic effects.
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1 Scope
This document specifies a procedure for the determination of five Alternaria toxins in wheat, tomato
puree and sunflower seeds by high-performance liquid chromatography (HPLC) coupled with tandem
mass spectrometry (MS/MS).
The method has been validated with both naturally contaminated and spiked samples of wheat, tomato
puree and sunflower seeds.
Validation levels for altenuene (ALT) ranged from 2,18 μg/kg to 13,8 μg/kg.
Validation levels for alternariol (AOH) ranged from 1,82 μg/kg to 46,7 μg/kg.
Validation levels for alternariol monomethyl ether (AME) ranged from 1,29 μg/kg to 47,2 μg/kg.
Validation levels for tentoxin (TEN) ranged from 5,29 μg/kg to 218 μg/kg.
Validation levels for tenuazonic acid (TEA) ranged from 41,8 μg/kg to 1 618 μg/kg.
Limits of quantification of 1 μg/kg for ALT (except in wheat and sunflower seeds), AOH and AME;
5 μg/kg for TEN and 10 μg/kg for TEA or lower are achievable using this method.
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:
• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp
4 Principle
A test portion of the sample spiked with the isotopically-labelled internal standards is extracted with a
methanol, water and acetic acid solution. The sample/extraction solvent mixture is centrifuged and an
aliquot of the supernatant is collected. The extract is diluted with an equal volume of 1 % aqueous
acetic acid solution, and concentrated on a polymeric solid-phase extraction (SPE) adsorbent. The
extract is eluted from the SPE column with a methanol and ethyl acetate solution. The eluate is then
evaporated, reconstituted, filtered through a polytetrafluoroethylene (PTFE) syringe filter and
subsequently analysed by HPLC-MS/MS.
5
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5 Reagents
Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696,
unless otherwise specified. Solutions shall be of quality for LC-MS analysis, unless otherwise specified.
Commercially available solutions with equivalent properties to those listed may be used.
5.1 Nitrogen compressed gas, purity equivalent to 99,99 % or better.
5.2 Water (H O), HPLC grade.
2
5.3 Water (H O), LC-MS grade.
2
5.4 Methanol, analytical grade.
5.5 Methanol, LC-MS grade.
5.6 Ethyl acetate, analytical grade or higher.
5.7 Ammonium hydroxide (NH OH), LC-MS grade, volume fraction φ(NH OH) = 25 %.
4 4
5.8 Ammonium hydroxide (NH OH), φ(NH OH) = 2,5 %.
4 4
Add 1 ml of ammonium hydroxide 25 % (5.7) to a 10 ml volumetric flask containing approximately 5 ml
of water (5.3) and fill up to the mark with water (5.3).
5.9 Acetic acid, φ ≥ 99,7 %.
5.10 Ammonium acetate (CH COONH ), LC-MS grade.
3 4
5.11 Ammonium acetate solution (CH COONH ), molar concentration c = 1 mol/l.
3 4
Dissolve 77,08 g of ammonium acetate (5.10) in 1 l water (5.3).
5.12 Extraction mixture, methanol +water + acetic acid (85+14+1, v+v+v) mixture.
Mix 850 ml of methanol (5.4) with 140 ml of water (5.2) and 10 ml of acetic acid (5.9).
5.13 Aqueous acetic acid solution, acetic acid + water (1+99, v+v), φ = 1 %.
Mix 10 ml of acetic acid (5.9) with 990 ml of water (5.2) and homogenize well.
5.14 Elution solution, methanol + ethyl acetate (75+25, v+v).
Mix 750 ml of methanol (5.4) with 250 ml of ethyl acetate (5.6) and homogenize well.
5.15 HPLC mobile phase A, 5 mmol/l ammonium acetate buffer at pH approximately 8,0.
Mix 5 ml of ammonium acetate solution (5.11) and approximately 200 µl of ammonium hydroxide 2,5 %
(5.8) with 900 ml of water (5.3). Adjust the volume with water (5.3) to 1 l and homogenize well.
Check the pH of the mobile phase A. The pH shall be between 7,95 and 8,05. Adjust with ammonium
hydroxide 2,5 % (5.8) to be within that range.
5.16 HPLC mobile phase B, methanol (5.5).
5.17 Alternaria toxins’ standards, e.g. crystalline, as a film or as a reference material.
5.17.1 Altenuene, at least 96 % purity (m/m).
5.17.2 Alternariol, at least 96 % purity (m/m).
5.17.3 Alternariol monomethyl ether, at least 96 % purity (m/m).
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5.17.4 Tentoxin, at least 96 % purity (m/m).
5.17.5 Tenuazonic acid, at least 96 % purity (m/m).
NOTE Laboratories can seek for an higher purity, if available on the market.
5.18 Isotopically-labelled internal standards, e.g. crystalline or as a standard solution.
5.18.1 Altenuene isotopically-labelled internal standard (ALT-ISTD), e.g. ALT-(methoxy-d3,
methyl-d3) (ALT-d6).
5.18.2 Alternariol isotopically-labelled internal standard (AOH-ISTD), e.g. AOH-(methyl-d3)
(AOH-d3).
5.18.3 Alternariol-monomethyl ether isotopically-labelled internal standard (AME-ISTD), e.g.
AME-(1-methyl-d3) (AME-d3).
5.18.4 Tentoxin isotopically-labelled internal standard (TEN-ISTD), e.g. TEN-d3.
13
5.18.5 Tenuazonic acid isotopically-labelled internal standard (TEA-ISTD), e.g. TEA-(acetyl- C )
2
13
(TEA C ), mixture of diastereomers in methanol.
2
NOTE Laboratories can seek for isotopically-labelled internal standards with a high degree of deuteration or
13
C-enrichment.
WARNING – Protective clothing, gloves and safety goggles should be worn at all times, and all standard
and sample preparation stages should be carried out in a fume hood.
5.19 Stock solutions of ALT, AOH, AME, TEN and TEA, e.g. at a mass concentration ρ = 100 µg/ml.
In case of crystalline powders, weigh in e.g. 5 mg of ALT (5.17.1), AOH (5.17.2), AME (5.17.3),
TEN (5.17.4) and TEA (5.17.5) to the nearest 0,1 mg into individual 50 ml volumetric flasks and fill up to
the mark with methanol (5.5). Homogenize vigorously.
In case of dried-down films, reconstitute the standard in the vial according to the certificate of each
individual standard.
The stock solutions are stable for at least six months if stored at ≤ 18 °C.
NOTE For the purpose of photometrically verifying the concentration of gravimetrically prepared standards,
molar extinction coefficients are available in the literature for AOH [5], AME [5], ALT [6], TEN [7] and TEA [8].
5.20 Standard solution 1.
Prepare a methanolic standard mixture that contains ALT (5.17.1), AOH (5.17.2), AME (5.17.3) in
500 ng/ml concentration, TEN (5.17.4) in 2 500 ng/ml concentration and TEA (5.17.5) in 5000 ng/ml
concentration.
For that purpose, transfer e.g. 25 µl of the stock solution of ALT, AOH and AME (5.19) into a 5 ml
volumetric flask. Transfer e.g. 125 µl of stock solution of TEN (5.19) and e.g. 250 µl of stock solution of
TEA (5.19) into the same 5 ml volumetric flask. Fill up to the calibration mark with methanol (5.5).
Homogenize vigorously. The standard solution 1 is stable for at least six months if stored at ≤ 18 °C.
NOTE The exact volumes to be measured to prepare the standard solution 1 (5.20) are derived from the exact
concentration of the stock solutions (5.19).
5.21 Standard solution 2.
Prepare a methanolic standard mixture that contains ALT (5.17.1), AOH (5.17.2), AME (5.17.3) in
100 ng/ml concentration, TEN (5.17.4) in 500 ng/ml concentration and TEA (5.17.5) in 1 000 ng/ml
concentration. Standard solution 2 is prepared by diluting standard solution 1 (5.20).
7
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Transfer 1 000 µl of the standard solution 1 (5.20) into a 5 ml volumetric flask. Fill up to the calibration
mark with methanol (5.5). Homogenize vigorously. Standard solution 2 is stable for at least six months
if stored at ≤ 18 °C.
5.22 Stock solutions of ALT-ISTD, AOH-ISTD, AME-ISTD, TEN-ISTD and TEA-ISTD, e.g. at a mass
concentration ρ = 750 µg/ml.
Prepare the stock solutions, e.g. dissolve 1 mg of crystalline powders of ALT-ISTD (5.18.1), AOH-ISTD
(5.18.2), AME-ISTD (5.18.3), TEN-ISTD (5.18.4) and TEA-ISTD (5.18.5) in individual vials with 1500 µl
of methanol (5.5). Homogenize vigorously. The stock solutions are stable for at least six months if
stored at ≤ 18 °C. TEA-ISTD might be supplied already as a methanolic solution with approximately the
same concentration.
5.23 Internal standard solution 1.
Prepare a methanolic standard mixture that contains AOH-ISTD (5.18.2), AME-ISTD (5.18.3) and TEN-
ISTD (5.18.4) in 5 μg/ml concentration, ALT-ISTD (5.18.1) in 10 μg/ml concentration and TEA-ISTD
(5.18.5) in 25 μg/ml concentration.
For that purpose, transfer e.g. 33,3 µl of the stock solutions of AOH-ISTD, AME-ISTD and TEN-ISTD
(5.22) into a 5 ml volumetric flask. Transfer e.g. 66,7 µl of stock solution of ALT-ISTD (5.22) and e.g.
166,7 µl of stock solution of TEA-ISTD (5.22) into the same 5 ml volumetric flask. Fill up to the
calibration mark with methanol (5.5). Homogenize vigorously. The internal standard solution 1 is stable
for at least six months if stored at ≤ 18 °C.
5.24 Internal standard solution 2.
Prepare a methanolic standard mixture that contains AOH-ISTD (5.18.2), AME-ISTD (5.18.3) and TEN-
ISTD (5.18.4) in 500 ng/ml concentration, ALT-ISTD (5.18.1) in 1 000 ng/ml concentration and TEA-
ISTD (5.18.5) in 2 500 ng/ml concentration. Internal standard solution 2 is prepared by diluting
internal standard solution 1 (5.23).
Transfer 1 000 µl of the internal standard solution 1 (5.23) into a 10 ml volumetric flask. Fill up to the
calibration mark with methanol (5.5). Homogenize vigorously. The internal standard solution 2 is stable
for at least six months if stored at ≤ 18 °C.
1
5.25 Polysorbate 20 (C H O ) (Tween®20 ), analytical grade.
58 114 26
5.26 Polysorbate 20 (C H O ) solution, 2 % (v+v) in water.
58 114 26
Pipette 2 ml of polysorbate 20 (5.25) into a 100 ml volumetric flask and fill up to the mark with water
(5.2). Homogenize well. This solution (polysorbate 20 + water, 2+98, v+v) can be used for three months
if stored at approximately 4 °C.
5.27 Calibration solutions.
Add different volumes of standard solution (5.20 resp. 5.21) and the internal standard solution 2 (5.24)
to five HPLC vials (6.18) e.g. as listed in Table 1. Add the methanol (5.5) volumes specified in Table 1
and mix gently. Add 600 µl of HPLC mobile phase A (5.15), cap the vial and mix it (6.15) for 20 s.
Prepare a sixth vial (blank) without standard solutions and without internal standard solution and use
it as an instrumental blank.
1
Tween®20 is a trade name of a polysorbate 20-type nonionic surfactant available from different suppliers.
This information is given for the convenience of users of this European standard and does not constitute an
endorsement by CEN of this product. Equivalent products may be used if they can be shown to lead to the
same results.
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Table 1 — Preparation of calibration solutions
ALT AOH AME TEN TEA
Calibration Standard Standard Internal Methanol Equivalent concentration
solution solution 1 solution 2 standard (5.5)
(5.20) (5.21) solution 2
(5.24)
µl µl µl µl µg/kg
1 10 50 340 1 1 1 5 10
2 50 50 300 5 5 5 25 50
3 100 50 250 10 10 10 50 100
4 50 – 50 300 25 25 25 125 250
5 200 – 50 150 100 100 100 500 1000
Blank – – – 400 – – – – –
6 Apparatus and equipment
Usual laboratory glassware and equipment and, in particular, the following:
6.1 pH meter.
6.2 Polypropylene (PP) centrifuge tube, (50 ml) with scale on it.
6.3 Laboratory balance, accuracy of 0,01 g.
6.4 Analytical balance, accuracy of 0,01 mg.
6.5 Adjustable mechanical vertical or horizontal shaker.
2
6.6 High speed blending device (e.g. Ultra-turrax® ).
6.7 Centrifuge, with temperature control and capable of generating a relative centrifugal force of
approximately 3 200 g.
6.8 Graduated volumetric pipettes, 10 ml capacity.
6.9 Displacement pipettes, e.g. 10 μl, 20 μl, 100 μl, 250 μl and 1 000 μl capacity, with appropriate
tips.
6.10 Solid-phase extraction (SPE) column, with hydrophilic modified styrene polymer with 6 ml
reservoir capacity, 200 mg adsorbent mass and, 100 μm particle size or smaller.
2
NOTE Phenomenex Strata-XL , with 6 ml reservoir capacity, 200 mg adsorbent mass and 100 µm particle size
have shown to meet these specifications.
6.11 PP reservoirs (approximately 25 ml), fit to SPE columns (6.10).
2
Ultra-turrax and Strata-XL are used as examples, available commercially from IKA and Phenomenex
respectively. This information is given for the convenience of users of this European Standard 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.
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6.12 Polytetrafluoroethylene (PTFE) syringe filter, 0,2 µm pore size and e.g. 13 mm or 15 mm of
diameter.
6.13 Syringe with needle, 1 ml.
6.14 Vacuum manifold, for SPE clean-up, with taps.
6.15 Mixer, with high shear rate.
6.16 Sample concentrator, with temperature control and gas supply.
6.17 Glass receiving tubes, for sample elution and evaporation.
6.18 Silanized glass HPLC vials, approximately 1,5 ml capacity and crimp caps or equivalent.
6.19 Beakers, 250 ml capacity.
6.20 Volumetric flasks, 5 ml, 10 ml, 50 ml, 100 ml and 1 l capacity.
6.21 HPLC-MS/MS system, with the following components:
6.21.1 HPLC pump, capable of maintaining a binary gradient at flow rates appropriate for the
analytical column in use with sufficient accuracy.
6.21.2 Degasser, optional, for degassing HPLC mobile phases.
6.21.3 Injection system, capable of injecting an appropriate volume of injection solution with
sufficient accuracy.
6.21.4 HPLC reversed phase column.
A suitable column and appropriate HPLC conditions with sufficient retention capacity of the first eluting
analyte. A capacity factor of 3 (k′ ≥ 3,0) and a minimum plate number for any of the analytes (N ≥ 2 000)
has shown to provide chromatograms as shown in Annex A.
Examples of suitable columns and gradients are reported in Annex B.
6.21.5 Pre-column, with the same stationary phase material as the analytical column (6.21.4).
6.21.6 Column oven, capable of maintaining a constant temperature.
6.21.7 Triple stage mass spectrometer.
Triple quadrupole, ion trap or quadrupole linear ion trap instrument, amongst others, equipped with an
electrospray ionization (ESI) interface and operated in multiple reaction monitoring (MRM) mode. Any
ionization mode (typically negative, or positive) giving sufficient yield may be employed.
6.21.8 Computer-based instrument control and data evaluation system.
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7 Procedure
7.1 Preparation of the test sample
In all instances everything shall be at room temperature before any kind of manipulation takes place.
Solid laboratory samples shall be finely ground and homogenized. Care shall be taken not to overheat
the sample during this process. Cryo-milling of fatty samples might be required.
Semi-solid samples shall be thoroughly homogenized by vigorous shaking for 10 min.
7.2 Extraction
Weigh 2,00 g to the nearest 0,01 g into a 50 ml centrifuge tube (6.2) for single analysis. Add 100 μl of
internal standard solution 2 (5.24).
For tomato puree: add 14,0 ml of extraction mixture (5.12). For wheat and sunflower seeds flour: add
15,0 ml of extraction mixture (5.12).
Cap the tube tightly and mix it for approximately 10 s to obtain a homogeneous suspension.
Sunflower seeds flour can get sticky and clog during manipulation. To allow for a complete extraction,
these samples need to be sufficiently dispersed in the extraction mixture using a high speed blender
(6.6) for 30 s prior to the extraction.
Shake it vigorously for 45 min with a shaker (6.5).
A small amount of the sunflower sample may stick to the wall of the tubes during extraction. In such
event, take the tubes out of the shaker and shake it by hand for 5 s to remove the material from the
walls. Bring the tubes back into the shaker and continue the extraction.
Centrifuge the sample at approximately 22 °C for at least 10 min at approximately 3 200 g (6.7) and
transfer 7,5 ml of the supernatant (equal to 1,0 g sample) into a new 50 ml centrifuge tube (6.2).
Dilute with an equal volume (7,5 ml) of 1 % aqueous acetic acid solution (5.13). Homogenize by mixing
for 5 s.
After dilution of the extract, turbidity may form in the solution depending on the sample. Centrifugation
may be performed should this turbidity cause the blockage of the SPE cartridge.
7.3 Solid phase extraction clean up
Connect the SPE column (6.10) to the vacuum manifold (6.14).
Condition the column by passing through 7 ml of methanol (5.4), followed by 7 ml of water (5.2) and
4 ml of 1 % acetic acid solution (5.13).
Close the tap under the column and pipette another 3 ml of 1 % acetic acid solution (5.13) into the SPE
column. Attach a reservoir (6.11) onto the SPE column.
Load the diluted sample into the reservoir. Wash the 50 ml centrifuge tube, which contained the diluted
sample, with 4 ml of 1 % acetic acid solution (5.13) and load it into the reservoir. Open the tap and let
the extract pass through at a flow rate of approximately 1 drop per second.
Remove the reservoir and wash the column with 4 ml of 2 % polysorbate 20 (C58H114O26) solution (5.26)
followed by 4 ml of 1 % aqueous acetic acid solution (5.13). Dry the column thoroughly with vacuum
(approximately 10 min).
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oSIST prEN 17521:2020
prEN 17521:2020 (E)
7.4 Preparation of the sample test solution
Place a glass receiving tube (6.17) in the vacuum manifold. Elute the extract with 7 ml of elution
solution (5.14) into the glass receiving tube. After the solution has passed through, dry the column for
10 s with vacuum and collect all effluent.
If the elution solution does not start dropping spontaneously, a gentle vacuum may be applied in order
to trigger the flow. After the first drops flow from the column, the vacuum is not needed.
Evaporate the eluate in the glass receiving tube (6.17) at 50 °C using the sample concentrator (6.16)
and a gentle stream of nitrogen (5.1).
Add 400 µl of methanol (5.5) to the dry extract and re-dissolve the residue on the tube walls by mixing
for at least 20 s.
Add 600 µl of HPLC mobile phase A (5.15) and mix for another 20 s.
Filter the extract through a PTFE syringe filter (6.12), with the aid of a 1 ml syringe (6.13), into an HPLC
vial (6.18).
Note If filtering does not result in a clear test solution, centrifugation of the test solution can be performed.
7.5 LC-MS/MS analysis
7.5.1 LC-MS/MS operating conditions
A combination of analytical column, mobile phase composition, gradient settings and injection volume
shall be such that it allows obtaining acceptable chromatographic separation of the analytes and
reliable results at the required levels, with sufficient selectivity.
Annex A illustrates an example chromatogram, and Annex B gives example conditions for suitable LC-
MS/MS systems.
7.5.2 Calibration curve
Before starting the injection sequence the LC-MS/MS instrument shall be equilibrated by injecting blank
solutions and matrix-free standard solutions until a stable response is reached.
Analyse once the calibration solutions (5.27) at the beginning of the injection sequence. Prepare
a calibration curve by plotting the response ratio (peak area of the analyte divided by the peak area of
the isotopically-labelled internal standard) against the respective concentration in the injected
calibration solutions (5.27). Follow the instructions given in section 7.7.
7.5.3 Injection sequence
Analyse the blank solutions and the sample test solutions in a sequence that avoids carry-over and cross
contamination. The following injection sequence fulfils this criterion:
• blank (5.27);
...
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