EN 17521:2021

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

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.

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

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.
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.

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 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.
5.3 Water (H O), LC-MS grade.
5.4 Methanol (CH OH), analytical grade.
5.5 Methanol (CH OH), LC-MS grade.
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 %.
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.
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 )
(AOH-d ).
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 T
...