EN ISO 18465:2017

SLOVENSKI STANDARD
01-april-2017
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Microbiology of the food chain - Quantitative determination of emetic toxin (cereulide)
using LC-MS/MS (ISO 18465:2017)
Mikrobiologie der Lebensmittelkette - Quantitative Bestimmung von emetischem Toxin
(Cereulid) mittels LC-MS/MS (ISO 18465:2017)
Microbiologie de la chaîne alimentaire - Détermination quantitative de la toxine émétique
(céreulide) par CL-SM/SM (ISO 18465:2017)
Ta slovenski standard je istoveten z: EN ISO 18465:2017
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 18465
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2017
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of the food chain - Quantitative
determination of emetic toxin (cereulide) using LC-MS/MS
(ISO 18465:2017)
Microbiologie de la chaîne alimentaire - Détermination Mikrobiologie der Lebensmittelkette - Quantitative
quantitative de la toxine émétique (céreulide) par CL- Bestimmung von emetischem Toxin (Cereulid) mittels
SM/SM (ISO 18465:2016) LC-MS/MS (ISO 18465:2017)
This European Standard was approved by CEN on 28 December 2016.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 18465:2017 E
worldwide for CEN national Members.

Contents Page
European Foreword . 3

European Foreword
This document (EN ISO 18465:2017) has been prepared by Technical Committee CEN/TC 275 “Food
analysis - Horizontal methods”, the secretariat of which is held by DIN, in collaboration with Technical
Committee ISO/TC 34 "Food products".
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 August 2017 and conflicting national standards shall
be withdrawn at the latest by August 2017.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent
rights.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,
Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands,
Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
the United Kingdom.
Endorsement notice
The text of ISO 18465:2017 has been approved by CEN as EN ISO 18465:2017 without any modification.
INTERNATIONAL ISO
STANDARD 18465
First edition
2017-01
Microbiology of the food chain —
Quantitative determination of emetic
toxin (cereulide) using LC-MS/MS
Microbiologie de la chaîne alimentaire — Détermination quantitative
de la toxine émétique (céreulide) par CL-SM/SM
Reference number
ISO 18465:2017(E)
©
ISO 2017
ISO 18465:2017(E)
© ISO 2017, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
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ii © ISO 2017 – All rights reserved

ISO 18465:2017(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General principle . 1
5 Reagents . 1
6 Apparatus and equipment . 3
7 Procedure. 4
7.1 Sample preparation . 4
7.2 Standard preparation . 4
7.3 LC-MS analysis . 5
7.3.1 LC conditions . . 5
7.3.2 MS conditions and tuning parameters . 5
7.3.3 Transitions (multiple reaction monitoring, MRM) . 6
8 Calculation . 6
9 Quality controls . 7
10 Precision . 8
10.1 General . 8
10.2 Repeatability . 8
10.3 Reproducibility . 9
Annex A (informative) Results of the interlaboratory study .10
[3]
Annex B (informative) Possible transitions of cereulide in MS .12
Bibliography .14
ISO 18465:2017(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment,
as well as information about ISO’s adherence to the World Trade Organization (WTO) principles in the
Technical Barriers to Trade (TBT) see the following URL: www . i so .org/ iso/ foreword .html.
This document was prepared by the European Committee for Standardization (CEN) Technical
Committee CEN/TC 275, Food Analysis — Horizontal methods, in collaboration with ISO Technical
Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology, in accordance with the
agreement on technical cooperation between ISO and CEN (Vienna Agreement).
iv © ISO 2017 – All rights reserved

ISO 18465:2017(E)
Introduction
Cereulide, the emetic toxin produced in foods by certain strains of Bacillus cereus, is a heat and acid
stable toxin that causes nausea and vomiting when ingested. In very rare cases, people can die after
ingestion of the toxin. Due to its stability, the toxin may still be present even when B. cereus can no
longer be detected. The presence of cereulide seems to be linked to starch-rich foods like rice (dishes)
and pasta (dishes). However, recent data suggest that the occurrence of food borne outbreaks due to
[9]
cereulide is more common to foods in general . The toxin has a cyclic structure and consists of in
total 12 monomers as a repeat of (D-O-Leucine-D-Alanine-L-O-Valine-L-Valine). Several methods
have been developed for the detection and/or quantification of the toxin. Some of these methods are
[3, 4]
nonspecific bio-assays and other methods are specifically based on the chemical analysis using
liquid chromatography with mass spectrometry (LC-MS/MS) for the detection and quantification of the
[5, 6, 7, 8]
toxin . The chemical methods are more specific for cereulide and have, therefore, been chosen as
the starting point for standardization of a method for the quantification of cereulide. Recently, research
has been done for the chemodiversity of
...