SIST EN ISO 18363-3:2022
(Main)Animal and vegetable fats and oils - Determination of fatty-acid-bound chloropropanediols (MCPDs) and glycidol by GC/MS - Part 3: Method using acid transesterification and measurement for 2-MCPD, 3-MCPD and glycidol (ISO 18363-3:2017)
Animal and vegetable fats and oils - Determination of fatty-acid-bound chloropropanediols (MCPDs) and glycidol by GC/MS - Part 3: Method using acid transesterification and measurement for 2-MCPD, 3-MCPD and glycidol (ISO 18363-3:2017)
This document specifies a procedure for the simultaneous determination of 2-MCPD esters (bound 2-MCPD), 3‐MCPD esters (bound 3‐MCPD) and glycidyl esters (bound glycidol) in a single assay, based on acid catalysed ester cleavage and derivatization of cleaved (free) analytes with phenylboronic acid (PBA) prior to GC/MS analysis.
This document is applicable to solid and liquid fats and oils. For all three analytes the limit of quantification (LOQ) is 0,1 mg/kg and the limit of detection (LOD) is 0,03 mg/kg.
Tierische und pflanzliche Fette und Öle - Bestimmung von fettsäuregebundenen Chlorpropandiol (MCPD) und Glycidol mittels GC/MS - Teil 3: Verfahren mittels Säureumesterung und Messung für 2 MCPD, 3 MCPD und Glycidol (ISO 18363 3:2017)
Dieses Dokument legt ein Verfahren für die gleichzeitige Bestimmung von 2 MCPD-Estern (gebundenem 2 MCPD), 3 MCPD-Estern (gebundenem 3 MCPD) und Glycidylestern (gebundenem Glycidol) in einem einzigen Assay fest, basierend auf einer vor der GC/MS-Analyse erfolgenden säurekatalysierten Esterspaltung und Derivatisierung der abgespaltenen (freien) Analyten mit Phenylboronsäure (en: phenylboronic acid, PBA).
Dieses Dokument ist auf feste und flüssige Fette und Öle anwendbar. Für alle drei Analyten liegt die Quantifizierungsgrenze (LOQ, en: limit of quantification) bei 0,1 mg/kg und die Nachweisgrenze (LOD, en: limit of detection) bei 0,03 mg/kg.
Corps gras d'origines animale et végétale - Détermination des esters de chloropropanediols (MCPD) et d'acides gras et des esters de glycidol et d'acides gras par CPG/SM - Partie 3: Méthode par transestérification acide et mesure du 2-MCPD, du 3-MCPD et du glycidol (ISO 18363-3:2017)
ISO 18363-3:2017 spécifie un mode opératoire permettant la détermination simultanée des esters de 2-MCPD (2-MCPD lié), des esters de 3-MCPD (3-MCPD lié) et des esters de glycidol (glycidol lié) en un seul essai, basé sur le clivage des esters par catalyse acide et la dérivatisation des analytes clivés (libres) à l'acide phénylboronique (PBA) avant analyse par CPG/SM.
ISO 18363-3:2017 est applicable aux corps gras solides et liquides. Pour l'ensemble des trois analytes, la limite de quantification (LOQ) est de 0,1 mg/kg et la limite de détection (LOD) est de 0,03 mg/kg.
Živalske in rastlinske maščobe ter olja - Določevanje maščobnokislinsko vezanih kloropropandiolov (MCPD) in glicidola z GC/MS - 3. del: Metoda z uporabo kislinske transesterifikacije in meritev 2-MCPD, 3-MCPD in glicidola (ISO 18363-3:2017)
Ta dokument določa postopek za hkratno določevanje estrov 2-MCPD (vezan 2-MCPD), estrov 3-MCPD (vezan 3-MCPD) in glicidil estrov (vezan glicidol) v enem samem testu, ki temelji na cepljenju estrov, kataliziranih s kislino, in derivatizaciji razcepljenih (prostih) analitov s fenilboronsko kislino (PBA) pred analizo GC/MS.
Ta dokument se uporablja za trdne in tekoče maščobe ter olja. Za vse tri analite je meja kvantifikacije (LOQ) 0,1 mg/kg, meja zaznavanja (LOD) pa 0,03 mg/kg.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 18363-3:2022
01-februar-2022
Živalske in rastlinske maščobe ter olja - Določevanje maščobnokislinsko vezanih
kloropropandiolov (MCPD) in glicidola z GC/MS - 3. del: Metoda z uporabo
kislinske transesterifikacije in meritev 2-MCPD, 3-MCPD in glicidola (ISO 18363-
3:2017)
Animal and vegetable fats and oils - Determination of fatty-acid-bound
chloropropanediols (MCPDs) and glycidol by GC/MS - Part 3: Method using acid
transesterification and measurement for 2-MCPD, 3-MCPD and glycidol (ISO 18363-
3:2017)
Tierische und pflanzliche Fette und Öle - Bestimmung von fettsäuregebundenen
Chlorpropandiol (MCPD) und Glycidol mittels GC/MS - Teil 3: Verfahren mittels
Säureumesterung und Messung für 2 MCPD, 3 MCPD und Glycidol (ISO 18363 3:2017)
Corps gras d'origines animale et végétale - Détermination des esters de
chloropropanediols (MCPD) et d'acides gras et des esters de glycidol et d'acides gras
par CPG/SM - Partie 3: Méthode par transestérification acide et mesure du 2-MCPD, du
3-MCPD et du glycidol (ISO 18363-3:2017)
Ta slovenski standard je istoveten z: EN ISO 18363-3:2021
ICS:
67.200.10 Rastlinske in živalske Animal and vegetable fats
maščobe in olja and oils
SIST EN ISO 18363-3:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 18363-3:2022
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SIST EN ISO 18363-3:2022
EN ISO 18363-3
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2021
EUROPÄISCHE NORM
ICS 67.200.10
English Version
Animal and vegetable fats and oils - Determination of fatty-
acid-bound chloropropanediols (MCPDs) and glycidol by
GC/MS - Part 3: Method using acid transesterification and
measurement for 2-MCPD, 3-MCPD and glycidol (ISO
18363-3:2017)
Corps gras d'origines animale et végétale - Tierische und pflanzliche Fette und Öle - Bestimmung
Détermination des esters de chloropropanediols von fettsäuregebundenen Chlorpropandiol (MCPD)
(MCPD) et d'acides gras et des esters de glycidol et und Glycidol mittels GC/MS - Teil 3: Verfahren mittels
d'acides gras par CPG/SM - Partie 3: Méthode par Säureumesterung und Messung für 2 MCPD, 3 MCPD
transestérification acide et mesure du 2-MCPD, du 3- und Glycidol (ISO 18363 3:2017)
MCPD et du glycidol (ISO 18363-3:2017)
This European Standard was approved by CEN on 5 December 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 ISO 18363-3:2021 E
worldwide for CEN national Members.
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SIST EN ISO 18363-3:2022
EN ISO 18363-3:2021 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 18363-3:2022
EN ISO 18363-3:2021 (E)
European foreword
The text of ISO 18363-3:2017 has been prepared by Technical Committee ISO/TC 34 "Food products” of
the International Organization for Standardization (ISO) and has been taken over as EN ISO 18363-
3:2021 by Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and oils and their by-
products - Methods of sampling and analysis” the secretariat of which is held by AFNOR.
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 June 2022, and conflicting national standards shall be
withdrawn at the latest by June 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.
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 organizations 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.
Endorsement notice
The text of ISO 18363-3:2017 has been approved by CEN as EN ISO 18363-3:2021 without any
modification.
3
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SIST EN ISO 18363-3:2022
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SIST EN ISO 18363-3:2022
INTERNATIONAL ISO
STANDARD 18363-3
First edition
2017-12
Animal and vegetable fats and oils —
Determination of fatty-acid-bound
chloropropanediols (MCPDs) and
glycidol by GC/MS —
Part 3:
Method using acid transesterification
and measurement for 2-MCPD,
3-MCPD and glycidol
Corps gras d'origines animale et végétale — Détermination des
esters de chloropropanediols (MCPD) et d'acides gras et des esters de
glycidol et d'acides gras par CPG/SM —
Partie 3: Méthode par transestérification acide et mesure pour le
2-MCPD, le 3-MCPD et le glycidol
Reference number
ISO 18363-3:2017(E)
©
ISO 2017
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SIST EN ISO 18363-3:2022
ISO 18363-3:2017(E)
COPYRIGHT PROTECTED DOCUMENT
© 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
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
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ISO copyright office
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Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved
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SIST EN ISO 18363-3:2022
ISO 18363-3:2017(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents . 2
5.1 Standard and reference compounds . 2
5.2 Standard solutions . 3
5.2.1 General. 3
5.2.2 Stock solutions (1 mg/ml) . 3
5.2.3 Working solutions . 3
5.3 Other reagents . 4
5.4 Reagent solutions . 4
6 Apparatus . 5
7 Sample . 5
7.1 Sampling . 5
7.2 Preparation of the test sample . 5
8 Procedure. 6
8.1 Test sample preparation . 6
8.2 Preparation of the calibration curve . 7
8.3 Gas chromatography/mass spectrometry references . 7
9 Expression of results . 8
9.1 Quantification of 3-MCPD esters . 8
9.2 Quantification of 2-MCPD esters . 9
9.3 Quantification of glycidyl esters . 9
10 Precision .10
10.1 General .10
10.2 Repeatability .10
10.3 Between-day reproducibility .10
11 Test report .11
Annex A (informative) Construction of the calibration curves .12
Annex B (informative) Results of the interlaboratory test .16
Bibliography .18
© ISO 2017 – All rights reserved iii
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SIST EN ISO 18363-3:2022
ISO 18363-3:2017(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 voluntary nature of standards, 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.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11,
Animal and vegetable fats and oils.
A list of all parts in the ISO 18363 series can be found on the ISO website.
iv © ISO 2017 – All rights reserved
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SIST EN ISO 18363-3:2022
ISO 18363-3:2017(E)
Introduction
The ISO 18363 series is a family of International Standards which can be used for the determination of
ester-bound MCPD and glycidol. This introduction describes the methods specified in the three documents
currently published or proposed so that the analyst can decide which methods are suitable for application.
The detailed application of each method is contained within the scope of the individual method.
ISO 18363-1 is a differential method equivalent to the DGF standard C-VI 18 (10) and identical to AOCS
Official Method Cd 29c-13. Briefly, it is based on a fast alkaline catalysed release of 3-MCPD and glycidol
from the ester derivatives. Glycidol is subsequently converted into induced 3-MCPD. It consists of two
parts. The first part (A) allows the determination of the sum of ester-bound 3-MCPD and ester-bound
glycidol, whereas the second part (B) determines ester-bound 3-MCPD only. Both assays are based
on the release of the target analytes 3-MCPD and glycidol from the ester-bound form by an alkaline
catalysed alcoholysis carried out at room temperature. In part A, an acidified sodium chloride solution
is used to stop the reaction and subsequently convert the glycidol into induced 3-MCPD. Thus, 3-MCPD
and glycidol become indistinguishable in part A. In part B, the reaction stop is achieved by the addition
of an acidified chloride-free salt solution which also prevents the conversion of glycidol into induced
MCPD. Thereby, part B allows the determination of the genuine 3-MCPD content. Finally, the glycidol
content of the sample is proportional to the difference of both assays (A – B) and can be calculated
when the transformation ratio from glycidol to 3-MCPD has been determined. ISO 18363-1 is applicable
to the fast determination of ester-bound 3-MCPD and glycidol in refined and non-refined vegetable oils
and fats. ISO 18363-1 can also apply to animal fats and used frying oils and fats, but a validation study
has to be undertaken before the analysis of these matrices. Any free analytes within the sample would
be included in the results, but the document does not allow the distinction between free and bound
analytes. However, as of publication, research has not shown any evidence of a free analyte content as
high as the esterified analyte content in refined vegetable oils and fats. In principle, ISO 18363-1 can
also be modified in such a way that the determination of 2-MCPD is feasible, but again, a validation
study has to be undertaken before the analysis of this analyte.
ISO 18363-2 (proposed) will represent AOCS Official Method Cd 29b-13. Briefly, it will be based on a slow
alkaline release of MCPD and glycidol from the ester derivatives. Glycidol is subsequently converted
into 3-MBPD. ISO 18363-2 will consist of two sample preparations that differ in the use of internal
standards. Both preparations will be used for the determination of ester-bound 2-MCPD and 3-MCPD.
In part A, a preliminary result for ester-bound glycidol will be determined. Because the 3-MCPD present
in the sample will be converted to some minor extent into induced glycidol by the sample preparation,
part B will serve to quantify this amount of induced glycidol that is subsequently subtracted from the
preliminary glycidol result of part A. By the use of isotopically labelled free MCPD isomers in assay A
and isotopically labelled ester-bound 2-MCPD and 3-MCPD in part B, the efficiency of ester cleavage will
be able to be monitored. Both assays A and B will be based on the release of the target analytes 2-MCPD,
3-MCPD, and glycidol from the ester-bound form by a slow alkaline catalysed alcoholysis in the cold.
In both sample preparations, the reaction will be stopped by the addition of an acidified concentrated
sodium bromide solution so as to convert the unstable and volatile glycidol into 3-MBPD which shows
comparable properties to 3-MCPD with regard to its stability and chromatographic performance.
Moreover, the major excess of bromide ions will prevent the undesired formation of 3-MCPD from
glycidol in the case of samples which contain naturally occurring amounts of chloride. ISO 18363-2
will be applicable to the determination of ester-bound 3-MCPD, 2-MCPD, and glycidol in refined and
unrefined vegetable oils and fats. It will also apply to animal fats and used frying oils and fats, but a
validation study will have to be undertaken before the analysis of these matrices. Any free analytes
within the sample would be included in the results, but the document will not allow the distinction
between free and bound analytes. However, as of publication of this document, research has not shown
any evidence of a free analyte content as high as the esterified analyte content in vegetable oils and fats.
This document represents AOCS Official Method Cd 29a-13. Briefly, it is based on the conversion
of glycidyl esters into 3-MBPD esters and a slow acid catalysed release of MCPD and MBPD from the
ester derivatives. This document is based on a single sample preparation in which glycidyl esters are
converted into MBPD monoesters, and subsequently, the free analytes 2-MCPD, 3-MCPD, and 3-MBPD
are released by a slow acid catalysed alcoholysis. The 3-MBPD represents the genuine content of bound
glycidol. This document can be applied for the determination of ester-bound 2-MCPD, 3-MCPD, and
© ISO 2017 – All rights reserved v
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SIST EN ISO 18363-3:2022
ISO 18363-3:2017(E)
glycidol in refined and non-refined vegetable oils and fats. It can also apply to animal fats and used
frying oils and fats, but a validation study has to be undertaken before the analysis of these matrices.
The method is suited for the analysis of bound (esterified) analytes, but if required, this document
can be also performed without the initial conversion of glycidyl esters. In such a setup, both free and
bound 2-MCPD and 3-MCPD forms would be included in the results and the amount of free analytes can
be calculated as a difference between two determinations performed in both setups. However, as of
publication of this document, research has not shown any evidence of a free analyte content as high as
the esterified analyte content in vegetable oils and fats.
vi © ISO 2017 – All rights reserved
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SIST EN ISO 18363-3:2022
INTERNATIONAL STANDARD ISO 18363-3:2017(E)
Animal and vegetable fats and oils — Determination of
fatty-acid-bound chloropropanediols (MCPDs) and glycidol
by GC/MS —
Part 3:
Method using acid transesterification and measurement
for 2-MCPD, 3-MCPD and glycidol
1 Scope
This document specifies a procedure for the simultaneous determination of 2-MCPD esters (bound
2-MCPD), 3-MCPD esters (bound 3-MCPD) and glycidyl esters (bound glycidol) in a single assay, based
on acid catalysed ester cleavage and derivatization of cleaved (free) analytes with phenylboronic
acid (PBA) prior to GC/MS analysis.
This document is applicable to solid and liquid fats and oils. For all three analytes the limit of
quantification (LOQ) is 0,1 mg/kg and the limit of detection (LOD) is 0,03 mg/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.
ISO 3696, Water for analytical laboratory use — Specification and test methods
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
3.1
bound 2-MCPD
amount of 2-MCPD cleaved from its esterified (bound) forms by acid catalysed transesterification
according to the reference method
Note 1 to entry: The content of 2-MCPD is calculated and reported as a mass fraction, in milligrams per
kilogram (mg/kg).
3.2
bound 3-MCPD
amount of 3-MCPD cleaved from its esterified (bound) forms by acid catalysed transesterification
according to the reference method
Note 1 to entry: The content of 3-MCPD is calculated and reported as a mass fraction, in milligrams per
kilogram (mg/kg).
© ISO 2017 – All rights reserved 1
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SIST EN ISO 18363-3:2022
ISO 18363-3:2017(E)
3.3
bound glycidol
amount of glycidol cleaved from its esterified (bound) forms by acid catalysed transesterification
according to the reference method
Note 1 to entry: The content of glycidol is calculated and reported as a mass fraction, in milligrams per
kilogram (mg/kg).
4 Principle
The oil/fat sample is dissolved in tetrahydrofuran, and the internal standards (pentadeuterated 3-MCPD
diester and pentadeuterated glycidyl ester) are added. During the first step of sample preparation,
glycidyl esters are converted into 3-MBPD monoesters by the addition of an acidified solution of sodium
bromide. Upon completion of the reaction, the organic phase, containing 2- and 3-MCPD esters and
3-MBPD esters, is separated and evaporated to dryness. In the second step the residue is dissolved
in tetrahydrofuran and the acid transesterification is initiated by the addition of an acid alcoholic
solution. After 16 h incubation at 40 °C, the sample mixture is neutralized and the fatty acid methyl
esters generated during the transesterification are removed. Finally, the purified sample [containing
cleaved (free) analytes] is derivatized with phenylboronic acid prior to GC/MS analysis.
The quantification of 2- and 3-MCPD esters (expressed as bound 2- and 3-MCPD) is based on the
2-MCPD/3-MCPD-d5 and 3-MCPD/3-MCPD-d5 signal ratio, respectively. The quantification of glycidyl
esters (expressed as bound glycidol) is based on the 3-MBPD/3-MBPD-d5 signal ratio.
This method allows the simultaneous quantification of all three analytes in a single assay.
5 Reagents
WARNING — This document requires handling of hazardous substances. Technical,
organizational, and personal safety measures shall be followed.
Unless otherwise stated, analytically pure reagents shall be used. Water shall comply with grade 3 of
ISO 3696.
5.1 Standard and reference compounds
5.1.1 1,2-Dipalmitoyl-3-chloropropanediol (PP-3-MCPD), purity ≥ 95 % (e.g. from a supplier or
synthesized from 3-MCPD and palmitoyl chloride as described by Reference [1]).
NOTE 1,2-Dipalmitoyl-3-chloropropanediol can be substituted by 1,2-dioleyl-3-chloropropanediol or other
fatty acid diesters of 3-MCPD with similar chain length (C16-C18 are preferred as they are the most abundant in
the majority of oils/fats).
5.1.2 1,3-Dipalmitoyl-2-chloropropanediol (PP-2-MCPD), purity ≥ 95 % (e.g. synthesized from
2-MCPD and palmitoyl chloride as described by Reference [1]).
NOTE In analogy with the recommendations given for PP-3-MCPD, 1,3-dipalmitoyl-2-chloropropanediol can
be substituted by other fatty acid diesters of 2-MCPD with similar chain length (C16-C18 are preferred as they
are the most abundant in the majority of oils/fats).
5.1.3 Pentadeuterated 1,2-dipalmitoyl-3-chloropropanediol (PP-3-MCPD-d5), purity ≥ 95 %.
NOTE The same consideration applied to 1,2-dipalmitoyl-3-chloropropanediol is valid also for its
pentadeuterated analogue, see the Note in 5.1.1.
5.1.4 Glycidyl palmitate (Gly-P), purity ≥ 98 %.
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SIST EN ISO 18363-3:2022
ISO 18363-3:2017(E)
NOTE Glycidyl palmitate can be substituted by glycidyl oleate or other fatty acid esters of glycidol with
similar chain length (C16-C18 are preferred as they are the most abundant in the majority of oils/fats).
5.1.5 Pentadeuterated glycidyl palmitate (Gly-P-d5), purity ≥ 98 %.
NOTE The same consideration applied to glycidyl palmitate is valid also for its pentadeuterated analogue,
see the Note in 5.1.4.
5.2 Standard solutions
5.2.1 General
All standard solutions can be prepared with either toluene (5.3.5) or tetrahydrofuran (5.3.1). Toluene is
preferred for standard solutions containing glycidyl esters.
5.2.2 Stock solutions (1 mg/ml)
a) Weigh 10 mg of PP-3-MCPD (5.1.1) in a 10 ml volumetric flask. Fill up to the mark, making sure that
the standard is completely dissolved in the solvent.
b) Weigh 10 mg of PP-2-MCPD (5.1.2) in a 10 ml volumetric flask. Fill up to the mark, making sure that
the standard is completely dissolved in the solvent.
c) Weigh 10 mg of PP-3-MCPD-d5 (5.1.3) in a 10 ml volumetric flask. Fill up to the mark, making sure
that the standard is completely dissolved in the solvent.
d) Weigh 10 mg of Gly-P (5.1.4) in a 10 ml volumetric flask. Fill up to the mark, making sure that the
standard is completely dissolved in the solvent.
e) W
...
SLOVENSKI STANDARD
oSIST prEN ISO 18363-3:2021
01-oktober-2021
Živalske in rastlinske maščobe ter olja - Določevanje maščobnokislinsko vezanih
kloropropandiolov (MCPD) in glicidola z GC/MS - 3. del: Metoda z uporabo
kislinske transesterifikacije in meritev 2-MCPD, 3-MCPD in glicidola (ISO 18363-
3:2017)
Animal and vegetable fats and oils - Determination of fatty-acid-bound
chloropropanediols (MCPDs) and glycidol by GC/MS - Part 3: Method using acid
transesterification and measurement for 2-MCPD, 3-MCPD and glycidol (ISO 18363-
3:2017)
Tierische und pflanzliche Fette und Öle - Bestimmung von fettsäuregebundenen
Chlorpropandiol (MCPD) und Glycidol mittels GC/MS - Teil 3: Verfahren mittels
Säureumesterung und Messung für 2 MCPD, 3 MCPD und Glycidol (ISO 18363 3:2017)
Corps gras d'origines animale et végétale - Détermination des esters de
chloropropanediols (MCPD) et d'acides gras et des esters de glycidol et d'acides gras
par CPG/SM - Partie 3: Méthode par transestérification acide et mesure du 2-MCPD, du
3-MCPD et du glycidol (ISO 18363-3:2017)
Ta slovenski standard je istoveten z: prEN ISO 18363-3
ICS:
67.200.10 Rastlinske in živalske Animal and vegetable fats
maščobe in olja and oils
oSIST prEN ISO 18363-3:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 18363-3:2021
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oSIST prEN ISO 18363-3:2021
INTERNATIONAL ISO
STANDARD 18363-3
First edition
2017-12
Animal and vegetable fats and oils —
Determination of fatty-acid-bound
chloropropanediols (MCPDs) and
glycidol by GC/MS —
Part 3:
Method using acid transesterification
and measurement for 2-MCPD,
3-MCPD and glycidol
Corps gras d'origines animale et végétale — Détermination des
esters de chloropropanediols (MCPD) et d'acides gras et des esters de
glycidol et d'acides gras par CPG/SM —
Partie 3: Méthode par transestérification acide et mesure pour le
2-MCPD, le 3-MCPD et le glycidol
Reference number
ISO 18363-3:2017(E)
©
ISO 2017
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oSIST prEN ISO 18363-3:2021
ISO 18363-3:2017(E)
COPYRIGHT PROTECTED DOCUMENT
© 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
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oSIST prEN ISO 18363-3:2021
ISO 18363-3:2017(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents . 2
5.1 Standard and reference compounds . 2
5.2 Standard solutions . 3
5.2.1 General. 3
5.2.2 Stock solutions (1 mg/ml) . 3
5.2.3 Working solutions . 3
5.3 Other reagents . 4
5.4 Reagent solutions . 4
6 Apparatus . 5
7 Sample . 5
7.1 Sampling . 5
7.2 Preparation of the test sample . 5
8 Procedure. 6
8.1 Test sample preparation . 6
8.2 Preparation of the calibration curve . 7
8.3 Gas chromatography/mass spectrometry references . 7
9 Expression of results . 8
9.1 Quantification of 3-MCPD esters . 8
9.2 Quantification of 2-MCPD esters . 9
9.3 Quantification of glycidyl esters . 9
10 Precision .10
10.1 General .10
10.2 Repeatability .10
10.3 Between-day reproducibility .10
11 Test report .11
Annex A (informative) Construction of the calibration curves .12
Annex B (informative) Results of the interlaboratory test .16
Bibliography .18
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oSIST prEN ISO 18363-3:2021
ISO 18363-3:2017(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 voluntary nature of standards, 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.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11,
Animal and vegetable fats and oils.
A list of all parts in the ISO 18363 series can be found on the ISO website.
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Introduction
The ISO 18363 series is a family of International Standards which can be used for the determination of
ester-bound MCPD and glycidol. This introduction describes the methods specified in the three documents
currently published or proposed so that the analyst can decide which methods are suitable for application.
The detailed application of each method is contained within the scope of the individual method.
ISO 18363-1 is a differential method equivalent to the DGF standard C-VI 18 (10) and identical to AOCS
Official Method Cd 29c-13. Briefly, it is based on a fast alkaline catalysed release of 3-MCPD and glycidol
from the ester derivatives. Glycidol is subsequently converted into induced 3-MCPD. It consists of two
parts. The first part (A) allows the determination of the sum of ester-bound 3-MCPD and ester-bound
glycidol, whereas the second part (B) determines ester-bound 3-MCPD only. Both assays are based
on the release of the target analytes 3-MCPD and glycidol from the ester-bound form by an alkaline
catalysed alcoholysis carried out at room temperature. In part A, an acidified sodium chloride solution
is used to stop the reaction and subsequently convert the glycidol into induced 3-MCPD. Thus, 3-MCPD
and glycidol become indistinguishable in part A. In part B, the reaction stop is achieved by the addition
of an acidified chloride-free salt solution which also prevents the conversion of glycidol into induced
MCPD. Thereby, part B allows the determination of the genuine 3-MCPD content. Finally, the glycidol
content of the sample is proportional to the difference of both assays (A – B) and can be calculated
when the transformation ratio from glycidol to 3-MCPD has been determined. ISO 18363-1 is applicable
to the fast determination of ester-bound 3-MCPD and glycidol in refined and non-refined vegetable oils
and fats. ISO 18363-1 can also apply to animal fats and used frying oils and fats, but a validation study
has to be undertaken before the analysis of these matrices. Any free analytes within the sample would
be included in the results, but the document does not allow the distinction between free and bound
analytes. However, as of publication, research has not shown any evidence of a free analyte content as
high as the esterified analyte content in refined vegetable oils and fats. In principle, ISO 18363-1 can
also be modified in such a way that the determination of 2-MCPD is feasible, but again, a validation
study has to be undertaken before the analysis of this analyte.
ISO 18363-2 (proposed) will represent AOCS Official Method Cd 29b-13. Briefly, it will be based on a slow
alkaline release of MCPD and glycidol from the ester derivatives. Glycidol is subsequently converted
into 3-MBPD. ISO 18363-2 will consist of two sample preparations that differ in the use of internal
standards. Both preparations will be used for the determination of ester-bound 2-MCPD and 3-MCPD.
In part A, a preliminary result for ester-bound glycidol will be determined. Because the 3-MCPD present
in the sample will be converted to some minor extent into induced glycidol by the sample preparation,
part B will serve to quantify this amount of induced glycidol that is subsequently subtracted from the
preliminary glycidol result of part A. By the use of isotopically labelled free MCPD isomers in assay A
and isotopically labelled ester-bound 2-MCPD and 3-MCPD in part B, the efficiency of ester cleavage will
be able to be monitored. Both assays A and B will be based on the release of the target analytes 2-MCPD,
3-MCPD, and glycidol from the ester-bound form by a slow alkaline catalysed alcoholysis in the cold.
In both sample preparations, the reaction will be stopped by the addition of an acidified concentrated
sodium bromide solution so as to convert the unstable and volatile glycidol into 3-MBPD which shows
comparable properties to 3-MCPD with regard to its stability and chromatographic performance.
Moreover, the major excess of bromide ions will prevent the undesired formation of 3-MCPD from
glycidol in the case of samples which contain naturally occurring amounts of chloride. ISO 18363-2
will be applicable to the determination of ester-bound 3-MCPD, 2-MCPD, and glycidol in refined and
unrefined vegetable oils and fats. It will also apply to animal fats and used frying oils and fats, but a
validation study will have to be undertaken before the analysis of these matrices. Any free analytes
within the sample would be included in the results, but the document will not allow the distinction
between free and bound analytes. However, as of publication of this document, research has not shown
any evidence of a free analyte content as high as the esterified analyte content in vegetable oils and fats.
This document represents AOCS Official Method Cd 29a-13. Briefly, it is based on the conversion
of glycidyl esters into 3-MBPD esters and a slow acid catalysed release of MCPD and MBPD from the
ester derivatives. This document is based on a single sample preparation in which glycidyl esters are
converted into MBPD monoesters, and subsequently, the free analytes 2-MCPD, 3-MCPD, and 3-MBPD
are released by a slow acid catalysed alcoholysis. The 3-MBPD represents the genuine content of bound
glycidol. This document can be applied for the determination of ester-bound 2-MCPD, 3-MCPD, and
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oSIST prEN ISO 18363-3:2021
ISO 18363-3:2017(E)
glycidol in refined and non-refined vegetable oils and fats. It can also apply to animal fats and used
frying oils and fats, but a validation study has to be undertaken before the analysis of these matrices.
The method is suited for the analysis of bound (esterified) analytes, but if required, this document
can be also performed without the initial conversion of glycidyl esters. In such a setup, both free and
bound 2-MCPD and 3-MCPD forms would be included in the results and the amount of free analytes can
be calculated as a difference between two determinations performed in both setups. However, as of
publication of this document, research has not shown any evidence of a free analyte content as high as
the esterified analyte content in vegetable oils and fats.
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oSIST prEN ISO 18363-3:2021
INTERNATIONAL STANDARD ISO 18363-3:2017(E)
Animal and vegetable fats and oils — Determination of
fatty-acid-bound chloropropanediols (MCPDs) and glycidol
by GC/MS —
Part 3:
Method using acid transesterification and measurement
for 2-MCPD, 3-MCPD and glycidol
1 Scope
This document specifies a procedure for the simultaneous determination of 2-MCPD esters (bound
2-MCPD), 3-MCPD esters (bound 3-MCPD) and glycidyl esters (bound glycidol) in a single assay, based
on acid catalysed ester cleavage and derivatization of cleaved (free) analytes with phenylboronic
acid (PBA) prior to GC/MS analysis.
This document is applicable to solid and liquid fats and oils. For all three analytes the limit of
quantification (LOQ) is 0,1 mg/kg and the limit of detection (LOD) is 0,03 mg/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.
ISO 3696, Water for analytical laboratory use — Specification and test methods
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
3.1
bound 2-MCPD
amount of 2-MCPD cleaved from its esterified (bound) forms by acid catalysed transesterification
according to the reference method
Note 1 to entry: The content of 2-MCPD is calculated and reported as a mass fraction, in milligrams per
kilogram (mg/kg).
3.2
bound 3-MCPD
amount of 3-MCPD cleaved from its esterified (bound) forms by acid catalysed transesterification
according to the reference method
Note 1 to entry: The content of 3-MCPD is calculated and reported as a mass fraction, in milligrams per
kilogram (mg/kg).
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3.3
bound glycidol
amount of glycidol cleaved from its esterified (bound) forms by acid catalysed transesterification
according to the reference method
Note 1 to entry: The content of glycidol is calculated and reported as a mass fraction, in milligrams per
kilogram (mg/kg).
4 Principle
The oil/fat sample is dissolved in tetrahydrofuran, and the internal standards (pentadeuterated 3-MCPD
diester and pentadeuterated glycidyl ester) are added. During the first step of sample preparation,
glycidyl esters are converted into 3-MBPD monoesters by the addition of an acidified solution of sodium
bromide. Upon completion of the reaction, the organic phase, containing 2- and 3-MCPD esters and
3-MBPD esters, is separated and evaporated to dryness. In the second step the residue is dissolved
in tetrahydrofuran and the acid transesterification is initiated by the addition of an acid alcoholic
solution. After 16 h incubation at 40 °C, the sample mixture is neutralized and the fatty acid methyl
esters generated during the transesterification are removed. Finally, the purified sample [containing
cleaved (free) analytes] is derivatized with phenylboronic acid prior to GC/MS analysis.
The quantification of 2- and 3-MCPD esters (expressed as bound 2- and 3-MCPD) is based on the
2-MCPD/3-MCPD-d5 and 3-MCPD/3-MCPD-d5 signal ratio, respectively. The quantification of glycidyl
esters (expressed as bound glycidol) is based on the 3-MBPD/3-MBPD-d5 signal ratio.
This method allows the simultaneous quantification of all three analytes in a single assay.
5 Reagents
WARNING — This document requires handling of hazardous substances. Technical,
organizational, and personal safety measures shall be followed.
Unless otherwise stated, analytically pure reagents shall be used. Water shall comply with grade 3 of
ISO 3696.
5.1 Standard and reference compounds
5.1.1 1,2-Dipalmitoyl-3-chloropropanediol (PP-3-MCPD), purity ≥ 95 % (e.g. from a supplier or
synthesized from 3-MCPD and palmitoyl chloride as described by Reference [1]).
NOTE 1,2-Dipalmitoyl-3-chloropropanediol can be substituted by 1,2-dioleyl-3-chloropropanediol or other
fatty acid diesters of 3-MCPD with similar chain length (C16-C18 are preferred as they are the most abundant in
the majority of oils/fats).
5.1.2 1,3-Dipalmitoyl-2-chloropropanediol (PP-2-MCPD), purity ≥ 95 % (e.g. synthesized from
2-MCPD and palmitoyl chloride as described by Reference [1]).
NOTE In analogy with the recommendations given for PP-3-MCPD, 1,3-dipalmitoyl-2-chloropropanediol can
be substituted by other fatty acid diesters of 2-MCPD with similar chain length (C16-C18 are preferred as they
are the most abundant in the majority of oils/fats).
5.1.3 Pentadeuterated 1,2-dipalmitoyl-3-chloropropanediol (PP-3-MCPD-d5), purity ≥ 95 %.
NOTE The same consideration applied to 1,2-dipalmitoyl-3-chloropropanediol is valid also for its
pentadeuterated analogue, see the Note in 5.1.1.
5.1.4 Glycidyl palmitate (Gly-P), purity ≥ 98 %.
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NOTE Glycidyl palmitate can be substituted by glycidyl oleate or other fatty acid esters of glycidol with
similar chain length (C16-C18 are preferred as they are the most abundant in the majority of oils/fats).
5.1.5 Pentadeuterated glycidyl palmitate (Gly-P-d5), purity ≥ 98 %.
NOTE The same consideration applied to glycidyl palmitate is valid also for its pentadeuterated analogue,
see the Note in 5.1.4.
5.2 Standard solutions
5.2.1 General
All standard solutions can be prepared with either toluene (5.3.5) or tetrahydrofuran (5.3.1). Toluene is
preferred for standard solutions containing glycidyl esters.
5.2.2 Stock solutions (1 mg/ml)
a) Weigh 10 mg of PP-3-MCPD (5.1.1) in a 10 ml volumetric flask. Fill up to the mark, making sure that
the standard is completely dissolved in the solvent.
b) Weigh 10 mg of PP-2-MCPD (5.1.2) in a 10 ml volumetric flask. Fill up to the mark, making sure that
the standard is completely dissolved in the solvent.
c) Weigh 10 mg of PP-3-MCPD-d5 (5.1.3) in a 10 ml volumetric flask. Fill up to the mark, making sure
that the standard is completely dissolved in the solvent.
d) Weigh 10 mg of Gly-P (5.1.4) in a 10 ml volumetric flask. Fill up to the mark, making sure that the
standard is completely dissolved in the solvent.
e) Weigh 10 mg of Gly-P-d5 (5.1.5) in a 10 ml volumetric flask. Fill up to the mark, making sure that
the standard is completely dissolved in the solvent.
NOTE Stock solutions are stable for at least three months when stored at −18 °C.
5.2.3 Working solutions
a) Calibration I (PP-3-MCPD, 55 μg/ml). Pipette 550 μl of the stock solution [5.2.2 a)] into a 10 ml
volumetric flask and fill up to the mark with the solvent.
b) Calibration II (PP-3-MCPD, 5,5 μg/ml). Pipette 1 ml of the Calibration I solution [5.2.3 a)] into a
10 ml volumetric flask and fill up to the mark with the solvent.
c) Calibration III (PP-2-MCPD, 55 μg/ml). Pipette 550 μl of the stock solution [5.2.2 b)] into a 10 ml
volumetric flask and fill up to the mark with the solvent.
d) Calibration IV (PP-2-MCPD, 5,5 µg/ml). Pipette 1 ml of the Calibration III solution [5.2.3 c)] into a
10 ml volumetric flask and fill up to the mark with the solvent.
e) Calibration V (Gly-P, 100 µg/ml). Pipette 1 ml of the stock solution [5.2.2 d)] into a 10 ml volumetric
flask and fill up to the mark with the solvent.
f) Calibration VI (Gly-P, 10 µg/ml). Pipette 1 ml of the Calibration V solution [5.2.3 e)] into a 10 ml
volumetric flask and fill up to the mark with the solvent.
g) Internal standard I (PP-3-MCPD-d5, 40 µg/ml). Pipette 400 µL of the stock solution [5.2.2 c)] into a
10 ml volumetric flask and fill up to the mark with the solvent.
h) Internal standard II (Gly-P-d5, 50 µg/ml). Pipette 500 µL of the stock solution [5.2.2 e)] into a 10 ml
volumetric flask and fill up to the mark with the solvent.
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NOTE 1 As an alternative to the preparation of separate standard solutions for each analyte, the three [5.2.3 a),
c), and e)] can be combined in one single standard solution at high concentration of all three analytes (“mixed
Calibration I”). To prepare the mixed solution, pipette 550 μl of PP-3-MCPD stock solution [5.2.2 a)], 550 μl of
PP-2-MCPD stock solution [5.2.2 b)], and 1 ml of Gly-P stock solution [5.2.2 d)] into a 10 ml volumetric flask and
fill up to the mark with the solvent. Also, the solutions 5.2.3 b), d), and f) can be combined in one single standard
solution at low concentration of all three analytes (“mixed Calibration II”). To prepare the mixed solution, pipette
1 ml of mixed Calibration I into a 10 ml volumetric flask and fill up to the mark with the solvent.
NOTE 2 The internal standard solutions [5.2.3 g and h)] can also be combined in a single solution (“mixed
internal standard”). To prepare the mixed solution, pipette 400 μl of PP-3-MCPD-d5 [5.2.2 c)] and 500 μl of
Gly-P-d5 [5.2.2 e)] into a 10 ml volumetric flask and fill up to the mark with solvent.
5.3 Other reagents
5.3.1 Tetrahydrofuran, anhydrous.
5.3.2 Methanol, analytical grade.
5.3.3 n-Heptane, analytical grade.
5.3.4 Acetone, analytical grade.
5.3.5 Toluene, analytical grade.
5.3.6 Water, ultra-pure (e.g. obtained by using a purification system).
5.3.7 Sulfuric acid, purity ≥ 95 %.
5.3.8 Sodium hydrogen carbonate, purity ≥ 99 %.
5.3.9 Sodium sulfate, purity ≥ 99 %.
5.3.10 Phenylboronic acid, purity ≥ 97 %.
5.3.11 Sodium bromide, purity ≥ 99,5 %.
5.4 Reagent solutions
5.4.1 Acid aqueous solution of sodium bromide [sodium bromide 3 mg/ml, sulfuric acid 5 %
(volume fraction)]. Prepare a concentrated aqueous solution of sodium bromide by dissolving 1 g of
sodium bromide (5.3.11) in 10 ml of ultrapure water (5.3.6). Transfer 180 μl of the concentrated solution
into an empty conical flask. Add 0.3 ml of sulfuric acid (5.3.7) and 5,5 ml of ultra-pure water (5.3.6).
Shake vigorously.
It is advisable to freshly prepare the solution on daily basis.
5.4.2 Sodium hydrogen carbonate solution (0,6 %, mass concentration). Weigh 0,6 g of sodium
hydrogen carbonate (5.3.8) in a 100 ml volumetric flask and fill up to the mark with ultra-pure water
(5.3.6). Use an ultrasonic bath (6.3) to ensure the complete dissolution of the reagent.
NOTE As an alternative, the solution can be prepared by dilution of the sodium hydrogen carbonate
saturated solution (5.4.4).
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5.4.3 Sulfuric acid/methanol solution (1,8 %,volume fraction). Pipette 1,8 ml of sulfuric acid (5.3.7)
in a 100 ml volumetric flask and fill up to the mark with methanol (5.3.2).
It is advisable to freshly prepare the solution on daily basis.
5.4.4
...
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