SIST EN ISO 18363-3:2024

SLOVENSKI STANDARD
01-september-2024
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-
3:2024)
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-
3:2024)
Tierische und pflanzliche Fette und Öle - Bestimmung von fettsäuregebundenem
Chlorpropandiol (MCPD) und Glycidol mittels GC/MS - Teil 3: Verfahren mittels
Säureumesterung und Messung für 2-MCPD, 3-MCPD und Glycidol (ISO 18363-3:2024)
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 18363-3:2024)
Ta slovenski standard je istoveten z: EN ISO 18363-3:2024
ICS:
67.200.10 Rastlinske in živalske Animal and vegetable fats
maščobe in olja and oils
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 18363-3
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2024
EUROPÄISCHE NORM
ICS 67.200.10 Supersedes EN ISO 18363-3:2021
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
18363-3:2024)
Corps gras d'origines animale et végétale - Tierische und pflanzliche Fette und Öle - Bestimmung
Détermination des esters de chloropropanediols von fettsäuregebundenem 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 18363-3:2024)
MCPD et du glycidol (ISO 18363-3:2024)
This European Standard was approved by CEN on 5 July 2024.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 18363-3:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 18363-3:2024) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with 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 January 2025, and conflicting national standards shall
be withdrawn at the latest by January 2025.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 18363-3:2021.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. 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, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 18363-3:2024 has been approved by CEN as EN ISO 18363-3:2024 without any
modification.
International
Standard
ISO 18363-3
Second edition
Animal and vegetable fats and
2024-06
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 du
2-MCPD, du 3-MCPD et du glycidol
Reference number
ISO 18363-3:2024(en) © ISO 2024

ISO 18363-3:2024(en)
© ISO 2024
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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 the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO 18363-3:2024(en)
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 .8
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 . 10
Annex A (informative) Construction of the calibration curves .11
Annex B (informative) Results of the interlaboratory test .15
Bibliography . 17

iii
ISO 18363-3:2024(en)
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of 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 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, in collaboration with the European Committee for Standardization (CEN)
Technical Committee CEN/TC 307, Oilseeds, vegetable and animal fats and oils and their by-products – Methods
of sampling and analysis, in accordance with the Agreement on technical cooperation between ISO and CEN
(Vienna Agreement).
This second edition cancels and replaces the first edition (ISO 18363-3:2017), of which it constitutes a minor
revision.
The main changes are as follows:
— the text of the Introduction has been revised to be consistent with ISO 18363-4:2021.
A list of all parts in the ISO 18363 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
ISO 18363-3:2024(en)
Introduction
[1]
The ISO 18363 series can be used for the determination of ester-bound MCPD and glycidol. This
introduction describes the methods specified in the different parts so that the analyst can decide which
methods are suitable for application. The detailed application of each method is contained within the scope
of each individual method.
[2]
ISO 18363-1 is a differential method equivalent to DGF standard C-VI 18 (10) and identical to AOCS Official
[3]
Method Cd 29c-13 . In brief, 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. Consequently, 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 must 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 of this document, 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 must be undertaken before the analysis of this analyte.
[4]
ISO 18363-2 represents AOCS Official Method Cd 29b-13 . In brief, it is based on a slow alkaline release of
MCPD and glycidol from the ester derivatives. Glycidol is subsequently converted into 3-MBPD. ISO 18363-2
consists of two sample preparations that differ in the use of internal standards. Both preparations are used
for the determination of ester-bound 2-MCPD and 3-MCPD. In part A, a preliminary result for ester-bound
glycidol is determined. Because the 3-MCPD present in the sample is converted to some minor extent into
induced glycidol by the sample preparation, part B serves 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 can be monitored. Both assays A and B are 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 is 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 prevents the undesired formation of 3-MCPD from glycidol in the case of samples
which contain naturally occurring amounts of chloride. ISO 18363-2 is applicable to the determination of
ester-bound 3-MCPD, 2-MCPD and glycidol in refined and unrefined vegetable oils and fats. It also applies
to animal fats and used frying oils and fats, but a validation study must be undertaken before the analysis
of these matrices. Any free analytes within the sample are included in the results, but the document does
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
refined vegetable oils and fats.
[5]
This document (i.e. ISO 18363-3) represents AOCS Official Method Cd 29a-13 . In brief, 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 is applicable to the determination of ester-bound 2-MCPD, 3-MCPD and glycidol in refined
and non-refined vegetable oils and fats. It also applies to animal fats and used frying oils and fats, but a
validation study must 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 also be performed without the

v
ISO 18363-3:2024(en)
initial conversion of glycidyl esters. In such a setup, both free and bound 2-MCPD and 3-MCPD forms are
included in the results and the amount of free analytes can be calculated as the 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 refined vegetable
oils and fats.
ISO 18363-4 specifies a rapid procedure based on fast alkaline cleavage of the MCPD and glycidyl esters.
The released glycidol is subsequently converted into 3-MBPD. The pH of the fast alkaline cleavage generally
causes the released MCPD to partially convert to glycidol during the cleavage of the esters, leading to
overestimation of the glycidyl ester content of the sample. By adding two distinct isotopically labelled ester-
bound 3-MCPD and glycidol internal standards, it is possible to quantify the amount of labelled glycidol
resulting from the degradation of the released internal standard. This information can be used to correct
for overestimation of the glycidyl ester induced glycidol by 3-MCPD induced glycidol. The same two internal
standards are used for quantification of the bound MCPD and glycidol, requiring a single sample preparation
to quantify bound 2-MCPD-, 3-MCPD- and glycidol esters. In analogue with ISO 18363-1, ISO 18363-2
and this document, the released MCPDs and 3-MBPD are derivatized with phenylboronic acid before GC-
MS/MS analysis. In contrast to the other parts of the ISO 18363 series, ISO 18363-4 requires GC-MS/MS
instrumentation to unambiguously detect each of the (isotopically labelled) MBPDs required for correct
quantification of the glycidyl ester induced glycidol. ISO 18363-4 is applicable to the determination of ester-
bound 3-MCPD, 2-MCPD and glycidol in refined and unrefined vegetable oils and fats. It also applies to
animal fats and used frying oils and fats, but a validation study must be undertaken before analysis of these
matrices. Any free analytes within the sample are included in the results, but ISO 18363-4 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 refined vegetable
oils and fats.
vi
International Standard ISO 18363-3:2024(en)
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 terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
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 18363-3:2024(en)
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 a
...