SIST EN ISO 18363-1:2022
(Main)Animal and vegetable fats and oils - Determination of fatty-acid-bound chloropropanediols (MCPDs) and glycidol by GC/MS - Part 1: Method using fast alkaline transesterification and measurement for 3-MCPD and differential measurement for glycidol (ISO 18363-1:2015)
Animal and vegetable fats and oils - Determination of fatty-acid-bound chloropropanediols (MCPDs) and glycidol by GC/MS - Part 1: Method using fast alkaline transesterification and measurement for 3-MCPD and differential measurement for glycidol (ISO 18363-1:2015)
This part of ISO 18363 describes a procedure for the indirect determination of 3-MCPD esters (bound 3-MCPD) and possible free 3-MCPD after alkaline catalysed ester cleavage and derivatization with phenylboronic acid (PBA). Furthermore, this part of ISO 18363 enables the indirect determination of glycidyl esters (bound glycidol) under the assumption that no other substances are present that react at room temperature with inorganic chloride to generate 3-MCPD.
This part of ISO 18363 is applicable to solid and liquid fats and oils. Milk and milk products (or fat coming from milk and milk products) are excluded from the scope of this part of ISO 18363.
Tierische und pflanzliche Fette und Öle - Bestimmung von fettsäuregebundenen Chlorpropandiol (MCPD) und Glycidol mittels GC/MS - Teil 1: Verfahren mittels schneller alkalischer Umesterung und Messung für 3 MCPD und Differenzmessung für Glycidol (ISO 18363 1:2015)
Dieser Teil von ISO 18363 beschreibt ein Verfahren für die indirekte Bestimmung von 3 MCPD-Estern (gebundenes 3 MCPD) und möglichem freiem 3 MCPD nach alkalisch katalysierter Esterspaltung und Derivatisierung mit Phenylboronsäure (en: phenylboronic acid, PBA). Außerdem ermöglicht dieser Teil von ISO 18363 die indirekte Bestimmung von Glycidylestern (gebundenes Glycidol) unter der Annahme, dass keine anderen Substanzen vorhanden sind, die bei Raumtemperatur mit anorganischem Chlorid zu 3 MCPD reagieren.
Dieser Teil von ISO 18363 ist auf feste und flüssige Fette und Öle anwendbar. Milch und Milcherzeugnisse (oder Fett aus Milch und Milcherzeugnissen) sind vom Anwendungsbereich dieses Teils von ISO 18363 ausgenommen.
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 1: Méthode par transestérification alcaline rapide et mesure pour le 3-MCPD et par mesure différentielle pour le glycidol (ISO 18363-1:2015)
L'ISO 18363-1:2015 décrit un mode opératoire permettant la détermination indirecte des esters de 3-MCPD (ou 3-MCPD lié) et des éventuels 3-MCPD libres après clivage des esters par catalyse alcaline et dérivatisation à l'acide phénylboronique (PBA). L'ISO 18363-1:2015 définit également la détermination indirecte des esters de glycidol (ou glycidol lié) en partant de l'hypothèse qu'aucune autre substance présente ne réagit, à température ambiante, avec le chlorure inorganique pour générer du 3-MCPD.
L'ISO 18363-1:2015 est applicable aux corps gras solides et liquides. Le lait et les produits laitiers (ou les corps gras issus du lait et des produits laitiers) sont exclus du domaine d'application de l'ISO 18363-1:2015.
Živalske in rastlinske maščobe ter olja - Določevanje maščobnokislinsko vezanih kloropropandiolov (MCPD) in glicidola z GC/MS - 1. del: Metoda z uporabo hitre alkalne transesterifikacije in meritve 3-MCPD ter diferencialne meritve glicidola (ISO 18363-1:2015)
Ta analitični postopek določa tekočinsko kromatografijo visoke ločljivosti z ultravijolično (UV) detekcijo (RP-HPLC-UV) z reverzno fazo za hkratno določevanje štirih dovoljenih karotenoidov v krmni mešanici za ribe, in sicer astaksantina (AXN), kantaksantina (CXN), adonirubina (ADR) in astaksantin dimetildisukcinata (AXN DMDS) ter šestih odobrenih karotenoidov v krmi za perutnino, in sicer kantaksantina (CXN); kapsantina (CSN), etil ester beta-apo-8'-karotenojske kisline (BACARE), citranaksantina (CIXN), luteina (LUT) in zeaksantina (ZEA), v ravneh od pribl. 2 do pribl. 4500 mg/kg (odvisno od karotenoida). V področje uporabe je dodan tudi beta-karoten (BCAR), dovoljen v krmnih mešanicah za vse živalske vrste. Analitski postopek je primeren za določanje količine navedenih karotenoidov in potrditev označevanja. Postopek se uporablja za naravne in sintetične krmne dodatke.
Ksantofilni estri, kot so lutein, zeaksantin in kapsantin, ki bi lahko bili prisotni v posamičnih krmilih, niso dovoljeni krmni dodatki in zato ne spadajo na področje uporabe te metode.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 18363-1:2022
01-februar-2022
Živalske in rastlinske maščobe ter olja - Določevanje maščobnokislinsko vezanih
kloropropandiolov (MCPD) in glicidola z GC/MS - 1. del: Metoda z uporabo hitre
alkalne transesterifikacije in meritve 3-MCPD ter diferencialne meritve glicidola
(ISO 18363-1:2015)Animal and vegetable fats and oils - Determination of fatty-acid-bound
chloropropanediols (MCPDs) and glycidol by GC/MS - Part 1: Method using fast alkaline
transesterification and measurement for 3-MCPD and differential measurement forglycidol (ISO 18363-1:2015)
Tierische und pflanzliche Fette und Öle - Bestimmung von fettsäuregebundenen
Chlorpropandiol (MCPD) und Glycidol mittels GC/MS - Teil 1: Verfahren mittels schneller
alkalischer Umesterung und Messung für 3 MCPD und Differenzmessung für Glycidol(ISO 18363 1:2015)
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 1: Méthode par transestérification alcaline rapide et mesure pour le
3-MCPD et par mesure différentielle pour le glycidol (ISO 18363-1:2015)Ta slovenski standard je istoveten z: EN ISO 18363-1:2021
ICS:
67.200.10 Rastlinske in živalske Animal and vegetable fats
maščobe in olja and oils
SIST EN ISO 18363-1:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 18363-1:2022
EN ISO 18363-1
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 1: Method using fast alkaline
transesterification and measurement for 3-MCPD and
differential measurement for glycidol (ISO 18363-1:2015)
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 1: Verfahren mittels
d'acides gras par CPG/SM - Partie 1: Méthode par schneller alkalischer Umesterung und Messung für 3
transestérification alcaline rapide et mesure pour le 3- MCPD und Differenzmessung für Glycidol (ISO 18363
MCPD et par mesure différentielle pour le glycidol (ISO 1:2015)18363-1:2015)
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 18363-1:2021 E
worldwide for CEN national Members.---------------------- Page: 3 ----------------------
SIST EN ISO 18363-1:2022
EN ISO 18363-1:2021 (E)
Contents Page
European foreword ....................................................................................................................................................... 3
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EN ISO 18363-1:2021 (E)
European foreword
The text of ISO 18363-1:2015 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-
1:2021 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 18363-1:2015 has been approved by CEN as EN ISO 18363-1:2021 without any
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SIST EN ISO 18363-1:2022
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SIST EN ISO 18363-1:2022
INTERNATIONAL ISO
STANDARD 18363-1
First edition
2015-08-15
Animal and vegetable fats and oils —
Determination of fatty-acid-bound
chloropropanediols (MCPDs) and
glycidol by GC/MS —
Part 1:
Method using fast alkaline
transesterification and measurement
for 3-MCPD and differential
measurement for 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 —
Partie 1: Méthode par transestérification alcaline rapide et
mesure pour le chloro-3 propane-1,2-diol (3-MCPD) et par mesure
différentielle pour le glycidol
Reference number
ISO 18363-1:2015(E)
ISO 2015
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SIST EN ISO 18363-1:2022
ISO 18363-1:2015(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2015, 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
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Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2015 – All rights reserved
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SIST EN ISO 18363-1:2022
ISO 18363-1:2015(E)
Contents Page
Foreword ........................................................................................................................................................................................................................................iv
Introduction ..................................................................................................................................................................................................................................v
1 Scope ................................................................................................................................................................................................................................. 1
2 Normative references ...................................................................................................................................................................................... 1
3 Terms and definitions ..................................................................................................................................................................................... 1
4 Principle ........................................................................................................................................................................................................................ 2
5 Reagents ........................................................................................................................................................................................................................ 3
5.1 General ........................................................................................................................................................................................................... 3
5.2 Standard and reference compounds .................................................................................................................................... 3
5.3 Solvents ......................................................................................................................................................................................................... 3
5.4 Other reagents ......................................................................................................................................................................................... 3
6 Apparatus ..................................................................................................................................................................................................................... 4
7 Sample ............................................................................................................................................................................................................................. 4
7.1 Sampling ....................................................................................................................................................................................................... 4
7.2 Preparation of the test sample .................................................................................................................................................. 4
8 Procedure..................................................................................................................................................................................................................... 5
8.1 Spiking with surrogate standard and homogenization ........................................................................................ 5
8.2 Ester cleavage and glycidol transformation ................................................................................................................... 5
8.3 Matrix clean up ....................................................................................................................................................................................... 5
8.4 Derivatization ........................................................................................................................................................................................... 5
8.5 Gas chromatography/mass spectrometry references ........................................................................................... 6
9 Expression of results ........................................................................................................................................................................................ 6
10 Precision ....................................................................................................................................................................................................................... 7
10.1 Interlaboratory test............................................................................................................................................................................. 7
10.2 Repeatability ............................................................................................................................................................................................. 8
10.3 Reproducibility ....................................................................................................................................................................................... 8
11 Test report ................................................................................................................................................................................................................... 8
Annex A (informative) Results of an collaborative trial ................................................................................................................... 9
Bibliography .............................................................................................................................................................................................................................11
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SIST EN ISO 18363-1:2022
ISO 18363-1:2015(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 WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information.
The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 11, Animal
and vegetable fats and oils.ISO 18363 consists of the following parts, under the general title Animal and vegetable fats and oils —
Determination of fatty-acid-bound chloropropanediols (MCPDs) and glycidol by GC/MS
— Part 1: Method using fast alkaline transesterification and measurement for 3-MCPD and differential
measurement for glycidolThe following parts are under preparation:
— Part 2: Method using alkaline transesterification and measurement for 2-MCPD, 3-MCPD and glycidol
— Part 3: Method using acid transesterification and measurement for 2-MCPD, 3-MCPD and glycidol
iv © ISO 2015 – All rights reserved---------------------- Page: 10 ----------------------
SIST EN ISO 18363-1:2022
ISO 18363-1:2015(E)
Introduction
ISO 18363 is a set of International Standards which can be used for the determination of ester-bound
MCPD and glycidol. There are currently three International Standards which have been proposed and
this introduction is a description of these methods, which can be used by the analyst to decide which
methods are suitable for their application. The detailed application of each method is contained within
the scope of the individual method.This part of ISO 18363 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. This part of
ISO 18363 is applicable for the fast determination of ester bound 3-MCPD and glycidol in refined and
non-refined vegetable oils and fats. This part of ISO 18363 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 standard 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, this part of ISO 18363 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.
The second part of the proposed International Standards for the determination of ester-bound MCPD
and glycidol represents the AOCS Official Method Cd 29b-13. Briefly, it is based on a slow alkaline
release of MCPD and glycidol from the ester derivatives. Glycidol is subsequently converted into
3-MBPD. The second part of the proposed International Standards consists of two sample preparations
that differ in the use of internal standards. Both parts can be 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 will be 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. The second part of the
proposed standards is applicable for the determination of ester bound 3-MCPD, 2-MCPD, and glycidol in
refined and unrefined vegetable oils and fats. The second part of the proposed International Standards
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 standard 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 vegetable oils and fats.The third part of the proposed International Standards for the determination of ester-bound MCPD
and glycidol represents the AOCS Official Method Cd 29a-13. Briefly, it is based on the conversion of
glycidyl esters into 3-MBPD esters and a slow acidic catalysed release of MCPD and MBPD from the
© ISO 2015 – All rights reserved v---------------------- Page: 11 ----------------------
SIST EN ISO 18363-1:2022
ISO 18363-1:2015(E)
ester derivatives. The third part of the proposed International Standards 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. The third part of the proposed International
Standards can be applied for the determination of ester bound 2-MCPD, 3-MCPD, and glycidol in refined
and non-refined vegetable oils and fats. The third part of the proposed International Standards 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, the third part of the proposed International Standards 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, 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 2015 – All rights reserved
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SIST EN ISO 18363-1:2022
INTERNATIONAL STANDARD ISO 18363-1:2015(E)
Animal and vegetable fats and oils — Determination of
fatty-acid-bound chloropropanediols (MCPDs) and glycidol
by GC/MS —
Part 1:
Method using fast alkaline transesterification and
measurement for 3-MCPD and differential measurement
for glycidol
1 Scope
This part of ISO 18363 describes a procedure for the indirect determination of 3-MCPD esters (bound
3-MCPD) and possible free 3-MCPD after alkaline catalysed ester cleavage and derivatization with
phenylboronic acid (PBA). Furthermore, this part of ISO 18363 enables the indirect determination of
glycidyl esters (bound glycidol) under the assumption that no other substances are present that react
at room temperature with inorganic chloride to generate 3-MCPD.This part of ISO 18363 is applicable to solid and liquid fats and oils. Milk and milk products (or fat
coming from milk and milk products) are excluded from the scope of this part of ISO 18363.
2 Normative referencesThe following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. 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 methods3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
bound 3-MCPD
sum of all 3-MCPD derivatives being cleaved by alkaline catalysed alcoholysis (especially fatty acid
esters) according to the reference methodNote 1 to entry: The content of 3-MCPD is calculated and reported as a mass fraction, in milligrams per kilogram
(mg/kg).Note 2 to entry: In fats and oils, the amount of free 3-MCPD which can possibly occur is generally negligibly low
but has a bearing on the result.3.2
bound glycidol
sum of all glycidyl derivatives being cleaved by alkaline catalysed alcoholysis (especially fatty acid
esters) according to the specified standardNote 1 to entry: The content of glycidol is calculated and reported as a mass fraction, in milligrams per kilogram
(mg/kg).© ISO 2015 – All rights reserved 1
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SIST EN ISO 18363-1:2022
ISO 18363-1:2015(E)
Note 2 to entry: The presence of free glycidol in fats and oils is unlikely because of the instability of this compound.
4 PrincipleFor the determination of the sum of bound 3-MCPD and bound glycidol as free 3-MCPD (assay A),
an aliquot of the sample is spiked with surrogate standard d -3-MCPD-1,2-bis-palmitoyl ester and
dissolved in tertiary-butyl methyl ether (tBME). The addition of a diluted solution of sodium hydroxide
or sodium methoxide in methanol will release free 3-MCPD and free glycidol. This reaction is stopped
by the addition of an excess amount of sodium chloride in acidic solution. Under acidic conditions, free
glycidol reacts with inorganic chloride to additional 3-MCPD and a small amount of 2-MCPD. Undesired
non-polar compounds in the sample are removed by double extraction of the aqueous phase with
isohexane. The analyte, together with the surrogate standard, is transferred into an organic phase by
multiple extraction of the aqueous phase with diethyl ether, ethyl acetate, or a mixture of both solvents.
Derivatization takes place in the organic phase by reaction with PBA. In order to remove excess amounts
of PBA, concentrate the analytes and transfer them into an inert organic solvent the sample extract is
then placed over a small amount of anhydrous sodium sulfate and evaporate until dry under a stream of
nitrogen before being finally re-dissolved in isooctane for the measurement by GC-MS.
For the determination of bound 3-MCPD, a second aliquot of the sample (assay B) is spiked with
-3-MCPD-1,2-bis-palmitoyl ester and dissolved in tBME. The addition of aa surrogate standard d
diluted solution of sodium hydroxide or sodium methoxide in methanol will release free 3-MCPD and
free glycidol. This reaction is stopped by the addition of an excess amount of an acidic chloride-free
salt solution (e.g. sodium sulfate, ammonium sulfate, sodium bromide). Under chloride-free acidic
conditions, free glycidol reacts to different products depending on the salt used, but does not generate
additional 3-MCPD. The additional sample preparation and measurement is performed as described for
assay A.For the calculation of bound glycidol, the difference of both determinations (assay A and B) is multiplied
by a non-stoichiometric factor reflecting the transformation of glycidol to 3-MCPD. This procedure is
based on the assumption that the difference between the determinations is caused only by the exclusive
occurrence of glycidyl esters.Quantification of the analytes is carried out using d -3-MCPD-1,2-bis-palmitoyl ester as surrogate
standard. Therefore, no control of the completeness of the ester cleavage is necessary. A matrix
calibration over the complete method is carried out periodically and serves for the determination
of method linearity, relative recovery and in the case of bound glycidol for the determination of the
transformation factor. As long as the method is not changed, this calibration does not necessarily have
to be done every working day.Samples which do contain 3-MCPD only and no glycidol result in lower values (approximately 2 %
to 10 %) when using procedure B instead of procedure A. The difference is due to an isotope effect
in procedure B. This effect can be determined by dosing a non-contaminated oil sample with equal
amounts of 3-MCPD-1,2-bis-dipalmitoylester and d -3-MCPD-1,2-bis-palmitoylester before being
divided into two aliquots. The aliquots are then analysed three times according to procedure A and
procedure B. From the determinations, a correction factor may be determined, which is the ratio of the
results from procedure A and B. The isotope effect also appears in the presence of bound glycidol.
As 3-MCPD can occur in certain polymers used for wet strengthening resins and for other purpose, it is
recommended that a blank sample be analysed every day in order to control undesired contamination.
The bla...
SLOVENSKI STANDARD
oSIST prEN ISO 18363-1:2021
01-oktober-2021
Živalske in rastlinske maščobe ter olja - Določevanje maščobnokislinsko vezanih
kloropropandiolov (MCPD) in glicidola z GC/MS - 1. del: Metoda z uporabo hitre
alkalne transesterifikacije in meritve 3-MCPD ter diferencialne meritve glicidola
(ISO 18363-1:2015)Animal and vegetable fats and oils - Determination of fatty-acid-bound
chloropropanediols (MCPDs) and glycidol by GC/MS - Part 1: Method using fast alkaline
transesterification and measurement for 3-MCPD and differential measurement forglycidol (ISO 18363-1:2015)
Tierische und pflanzliche Fette und Öle - Bestimmung von fettsäuregebundenen
Chlorpropandiol (MCPD) und Glycidol mittels GC/MS - Teil 1: Verfahren mittels schneller
alkalischer Umesterung und Messung für 3 MCPD und Differenzmessung für Glycidol(ISO 18363 1:2015)
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 1: Méthode par transestérification alcaline rapide et mesure pour le
3-MCPD et par mesure différentielle pour le glycidol (ISO 18363-1:2015)Ta slovenski standard je istoveten z: prEN ISO 18363-1
ICS:
67.200.10 Rastlinske in živalske Animal and vegetable fats
maščobe in olja and oils
oSIST prEN ISO 18363-1:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 18363-1:2021
INTERNATIONAL ISO
STANDARD 18363-1
First edition
2015-08-15
Animal and vegetable fats and oils —
Determination of fatty-acid-bound
chloropropanediols (MCPDs) and
glycidol by GC/MS —
Part 1:
Method using fast alkaline
transesterification and measurement
for 3-MCPD and differential
measurement for 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 —
Partie 1: Méthode par transestérification alcaline rapide et
mesure pour le chloro-3 propane-1,2-diol (3-MCPD) et par mesure
différentielle pour le glycidol
Reference number
ISO 18363-1:2015(E)
ISO 2015
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oSIST prEN ISO 18363-1:2021
ISO 18363-1:2015(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2015, 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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copyright@iso.org
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ii © ISO 2015 – All rights reserved
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oSIST prEN ISO 18363-1:2021
ISO 18363-1:2015(E)
Contents Page
Foreword ........................................................................................................................................................................................................................................iv
Introduction ..................................................................................................................................................................................................................................v
1 Scope ................................................................................................................................................................................................................................. 1
2 Normative references ...................................................................................................................................................................................... 1
3 Terms and definitions ..................................................................................................................................................................................... 1
4 Principle ........................................................................................................................................................................................................................ 2
5 Reagents ........................................................................................................................................................................................................................ 3
5.1 General ........................................................................................................................................................................................................... 3
5.2 Standard and reference compounds .................................................................................................................................... 3
5.3 Solvents ......................................................................................................................................................................................................... 3
5.4 Other reagents ......................................................................................................................................................................................... 3
6 Apparatus ..................................................................................................................................................................................................................... 4
7 Sample ............................................................................................................................................................................................................................. 4
7.1 Sampling ....................................................................................................................................................................................................... 4
7.2 Preparation of the test sample .................................................................................................................................................. 4
8 Procedure..................................................................................................................................................................................................................... 5
8.1 Spiking with surrogate standard and homogenization ........................................................................................ 5
8.2 Ester cleavage and glycidol transformation ................................................................................................................... 5
8.3 Matrix clean up ....................................................................................................................................................................................... 5
8.4 Derivatization ........................................................................................................................................................................................... 5
8.5 Gas chromatography/mass spectrometry references ........................................................................................... 6
9 Expression of results ........................................................................................................................................................................................ 6
10 Precision ....................................................................................................................................................................................................................... 7
10.1 Interlaboratory test............................................................................................................................................................................. 7
10.2 Repeatability ............................................................................................................................................................................................. 8
10.3 Reproducibility ....................................................................................................................................................................................... 8
11 Test report ................................................................................................................................................................................................................... 8
Annex A (informative) Results of an collaborative trial ................................................................................................................... 9
Bibliography .............................................................................................................................................................................................................................11
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oSIST prEN ISO 18363-1:2021
ISO 18363-1:2015(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 WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information.
The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 11, Animal
and vegetable fats and oils.ISO 18363 consists of the following parts, under the general title Animal and vegetable fats and oils —
Determination of fatty-acid-bound chloropropanediols (MCPDs) and glycidol by GC/MS
— Part 1: Method using fast alkaline transesterification and measurement for 3-MCPD and differential
measurement for glycidolThe following parts are under preparation:
— Part 2: Method using alkaline transesterification and measurement for 2-MCPD, 3-MCPD and glycidol
— Part 3: Method using acid transesterification and measurement for 2-MCPD, 3-MCPD and glycidol
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oSIST prEN ISO 18363-1:2021
ISO 18363-1:2015(E)
Introduction
ISO 18363 is a set of International Standards which can be used for the determination of ester-bound
MCPD and glycidol. There are currently three International Standards which have been proposed and
this introduction is a description of these methods, which can be used by the analyst to decide which
methods are suitable for their application. The detailed application of each method is contained within
the scope of the individual method.This part of ISO 18363 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. This part of
ISO 18363 is applicable for the fast determination of ester bound 3-MCPD and glycidol in refined and
non-refined vegetable oils and fats. This part of ISO 18363 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 standard 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, this part of ISO 18363 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.
The second part of the proposed International Standards for the determination of ester-bound MCPD
and glycidol represents the AOCS Official Method Cd 29b-13. Briefly, it is based on a slow alkaline
release of MCPD and glycidol from the ester derivatives. Glycidol is subsequently converted into
3-MBPD. The second part of the proposed International Standards consists of two sample preparations
that differ in the use of internal standards. Both parts can be 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 will be 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. The second part of the
proposed standards is applicable for the determination of ester bound 3-MCPD, 2-MCPD, and glycidol in
refined and unrefined vegetable oils and fats. The second part of the proposed International Standards
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 standard 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 vegetable oils and fats.The third part of the proposed International Standards for the determination of ester-bound MCPD
and glycidol represents the AOCS Official Method Cd 29a-13. Briefly, it is based on the conversion of
glycidyl esters into 3-MBPD esters and a slow acidic catalysed release of MCPD and MBPD from the
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oSIST prEN ISO 18363-1:2021
ISO 18363-1:2015(E)
ester derivatives. The third part of the proposed International Standards 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. The third part of the proposed International
Standards can be applied for the determination of ester bound 2-MCPD, 3-MCPD, and glycidol in refined
and non-refined vegetable oils and fats. The third part of the proposed International Standards 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, the third part of the proposed International Standards 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, 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 2015 – All rights reserved
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oSIST prEN ISO 18363-1:2021
INTERNATIONAL STANDARD ISO 18363-1:2015(E)
Animal and vegetable fats and oils — Determination of
fatty-acid-bound chloropropanediols (MCPDs) and glycidol
by GC/MS —
Part 1:
Method using fast alkaline transesterification and
measurement for 3-MCPD and differential measurement
for glycidol
1 Scope
This part of ISO 18363 describes a procedure for the indirect determination of 3-MCPD esters (bound
3-MCPD) and possible free 3-MCPD after alkaline catalysed ester cleavage and derivatization with
phenylboronic acid (PBA). Furthermore, this part of ISO 18363 enables the indirect determination of
glycidyl esters (bound glycidol) under the assumption that no other substances are present that react
at room temperature with inorganic chloride to generate 3-MCPD.This part of ISO 18363 is applicable to solid and liquid fats and oils. Milk and milk products (or fat
coming from milk and milk products) are excluded from the scope of this part of ISO 18363.
2 Normative referencesThe following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. 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 methods3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
bound 3-MCPD
sum of all 3-MCPD derivatives being cleaved by alkaline catalysed alcoholysis (especially fatty acid
esters) according to the reference methodNote 1 to entry: The content of 3-MCPD is calculated and reported as a mass fraction, in milligrams per kilogram
(mg/kg).Note 2 to entry: In fats and oils, the amount of free 3-MCPD which can possibly occur is generally negligibly low
but has a bearing on the result.3.2
bound glycidol
sum of all glycidyl derivatives being cleaved by alkaline catalysed alcoholysis (especially fatty acid
esters) according to the specified standardNote 1 to entry: The content of glycidol is calculated and reported as a mass fraction, in milligrams per kilogram
(mg/kg).© ISO 2015 – All rights reserved 1
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oSIST prEN ISO 18363-1:2021
ISO 18363-1:2015(E)
Note 2 to entry: The presence of free glycidol in fats and oils is unlikely because of the instability of this compound.
4 PrincipleFor the determination of the sum of bound 3-MCPD and bound glycidol as free 3-MCPD (assay A),
an aliquot of the sample is spiked with surrogate standard d -3-MCPD-1,2-bis-pal...
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