SIST EN ISO 18363-4:2021
(Main)Animal and vegetable fats and oils - Determination of fatty-acid-bound chloropropanediols (MCPDs) and glycidol by GC/MS - Part 4: Method using fast alkaline transesterification and measurement for 2-MCPD, 3-MCPD and glycidol by GC-MS/MS (ISO 18363-4:2021)
Animal and vegetable fats and oils - Determination of fatty-acid-bound chloropropanediols (MCPDs) and glycidol by GC/MS - Part 4: Method using fast alkaline transesterification and measurement for 2-MCPD, 3-MCPD and glycidol by GC-MS/MS (ISO 18363-4:2021)
This part of ISO 18363 describes a rapid 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 alkaline catalysed ester cleavage and derivatization of cleaved (free) analytes with phenylboronic acid (PBA) prior to GC-MS/MS analysis.
This method is applicable to solid and liquid fats and oils. This part of ISO 18363 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.
Milk and milk products (or fat coming from milk and milk products) are excluded from the scope of this international standard.
Tierische und pflanzliche Fette und Öle - Bestimmung von fettsäuregebundenem Chlorpropandiol (MCPD) und Glycidol mittels GC/MS - Teil 4: Verfahren mittels schneller alkalischer Umesterung und Messung für 2-MCPD, 3-MCPD und Glycidol (ISO 18363-4:2021)
Dieser Teil von ISO 18363 beschreibt ein schnelles Verfahren für die gleichzeitige Bestimmung von 2-MCPD-Estern (gebundenes 2-MCPD), 3-MCPD-Estern (gebundenes 3-MCPD) und Glycidylestern (gebundenes Glycidol) in einer einzigen Prüfung auf der Grundlage einer alkalisch katalysierten Esterspaltung und der Derivatisierung der abgespaltenen (freien) Analyten vor der GC-MS/MS-Analyse mit Phenylboronsäure (PBA).
Das Verfahren ist auf feste und flüssige Fette und Öle anwendbar. Dieser Teil von ISO 18363 kann auch auf tierische Fette und gebrauchte Frittieröle und -fette angewendet werden, vor der Analyse dieser Matrices muss jedoch eine Validierungsstudie erfolgen. Für alle drei Analyten beträgt die Bestimmungsgrenze (LOQ, en: limit of quantification) 0,1 mg/kg und die Nachweisgrenze (LOD, en: limit of detection) 0,03 mg/kg.
Milch- und Milcherzeugnisse (oder Fett aus Milch und Milcherzeugnissen), Säuglings(anfangs)nahrung, Emulgatoren, freie Fettsäuren sowie andere aus Fetten und Ölen gewonnene Matrices sind vom Anwendungsbereich dieser Internationalen Norm ausgenommen.
WARNUNG Es wird auf die Vorschriften hingewiesen, in denen die Handhabung von gefährlichen Stoffen festgelegt ist. Technische, organisatorische und persönliche Sicherheitsvorkehrungen müssen eingehalten werden
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 4: Méthode par transestérification alcaline rapide et mesure pour le 2-MCPD, le 3-MCPD et le glycidol par CPG-SM/SM (ISO 18363-4:2021)
Le présent document spécifie un mode opératoire rapide de 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 une seule analyse, reposant sur un clivage des esters en présence d’un catalyseur alcalin et une dérivation des analytes clivés (libres) avec de l’acide phénylboronique (PBA) avant l’analyse par CPG-SM/SM. La surestimation de la teneur en esters de glycidol est corrigée par l’ajout d’un ester de 3-MCPD marqué aux isotopes, ce qui permet la quantification du glycidol issu du 3-MCPD pendant le mode opératoire.
Cette méthode est applicable aux corps gras solides et liquides. Le présent document s’applique également aux graisses animales et aux corps gras de friture usagés, mais ces matrices n’étaient pas incluses dans la validation. Pour ces trois analytes, la limite de quantification (LQ) est de 0,1 mg/kg et la limite de détection (LOD) est de 0,03 mg/kg.
Le lait et les produits laitiers (ou les corps gras issus du lait et des produits laitiers), les formules infantiles, les émulsifiants, les acides gras libres et autres matrices dérivées de corps gras sont exclus du domaine d’application du présent document.
Živalske in rastlinske maščobe ter olja - Določevanje maščobno kislinsko vezanih kloropropandiolov (MCPDs) in glicidola z GC/MS - 4. del: Metoda z uporabo hitre alkalne transesterifikacije in meritev 2- MCPD, 3-MCPD in glicidolov z GC-MS/MS (ISO 18363-4:2021)
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 18363-4:2021
01-november-2021
Živalske in rastlinske maščobe ter olja - Določevanje maščobno kislinsko vezanih
kloropropandiolov (MCPDs) in glicidola z GC/MS - 4. del: Metoda z uporabo hitre
alkalne transesterifikacije in meritev 2- MCPD, 3-MCPD in glicidolov z GC-MS/MS
(ISO 18363-4:2021)
Animal and vegetable fats and oils - Determination of fatty-acid-bound
chloropropanediols (MCPDs) and glycidol by GC/MS - Part 4: Method using fast alkaline
transesterification and measurement for 2-MCPD, 3-MCPD and glycidol by GC-MS/MS
(ISO 18363-4:2021)
Tierische und pflanzliche Fette und Öle - Bestimmung von fettsäuregebundenem
Chlorpropandiol (MCPD) und Glycidol mittels GC/MS - Teil 4: Verfahren mittels schneller
alkalischer Umesterung und Messung für 2-MCPD, 3-MCPD und Glycidol (ISO 18363-
4:2021)
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 4: Méthode par transestérification alcaline rapide et mesure pour le
2-MCPD, le 3-MCPD et le glycidol par CPG-SM/SM (ISO 18363-4:2021)
Ta slovenski standard je istoveten z: EN ISO 18363-4:2021
ICS:
67.200.10 Rastlinske in živalske Animal and vegetable fats
maščobe in olja and oils
SIST EN ISO 18363-4:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 18363-4:2021
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SIST EN ISO 18363-4:2021
EN ISO 18363-4
EUROPEAN STANDARD
NORME EUROPÉENNE
August 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 4: Method using fast alkaline
transesterification and measurement for 2-MCPD, 3-MCPD
and glycidol by GC-MS/MS (ISO 18363-4:2021)
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 4: Verfahren mittels
d'acides gras par CPG/SM - Partie 4: Méthode par schneller alkalischer Umesterung und Messung für 2-
transestérification alcaline rapide et mesure pour le 2- MCPD, 3-MCPD und Glycidol (ISO 18363-4:2021)
MCPD, le 3-MCPD et le glycidol par CPG-SM/SM (ISO
18363-4:2021)
This European Standard was approved by CEN on 26 July 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 18363-4:2021 E
worldwide for CEN national Members.
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SIST EN ISO 18363-4:2021
EN ISO 18363-4:2021 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 18363-4:2021
EN ISO 18363-4:2021 (E)
European foreword
This document (EN ISO 18363-4:2021) 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 February 2022, and conflicting national standards
shall be withdrawn at the latest by February 2022.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
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 websites.
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 18363-4:2021 has been approved by CEN as EN ISO 18363-4:2021 without any
modification.
3
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SIST EN ISO 18363-4:2021
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SIST EN ISO 18363-4:2021
INTERNATIONAL ISO
STANDARD 18363-4
First edition
2021-08
Animal and vegetable fats and oils —
Determination of fatty-acid-bound
chloropropanediols (MCPDs) and
glycidol by GC/MS —
Part 4:
Method using fast alkaline
transesterification and measurement
for 2-MCPD, 3-MCPD and glycidol by
GC-MS/MS
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 4: Méthode par transestérification alcaline rapide et mesure
pour le 2-MCPD, le 3-MCPD et le glycidol par CPG-SM/SM
Reference number
ISO 18363-4:2021(E)
©
ISO 2021
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SIST EN ISO 18363-4:2021
ISO 18363-4:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021
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
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Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved
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SIST EN ISO 18363-4:2021
ISO 18363-4:2021(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 General . 2
5.2 Standard and reference compounds . 2
5.3 Standard solutions . 3
5.3.1 General. 3
5.3.2 Stock solutions . 3
5.3.3 Working solutions . 3
5.4 Other reagents . 4
5.5 Reagent solutions . 4
6 Apparatus . 5
7 Sample and storage . 5
7.1 Sampling . 5
7.2 Preparation of the test sample . 5
7.3 Storage conditions . 6
8 Procedure. 6
8.1 Test sample preparation . 6
8.2 Preparation of the calibration curve . 7
8.3 Gas chromatography and mass spectrometry settings . 7
9 Expression of results . 8
9.1 General . 8
9.2 Quantification of 2-MCPD- and 3-MCPD esters . 8
9.3 Quantification of glycidyl esters .10
9.4 Quality control .12
10 Notes .13
11 Precision .14
11.1 General .14
11.2 Repeatability .14
11.3 Between-day reproducibility .14
Annex A (normative) Supporting tables .15
Annex B (informative) Statistical results of the ISO collaborative study .17
Annex C (informative) Chromatograms .21
Bibliography .23
© ISO 2021 – All rights reserved iii
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SIST EN ISO 18363-4:2021
ISO 18363-4:2021(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 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).
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 2021 – All rights reserved
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SIST EN ISO 18363-4:2021
ISO 18363-4:2021(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 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 the individual method.
[9]
ISO 18363-1 is a differential method equivalent to the DGF standard C-VI 18 (10) and identical to
[6]
AOCS Official 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, 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.
[5]
ISO 18363-2 represents the 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 a 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.
[4]
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 acidic catalysed release of MCPD and MBPD from the ester
derivatives. ISO 18363-3 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.
ISO 18363-3 is applicable to the determination of ester-bound 2-MCPD, 3-MCPD and glycidol in refined
© ISO 2021 – All rights reserved v
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SIST EN ISO 18363-4:2021
ISO 18363-4:2021(E)
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 ISO 18363-3 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
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, 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 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 ISO 18363-3, 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, this document requires GC-MS/MS instrumentation to unambiguously detect each
of the (isotopically labelled) MBPDs required for correct quantification of the glycidyl ester induced
glycidol. This document 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 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.
vi © ISO 2021 – All rights reserved
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SIST EN ISO 18363-4:2021
INTERNATIONAL STANDARD ISO 18363-4:2021(E)
Animal and vegetable fats and oils — Determination of
fatty-acid-bound chloropropanediols (MCPDs) and glycidol
by GC/MS —
Part 4:
Method using fast alkaline transesterification and
measurement for 2-MCPD, 3-MCPD and glycidol by GC-MS/
MS
1 Scope
This document specifies a rapid 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 alkaline catalysed ester cleavage and derivatization of cleaved (free) analytes with phenylboronic
acid (PBA) prior to GC-MS/MS analysis. Glycidyl ester overestimation is corrected by addition of an
isotopic labelled ester bound 3-MCPD which allows the quantification of 3-MCPD induced glycidol
during the procedure.
This method is applicable to solid and liquid fats and oils. This document also applies to animal fats and
used frying oils and fats, but these matrices were not included in the validation. 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.
Milk and milk products (or fat coming from milk and milk products), infant formulas, emulsifiers, free
fatty acids and other fats- and oils-derived matrices are excluded from the scope of this document.
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:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
bound 2-MCPD
amount of 2-MCPD cleaved from its esterified (bound) forms by alkaline-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).
© ISO 2021 – All rights reserved 1
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SIST EN ISO 18363-4:2021
ISO 18363-4:2021(E)
3.2
bound 3-MCPD
amount of 3-MCPD cleaved from its esterified (bound) forms by alkaline-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).
3.3
bound glycidol
amount of glycidol cleaved from its esterified (bound) forms by alkaline-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 or fat sample is dissolved in toluene and tert-butyl-methyl-ether, and the internal standards
13
(3-MCPD- C diester and pentadeuterated glycidyl ester) are added. The sample is then cooled down to
3
10 °C before the alkaline transesterifi
...
SLOVENSKI STANDARD
oSIST prEN ISO 18363-4:2021
01-februar-2021
Živalske in rastlinske maščobe in olja - Ugotavljanje na maščobno kislino vezanih
kloropropanediolov (MCPD) in glicidola z GC/MS - 4. del: Metoda z uporabo hitrega
alkalnega preestrenja in meritev 2-MCPD, 3-MCPD in glicidola z GC-MS/MS
(ISO/DIS 18363-4:2020)
Animal and vegetable fats and oils - Determination of fatty-acid-bound
chloropropanediols (MCPDs) and glycidol by GC/MS - Part 4: Method using fast alkaline
transesterification and measurement for 2-MCPD, 3-MCPD and glycidol by GC-MS/MS
(ISO/DIS 18363-4:2020)
Tierische und pflanzliche Fette und Öle - Bestimmung von fettsäuregebundenem
Chlorpropandiol (MCPD) und Glycidol mittels GC/MS - Teil 4: Verfahren mittels schneller
alkalischer Umesterung und Messung für 2-MCPD, 3-MCPD und Glycidol (ISO/DIS
18363-4:2020)
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 4: Méthode par transestérification alcaline rapide et mesure pour le
2-MCPD, le 3-MCPD et le glycidol par CPG-SM/SM (ISO/DIS 18363-4:2020)
Ta slovenski standard je istoveten z: prEN ISO 18363-4
ICS:
67.200.10 Rastlinske in živalske Animal and vegetable fats
maščobe in olja and oils
oSIST prEN ISO 18363-4:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 18363-4:2021
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oSIST prEN ISO 18363-4:2021
DRAFT INTERNATIONAL STANDARD
ISO/DIS 18363-4
ISO/TC 34/SC 11 Secretariat: BSI
Voting begins on: Voting terminates on:
2020-12-10 2021-03-04
Animal and vegetable fats and oils — Determination of
fatty-acid-bound chloropropanediols (MCPDs) and glycidol
by GC/MS —
Part 4:
Method using fast alkaline transesterification and
measurement for 2-MCPD, 3-MCPD and glycidol by GC-MS/
MS
ICS: 67.200.10
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
ISO/CEN PARALLEL PROCESSING
BEING ACCEPTABLE FOR INDUSTRIAL,
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USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 18363-4:2020(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
©
PROVIDE SUPPORTING DOCUMENTATION. ISO 2020
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oSIST prEN ISO 18363-4:2021
ISO/DIS 18363-4:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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ii © ISO 2020 – All rights reserved
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oSIST prEN ISO 18363-4:2021
ISO/DIS 18363-4:2020(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 General . 2
5.2 Standard and reference compounds . 2
5.3 Standard solutions . 3
5.3.1 Stock solutions . 3
5.3.2 Working solutions . 4
5.4 Other reagents . 4
5.5 Reagent solutions . 5
6 Apparatus . 5
7 Sample and storage . 5
7.1 Sampling . 5
7.2 Preparation of the test sample . 5
7.3 Storage conditions . 6
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 2-MCPD- and 3-MCPD esters . 8
9.2 Quantification of glycidyl esters .10
9.3 Quality control .12
10 Notes .12
11 Precision .14
11.1 General .14
11.2 Repeatability .14
11.3 Between-day reproducibility .14
Annex A (Supporting tables) .15
Annex B (informative) Statistical results of the ISO Collaborative study.17
Annex C (informative) Chromatograms .21
Bibliography .23
© ISO 2020 – All rights reserved iii
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oSIST prEN ISO 18363-4:2021
ISO/DIS 18363-4:2020(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. 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. 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 glycidol
— Part 2: Method using alkaline transesterification and measurement of 2-MCPD, 3-MCPD and glycidol
— Part 3: Method using acid transesterification and measurement of 2-MCPD, 3-MCPD and glycidol
— Part 4: Method using fast alkaline transesterification and measurement for 2-MCPD, 3-MCPD and
glycidol (MS/MS)
iv © ISO 2020 – All rights reserved
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oSIST prEN ISO 18363-4:2021
ISO/DIS 18363-4:2020(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 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, this 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 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. ISO 18363-2 consists 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 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. 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 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.
ISO 18363-3 represents 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
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 2020 – All rights reserved v
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oSIST prEN ISO 18363-4:2021
ISO/DIS 18363-4:2020(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, research has not shown any evidence of a free analyte content as high as the esterified
analyte content in vegetable oils and fats.
ISO 18363-4 describes 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
isoptopically 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 Parts 1 to 3, the released MCPD’s and 3-MBPD are derivatized with phenylboronic
acid before GC-MS/MS analysis. In contrast to the other parts of ISO 18363, part 4 requires GC-MS/MS
instrumentation to unambiguously detect each of the (isotopically labelled) MBPD’s required for correct
quantification of the glycidyl ester induced glycidol. ISO-1363-4 is 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 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.
vi © ISO 2020 – All rights reserved
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oSIST prEN ISO 18363-4:2021
DRAFT INTERNATIONAL STANDARD ISO/DIS 18363-4:2020(E)
Animal and vegetable fats and oils — Determination of
fatty-acid-bound chloropropanediols (MCPDs) and glycidol
by GC/MS —
Part 4:
Method using fast alkaline transesterification and
measurement for 2-MCPD, 3-MCPD and glycidol by GC-MS/
MS
1 Scope
This part of ISO 18363 describes a rapid 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 alkaline catalysed ester cleavage and derivatization of cleaved (free) analytes with
phenylboronic acid (PBA) prior to GC-MS/MS analysis.
This method is applicable to solid and liquid fats and oils. This part of ISO 18363 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. 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.
Milk and milk products (or fat coming from milk and milk products), infant formulas, emulsifiers, free
fatty acids and other fats and oils derived matrices are excluded from the scope of this international
standard.
2 Normative references
The 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 methods
ISO 5555, Animal and vegetable fats and oils — Sampling
ISO 661, Animal and vegetable fats and oils — Preparation of test sample
3 Terms and definitions
3.1
Bound 2-MCPD
The amount of 2-MCPD cleaved from its esterified (bound) forms by alkaline-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).
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oSIST prEN ISO 18363-4:2021
ISO/DIS 18363-4:2020(E)
3.2
Bound 3-MCPD
The amount of 3-MCPD cleaved from its esterified (bound) forms by alkaline-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).
3.3
Bound glycidol
The amount of glycidol cleaved from its esterified (bound) forms by alkaline-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 toluene and tert-butyl-methyl-ether, and the internal standards
13
(3-MCPD- C diester and pentadeuterated glycidyl ester) are added. The sample is then cooled down to
3
10 ºC before the alkaline transesterification is initiated by the addition of a sodium methoxide solution
in methanol. After 12 min incubation at 10 ºC, the sample mixture is acidified with an acidic solution
of sodium bromide to convert the released glycidol to 3-MBPD. The fatty acid methyl esters generated
during the transesterification are removed by duplicate extraction of the organic layer. Finally, the
purified sample – containing cleaved (free) analytes – is derivatized with phenylboronic acid prior to
[1]
GC-MS/MS analysis .
13
The quantification of ester bound 2- and 3-MCPD is based on the 2-MCPD/3-MCPD- C and
3
13
3-MCPD/3-MCPD- C signal ratio, respectively. The quantification of ester bound glycidol is based on
3
13
the 3-MBPD/3-MBPD-d5 signal ratio. The amount of 3-MBPD- C formed after the transesterification
3
13
reaction signifies the amount of released 3-MCPD- C that has degraded to glycidol due to the
3
conditions of the alkaline transesterification. Because no difference in degradation speed between
13
3-MCPD and 3-MCPD- C has been observed, it is then used to correct for overestimation of the glycidyl
3
ester induced glycidol caused by this degradation of 3-MCPD. Under the conditions used the 2-MCPD is
[2]
considered stable and thus will not significantly contribute to possible glycidol overestimation .
This method allows the simultaneous quantification of all three analytes in a single assay.
5 Reagents
5.1 General
WARNING — Attention is drawn to the regulations which specify the 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.
5.2 Standard and reference compounds
5.2.1 1,2-Dipalmitoyl-3-chloropropanediol (PP-3-MCPD), purity ≥ 95% (e.g., supplied by Toronto
1)
Research Chemicals Inc., North York, ON, Canada or synthesized from 3-MCPD and palmitoyl chloride
[1]
as described by Kraft et al. ).
1) The Standard and reference compound supplier is an example of a suitable supplier of reference standards
that is commercially available. This information is given for the convenience of users of this document and does not
constitute an endorsement by ISO of this product.
2 © ISO 2020 – All rights reserved
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oSIST prEN ISO 18363-4:2021
ISO/DIS 18363-4:2020(E)
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 should be preferred as they are the most
abundant in the majority of oils/fats).
5.2.2 1,3-Distearoyl-2-chloropropanediol (SS-2-MCPD), purity ≥ 95% (e.g., supplied by Toronto
1)
Research Chemicals Inc., North York, ON, Canada ).
NOTE In analogy with the recommendations given for PP-3-MCPD, 1,3-distearoyl-2-chloropropanediol can
be substituted by other fatty acid diesters of 2-MCPD with similar chain length (C16-C18 should be preferred as
they are the most abundant in the majority of oils/fats).
13
5.2.3 Carbon-13 labelled- 1,2-dipalmitoyl-3-chloropropanediol (PP-3-MCPD- C ), purity ≥ 95%
3
1)
(e.g., supplied by Toronto Research Chemicals Inc., North York, ON, Canada ).
NOTE The same consideration applied to 1,2-dipalmitoyl-3-chloropropanediol is valid also for its carbon-13
labelled analogue, see Note in 5.2.1.
5.2.4 Glycidyl stearate (Gly-S), purity ≥ 98% (e.g., supplied by Toronto Research Chemicals Inc., North
1)
York, ON, Canada ).
NOTE Glycidyl stearate can be substituted by glycidyl oleate or other fatty acid esters of glycidol with similar
chain length (C16-C18 should be preferred as they are the most abundant in the majority of oils/fats).
5.2.5 Pentadeuterated glycidyl stearate (Gly-S-d5), purity ≥ 98% (e.g., supplied by Toronto Research
1)
Chemicals Inc., North York, ON, Canada ).
NOTE The same consideration applied to glycidyl palmitate is valid also for its pentadeuterated analogue,
see Note in 5.2.4.
5.3 Standard solutions
All standard solutions are prepared with toluene (5.4.4). All standards are prepared using ester-bound
reference compounds (5.2). Concentrations are given in the free component equivalent concentration. For
an exemplary calculation of ester-bound to free equivalent concentration conversion, please see Note 3.
5.3.1 Stock solutions
a) Calibration Stock (3-MCPD: 52.7µg/mL, glycidol: 52.2 µg/mL, 2-MCPD: 48.1 µg/mL): Weigh
14.0 mg of PP-3-MCPD (5.2.1), 12.0 mg of Gly-S (5.2.4) and 14.0 mg of SS-2-MCPD (5.2.2) in a 50 mL
volumetric flask. Fill up to the mark, making sure that the standards are completely dissolved in
the sol
...
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