SIST EN ISO 18363-2:2025

SLOVENSKI STANDARD
01-marec-2025
Nadomešča:
SIST EN ISO 18363-2:2018
Živalske in rastlinske maščobe ter olja - Določevanje maščobno-kislinsko vezanih
kloropropandiolov (MCPD) in glicidola z GC/MS - 2. del: Metoda z uporabo
počasne alkalne transesterifikacije in meritev 2-MCPD, 3-MCPD in glicidola (ISO
18363-2:2025)
Animal and vegetable fats and oils - Determination of fatty-acid-bound
chloropropanediols (MCPDs) and glycidol by GC/MS - Part 2: Method using slow alkaline
transesterification and measurement for 2-MCPD, 3-MCPD and glycidol (ISO 18363-
2:2025)
Tierische und pflanzliche Fette und Öle - Bestimmung von fettsäuregebundenem
Chlorpropandiol (MCPD) und Glycidol mittels GC/MS - Teil 2: Verfahren mittels
langsamer alkalischer Umesterung und Messung für 2-MCPD, 3-MCPD und Glycidol
(ISO 18363-2:2025)
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 2: Méthode par transestérification alcaline lente et mesure pour le 2
-MCPD, le 3-MCPD et le glycidol (ISO 18363-2:2025)
Ta slovenski standard je istoveten z: EN ISO 18363-2:2025
ICS:
67.200.10 Rastlinske in živalske Animal and vegetable fats
maščobe in olja and oils
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 18363-2
EUROPEAN STANDARD
NORME EUROPÉENNE
January 2025
EUROPÄISCHE NORM
ICS 67.200.10 Supersedes EN ISO 18363-2:2018
English Version
Animal and vegetable fats and oils - Determination of fatty-
acid-bound chloropropanediols (MCPDs) and glycidol by
GC/MS - Part 2: Method using slow alkaline
transesterification and measurement for 2-MCPD, 3-MCPD
and glycidol (ISO 18363-2:2025)
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 2: Verfahren mittels
d'acides gras par CPG/SM - Partie 2: Méthode par langsamer alkalischer Umesterung und Messung für 2-
transestérification alcaline lente et mesure pour le 2- MCPD, 3-MCPD und Glycidol (ISO 18363-2:2025)
MCPD, le 3-MCPD et le glycidol (ISO 18363-2:2025)
This European Standard was approved by CEN on 21 December 2024.

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

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

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2025 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 18363-2:2025 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 18363-2:2025) 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 July 2025, and conflicting national standards shall be
withdrawn at the latest by July 2025.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 18363-2:2018.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 18363-2:2025 has been approved by CEN as EN ISO 18363-2:2025 without any
modification.
International
Standard
ISO 18363-2
Second edition
Animal and vegetable fats and
2025-01
oils — Determination of fatty-acid-
bound chloropropanediols (MCPDs)
and glycidol by GC/MS —
Part 2:
Method using slow alkaline
transesterification and
measurement for 2-MCPD, 3-MCPD
and glycidol
Corps gras d’origines animale et végétale — Détermination des
esters de chloropropanediols (MCPD) et d’acides gras et des
esters de glycidol et d’acides gras par CPG/SM —
Partie 2: Méthode par transestérification alcaline lente et mesure
pour le 2-MCPD, le 3-MCPD et le glycidol
Reference number
ISO 18363-2:2025(en) © ISO 2025

ISO 18363-2:2025(en)
© ISO 2025
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
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Email: copyright@iso.org
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Published in Switzerland
ii
ISO 18363-2:2025(en)
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 Solvents and chemicals .3
5.3 Standard and reference compounds .3
5.4 Working solutions .4
5.5 Other solutions .4
6 Apparatus . 4
7 Sample . 5
7.1 Sampling .5
7.2 Preparation of the test sample .5
8 Procedure . 5
8.1 Spiking with surrogate standard and homogenization .5
8.2 Ester cleavage and glycidol transformation .5
8.3 Matrix removal .6
8.4 Derivatization .6
8.5 Gas chromatography/mass spectrometry references .6
9 Expression of results . 7
9.1 Determination of bound glycidol . .7
9.2 Determination of bound 2-MCPD .8
9.3 Determination of bound 3-MCPD .9
9.4 Determination of the degree of diester cleavage .9
9.5 Quality control .9
10 Notes . . 10
Annex A (informative) Examples of relevant chromatograms and data evaluation using “low-
MCPD” palm oil .12
Annex B (informative) Results of interlaboratory tests . 19
Bibliography .21

iii
ISO 18363-2:2025(en)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
ISO technical committees. Each member body interested in a subject for which a technical committee
has been established has the right to be represented on that committee. International organizations,
governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely
with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types
of ISO document should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO’s adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11,
Animal and vegetable fats and oils, in collaboration with the European Committee for Standardization (CEN)
Technical Committee CEN/TC 307, Oilseeds, vegetable and animal fats and oils and their by-products - Methods
of sampling and analysis, in accordance with the Agreement on technical cooperation between ISO and CEN
(Vienna Agreement).
This second edition cancels and replaces the first edition (ISO 18363-2:2018), of which it constitutes a minor
revision to align the Introduction with ISO 18363-4:2021.
A list of all parts in the ISO 18363 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
ISO 18363-2:2025(en)
Introduction
[1]
The ISO 18363 series can be used for the determination of ester-bound MCPD and glycidol. This
introduction describes the methods specified in the different parts so that the analyst can decide which
methods are suitable for application. The detailed application of each method is contained within the scope
of each individual method.
[2] [3]
ISO 18363-1 is a differential method equivalent to the DGF standard C-VI 18 (10) and identical to AOCS
[4]
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 of this document, research has not
shown any evidence of a free analyte content as high as the esterified analyte content in refined vegetable
oils and fats. In principle, ISO 18363-1 can also be modified in such a way that the determination of 2-MCPD
[5]
is feasible, but again, a validation study must be undertaken before the analysis of this analyte.
[6][7]
This document (i.e. ISO 18363-2) represents AOCS Official Method Cd 29b-13. For information on
corresponding validation data, see Annex B. 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. This document 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 isotope-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. 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 the analysis
of these matrices. Any free analytes within the sample are included in the results, but the document does
not allow the distinction between free and bound analytes. However, as of publication of this document,
research has not shown any evidence of a free analyte content as high as the esterified analyte content in
refined vegetable oils and fats.
[8] [9][10]
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 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

v
ISO 18363-2:2025(en)
(esterified) analytes, but if required, ISO 18363-3 can also be 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 of this document, research has not shown any evidence of a free
analyte content as high as the esterified analyte content in refined vegetable oils and fats.
[11]
ISO 18363-4 specifies a rapid procedure based on fast alkaline cleavage of the MCPD and glycidyl
esters. The released glycidol is subsequently converted into 3-MBPD. The pH of the fast alkaline cleavage
generally causes the released MCPD to partially convert to glycidol during the cleavage of the esters, leading
to overestimation of the glycidyl ester content of the sample. By adding two distinct isotopically labelled
ester-bound 3-MCPD and glycidol internal standards, it is possible to quantify the amount of labelled
glycidol resulting from the degradation of the released internal standard. This information can be used to
correct for overestimation of the glycidyl ester induced glycidol by 3-MCPD induced glycidol. The same two
internal standards are used for quantification of the bound MCPD and glycidol, requiring a single sample
preparation to quantify bound 2-MCPD-, 3-MCPD- and glycidol esters. In analogue with ISO 18363-1, this
document 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, ISO 18363-4 requires GC-MS/
MS instrumentation to unambiguously detect each of the (isotopically labelled) MBPDs required for correct
quantification of the glycidyl ester induced glycidol. ISO 18363-4 is applicable to the determination of ester-
bound 3-MCPD, 2-MCPD and glycidol in refined and unrefined vegetable oils and fats. It also applies to
animal fats and used frying oils and fats, but a validation study must be undertaken before analysis of these
matrices. Any free analytes within the sample are included in the results, but ISO 18363-4 will not allow the
distinction between free and bound analytes. However, as of publication of this document, research has not
shown any evidence of a free analyte content as high as the esterified analyte content in refined vegetable
oils and fats.
vi
International Standard ISO 18363-2:2025(en)
Animal and vegetable fats and oils — Determination of
fatty-acid-bound chloropropanediols (MCPDs) and glycidol
by GC/MS —
Part 2:
Method using slow alkaline transesterification and
measurement for 2-MCPD, 3-MCPD and glycidol
1 Scope
This document specifies a procedure for the parallel determination of glycidol together with 2-MCPD and
3-MCPD present in bound or free form in oils and fats. The method is based on alkaline-catalysed ester
cleavage, transformation of the released glycidol into monobromopropanediol (MBPD) and derivatisation of
the derived free diols (MCPD and MBPD) with phenylboronic acid (PBA). Though free MCPD and glycidol are
supposed to be present in fats and oils in low to negligible quantities only, in the event that free analytes are
present, they would contribute proportionately to the results. The results always being the sum of the free
and the bound form of a single analyte.
This method is applicable to solid and liquid fats and oils. This document can also apply to animal fats and
used frying oils and fats, but a validation study is 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
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 https:// www .electropedia .org/
3.1
bound 2-MCPD
sum of all 2-MCPD-derivatives that are cleaved by alkaline-catalysed alcoholysis
Note 1 to entry: The content of bound 2-MCPD is reported in milligrams per kilogram (mg/kg).

ISO 18363-2:2025(en)
3.2
bound 3-MCPD
sum of all 3-MCPD-derivatives that are cleaved by alkaline-catalysed alcoholysis
Note 1 to entry: The content of bound 3-MCPD is reported in milligrams per kilogram (mg/kg).
3.3
bound glycidol
sum of all glycidyl derivatives that are cleaved by alkaline-catalysed alcoholysis
Note 1 to entry: The content of bound glycidol is reported in milligrams per kilogram (mg/kg).
4 Principle
For the determination of bound 2-MCPD, bound 3-MCPD and bound glycidol as free 2-MCPD, free 3-MCPD and
free 3-MBPD (3-Monobromopropanediol), two aliquots (A and B) of the sample are spiked with surrogate
standards (d -2-MCPD, d -3-MCPD, d -glycidylester in assay A and d -2-MCPD-1,3-diester, d -3-MCPD-1,2-
5 5 5 5 5
diester in assay B) and dissolved in diethyl ether. Both assays are processed in parallel. The addition of a
diluted solution of sodium hydroxide or sodium methoxide in methanol in the cold will release free 2-MCPD,
free 3-MCPD and free glycidol over a period of 8 h to 12 h. This reaction is stopped by the addition of an excess
amount of sodium bromide in acidic solution. Under acidic conditions, free glycidol reacts with inorganic
bromide to form 3-MBPD and a small amount of 2-MBPD. Undesired non-polar compounds in the sample
are removed by double extraction of the aqueous phase with isohexane. The analytes, together with the
surrogate standards, are 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, the analytes are concentrated and transferred
into an inert organic solvent. The sample extract is then placed over a small amount of anhydrous sodium
sulfate and evaporated to dryness under a stream of nitrogen before being finally redissolved in iso-octane
for the measurement by GC/MS.
The alkaline catalysed transesterification in the cold minimizes the undesired transformation of 3-MCPD
into glycidol that proceeds to a significant extend when the reaction is carried out at room temperature.
Nevertheless, in case of large amounts of 3-MCPD being present, even a minor transformation into glycidol
might increase the glycidol results from assay A artificially. In order to achieve the correct glycidol results,
even in the presence of high 3-MCPD content, assay B serves for the determination of the undesired
3-MCPD–glycidol transformation by determining the amount of d -glycidol that has been generated from d -
5 5
3-MCPD-diester by the sample preparation. The corresponding transformation ratio is used for correcting
the glycidol value derived from assay A. Another point to be taken into account is that 3-MCPD is converted
approximately 1,2-fold faster via glycidol into 3-MBPD than 3-MCPD-d via glycidol-d into 3-MBPD-d .
5 5 5
Consequently, the isotopic factor I = 1,2 has to be considered for the quantitative determination of the
amount of glycidol that has been generated accidentally from the non-labelled 3-MCPD by alkaline treatment
in assay A.
Quantification of the analytes is carried out by internal one-point-calibration using the corresponding
d -esters as surrogate standards. Therefore, no external calibration is necessary. Likewise, no analyte
recoveries have to be considered. However, the cleaving rates of MCPD mono- and diesters might be
different and as only d -MCPD-diesters serve as internal standards, the degree of ester cleavage should have
proceeded on a large scale. Therefore, the degree of variations in ester cleavage is monitored by calculating
the differences in 3-MCPD results between assay A and B. For information on deriving quantitative data
[9]
from corresponding chromatograms, see expression of results and Annex A.
As 3-MCPD can occur in certain polymers used for wet strengthening resins and for other purposes, it might
also occur from the use of consumables, e.g. screw lid vials or filter. Baking the glassware at 400 °C to 500 °C
can reduce this problem. A better solution is the use of non-contaminated materials.

ISO 18363-2:2025(en)
5 Reagents
5.1 General
WARNING — This document requires handling of hazardous substances. Technical, organizational
and personal safety measures shall be followed.
Unless otherwise stated analytically pure reagents shall be used. Water shall comply with grade 3 of
ISO 3696.
5.2 Solvents and chemicals
5.2.1 Toluene.
5.2.2 tertiary-Butyl methyl ether (tBME), (2-Methoxy-2-methylpropane).
5.2.3 Methanol.
5.2.4 iso-Hexane (2-methyl pentane).
5.2.5 Ethyl acetate.
5.2.6 Diethyl ether.
5.2.7 iso-Octane.
5.2.8 Sodium sulfate anhydrous, granular.
5.3 Standard and reference compounds
5.3.1 2-MCPD.
5.3.2 2-MCPD-d .
1)
5.3.3 2-MCPD-1,3-bis-stearoylester.
1)
5.3.4 2-MCPD-d -1,3-bis-stearoylester.
5.3.5 3-MCPD.
5.3.6 3-MCPD-d .
1)
5.3.7 3-MCPD-1,2-bis-palmitoylester.
1)
5.3.8 3-MCPD-d -1,2-bis-palmitoylester.
1)
5.3.9 Glycidyl oleate.
1)
5.3.10 Glycidyl-d oleate.
1) Other commercially available fatty acid esters of the analytes may be substituted.

ISO 18363-2:2025(en)
2)
5.4 Working solutions
5.4.1 2-MCPD: 10,0 μg/ml in methanol.
5.4.2 2-MCPD-d : 10,0 μg/ml in methanol.
5.4.3 3-MCPD: 10,0 μg/ml in methanol.
5.4.4 3-MCPD-d : 10,0 μg/ml in methanol.
5.4.5 2-MCPD-1,3-bis-stearoyl ester: 29,1 μg/ml in toluene; equivalent to 5,0 μg/ml free 2-MCPD.
5.4.6 2-MCPD-d -1,3-bis-stearoyl ester: 29,3 μg/ml in toluene; equivalent to 5,0 μg/ml free 2-MCPD.
5.4.7 3-MCPD-1,2-bis-palmitoyl ester: 26,6 μg/ml in toluene; equivalent to 5,0 μg/ml free 2-MCPD.
5.4.8 3-MCPD-d -1,2-bis-palmitoyl ester: 26,8 μg/ml in toluene; equivalent to 5,0 μg/ml free 2-MCPD.
5.4.9 Glycidyl oleate: 22,8 μg/ml in toluene; equivalent to 5,0 μg/ml free glycidol.
5.4.10 Glycidyl-d oleate: 23,2 μg/ml in toluene; equivalent to 5,0 μg/ml free glycidol.
5.5 Other solutions
5.5.1 Sodium hydroxide solution. Weigh 0,25 g freshly ground sodium hydroxide in a 100 ml plastic
bottle. Add 100 ml methanol and tightly seal the bottle. Shake vigorously (vortex) until the sodium hydroxide
has been dissolved completely. Store in a freezer at –22 °C to –25 °C (see 10.1).
5.5.2 Acidified sodium bromide solution. Weigh 600 g sodium bromide in a 1 l screw cap glass
volumetric flask, add deionised water up to the 1 l mark. Acidify the mixture with 3 ml of ortho-phosphoric
acid (85 %), seal tightly and shake (vortex) until the solution is clear. 600 μl of this solution must neutralize
350 μl of sodium hydroxide solution (5.5.1). Adjust the pH-value to the acidic range (pH 3 to pH 1). Store the
solution in a freezer at –22 °C to –25 °C (see 10.1).
5.5.3 Saturated solution of phenylboronic acid (PBA) in diethyl ether. Add approximately 200 mg
PBA to 10 ml diethyl ether in a screw cap vial. Shake well, allow non-dissolved PBA to settle and remain as
precipitate. For derivatization purposes, use only the clear supernatant.
6 Apparatus
6.1 Micropipettes (e.g. 10 µl to 100 µl, 10 µl to 200 µl, 100 µl to 1 000 µl).
6.2 Piston stroke and volumetric pipettes, various sizes.
6.3 Volumetric flasks, various sizes.
6.4 Analytical balance, readability 0,000 1 g, weighing precision 0,001 g.
6.5 Screw cap vials (approximately 2 ml in capacity) and screw caps with polytetrafluoroethylene
(PTFE)-coated septa.
2) Other concentrations of working solutions may be substituted.

ISO 18363-2:2025(en)
6.6 Pasteur pipettes and pipette bulbs.
6.7 Micro inserts (approximately 200 µl in capacity) for screw cap vials (approximately 2 ml in capacity).
6.8 Nitrogen blow-off equipment.
6.9 GC/MS-system with temperature programmable injector.
6.10 Fused-silica-GC-column, stationary phase 50 % diphenyl/50 % dimethyl polysiloxane, length 30 m,
ID 0,25 mm, film thickness 0,25 μm, low bleed for MS purpose, with pre-column.
The precolumn, that should be exchanged periodically, retards non-volatile components and thereby serves
to prolong the lifetime of the main column.
7 Sample
7.1 Sampling
[12]
Sampling is not part of this method. A recommended sampling method is given in ISO 5555 .
7.2 Preparation of the test sample
Aliquot liquid samples directly. Melt solid or semi-solid fats at approximately 80 °C in a drying oven or
water bath. For high-melting point fats, the temperature can be increased in 10 °C steps until the melting
process starts. Aliquot solid samples that contain higher amounts of water without melting to avoid phase
separation.
8 Procedure
NOTE See 10.2.
8.1 Spiking with surrogate standard and homogenization
Weigh two (100 ± 0,5) mg aliquots of the sample into two screw cap vials, approximately 2 ml capacity,
or weigh accurately approximatively two 100 mg aliquots of the sample and adopt measures to ensure the
correct mass balance for quantification. To assay A, add 50 μl each 2- and 3-MCPD-d standard working
solutions (5.4.2 and 5.4.4). To assay A, pipette 100 μl glycidyl-d ester standard working solution (5.4.10).
To assay B, transfer 100 μL 2-MCPD-d -bis-ester standard working solution (5.4.6) and 100 μl 3-MCPD-d -
5 5
bis-ester standard working solution (5.4.8). To each sample, add 600 μl diethyl ether and shake (vortex) the
mixtures until completely dissolved. In the case of high melting sample material, the reaction vessels may
require gentle warming.
8.2 Ester cleavage and glycidol transformation
Place both assays for a minimum of 15 min in a freezer to cool down to –22 °C to –25 °C (precipitation of
sample material at this stage of sample preparation is not a problem). To each assay, add 350 μl methanolic
sodium hydroxide solution (5.5.1). To complete the ester cleavage, seal the vials, briefly shake (vortex) and
keep for at least 16 h at –22 °C to –25 °C. Stop the reactions with 600 μl acidified sodium bromide solution
(5.5.2) kept at –22 °C to –25 °C. Briefly shake (vortex) the mixtures and place under a gentle stream of
nitrogen to reduce the volume of the organic phase to approximately 100 μl. To both assays, add 600 μl
iso-hexane, seal the vials and shake vigorously (vortex). Leave the mixtures at room temperature for
approximately 5 min to 10 min to complete the transformation of glycidol into MBPD (see 10.3).
NOTE Other temperature ranges for the procedure of ester-cleavage can be applied if a method validation has
been carried out. It is important to consider that working at temperature ranges above –22 °C causes an increasing
conversion of 3-MCPD into glycidol. As a consequence the uncertainty of glycidol determination can increase.

ISO 18363-2:2025(en)
8.3 Matrix removal
Remove the organic phase by Pasteur pipette. Wash the aqueous phase in each assay with a second 600 μl of
iso-hexane.
8.4 Derivatization
Using new Pasteur pipettes, extract each essay three times with 600 μl of 3:2 (v:v) mixture of diethyl ether/
ethyl acetate. Take care that the pipette does not touch the aqueous phase. For each assay A and B, combine
the organic extracts in a new screw cap vial containing a small amount of anhydrous sodium sulfate. If the
drying agent gets sticky, transfer the solution into a new screw cap vial with fresh sodium sulfate. Add 20 μl
of the derivatization reagent (5.5.3) to both organic extracts to achieve derivatization. If the derivatization
reaction results in a small analyte signal response, increase the amount of derivatization agent until the
signal to noise ratio in the corresponding chromatogram does not increase. The maximum amount of
derivatization reagent that can be used is limited by the individual capacity of the gas chromatographic–
mass spectrometric system to exclude excess PBA. To complete the derivatization reaction and to remove
excess reagent, evaporate to dryness both assays using a gentle stream of nitrogen. Re-dissolve soluble
fractions in approximately 300 μl to 500 μl iso-octane and vigorously shake (vortex) the mixture for a few
seconds. For GC/MS measurement, transfer a portion of each solution into a 200 μl micro-insert.
8.5 Gas chromatography/mass spectrometry references
8.5.1 Gas chromatography: injection volume: 1 μl to 2 μl.
8.5.2 Carrier gas: helium 4.6 (99,996 %) or helium 5.0 (99,999 %), const. flow 1 ml/min to 1,5 ml/min.
8.5.3 Programmed temperature vaporizer (PTV) temperature programme: e.g. 85 °C, increase
temperature at 300 °C/min to 165 °C, hold 10 min isothermal, increase at 300 °C/min to 320 °C, hold 8 min
isothermal.
8.5.4 Injector: e.g. split-less, purge flow 50 ml/min after 0,5 min to 1 min, septum purge 3 ml/min.
8.5.5 GC oven temperature programme: e.g. 85 °C, isothermal 0,5 min, increase temperature at 6 °C/min
to 150 °C, then 12 °C/min to 180 °C, then 25 °C/min to 280 °C, hold isothermal 8 min.
8.5.6 Mass spectrometry: electron-impact (EI), selected ion monitoring (SIM).
8.5.7 Detected ion traces related to the mass to charge ratios (m/z) from the phenylboronic derivatives
of the analytes are summarized in Table 1. See examples for the resulting chromatograms as shown in
Figures A.1 to A.10.
Table 1 — Summary of detected ion traces related to the mass to charge ratios (m/z) from the
phenylboronic derivatives of the analytes
m/z m/z m/z m/z
3-MCPD-d 149 150 201 203
3-MCPD 146 147 196 198
2-MCPD-d 201 203
2-MCPD 196 198
3-MBPD-d (glycidol-d transformation product) 245 247
5 5
3-MBPD (glycidol transformation product) 240 242
Target ions of labelled and unlabelled analytes should correspond for quantification purposes, e.g. m/z = 150
versus 147 (3-MCPD-d versus 3-MCPD), m/z = 201 versus 196 (2-MCPD-d versus 2-MCPD), m/z = 245
5 5
versus 240 (3-MBPD-d versus 3-MBPD).
ISO 18363-2:2025(en)
8.5.8 Suitable software with automatic data analysis is recommended for quantification.
9 Expression of results
NOTE See 10.4.
9.1 Determination of bound glycidol
9.1.1 Bound glycidol in assay A is quantified in 9.1.2 without correction, when 3-MCPD is low in proportion
to the glycidol content. In the presence of larger amounts of 3-MCPD, 3-MCPD conversion into glycidol may be
incomplete leading to overestimation of glycidol. To compensate for this, the conversion ratio of 3-MCPD-d -
diester into 3-MBPD-d in assay B is determined and a factor is applied to quantify the potential conversion
of 3-MCPD into 3-MBPD in assay A. This procedure is described in 9.1.3 to 9.1.5.
9.1.2 For the quantitative determination of bound glycidol, the signal area of the phenylboronic derivative
of 3-MBPD in assay A is multiplied by the spiking level of the internal standard glycidol-d (introduced
as glycidyl-d ester) related to its concentration in the sample and divided by the signal area of the
phenylboronic derivative of 3-MBPD-d , as shown by Formula (1):
SA ×w
3-MBPD(A) glycidol-d5(A)
w = (1)
glycidol uncorrected(A)
SA
3-MBPDD-d5(A)
where
w is the uncorrected mass fraction of glycidol in assay A, in mg/kg;
glycidol uncorrected(A)
w is the mass fraction of glycidol-d in assay A, in mg/kg;
glycidol-d5(A) 5
SA is the signal area of the phenylboronic derivative of 3-MBPD in assay A;
3-MBPD(A)
SA is the signal area of the phenylboronic derivative of 3-MBPD-d in assay A.
3-MBPD-d5(A) 5
If the sample contains significant amounts of 3-MCPD, the uncorrected glycidol value can be adjusted by a
factor as follows.
9.1.3 To determine the undesired transformation of 3-MCPD via glycidol into 3-MBPD during sample
preparation, the corresponding transformation ratio 3-MCPD-d → 3-MBPD-d in assay B is calculated by
5 5
dividing the signal area of the phenylboronic derivative of 3-MBPD-d by t
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