SIST EN ISO 18363-4:2021

SLOVENSKI STANDARD
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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.

Contents Page
European foreword . 3

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.
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
ISO 18363-4:2021(E)
© ISO 2021
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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Published in Switzerland
ii © ISO 2021 – All rights reserved

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 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

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 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

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 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
(3-MCPD- C diester and pentadeuterated glycidyl ester) are added. The sample is then cooled down to
10 °C before the alkaline transesterification is initiated by the addition of a sodium methoxide solution
in methanol. After 12 min incubation at 10 °C, 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
GC-MS/MS analysis.
The quantification of ester bound 2-MCPD and 3-MCPD is based on the 2-MCPD/3-MCPD- C and
3-MCPD/3-MCPD- C signal ratio, respectively. The quantification of ester-bound glycidol is based on
the 3-MBPD/3-MBPD-d5 signal ratio. The amount of 3-MBPD- C formed after the transesterification
reaction signifies the amount of released 3-MCPD- C that has degraded to glycidol due to the
conditions of the alkaline transesterification. Because no difference in degradation speed between
3-MCPD and 3-MCPD- C has been observed, it is then used to correct for overestimation of the glycidyl
ester induced glycidol caused by this degradation of 3-MCPD. Under the conditions used the 2-MCPD is
[7][8]
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 conform to grade 3 of
ISO 3696.
5.2 Standard and reference compounds
5.2.1 1,2-Dipalmitoyl-3-chloropropanediol (PP-3-MCPD), purity ≥ 95 %.
NOTE 1,2-Dipalmitoyl-3-chloropropanediol can be substituted with 1,2-dioleyl-3-chloropropanediol or
other fatty acid diesters of 3-MCPD with similar chain length (C16 to C18 are preferred as they are the most
abundant in the majority of oils or fats).
5.2.2 1,3-Distearoyl-2-chloropropanediol (SS-2-MCPD), purity ≥ 95 %.
2 © ISO 2021 – All rights reserved

ISO 18363-4:2021(E)
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 to C18 are preferred as they
are the most abundant in the majority of oils or fats).
5.2.3 Carbon-13 labelled 1,2-dipalmitoyl-3-chloropropanediol (PP-3-MCPD- C ), purity ≥ 95 %.
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 %.
NOTE Glycidyl stearate can be substituted by glycidyl oleate or other fatty acid esters of glycidol with similar
chain length (C16 to C18 are preferred as they are the most abundant in the majority of oils or fats).
5.2.5 Pentadeuterated glycidyl stearate (Gly-S-d5), purity ≥ 98 %.
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
5.3.1 General
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
and shall be corrected for reference compounds (5.2) purity. For an example calculation of ester-bound
to free equivalent concentration conversion, see 10.2.
5.3.2 Stock solutions
NOTE Stock solutions are stable for at least 12 months when stored at −18 °C. Using an ultrasonic bath can
help to ensure all standards are completely dissolved.
5.3.2.1 Calibration stock (3-MCPD: 52,7 µg/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 solvent.
5.3.2.2 Spike stock (3-MCPD: 52,7 µg/ml, glycidol: 52,2 µg/ml, 2-MCPD: 34,4 µg/ml). Weigh 14,0 mg
of PP-3-MCPD (5.2.1), 12,0 mg of Gly-S (5.2.4) and 10,0 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 solvent.
13 13 13
5.3.2.3 PP-3-MCPD- C stock (3-MCPD- C : 38,5 µg/ml). Weigh 20 mg of PP-3-MCPD- C (5.2.3) in
3 3 3
a 100 ml volumetric flask. Fill up to the mark, making sure that the standard is completely dissolved in
the solvent.
5.3.2.4 Gly-S-d5 stock (Glycidol-d5: 45,8 µg/ml). Weigh 10 mg of Gly-S-d5 (5.2.5) in a 50 ml volumetric
flask. Fill up to the mark, making sure that the standard is completely dissolved in the solvent.
5.3.3 Working solutions
It is advisable to freshly prepare the calibration working solutions on the day they are to be used.
ISO 18363-4:2021(E)
The concentrations of all stock and standard solutions shall be corrected for the purity of the used
standards.
NOTE The spike solution (5.3.3.4) and internal standard solution (5.3.3.5) can be stored in the refrigerator
for at least three months.
5.3.3.1 Calibration working solution I (3-MCPD: 7,9 µg/ml, glycidol: 7,8 µg/ml, 2-MCPD: 7,2 µg/ml).
Pipette 300 μl of the stock solution (5.3.2.1) into a 2,5 ml GC vial containing 1 700 µl of toluene (5.4.4)
and homogenize using a vortex mixer.
5.3.3.2 Calibration working solution II (3-MCPD: 3,2 µg/ml, glycidol: 3,1 µg/ml, 2-MCPD: 2,9 µg/ml).
Pipette 120 μl of the stock solution (5.3.2.1) into a 2,5 ml GC vial containing 1 880 µl of toluene (5.4.4)
and homogenize using a vortex mixer.
5.3.3.3 Calibration working solution III (3-MCPD: 0,16 µg/ml, glycidol: 0,16 µg/ml, 2-MCPD: 0,14 µg/
ml). Pipette 40 μl of calibration working solution I (5.3.3.1) into a 2,5 ml GC vial containing 1 960 µl of
toluene (5.4.4) and homogenize using a vortex mixer.
5.3.3.4 Spike solution (3-MCPD: 1,05 µg/ml, glycidol: 1,04 µg/ml, 2-MCPD: 0,69 µg/ml). Pipette 5,0 ml
of the spike stock solution (5.3.2.2) into a 250 ml volumetric flask and fill up to the mark with the solvent.
5.3.3.5 Internal standard solution (3-MCPD- C : 1,54 µg/ml, glycidol-d5: 0,92 µg/ml). Pipette
5,0 ml of the Gly-S-d5 (5.3.2.4) and 10,0 ml of PP-3-MCPD- C (5.3.2.3) into a 250 ml volumetric flask
and fill up to the mark with the solvent.
5.4 Other reagents
5.4.1 Methanol, analytical grade.
5.4.2 Iso-octane, analytical grade.
5.4.3 Acetone, analytical grade.
5.4.4 Toluene, analytical grade.
5.4.5 Tert-butyl-methyl-ether, analytical grade.
5.4.6 Water, ultra-pure.
5.4.7 Sulfuric acid (purity ≥ 95 %).
5.4.8 Phenylboronic acid (purity ≥ 97 %).
5.4.9 Sodium bromide (purity ≥ 99,5 %).
5.4.10 Sodium methoxide solution in methanol (mass fraction of 25 %).
5.4.11 Non-thermally treated, cold pressed vegetable oil (blank oil, see 9.4).
5.5 Reagent solutions
5.5.1 Aqueous sulfuric acid solution (25 %). Transfer 25 ml of sulfuric acid (5.4.7) to a 100 ml
volumetric flask containing 50 ml of H O (5.4.6). Fill up to the mark with H O (5.4.6) and homogenize.
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4 © ISO 2021 – All rights reserved

ISO 18363-4:2021(E)
5.5.2 Acid aqueous solution of sodium bromide (sodium bromide 600 mg/ml, sulfuric acid volume
fraction of 0,9 %). Dissolve 600 g of sodium bromide (5.4.9) into 700 ml of ultrapure water (5.4.6).
Transfer the sodium bromide solution to a 1 000 ml volumetric flask containing 36 ml of sulfuric acid
solution (5.5.1). Fill up to the mark with H O (5.4.6) and homogenize.
5.5.3 Sodium methoxide solution (0,35M). Transfer 20 ml of sodium methoxide (mass fraction of
25 %) (5.4.10) to a 250 ml volumetric flask, fill up to the mark with methanol (5.4.1) and homogenize.
NOTE The sodium methoxide solution (0,35M) can be stored in a refrigerator for at least three months.
5.5.4 Phenylboronic acid solution (saturated). Weigh 12,0 g of phenylboronic acid (5.4.8) and add
100 ml volume fraction of 5 % water (5.4.6) in acetone (5.4.3) mixture. Shake vigorously.
NOTE The phenylboronic acid does not dissolve completely in the solvent mixture. Only the supernatant
is used for the derivatization step (see 8.1.11). The solution can be stored at room temperature for at least
three months.
6 Apparatus
6.1 Vortex mixer.
6.2 Cooled sample tray, set to 10 °C ± 0,5 °C.
6.3 Heated sample tray, with agitator capabilities set to 80 °C ± 4,0 °C.
6.4 Ultrasonic bath.
6.5 GC-MS/MS system, with split/splitless injector and backflush option.
6.6 Fused-silica-GC-column, stationary phase 5 % diphenyl to 95 % dimethylpolysiloxane or similar
polarity, length 20 m, ID 0,18 mm, film thickness 0,18 µm. Pre-column: stationary phase 5 % diphenyl to
95 % dimethylpolysiloxane or similar polarity, length 2 m, ID 0,53 mm, film thickness 0,10 µm.
The pre-column is periodically exchanged to retain good peak shape and sensitivity.
6.7 Electronic pipette, capable of pipetting volumes of 1,0 µl to 1 000 µl.
Using an electronic pipette is recommended for the sequential addition of accurate amounts of internal
standard solutions or the dilution of standards for calibration.
7 Sample and storage
7.1 Sampling
Sampling is not part of this method. A recommended sampling method is given in ISO 5555.
7.2 Preparation of the test sample
Liquid samples shall be used without additional treatment. Solid or turbid fats shall be carefully melted
at approximately 60 °C in a drying oven or water bath. For high-melting fats, the temperature shall be
carefully increased in 10 °C steps until the melting process starts. Samples containing high amounts of
water shall be dried (e.g. by anhydrous Na SO ) before sampling.
2 4
Oils and fats with higher melting points > 60 °C often show solidification when incubated at 10 °C in the
presence of the reaction medium (see 8.1.4). This influences the completeness of the ester cleavage as
ISO 18363-4:2021(E)
the reaction speed decreases substantially. It is important that high melting fats form a milky solution
to keep the reaction going. Samples that completely solidify typically show low signal intensities and
the results are not reproducible. The internal standard signals are effective markers to spot samples
that did not readily undergo the cleavage reaction. If the internal standard signals of the sample
are < 50 % of the mean internal standard signals of the recovery samples, the sample preparation shall
be restarted with less fat or oil.
7.3 Storage conditions
The glycidyl ester concentrations are subject to storage conditions whereas the MCPD esters are not.
Room temperature (22 °C) has proven to provide the best stability for both glycidyl and MCPD esters
and thus samples should be stored under such conditions. Samples cannot be stored under refrigerated
conditions (4 °C) as the degradation of glycidyl esters is likely to occur over time.
8 Procedure
8.1 Test sample preparation
NOTE The procedure has been given for a single sample. A timetable for the parallel sample preparation of a
batch of 20 samples for steps 8.1.5 and 8.1.6 is given in Table A.2.
8.1.1 Weigh 100 mg to 120 mg of oil or fat sample (to a precision of 0,01 mg) in a 2,5 ml GC vial.
For fats or oil matrices with melting points > 60 °C, a maximum of 100 mg of fat is weighed to prevent
solidification of the reaction mixture during transesterification (see 10.1).
8.1.2 Add 100 μl of toluene (5.4.4) and 200 μl of tBME (5.4.5) followed by 100 μl of internal standard
working solution (5.3.3.5) to all samples.
Steps 8.1.3 to 8.1.9 shall be executed uninterrupted to ensure correct quantification.
8.1.3 Agitate all samples using a heated agitator (80 °C, 250 r/min) for 120 s or until all fat has been
melted and dissolved.
8.1.4 Homogenize for 10 s using a vortex mixer and place the vials in a cooled sample tray (10 °C).
Leave them to cool for exactly 240 s.
8.1.5 Start the transesterification by adding 200 μl of sodium methoxide solution (5.5.3). Homogenize
for 10 s after addition of the sodium methoxide solution (5.5.3) using a vortex mixer and place the sample
back in the cooled sample tray.
8.1.6 After exactly 12 min, stop the reaction by adding 700 μl of aqueous acidified sodium bromide
solution (5.5.2). Homogenize for 10 s using a vortex mixer and let the temperature stabilize at room
temperature for at least 5 min for the complete conversion of all glycidol to 3-MBPD.
8.1.7 Add to all samples 300 μl of iso-octane (5.4.2) and homogenize using a vortex mixer for 10 s.
8.1.8 Agitate all samples using a heated agitator (80 °C, 400 r/min) for 270 s ± 10 s or until the
(partially) solidified or jellified upper layer has been completely dissolved and homogenized (see 7.2).
8.1.9 Let the samples cool down for 3 min before extracting the organic layer, while being careful not
to extract any of the water layer and discard the extract.
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ISO 18363-4:2021(E)
8.1.10 Add 600 μl of fresh iso-octane (5.4.2) and homogenize using a vortex mixer for 10 s followed
by extraction of the organic layer, while being careful not to extract any of the water layer. Discard the
extract.
8.1.11 Add 100 μl of PBA solution (5.5.4) and homogenize using a vortex mixer for 10 s.
8.1.12 Add 600 μl of fresh iso-octane (5.4.2) and homogenize using a vortex mixer for 10 s to extract the
derivatives and analyse using a GC-MS/MS system by injection 2,0 µl of the organic phase.
8.2 Preparation of the calibration curve
8.2.1 Weigh 100 mg to 120 mg of blank oil (5.4.11) into 10 separate 2,5 ml GC vials. Add toluene (5.4.4),
tBME (5.4.5), internal standard solution (5.3.3.5) and the corresponding calibration solution (5.3.3.1 to
5.3.3.3) as indicated in Table A.1.
8.2.2 Treat the calibration samples according to the procedure used for the test samples, starting with
the dissolution of the sample (see 8.1.3).
All ratios of solvent and reagents should be equal for all calibration samples and unknown samples as
described in 8.1.
8.3 Gas chromatography and mass spectrometry settings
8.3.1 Injection volume: 2,0 μl.
8.3.2 Injection mode: splitless, splitless time = 2,0 min.
8.3.3 Injection temperature: 350 °C.
8.3.4 Carrier gas: helium, flow rate: 1,7 ml/min, constant septum purge flow: 5,0 ml/min, split
flow = 100 ml/min.
8.3.5 Backflush initiation time: 6,0 min. The backflush initiation setting shall be redetermined after
each change to the analytical or pre-column.
8.3.6 GC oven temperature
...