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
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
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.
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
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.
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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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ISO 18363-4:2021(E)
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© ISO 2021
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ii © ISO 2021 – All rights reserved
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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
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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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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
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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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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).
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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 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.
13
The quantification of ester bound 2-MCPD 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
[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
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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).
13
5.2.3 Carbon-13 labelled 1,2-dipalmitoyl-3-chloropropanediol (PP-3-MCPD- C ), purity ≥ 95 %.
3
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.
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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.
13
5.3.3.5 Internal standard solution (3-MCPD- C : 1,54 µg/ml, glycidol-d5: 0,92 µg/ml). Pipet
...
NORME ISO
INTERNATIONALE 18363-4
Première édition
2021-08
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
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
Numéro de référence
ISO 18363-4:2021(F)
©
ISO 2021
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ISO 18363-4:2021(F)
DOCUMENT PROTÉGÉ PAR COPYRIGHT
© ISO 2021
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Publié en Suisse
ii © ISO 2021 – Tous droits réservés
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ISO 18363-4:2021(F)
Sommaire Page
Avant-propos .iv
Introduction .v
1 Domaine d’application . 1
2 Références normatives . 1
3 Termes et définitions . 1
4 Principe . 2
5 Réactifs . 2
5.1 Généralités . 2
5.2 Composés étalons et composés de référence . 3
5.3 Solutions étalons . 3
5.3.1 Généralités . 3
5.3.2 Solutions mères . 3
5.3.3 Solutions de travail . 4
5.4 Autres réactifs . 4
5.5 Solutions de réactifs . 5
6 Appareillage . 5
7 Transport et stockage. 6
7.1 Échantillonnage . 6
7.2 Préparation de l’échantillon pour essai . 6
7.3 Conditions de conservation . 6
8 Mode opératoire. 6
8.1 Préparation de l’échantillon pour essai . 6
8.2 Préparation de la droite d’étalonnage . 7
8.3 Paramétrages relatifs à la chromatographie en phase gazeuse et spectrométrie de
masse (CPG/SM) . 8
9 Expression des résultats. 9
9.1 Généralités . 9
9.2 Quantification des esters de 2-MCPD et 3-MCPD . 9
9.3 Quantification des esters de glycidol .11
9.4 Contrôle qualité .13
10 Notes .14
11 Fidélité .15
11.1 Généralités .15
11.2 Répétabilité .15
11.3 Reproductibilité entre jours .15
Annexe A (normative) Tableaux explicatifs .16
Annexe B (informative) Résultats statistiques de l’étude comparative interlaboratoires ISO .18
Annexe C (informative) Chromatogrammes .22
Bibliographie .24
© ISO 2021 – Tous droits réservés iii
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ISO 18363-4:2021(F)
Avant-propos
L’ISO (Organisation internationale de normalisation) est une fédération mondiale d’organismes
nationaux de normalisation (comités membres de l’ISO). L’élaboration des Normes internationales est
en général confiée aux comités techniques de l’ISO. Chaque comité membre intéressé par une étude
a le droit de faire partie du comité technique créé à cet effet. Les organisations internationales,
gouvernementales et non gouvernementales, en liaison avec l’ISO participent également aux travaux.
L’ISO collabore étroitement avec la Commission électrotechnique internationale (IEC) en ce qui
concerne la normalisation électrotechnique.
Les procédures utilisées pour élaborer le présent document et celles destinées à sa mise à jour sont
décrites dans les Directives ISO/IEC, Partie 1. Il convient, en particulier, de prendre note des différents
critères d’approbation requis pour les différents types de documents ISO. Le présent document a été
rédigé conformément aux règles de rédaction données dans les Directives ISO/IEC, Partie 2 (voir www
.iso .org/ directives).
L’attention est attirée sur le fait que certains des éléments du présent document peuvent faire l’objet de
droits de propriété intellectuelle ou de droits analogues. L’ISO ne saurait être tenue pour responsable
de ne pas avoir identifié de tels droits de propriété et averti de leur existence. Les détails concernant
les références aux droits de propriété intellectuelle ou autres droits analogues identifiés lors de
l’élaboration du document sont indiqués dans l’Introduction et/ou dans la liste des déclarations de
brevets reçues par l’ISO (voir www .iso .org/ brevets).
Les appellations commerciales éventuellement mentionnées dans le présent document sont données
pour information, par souci de commodité, à l’intention des utilisateurs et ne sauraient constituer un
engagement.
Pour une explication de la nature volontaire des normes, la signification des termes et expressions
spécifiques de l’ISO liés à l’évaluation de la conformité, ou pour toute information au sujet de l’adhésion
de l’ISO aux principes de l’Organisation mondiale du commerce (OMC) concernant les obstacles
techniques au commerce (OTC), voir www .iso .org/ avant -propos.
Le présent document a été élaboré par le comité technique ISO/TC 34, Produits alimentaires, sous-comité
SC 11, Corps gras d’origines animale et végétale, en collaboration avec le comité technique CEN/TC 307,
Oléagineux, corps gras d’origines végétale et animale et leurs co-produits – Méthodes d’échantillonnage
et d’analyse, du Comité européen de normalisation (CEN), conformément à l’Accord de coopération
technique entre l’ISO et le CEN (Accord de Vienne).
Une liste de toutes les parties de la série ISO 18363 se trouve sur le site web de l’ISO.
Il convient que l’utilisateur adresse tout retour d’information ou toute question concernant le présent
document à l’organisme national de normalisation de son pays. Une liste exhaustive desdits organismes
se trouve à l’adresse www .iso .org/ fr/ members .html.
iv © ISO 2021 – Tous droits réservés
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ISO 18363-4:2021(F)
Introduction
La série ISO 18363 est un ensemble de Normes internationales pouvant être utilisé pour la
détermination des esters de MCPD et de glycidol. Cette introduction décrit les méthodes spécifiées
dans les différentes parties pour que les analystes puissent décider des méthodes qui conviennent à
leurs applications. L’application détaillée de chaque méthode figure dans le domaine d’application de la
méthode concernée.
[9]
L’ISO 18363-1 est une méthode différentielle équivalente à la norme C-VI 18 (10) de la DGF et identique
[6]
à la méthode officielle Cd 29c-13 de l’AOCS. En résumé, elle repose sur la libération rapide par catalyse
alcaline de 3-MCPD et de glycidol à partir de leurs dérivés esters. Le glycidol est par la suite converti
en 3-MCPD. Elle comprend deux parties. La première partie (A) permet de déterminer la somme des
esters de 3-MCPD et des esters de glycidol, tandis que la seconde partie (B) ne détermine que les esters
de 3-MCPD. Les deux analyses reposent sur la libération des analytes cibles, le 3-MCPD et le glycidol,
à partir des esters par alcoolyse en présence d’un catalyseur alcalin à température ambiante. Dans
la partie A, une solution acidifiée de chlorure de sodium est utilisée pour interrompre la réaction et
induire par la suite la conversion du glycidol en 3-MCPD. Il n’est par conséquent plus possible de faire la
distinction entre le 3-MCPD et le glycidol dans la partie A. Dans la partie B, la réaction est interrompue
par ajout d’une solution saline acidifiée exempte de chlorure qui évite aussi la conversion du glycidol en
MCPD. La partie B permet ainsi de déterminer la véritable teneur en 3-MCPD. Enfin, la teneur en glycidol
de l’échantillon est proportionnelle à la différence entre les deux analyses (A – B) et peut être calculée
lorsque le coefficient de transformation du glycidol en 3-MCPD a été déterminé. L’ISO 18363-1 s’applique
à la détermination rapide des esters de 3-MCPD et de glycidol dans les corps gras d’origine végétale
raffinés et non raffinés. L’ISO 18363-1 peut également s’appliquer aux graisses animales et aux corps
gras de friture usagés, mais une étude de validation doit être menée avant de procéder à l’analyse de
ces matrices. Les analytes libres éventuellement contenus dans l’échantillon sont habituellement inclus
dans les résultats, mais le document ne permet pas de faire la distinction entre les analytes libres et
les analytes liés. Néanmoins, les études disponibles au moment de la publication de la présente norme
ne révèlent aucune teneur en analytes libres aussi élevée que la teneur en analytes estérifiés dans les
corps gras d’origine végétale raffinés. En principe, l’ISO 18363-1 peut également être modifiée de façon
à permettre la détermination du 2-MCPD, mais une fois encore, une étude de validation doit être menée
avant d’analyser cet analyte.
[5]
L’ISO 18363-2 correspond à la méthode officielle Cd 29b-13 de l’AOCS. En résumé, elle repose sur une
libération alcaline lente de MCPD et de glycidol à partir des formes esters. Le glycidol est par la suite
converti en 3-MBPD. L’ISO 18363-2 comprend deux préparations d’échantillons qui se distinguent par
l’utilisation d’étalons internes. Les deux préparations sont utilisées pour la détermination des esters de
2-MCPD et de 3-MCPD. Un résultat préliminaire pour les esters de glycidol est déterminé dans la partie A.
Comme le 3-MCPD présent dans l’échantillon est partiellement converti en glycidol lors de la préparation
de l’échantillon, la partie B sert à quantifier la teneur en glycidol issue de cette transformation qui
est ensuite soustraite du résultat préliminaire obtenu pour le glycidol dans la partie A. L’utilisation
d’isomères isotopiques de MCPD libres dans l’analyse A et de formes isotopiques d’esters de 2-MCPD
et de 3-MCPD dans la partie B permet de surveiller le rendement du clivage des esters. Les analyses A
et B reposent toutes les deux sur la libération des analytes cibles, 2-MCPD, 3-MCPD et glycidol à partir
des esters dans le cadre d’une alcoolyse lente en présence d’un catalyseur alcalin dans le froid. Dans
les deux préparations d’échantillons, la réaction est interrompue par l’ajout d’une solution acidifiée
et concentrée de bromure de sodium de façon à transformer le glycidol instable et volatil en 3-MBPD
qui présente des propriétés comparables au 3-MCPD en matière de stabilité et de performances
chromatographiques. De plus, l’excès important d’ions bromure empêche la formation non souhaitée de
3-MCPD à partir du glycidol dans les échantillons contenant naturellement des quantités de chlorure.
L’ISO 18363-2 est applicable à la détermination des esters de 3-MCPD, de 2-MCPD et de glycidol dans les
corps gras d’origine végétale raffinés et non raffinés. Elle s’applique également aux graisses animales
et aux corps gras de friture usagés, mais une étude de validation doit être menée avant de procéder
à l’analyse de ces matrices. Les analytes libres éventuellement contenus dans l’échantillon sont inclus
dans les résultats, mais le document ne permet pas de faire la distinction entre les analytes libres et
les analytes liés. Néanmoins, les études disponibles au moment de la publication du présent document
ne révèlent aucune teneur en analytes libres aussi élevée que la teneur en analytes estérifiés dans les
corps gras d’origine végétale.
© ISO 2021 – Tous droits réservés v
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ISO 18363-4:2021(F)
[4]
L’ISO 18363-3 correspond à la méthode officielle Cd 29a-13 de l’AOCS. En résumé, elle repose sur la
transformation des esters de glycidol en esters de 3-MBPD et une libération lente par catalyse acide du
MCPD et du MBPD à partir des formes esters. L’ISO 18363-3 repose sur la préparation d’un échantillon
unique dans lequel les esters de glycidol sont transformés en monoesters de MBPD puis les analytes
libres 2-MCPD, 3-MCPD et 3-MBPD sont libérés par alcoolyse lente en présence d’un catalyseur acide.
Le 3-MBPD représente la véritable teneur en glycidol issu des formes esters. L’ISO 18363-3 s’applique à
la détermination des esters de 2-MCPD, de 3-MCPD et de glycidol dans les corps gras d’origine végétale
raffinés et non raffinés. Elle s’applique également aux graisses animales et aux corps gras de friture
usagés, mais une étude de validation doit être menée avant de procéder à l’analyse de ces matrices.
La méthode est adaptée à l’analyse d’analytes liés (estérifiés), mais si cela est exigé, l’ISO 18363-3
peut également être mise en œuvre sans la transformation initiale des esters de glycidol. Dans
cette configuration, les formes libres et liées du 2-MCPD et du 3-MCPD sont toutes deux incluses
dans les résultats et la teneur en analytes libres peut être calculée comme la différence entre deux
déterminations réalisées dans les deux configurations. Néanmoins, les études disponibles au moment
de la publication ne révèlent aucune teneur en analytes libres aussi élevée que la teneur en analytes
estérifiés dans les corps gras d’origine végétale.
Le présent document spécifie un mode opératoire rapide reposant sur un clivage alcalin rapide des
esters de MCPD et de glycidol. Le glycidol libéré est par la suite converti en 3-MBPD. Le pH du clivage
alcalin rapide entraîne généralement la transformation partielle des MCPD libérés en glycidol au cours
du clivage des esters, conduisant à une surestimation de la teneur en esters de glycidol de l’échantillon.
En ajoutant deux étalons internes distincts d’ester de 3-MCPD et d’ester de glycidol marqués aux
isotopes, il est possible de quantifier la quantité de glycidol marqué résultant de la dégradation de
l’étalon interne libéré. Cette information peut être utilisée pour corriger la surestimation du glycidol
issu des esters de glycidol par le glycidol issu du 3-MCPD. Les deux mêmes étalons internes sont
utilisés pour la quantification des esters de MCPD et des esters de glycidol, nécessitant la préparation
d’un seul échantillon pour quantifier les esters de 2-MCPD, 3-MCPD et de glycidol. De même que dans
l’ISO 18363-1, l’ISO 18363-2 et l’ISO 18363-3, les MCPD et 3-MBPD libérés sont dérivés avec de l’acide
phénylboronique avant l’analyse par CPG-SM/SM. Au contraire des autres parties de la série ISO 18363,
le présent document exige un appareil de type CPG-SM/SM pour détecter sans ambiguïté chaque MBPD
(marqué aux isotopes) requis pour une quantification exacte du glycidol issu des esters de glycidol. Le
présent document est applicable à la détermination des esters de 3-MCPD, de 2-MCPD et de glycidol
dans les corps gras d’origine végétale raffinés et non raffinés. Elle s’applique également aux graisses
animales et aux corps gras de friture usagés, mais une étude de validation doit être menée avant de
procéder à l’analyse de ces matrices. Les analytes libres éventuellement contenus dans l’échantillon sont
inclus dans les résultats, mais le document ne permettra pas de faire la distinction entre les analytes
libres et les analytes liés. Néanmoins, les études disponibles au moment de la publication du présent
document ne révèlent aucune teneur en analytes libres aussi élevée que la teneur en analytes estérifiés
dans les corps gras d’origine végétale.
vi © ISO 2021 – Tous droits réservés
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NORME INTERNATIONALE ISO 18363-4:2021(F)
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
1 Domaine d’application
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.
2 Références normatives
Les documents suivants sont cités dans le texte de sorte qu’ils constituent, pour tout ou partie de leur
contenu, des exigences du présent document. Pour les références datées, seule l’édition citée s’applique.
Pour les références non datées, la dernière édition du document de référence s’applique (y compris les
éventuels amendements).
ISO 3696, Eau pour laboratoire à usage analytique — Spécification et méthodes d’essai
3 Termes et définitions
Pour les besoins du présent document, les termes et définitions suivants s’appliquent.
L’ISO et l’IEC tiennent à jour des bases de données terminologiques destinées à être utilisées en
normalisation, consultables aux adresses suivantes:
— ISO Online browsing platform: disponible à l’adresse https:// www .iso .org/ obp
— IEC Electropedia: disponible à l’adresse http:// www .electropedia .org/
© ISO 2021 – Tous droits réservés 1
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ISO 18363-4:2021(F)
3.1
2-MCPD lié
quantité de 2-MCPD obtenu par clivage de ses formes estérifiées (liées) par transestérification en
présence d’un catalyseur alcalin conformément à la méthode de référence
Note 1 à l'article: La teneur en 2-MCPD est calculée et consignée sous forme de fraction massique, exprimée en
milligrammes par kilogramme (mg/kg).
3.2
3-MCPD lié
quantité de 3-MCPD obtenu par clivage de ses formes estérifiées (liées) par transestérification en
présence d’un catalyseur alcalin conformément à la méthode de référence
Note 1 à l'article: La teneur en 3-MCPD est calculée et consignée sous forme de fraction massique, exprimée en
milligrammes par kilogramme (mg/kg).
3.3
glycidol lié
quantité de glycidol obtenu par clivage de ses formes estérifiées (liées) par transestérification en
présence d’un catalyseur alcalin conformément à la méthode de référence
Note 1 à l'article: La teneur en glycidol est calculée et consignée sous forme de fraction massique, exprimée en
milligrammes par kilogramme (mg/kg).
4 Principe
L’échantillon de corps gras est dissous dans du toluène et du tert-butyl-méthyl-éther, et les étalons
13
internes (diester 3-MCPD- C et ester de glycidol pentadeutéré) sont ajoutés. L’échantillon est ensuite
3
refroidi à 10 °C avant de commencer la transestérification alcaline par l’ajout d’une solution de
méthylate de sodium dans du méthanol. Après 12 min d’incubation à 10 °C, le mélange échantillon est
acidifié avec une solution acide de bromure de sodium pour transformer le glycidol libéré en 3-MBPD.
Les esters méthyliques d’acides gras produits au cours de la transestérification sont éliminés par une
double extraction de la phase organique. Pour finir, l’échantillon purifié (contenant les analytes clivés
[libres]) est dérivé avec de l’acide phénylboronique avant l’analyse par CPG-SM/SM.
La quantification des esters de 2-MCPD et de 3-MCPD repose sur le rapport des signaux 2-MCPD/3-
13 13
MCPD- C et 3-MCPD/3-MCPD- C , respectivement. La quantification des esters de glycidol repose sur
3 3
13
le rapport des signaux 3-MBPD/3-MBPD-d5. La quantité de 3-MBPD- C formée après la réaction de
3
13
transestérification correspond à la quantité de 3-MCPD- C libérée qui s’est transformée en glycidol du
3
fait des conditions de la transestérification alcaline. Comme aucune différence de vitesse de dégradation
13
n’a été observée entre le 3-MCPD et le 3-MCPD- C , cette quantité est ensuite utilisée pour corriger la
3
surestimation du glycidol issu des esters de glycidol, causée par cette dégradation du 3-MCPD. Dans
les conditions appliquées, le 2-MCPD est considéré comme stable et ne contribue donc pas de manière
[7][8]
significative à une éventuelle surestimation du glycidol .
Cette méthode permet la quantification simultanée des trois analytes en une seule analyse.
5 Réactifs
5.1 Généralités
AVERTISSEMENT — L’attention du lecteur est attirée sur les règlements qui régissent la
manipulation des substances dangereuses. Les mesures de sécurité sur les plans technique,
organisationnel et du personnel doivent être suivies.
Sauf indication contraire, des réactifs analytiquement purs doivent être utilisés. L’eau doit être de
qualité 3 conformément à l’ISO 3696.
2 © ISO 2021 – Tous droits réservés
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ISO 18363-4:2021(F)
5.2 Composés étalons et composés de référence
5.2.1 1,2-Dipalmitoyl-3-chloropropanediol (PP-3-MCPD), pureté ≥ 95 %.
NOTE Le 1,2-dipalmitoyl-3-chloropropanediol peut être remplacé par le 1,2-dioléyl-3-chloropropanediol ou
d’autres diesters d’acide gras du 3-MCPD de longueur de chaîne similaire (les C16 à C18 sont préférés, car ils sont
les plus abondants dans la plupart des corps gras).
5.2.2 1,3-Distéaroyl-2-chloropropanediol (SS-2-MCPD), pureté ≥ 95 %.
NOTE Par analogie avec les recommandations fournies pour le PP-3-MCPD, le 1,3-distéaroyl-2-
chloropropanediol peut être remplacé par d’autres diesters d’acide gras du 2-MCPD de longueur de chaîne
similaire (les C16 à C18 sont préférés, car ils sont les plus abondants dans la plupart des corps gras).
13
5.2.3 1,2-Dipalmitoyl-3-chloropropanediol marqué au carbone 13 (PP-3-MCPD- C ),
3
pureté ≥ 95 %.
NOTE La même considération que celle appliquée au 1,2-dipalmitoyl-3-chloropropanediol est également
valable pour son analogue marqué au carbone 13; voir Note en 5.2.1.
5.2.4 Stéarate de glycidyle (Gly-S), pureté ≥ 98 %.
NOTE Le stéarate de glycidyle peut être remplacé par l’oléate de glycidyle ou d’autres esters d’acide gras
du glycidol de longueur de chaîne similaire (les C16 à C18 sont préférés, car ils sont les plus abondants dans la
plupart des corps gras).
5.2.5 Stéarate de glycidyle pentadeutéré (Gly-S-d5), pureté ≥ 98 %.
NOTE La même considération que celle appliquée au palmitate de glycidyle est également valable pour son
analogue pentadeutéré; voir Note en 5.2.4.
5.3 Solutions étalons
5.3.1 Généralités
Toutes les solutions étalons sont préparées avec du toluène (5.4.4). Tous les étalons sont préparés
en utilisant des composés d’ester de référence (5.2). Les concentrations sont indiquées dans la
concentration équivalente en composants libres et elles doivent être corrigées en fonction de la pureté
des composés de référence (5.2). Pour un exemple de calcul de conversion de la concentration des esters
en concentration équivalente libre, voir 10.2.
5.3.2 Solutions mères
NOTE Les solutions mères sont stables pendant au moins 12 mois en ét
...
INTERNATIONAL ISO
STANDARD 18363-4
First edition
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
PROOF/ÉPREUVE
Reference number
ISO 18363-4:2021(E)
©
ISO 2021
---------------------- Page: 1 ----------------------
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
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Published in Switzerland
ii PROOF/ÉPREUVE © ISO 2021 – All rights reserved
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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 or mass spectrometry references . 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 PROOF/ÉPREUVE iii
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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.
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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.
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. 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.
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.
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
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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.
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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).
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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 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 (see ISO 3696).
13
The quantification of ester bound 2-MCPD 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 considered stable and thus will not significantly contribute to possible glycidol overestimation (see
ISO 5555).
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 %.
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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).
13
5.2.3 Carbon-13 labelled 1,2-dipalmitoyl-3-chloropropanediol (PP-3-MCPD- C ), purity ≥ 95 %.
3
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 exemplary 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.
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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 the stock solution (5.3.2.1) 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 s
...
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