Animal and vegetable fats and oils - Determination of fatty-acid-bound chloropropanediols (MCPDs) and glycidol by GC/MS - Part 2: Method using slow alkaline transesterification and measurement for 2-MCPD, 3-MCPD and glycidol (ISO 18363-2:2018)

This part of ISO 18363 describes a procedure for the parallel determination of glycidol together with 2-MCPD and 3-MCPD present in bound or free form in oils and fats. The method is based on alkalinecatalyzed ester cleavage, transformation of the released glycidol into monobromopropanediol (MBPD) and derived free diols (MCPD and MBPD) with phenylboronic acid (PBA). Though free MCPD and glycidol are supposed to be present in fats and oils in low to negligible quantities only, significant content would increase proportionately the determination of bound analytes.
This method is applicable to solid and liquid fats and oils. This part of ISO 18363 can also apply to animal fats and used frying oils and fats, but a validation study must be undertaken before the analysis of these matrices.
Milk and milk products (or fat coming from milk and milk products) are excluded from the scope of this international standard.

Tierische und pflanzliche Fette und Öle - Bestimmung von fettsäuregebundenem Chlorpropandiol (MCPD) und Glycidol mittels GC/MS - Teil 2: Verfahren mittels langsamer alkalischer Umesterung und Messung für 2-MCPD, 3-MCPD und Glycidol (ISO 18363-2:2018)

Corps gras d'origines animale et végétale - Détermination des esters de chloropropanediols (MCPD) et d'acides gras et des esters de glycidol et d'acides gras par CPG/SM - Partie 2: Méthode par transestérification alcaline et mesure pour le 2-MCPD, le 3-MCPD et le glycidol (ISO 18363-2:2018)

Le présent document décrit un mode opératoire permettant la détermination parallèle du glycidol ainsi que du 2-MCPD et du 3-MCPD présents sous forme liée ou libre dans les corps gras. La méthode repose sur un clivage des esters par catalyse alcaline, une transformation du glycidol ainsi libéré en monobromopropanediol (MBPD) et une dérivatisation des diols libres (MCPD et MBPD) en présence d'acide phénylboronique (PBA). Même si les corps gras ne sont censés contenir que des quantités faibles voire négligeables de MCPD libre et de glycidol libre, une teneur importante augmenterait proportionnellement la détermination des analytes liés.
La présente méthode est applicable aux corps gras solides et liquides. Le présent document peut également s'appliquer aux graisses animales et aux huiles de friture usagées, mais une étude de validation est menée avant de procéder à l'analyse de ces matrices.
Le lait et les produits laitiers (ou les corps gras issus du lait et des produits laitiers) sont exclus du domaine d'application du présent document.

Živalske in rastlinske maščobe in olja - Ugotavljanje na maščobno kislino vezanih kloropropanediolov (MCPD) in glicidola z GC/MS - 2. del: Metoda z uporabo počasnega alkalnega preestrenja in meritev 2-MCPD, 3-MCPD in glicidola (ISO 18363-2:2018)

Ta del standarda ISO 18363 opisuje postopek za vzporedno ugotavljanje glicidola z 2-MCPD in 3-MCPD, prisotnega v vezanih ali prostih oljih in maščobah. Ta metoda temelji na alkalno katalizirani cepitvi estra, pretvarjanju sproščenega glicidola v monobromopropandiol (MBPD) in izpeljane proste diole (MCPD in MBPD) s fenilborovo kislino (PBA). Čeprav bi naj bila prosti MCPD in glicidol prisotna v maščobah ter oljih samo v nizkih do zanemarljivih količinah, bi velika vsebnost sorazmerno povečala ugotavljanje vezanih analitov.
Ta metoda se uporablja za trdne in tekoče maščobe ter olja. Ta del standarda ISO 18363 se lahko uporablja tudi za živalske maščobe ter uporabljena olja in maščobe za cvrtje, vendar je treba pred analizo teh matrik izvesti predhodno validacijsko študijo.
Mleko in mlečni izdelki (ali maščoba iz mleka in mlečnih izdelkov) niso zajeti v tem mednarodnem standardu.

General Information

Status
Published
Public Enquiry End Date
04-Feb-2018
Publication Date
12-Nov-2018
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
22-Oct-2018
Due Date
27-Dec-2018
Completion Date
13-Nov-2018

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SLOVENSKI STANDARD
SIST EN ISO 18363-2:2018
01-december-2018

äLYDOVNHLQUDVWOLQVNHPDãþREHLQROMD8JRWDYOMDQMHQDPDãþREQRNLVOLQRYH]DQLK

NORURSURSDQHGLRORY 0&3' LQJOLFLGROD]*&06GHO0HWRGD]XSRUDER
SRþDVQHJDDONDOQHJDSUHHVWUHQMDLQPHULWHY0&3'0&3'LQJOLFLGROD ,62

Animal and vegetable fats and oils - Determination of fatty-acid-bound

chloropropanediols (MCPDs) and glycidol by GC/MS - Part 2: Method using slow alkaline

transesterification and measurement for 2-MCPD, 3-MCPD and glycidol (ISO 18363-
2:2018)
Tierische und pflanzliche Fette und Öle - Bestimmung von fettsäuregebundenem
Chlorpropandiol (MCPD) und Glycidol mittels GC/MS - Teil 2: Verfahren mittels
langsamer alkalischer Umesterung und Messung für 2-MCPD, 3-MCPD und Glycidol
(ISO 18363-2:2018)
Corps gras d'origines animale et végétale - Détermination des esters de

chloropropanediols (MCPD) et d'acides gras et des esters de glycidol et d'acides gras

par CPG/SM - Partie 2: Méthode par transestérification alcaline et mesure pour le 2-

MCPD, le 3-MCPD et le glycidol (ISO 18363-2:2018)
Ta slovenski standard je istoveten z: EN ISO 18363-2:2018
ICS:
67.200.10 5DVWOLQVNHLQåLYDOVNH Animal and vegetable fats
PDãþREHLQROMD and oils
SIST EN ISO 18363-2:2018 en

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

---------------------- Page: 1 ----------------------
SIST EN ISO 18363-2:2018
---------------------- Page: 2 ----------------------
SIST EN ISO 18363-2:2018
EN ISO 18363-2
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2018
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 2: Method using slow alkaline
transesterification and measurement for 2-MCPD, 3-MCPD
and glycidol (ISO 18363-2:2018)

Corps gras d'origines animale et végétale - Tierische und pflanzliche Fette und Öle - Bestimmung

Détermination des esters de chloropropanediols von fettsäuregebundenem Chlorpropandiol (MCPD)

(MCPD) et d'acides gras et des esters de glycidol et und Glycidol mittels GC/MS - Teil 2: Verfahren mittels

d'acides gras par CPG/SM - Partie 2: Méthode par langsamer alkalischer Umesterung und Messung für 2-

transestérification alcaline et mesure pour le 2-MCPD, MCPD, 3-MCPD und Glycidol (ISO 18363-2:2018)

le 3-MCPD et le glycidol (ISO 18363-2:2018)
This European Standard was approved by CEN on 22 September 2018.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, 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

© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 18363-2:2018 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
SIST EN ISO 18363-2:2018
EN ISO 18363-2:2018 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 18363-2:2018
EN ISO 18363-2:2018 (E)
European foreword

This document (EN ISO 18363-2:2018) 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 April 2019, and conflicting national standards shall be

withdrawn at the latest by April 2019.

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.

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, Former Yugoslav Republic of Macedonia,

France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,

Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and the United Kingdom.
Endorsement notice

The text of ISO 18363-2:2018 has been approved by CEN as EN ISO 18363-2:2018 without any

modification.
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SIST EN ISO 18363-2:2018
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SIST EN ISO 18363-2:2018
INTERNATIONAL ISO
STANDARD 18363-2
First edition
2018-08
Animal and vegetable fats and oils —
Determination of fatty-acid-bound
chloropropanediols (MCPDs) and
glycidol by GC/MS —
Part 2:
Method using slow alkaline
transesterification and measurement
for 2-MCPD, 3-MCPD and glycidol
Corps gras d'origines animale et végétale — Détermination des
esters de chloropropanediols (MCPD) et d'acides gras et des esters de
glycidol et d'acides gras par CPG/SM —
Partie 2: Méthode par transestérification alcaline lente et mesure
pour le 2-MCPD, le 3-MCPD et le glycidol
Reference number
ISO 18363-2:2018(E)
ISO 2018
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SIST EN ISO 18363-2:2018
ISO 18363-2:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018

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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved
---------------------- Page: 8 ----------------------
SIST EN ISO 18363-2:2018
ISO 18363-2:2018(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

5 Reagents ........................................................................................................................................................................................................................ 3

5.1 General ........................................................................................................................................................................................................... 3

5.2 Solvents and chemicals .................................................................................................................................................................... 3

5.3 Standard and reference compounds .................................................................................................................................... 3

5.4 Working solutions** ........................................................................................................................................................................... 4

5.5 Other solutions ....................................................................................................................................................................................... 4

6 Apparatus ..................................................................................................................................................................................................................... 4

7 Sample ............................................................................................................................................................................................................................. 5

7.1 Sampling ....................................................................................................................................................................................................... 5

7.2 Preparation of the test sample .................................................................................................................................................. 5

8 Procedure..................................................................................................................................................................................................................... 5

8.1 Spiking with surrogate standard and homogenization ........................................................................................ 5

8.2 Ester cleavage and glycidol transformation ................................................................................................................... 5

8.3 Matrix removal ........................................................................................................................................................................................ 6

8.4 Derivatization ........................................................................................................................................................................................... 6

8.5 Gas chromatography/mass spectrometry references ........................................................................................... 6

9 Expression of results ........................................................................................................................................................................................ 7

9.1 Determination of bound glycidol ............................................................................................................................................. 7

9.2 Determination of bound 2-MCPD ........................................................................................................................................... 8

9.3 Determination of bound 3-MCPD ........................................................................................................................................... 9

9.4 Determination of the degree of diester cleavage ....................................................................................................... 9

9.5 Quality control ......................................................................................................................................................................................10

10 Notes ...............................................................................................................................................................................................................................10

Annex A (informative) Examples of relevant chromatograms and data evaluation using

“low-MCPD” palm oil ......................................................................................................................................................................................12

Annex B (informative) Results of interlaboratory tests.................................................................................................................19

Bibliography .............................................................................................................................................................................................................................21

© ISO 2018 – All rights reserved iii
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SIST EN ISO 18363-2:2018
ISO 18363-2:2018(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.o rg/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.
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 2018 – All rights reserved
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SIST EN ISO 18363-2:2018
ISO 18363-2:2018(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 three documents

currently published or proposed so that the analyst can decide which methods are suitable for application.

The detailed application of each method is contained within the scope of the individual method.

[1] [2]

ISO 18363-1 is a differential method equivalent to the DGF standard C-VI 18 (10) and identical to

[3]

AOCS Official Method Cd 29c-13 . Briefly, it is based on a fast alkaline catalysed release of 3-MCPD

and glycidol from the ester derivatives. Glycidol is subsequently converted into induced 3-MCPD. It

consists of two parts. The first part (A) allows the determination of the sum of ester-bound 3-MCPD and

ester-bound glycidol, whereas the second part (B) determines ester-bound 3-MCPD only. Both assays

are based on the release of the target analytes 3-MCPD and glycidol from the ester-bound form by an

alkaline catalysed alcoholysis carried out at room temperature. In part A, an acidified sodium chloride

solution is used to stop the reaction and subsequently convert the glycidol into induced 3-MCPD. Thus,

3-MCPD and glycidol become indistinguishable in part A. In part B, the reaction stop is achieved by

the addition of an acidified chloride-free salt solution which also prevents the conversion of glycidol

into induced MCPD. Thereby, part B allows the determination of the genuine 3-MCPD content. Finally,

the glycidol content of the sample is proportional to the difference of both assays (A – B) and can be

calculated when the transformation ratio from glycidol to 3-MCPD has been determined. ISO 18363-1

is applicable to the fast determination of ester-bound 3-MCPD and glycidol in refined and non-refined

vegetable oils and fats. ISO 18363-1 can also apply to animal fats and used frying oils and fats, but a

validation study has to be undertaken before the analysis of these matrices. Any free analytes within

the sample would be included in the results, but the document does not allow the distinction between

free and bound analytes. However, as of publication, research has not shown any evidence of a free

analyte content as high as the esterified analyte content in refined vegetable oils and fats. In principle,

[4]

ISO 18363-1 can also be modified in such a way that the determination of 2-MCPD is feasible , but

again, a validation study has to be undertaken before the analysis of this analyte.

[5][6]

This document represents AOCS Official Method Cd 29b-13 . For information on corresponding

validation data, see Annex B. Briefly, it is based on a slow alkaline release of MCPD and glycidol from

the ester derivatives. Glycidol is subsequently converted into 3-MBPD. This document consists of

two sample preparations that differ in the use of internal standards. Both preparations will be used

for the determination of ester-bound 2-MCPD and 3-MCPD. In part A, a preliminary result for ester-

bound glycidol is determined. Because the 3-MCPD present in the sample will be converted to some

minor extent into induced glycidol by the sample preparation, part B serves to quantify this amount

of induced glycidol that is subsequently subtracted from the preliminary glycidol result of part A. By

the use of isotope-labelled free MCPD isomers in assay A and isotope-labelled ester-bound 2-MCPD and

3-MCPD in part B, the efficiency of ester cleavage can be monitored. Both assays A and B are based on

the release of the target analytes 2-MCPD, 3-MCPD, and glycidol from the ester-bound form by a slow

alkaline catalysed alcoholysis in the cold. In both sample preparations, the reaction is stopped by the

addition of an acidified concentrated sodium bromide solution so as to convert the unstable and volatile

glycidol into 3-MBPD which shows comparable properties to 3-MCPD with regard to its stability and

chromatographic performance. Moreover, the major excess of bromide ions prevents the undesired

formation of 3-MCPD from glycidol in the case of samples which contain naturally occurring amounts

of chloride. This document is applicable to the determination of ester-bound 3-MCPD, 2-MCPD, and

glycidol in refined and unrefined vegetable oils and fats. It also applies to animal fats and used frying

oils and fats, but a validation study will have to be undertaken before the analysis of these matrices. Any

free analytes within the sample would be included in the results, but the document does not allow the

distinction between free and bound analytes. However, as of publication of this document, research has

not shown any evidence of a free analyte content as high as the esterified analyte content in vegetable

oils and fats.
[7] [8][9]

ISO 18363-3 represents AOCS Official Method Cd 29a-13 . Briefly, it is based on the conversion

of glycidyl esters into 3-MBPD esters and a slow acidic catalysed release of MCPD and MBPD from the

ester derivatives. 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

© ISO 2018 – All rights reserved v
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SIST EN ISO 18363-2:2018
ISO 18363-2:2018(E)

glycidol. ISO 18363-3 can be applied for the determination of ester-bound 2-MCPD, 3-MCPD, and glycidol

in refined and non-refined vegetable oils and fats. It can also apply to animal fats and used frying

oils and fats, but a validation study has to be undertaken before the analysis of these matrices. The

method is suited for the analysis of bound (esterified) analytes, but if required, ISO 18363-3 can also be

performed without the initial conversion of glycidyl esters. In such a setup, both free and bound 2-MCPD

and 3-MCPD forms would be included in the results and the amount of free analytes can be calculated

as a difference between two determinations performed in both setups. However, as of publication 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 2018 – All rights reserved
---------------------- Page: 12 ----------------------
SIST EN ISO 18363-2:2018
INTERNATIONAL STANDARD ISO 18363-2:2018(E)
Animal and vegetable fats and oils — Determination of
fatty-acid-bound chloropropanediols (MCPDs) and glycidol
by GC/MS —
Part 2:
Method using slow alkaline transesterification and
measurement for 2-MCPD, 3-MCPD and glycidol
1 Scope

This document specifies a procedure for the parallel determination of glycidol together with 2-MCPD

and 3-MCPD present in bound or free form in oils and fats. The method is based on alkaline-catalysed

ester cleavage, transformation of the released glycidol into monobromopropanediol (MBPD) and

derivatisation of the derived free diols (MCPD and MBPD) with phenylboronic acid (PBA). Though free

MCPD and glycidol are supposed to be present in fats and oils in low to negligible quantities only, in the

event that free analytes are present, they would contribute proportionately to the results. The results

always being the sum of the free and the bound form of a single analyte.

This method is applicable to solid and liquid fats and oils. This document can also apply to animal fats

and used frying oils and fats, but a validation study is undertaken before the analysis of these matrices.

Milk and milk products (or fat coming from milk and milk products) are excluded from the scope of this

document.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 3696, Water for analytical laboratory use — Specification and test methods
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
bound 2-MCPD
sum of all 2-MCPD-derivatives that are cleaved by alkaline-catalysed alcoholysis

Note 1 to entry: The content of bound 2-MCPD is reported in milligrams per kilogram (mg/kg).

© ISO 2018 – All rights reserved 1
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SIST EN ISO 18363-2:2018
ISO 18363-2:2018(E)
3.2
bound 3-MCPD
sum of all 3-MCPD-derivatives that are cleaved by alkaline-catalysed alcoholysis

Note 1 to entry: The content of bound 3-MCPD is reported in milligrams per kilogram (mg/kg).

3.3
bound glycidol

sum of all glycidyl derivatives that are cleaved by alkaline-catalysed alcoholysis

Note 1 to entry: The content of bound glycidol is reported in milligrams per kilogram (mg/kg).

4 Principle

For the determination of bound 2-MCPD, bound 3-MCPD and bound glycidol as free 2-MCPD, free

3-MCPD and free 3-MBPD (3-Monobromopropanediol), two aliquots (A and B) of the sample are spiked

with surrogate standards (d -2-MCPD, d -3-MCPD, d -glycidylester in assay A and d -2-MCPD-1,3-

5 5 5 5

diester, d -3-MCPD-1,2-diester in assay B) and dissolved in diethyl ether. Both assays are processed in

parallel. The addition of a diluted solution of sodium hydroxide or sodium methoxide in methanol in the

cold will release free 2-MCPD, free 3-MCPD and free glycidol over a period of 8 h to 12 h. This reaction

is stopped by the addition of an excess amount of sodium bromide in acidic solution. Under acidic

conditions, free glycidol reacts with inorganic bromide to form 3-MBPD and a small amount of 2-MBPD.

Undesired non-polar compounds in the sample are removed by double extraction of the aqueous phase

with isohexane. The analytes, together with the surrogate standards, are transferred into an organic

phase by multiple extraction of the aqueous phase with diethyl ether, ethyl acetate or a mixture of

both solvents. Derivatization takes place in the organic phase by reaction with PBA. In order to remove

excess amounts of PBA, the analytes are concentrated and transferred into an inert organic solvent.

The sample extract is then placed over a small amount of anhydrous sodium sulfate and evaporated to

dryness under a stream of nitrogen before being finally redissolved in iso-octane for the measurement

by GC/MS.

The alkaline catalysed transesterification in the cold minimizes the undesired transformation of 3-MCPD

into glycidol that proceeds to a significant extend when the reaction is carried out at room temperature.

Nevertheless, in case of large amounts of 3-MCPD being present, even a minor transformation into

glycidol might increase the glycidol results from assay A artificially. In order to achieve the correct

glycidol results, even in the presence of high 3-MCPD content, assay B serves for the determination of

the undesired 3-MCPD–glycidol transformation by determining the amount of d -glycidol that has been

generated from d -3-MCPD-diester by the sample preparation. The corresponding transformation ratio

is used for correcting the glycidol value derived from assay A. Another point to be taken into account

is that 3-MCPD is converted approximately 1,2-fold faster via glycidol into 3-MBPD than 3-MCPD-d

via glycidol-d into 3-MBPD-d . Consequently, the isotopic factor I = 1,2 has to be considered for the

5 5

quantitative determination of the amount of glycidol that has been generated accidentally from the

non-labelled 3-MCPD by alkaline treatment in assay A.

Quantification of the analytes is carried out by internal one-point-calibration using the corresponding

d -esters as surrogate standards. Therefore, no external calibration is necessary. Likewise, no analyte

recoveries have to be considered. However, the cleaving rates of MCPD mono- and diesters might be

different and as only d -MCPD-diesters serve as internal standards, the degree of ester cleavage should

have proceeded on a large scale. Therefore, the degree of variations in ester cleavage is monitored

by calculating the differences in 3-MCPD results between assay A and B. For information on deriving

[9]

quantitative data from corresponding chromatograms, see expression of results and Annex A.

As 3-MCPD can occur in certain polymers used for wet strengthening resins and for other purposes,

it might also occur from the use of consumables, e.g. screw lid vials or filter. Baking the glassware at

400 °C to 500 °C can reduce this problem. A better solution is the use of non-contaminated materials.

2 © ISO 2018 – All rights reserved
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SIST EN ISO 18363-2:2018
ISO 18363-2:2018(E)
5 Reagents
5.1 General
WARNING — This document requires handling of hazardous substances. Technical,
organizational and personal safety measures shall be followed.
...

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