Coffee and coffee products - Determination of acrylamide - Methods using HPLC-MS/MS and GC-MS after derivatization (ISO 18862:2016)

ISO 18862:2016 specifies methods for the determination of acrylamide in coffee and coffee products by extraction with water, clean-up by solid-phase extraction and determination by HPLC-MS/MS and GC-MS. It was validated in a method validation study on roasted coffee, soluble coffee, coffee substitutes and coffee products with ranges from 53 μg/kg to 612,1 μg/kg.

Kaffee und Kaffee-Erzeugnisse - Bestimmung von Acrylamid - Verfahren mittels HPLC-MS/MS und mittels GC-MS nach Derivatisierung (ISO 18862:2016)

Dieses Dokument legt Verfahren zur Bestimmung des Acrylamidgehalts in Kaffee und Kaffee Erzeugnissen durch Wasserextraktion, Aufreinigung durch Festphasen Extraktion und Bestimmung mittels HPLC MS/MS und GC MS fest. Es wurde in einer Verfahrensvalidierungsstudie an Röstkaffee, löslichem Kaffee, Kaffeesurrogaten und Kaffee Erzeugnissen mit Bereichen von 53 μg/kg bis 612,1 μg/kg validiert.

Café et derivés du café - Détermination de la teneur en acrylamide - Méthodes par CLHP-SM/SM et CG-SM après dérivation (ISO 18862:2016)

Le présent document spécifie des méthodes de dosage de l'acrylamide dans le café et les dérivés du café par extraction à l'eau, purification par extraction en phase solide et dosage par CLHP-SM/SM et CG-SM. Il a été validé au cours d'une étude de validation de la méthode réalisée sur du café torréfié, du café soluble, des substituts de café et des dérivés du café dans des plages de concentration allant de 53 μg/kg à 612,1 μg/kg.

Kava in proizvodi iz kave - Določevanje akrilamida - Metode z uporabo HPLC-MS/MS in GC-MS po derivatizaciji (ISO 18862:2016)

ISO 18862:2016 določa metode za določevanje akrilamida v kavi in kavnih izdelkih z ekstrakcijo z vodo, čiščenje s trdnofazno ekstrakcijo ter določevanje s HPLC-MS/MS in GC-MS. Potrjena je bila v študiji potrjevanja metode na praženi kavi, topni kavi, kavnih nadomestkih in kavnih izdelkih z obsegom od 53 μg/kg do 612,1 µg/kg.

General Information

Status
Published
Public Enquiry End Date
29-Sep-2018
Publication Date
10-Dec-2019
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
21-Nov-2019
Due Date
26-Jan-2020
Completion Date
11-Dec-2019

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SLOVENSKI STANDARD
SIST EN ISO 18862:2020
01-januar-2020
Kava in proizvodi iz kave - Določevanje akrilamida - Metode z uporabo HPLC-
MS/MS in GC-MS po derivatizaciji (ISO 18862:2016)
Coffee and coffee products - Determination of acrylamide - Methods using HPLC-MS/MS
and GC-MS after derivatization (ISO 18862:2016)
Kaffee und Kaffee-Erzeugnisse - Bestimmung von Acrylamid - Verfahren mittels HPLC-
MS/MS und mittels GC-MS nach Derivatisierung (ISO 18862:2016)
Café et derivés du café - Détermination de la teneur en acrylamide - Méthodes par
CLHP-SM/SM et CG-SM après dérivation (ISO 18862:2016)
Ta slovenski standard je istoveten z: EN ISO 18862:2019
ICS:
67.140.20 Kava in kavni nadomestki Coffee and coffee substitutes
SIST EN ISO 18862:2020 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 18862:2020

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SIST EN ISO 18862:2020


EN ISO 18862
EUROPEAN STANDARD

NORME EUROPÉENNE

October 2019
EUROPÄISCHE NORM
ICS 67.140.20
English Version

Coffee and coffee products - Determination of acrylamide -
Methods using HPLC-MS/MS and GC-MS after
derivatization (ISO 18862:2016)
Café et derivés du café - Dosage de l'acrylamide - Kaffee und Kaffee-Erzeugnisse - Bestimmung von
Méthodes par CLHP-SM/SM et CG-SM après dérivation Acrylamid - Verfahren mittels HPLC-MS/MS und
(ISO 18862:2016) mittels GC-MS nach Derivatisierung (ISO 18862:2016)
This European Standard was approved by CEN on 5 November 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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 18862:2019 E
worldwide for CEN national Members.

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SIST EN ISO 18862:2020
EN ISO 18862:2019 (E)
Contents Page
European foreword . 3

2

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SIST EN ISO 18862:2020
EN ISO 18862:2019 (E)
European foreword
The text of ISO 18862:2016 has been prepared by Technical Committee ISO/TC 34 "Food products” of
the International Organization for Standardization (ISO) and has been taken over as EN ISO 18862:2019
by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods” the secretariat of which is
held by DIN.
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 2020, and conflicting national standards shall be
withdrawn at the latest by April 2020.
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, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 18862:2016 has been approved by CEN as EN ISO 18862:2019 without any modification.


3

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SIST EN ISO 18862:2020

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SIST EN ISO 18862:2020
INTERNATIONAL ISO
STANDARD 18862
First edition
2016-07-15
Coffee and coffee products —
Determination of acrylamide —
Methods using HPLC-MS/MS and GC-
MS after derivatization
Café et de ses dérivés — Dosage de l’acrylamide — Méthodes utilisant
CLHP-MS/MS et CG-MS après dérivation
Reference number
ISO 18862:2016(E)
©
ISO 2016

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SIST EN ISO 18862:2020
ISO 18862:2016(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, 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
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved

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SIST EN ISO 18862:2020
ISO 18862:2016(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 1
6 Apparatus . 3
7 Sampling . 4
8 Procedure. 4
8.1 General . 4
8.2 Preparation of the sample extract . 4
8.3 Clean-up of the extracts . 5
8.3.1 Carrez precipitation . 5
8.3.2 Solid phase extraction . 5
8.4 HPLC-MS/MS measurement . 5
8.4.1 High-performance liquid chromatography (HPLC) . 5
8.4.2 Identification and quantification by mass spectrometry (HPLC-MS/MS) . 6
8.5 Measurement with GC-MS . 6
8.5.1 Derivatization and sample preparation for gas chromatography . 6
8.5.2 Gas chromatography . . 7
8.5.3 Identification and quantification by mass spectrometry . 7
9 Calibration . 7
9.1 General advice . 7
9.2 Determination of linearity and definition of the working range . 7
9.3 Calibration with internal standard solution . 7
9.4 Determination of the laboratory specific recovery. 8
10 Evaluation . 8
10.1 Criteria for identification . 8
10.2 Calculation and final results . 8
11 Precision data . 9
11.1 General . 9
11.2 Repeatability . 9
11.3 Reproducibility . 9
11.4 Recovery . 9
12 Measurement uncertainty . 9
13 Test report .10
Annex A (informative) Performance characteristics .11
Annex B (informative) Examples of absorber materials .12
Annex C (informative) Examples of columns and analysis conditions .13
Bibliography .19
© ISO 2016 – All rights reserved iii

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SIST EN ISO 18862:2020
ISO 18862:2016(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 on 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 the following URL: www.iso.org/iso/foreword.html.
The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 15, Coffee.
iv © ISO 2016 – All rights reserved

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SIST EN ISO 18862:2020
ISO 18862:2016(E)

Introduction
When applying this document, all existing safety regulations have to be followed.
© ISO 2016 – All rights reserved v

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SIST EN ISO 18862:2020

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SIST EN ISO 18862:2020
INTERNATIONAL STANDARD ISO 18862:2016(E)
Coffee and coffee products — Determination of acrylamide
— Methods using HPLC-MS/MS and GC-MS after
derivatization
WARNING — The use of this document can involve hazardous materials, operations and
equipment. This document does not purport to address all the safety problems associated with
its use. It is the responsibility of the user of this document to take appropriate measures for
ensuring the safety and health of the personnel prior to application of this document and to
fulfil statutory requirements for this purpose.
1 Scope
This document specifies methods for the determination of acrylamide in coffee and coffee products by
extraction with water, clean-up by solid-phase extraction and determination by HPLC-MS/MS and GC-
MS. It was validated in a method validation study on roasted coffee, soluble coffee, coffee substitutes
and coffee products with ranges from 53 μg/kg to 612,1 μg/kg.
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
No terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
4 Principle
The coffee sample is extracted with water or, in the case of soluble products, dissolved in water. A clean-
up by solid phase extraction is employed to remove interfering matrix compounds. Two alternative
methods can be used for the determination: high-performance liquid chromatography with mass
spectrometric detection (HPLC-MS/MS) or, after a bromination of the acrylamide, gas chromatography
with mass spectrometric detection (GC-MS). In both cases, isotopic labelled internal standard solutions
are used.
5 Reagents
WARNING — In view of health risks when working with acrylamide, appropriate preventive
and protection measures shall be taken, such as using a fume cupboard, aspirating acrylamide-
containing solutions only with a pipette, and avoiding skin and eye contact or inhalation of
acrylamide-containing vapour.
© ISO 2016 – All rights reserved 1

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SIST EN ISO 18862:2020
ISO 18862:2016(E)

If available, reagents of “residue analysis grade” or “analytical reagent grade” shall be used. The level
of impurities in the reagents that contribute to the blank should be negligibly small. The blank shall be
checked regularly.
5.1 Water, of grade 1 according to ISO 3696, MS-grade is recommended.
5.2 Operating gases of high purity, suitable for GC and mass spectrometry according to the
instructions of the manufacturer of the apparatus.
5.3 Solvents, such as methanol, ethyl acetate, acetonitrile, n-hexane, MS-grade is recommended.
5.4 Acrylamide, C H NO, purity >98 %, reference substance.
3 5
5.4.1 Acrylamide stock solution, mass concentration ρ = 1 000 μg/ml.
Weigh (0,10 ± 0,001) g of acrylamide into a 100 ml one-mark volumetric flask and swirl it in 30 ml
of water in order to dissolve the acrylamide. Fill up to the mark with water and mix well. The stock
solution is stable for at least 3 months when stored protected from light at a maximum of 6 °C.
Alternatively, a commercially available solution with a mass concentration of ρ = 1 000 µg/ml may be
used. The information of the manufacturer regarding the stability of the solution shall be observed.
5.4.2 Acrylamide calibration solution, ρ = 10 μg/ml.
Using a pipette, transfer (1,0 ± 0,001) ml of the acrylamide stock solution (5.4.1) into a 100 ml one-mark
volumetric flask and fill up to the mark with water. This solution shall be stored protected from light at
a maximum of 6 °C and shall be freshly prepared every working day. Depending on the working range,
more dilution steps might be necessary.
5.5 D3-acrylamide (acrylamide-2,3,3-d3) internal standard solution, C H D NO, purity >98 %,
3 2 3
reference substance.
5.5.1 D3-acrylamide stock solution (internal standard solution).
Weigh (0,10 ± 0,001) g of D3-acrylamide into a 100 ml one-mark volumetric flask and swirl it in 30 ml
of water in order to dissolve the D3-acrylamide. Fill up to the mark with water and mix well. The stock
solution is stable for at least 3 months when stored protected from light at a maximum of 6 °C.
Alternatively, a commercially available solution with a mass concentration of ρ = 1 000 µg/ml may be
used. The information of the manufacturer regarding the stability of the solution shall be observed.
5.5.2 D3-acrylamide internal standard solution.
Using a pipette, transfer (1,0 ± 0,001) ml of the D3-acrylamide stock solution (5.5.1) into a 100 ml one-
mark volumetric flask and fill up to the mark with water. This solution shall be stored protected from
light at a maximum of 6 °C and shall be freshly prepared every working day. Depending on the working
range, more dilution steps might be necessary.
NOTE 1 For HPLC-MS/MS, the solutions according to 5.4.1 to 5.5.2 can be prepared using the HPLC eluent as a
solvent. The stability of these solutions depends on the mobile phase used and has to be validated.
When using GC-MS, all standard solutions according to 5.4.2 and 5.5.2 shall be subjected to the
derivatization step according to 8.5.1.
13
NOTE 2 Instead of D3-acrylamide, it is also possible to use C acrylamide for the preparation of the internal
3
standard solution. However, in the following clauses, the procedure and calculation are described for D3-
acrylamide only.
2 © ISO 2016 – All rights reserved

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SIST EN ISO 18862:2020
ISO 18862:2016(E)

5.6 Saturated bromine water.
Saturate distilled water with bromine in a 100 ml one-mark volumetric flask (with a glass stopper)
until a phase of bromine is formed at the bottom of the flask (around 3,5 % of bromine at 4 °C). Acidify
the bromine water to a pH of about 1 using concentrated hydrobromic acid, (HBr, with a specific gravity
3
of 1,48 g/cm ).
If stored at 4 °C and protected from light, the solution can be used for about 4 weeks.
5.7 Potassium bromide, KBr.
5.8 Sodium thiosulfate (pentahydrate), Na S O · 5 H O.
2 2 3 2
5.9 Triethylamine, (C H ) N.
2 5 3
5.10 Sodium sulfate (anhydrous, granular), Na SO .
2 4
5.11 Carrez solution I.
Dissolve 10,6 g of potassium hexacyanoferrate trihydrate (II) K [Fe(CN) ] · 3 H O in 100 ml of water. If
4 6 2
stored at 4 °C and protected from light, the solution is stable for 6 months.
5.12 Carrez solution II.
Dissolve 21,9 g of zinc acetate dihydrate Zn(CH COO) · 2 H O in 100 ml of water. If stored at 4 °C and
3 2 2
protected from light, the solution is stable for 6 months.
5.13 Borate buffer, pH 8,6.
Mix 68 ml of a 0,1 molar sodium borate solution (20,12 g Na B O per litre of water) and 32 ml of
2 4 7
0,1 molar hydrochloric acid, c(HCl) = 0,1 mol/l, in a 100 ml one-mark volumetric flask.
6 Apparatus
Usual laboratory apparatus and, in particular, apparatus according to 6.1 to 6.14 are required.
Apparatus and parts of the apparatus which come into contact with the sample and extract shall be free
of residues which can cause blank values. Preferably glassware or equipment made of stainless steel or
PTFE (polytetrafluoroethylene) shall be used.
6.1 Analytical balance, capable of weighing to an accuracy of 0,1 mg.
6.2 Coffee mill, suitable for grinding roasted coffee beans.
6.3 Glassware, for collecting and storing the extracts, preferably made of amber glass, as sample vials
for manual or automatic use, equipped with an inert seal (e.g. vials with PTFE coated septum).
6.4 Ultrasonic bath, capable of being maintained at 40 °C.
6.5 Laboratory centrifuge, suitable for 15 ml and 50 ml centrifugal tubes and with a minimum g-force
of 2 000 g.
6.6 Centrifuge tubes, of 15 ml and 50 ml.
6.7 One-mark volumetric flask, of 20 ml and 100 ml.
© ISO 2016 – All rights reserved 3

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SIST EN ISO 18862:2020
ISO 18862:2016(E)

6.8 Pipettes, glass or automatic, suitable for measuring volume ranges of standard solutions and
sample extract dilutions.
6.9 Glass or polypropylene cartridges, with sorbents for the solid phase extraction (SPE), and for
the clean-up of extracts in 8.3.2 and 8.5.1 (examples are given in Table B.1).
6.10 High performance liquid chromatograph (for the test procedure according to 8.4), equipped
with ESI and mass spectrometric detector (HPLC-MS/MS); gas supply as specified by the manufacturer.
6.11 HPLC column (for the test procedure according to 8.4), suitable for acrylamide chromatography
(examples are given in Table C.1).
6.12 Gas chromatograph (for the test procedure according to 8.5) with mass spectrometric detector
(GC-MS) and operating gas supply (5.2) as specified by the manufacturer.
6.13 GC column, (for the test procedure according to 8.5) capillary column, suitable for acrylamide
chromatography (examples are given in Table C.2).
6.14 Membrane filter units, syringe filter (e.g. cellulose acetate filters 0,45 µm pore size)
suitable for filtration of sample eluate obtained by solid phase extraction before injection into the
chromatographic system.
7 Sampling
Sampling is not part of the method specified in this document. The sampling procedure shall be subject
to agreement by the interested parties. A representative, thoroughly mixed sample shall be used, which
has not been damaged or adulterated during transport or storage.
In order to exclude changes in the acrylamide levels, the analysis shall be performed shortly after
reception of the sample. The samples shall be stored under cool conditions below 6 °C at a maximum of
6 months, under the exclusion of light and they shall be exposed to room temperature only for analysis.
The date of receipt of the sample, as well as the date of roasting or the best-before date, shall be
documented along with the date of analysis.
8 Procedure
8.1 General
To avoid losses of the analyte, it is necessary that the samples are protected from light during extraction
and further preparation. For this reason, amber glassware shall always be used. Otherwise, the content
of the vessels and flasks shall be protected from incident light using aluminium foil.
8.2 Preparation of the sample extract
If necessary, grind the sample in a coffee mill (6.2) and homogenize thoroughly.
Weigh 2 g of the homogenized sample of roasted coffee, soluble coffee or coffee substitute or 5 g of
liquid coffee beverage to the nearest 1 mg using an analytical balance (6.1) and transfer it into a 50 ml
centrifuge tube (6.6).
4 © ISO 2016 – All rights reserved

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SIST EN ISO 18862:2020
ISO 18862:2016(E)

Add 2 ml of n-hexane to the test sample and shake briefly. Then spike the test sample with D3-acrylamide
as the internal standard solution in a concentration corresponding to the expected acrylamide level of
the sample.
EXAMPLE Weigh 2 g of coffee and add 100 µl internal standard solution (ρ = 10 µg/ml), which is equivalent
to an acrylamide mass fraction of 500 µg/kg in the coffee sample.
Add 20 ml of distilled water, shake briefly but vigorously, and sonicate (6.4) for 15 min at
approximately 40 °C.
Allow a few minutes for precipitation and in the case of non-sedimenting samples centrifuge (6.5) for
15 min at 2 000 g to separate suspended solids. Before liquid chromatography (8.4) or derivatization
and gas chromatographic separation (8.5), take 10 ml from the lower aqueous phase and use it for a
further clean-up according to 8.3. Take the lower aqueous phase through the upper hexane phase using
a pipette without removing the hexane phase. If necessary, the hexane phase may also be removed
cautiously using a Pasteur pipette.
8.3 Clean-up of the extracts
8.3.1 Carrez precipitation
Clean-up the sample extract prepared according to 8.2 by Carrez precipitation. Add 1 000 µl of Carrez
solution I (5.11) and shake. Add 1 000 µl of Carrez solution II (5.12) and shake again. After a short
exposure time, centrifuge for 4 min at 2 000 g. Decant the supernatant, wash the residue with 2 ml
to 3 ml of water, centrifuge and decant again. Combine both aqueous solutions.
8.3.2 Solid phase extraction
Clean-up the sample extract after Carrez precipitation (8.3.1) by solid phase extraction (SPE) using
two sequential cartridges with adsorber material (examples are given in Table B.1). The first cartridge
contains 500 mg of C18 material, the second cartridge 500 mg of ion exchanger. The cartridges can be
used in a serial alignment. If appropriate, a combined cartridge can be used.
Condition both SPE columns according to the manufacturer’s instructions successively with methanol
and distilled water. Place the complete sample extract (8.3.1) on top of the upper (first) SPE column,
allow to soak and add 2 ml to 3 ml of wate
...

SLOVENSKI STANDARD
oSIST prEN ISO 18862:2018
01-september-2018
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0606LQ*&06SRGHULYDWL]DFLML ,62
Coffee and coffee products - Determination of acrylamide - Methods using HPLC-MS/MS
and GC-MS after derivatization (ISO 18862:2016)
Kaffee und Kaffee-Erzeugnisse - Bestimmung von Acrylamid - Verfahren mittels HPLC-
MS/MS und mittels GC-MS nach Derivatisierung (ISO 18862:2016)
Café et derivés du café - Détermination de la teneur en acrylamide - Méthodes par CLHP
-SM/SM et CG-SM après dérivation (ISO 18862:2016)
Ta slovenski standard je istoveten z: prEN ISO 18862
ICS:
67.140.20 Kava in kavni nadomestki Coffee and coffee substitutes
oSIST prEN ISO 18862:2018 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
oSIST prEN ISO 18862:2018

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oSIST prEN ISO 18862:2018
INTERNATIONAL ISO
STANDARD 18862
First edition
2016-07-15
Coffee and coffee products —
Determination of acrylamide —
Methods using HPLC-MS/MS and GC-
MS after derivatization
Café et de ses dérivés — Dosage de l’acrylamide — Méthodes utilisant
CLHP-MS/MS et CG-MS après dérivation
Reference number
ISO 18862:2016(E)
©
ISO 2016

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oSIST prEN ISO 18862:2018
ISO 18862:2016(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, 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
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved

---------------------- Page: 4 ----------------------
oSIST prEN ISO 18862:2018
ISO 18862:2016(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 1
6 Apparatus . 3
7 Sampling . 4
8 Procedure. 4
8.1 General . 4
8.2 Preparation of the sample extract . 4
8.3 Clean-up of the extracts . 5
8.3.1 Carrez precipitation . 5
8.3.2 Solid phase extraction . 5
8.4 HPLC-MS/MS measurement . 5
8.4.1 High-performance liquid chromatography (HPLC) . 5
8.4.2 Identification and quantification by mass spectrometry (HPLC-MS/MS) . 6
8.5 Measurement with GC-MS . 6
8.5.1 Derivatization and sample preparation for gas chromatography . 6
8.5.2 Gas chromatography . . 7
8.5.3 Identification and quantification by mass spectrometry . 7
9 Calibration . 7
9.1 General advice . 7
9.2 Determination of linearity and definition of the working range . 7
9.3 Calibration with internal standard solution . 7
9.4 Determination of the laboratory specific recovery. 8
10 Evaluation . 8
10.1 Criteria for identification . 8
10.2 Calculation and final results . 8
11 Precision data . 9
11.1 General . 9
11.2 Repeatability . 9
11.3 Reproducibility . 9
11.4 Recovery . 9
12 Measurement uncertainty . 9
13 Test report .10
Annex A (informative) Performance characteristics .11
Annex B (informative) Examples of absorber materials .12
Annex C (informative) Examples of columns and analysis conditions .13
Bibliography .19
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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 on 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 the following URL: www.iso.org/iso/foreword.html.
The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 15, Coffee.
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Introduction
When applying this document, all existing safety regulations have to be followed.
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oSIST prEN ISO 18862:2018
INTERNATIONAL STANDARD ISO 18862:2016(E)
Coffee and coffee products — Determination of acrylamide
— Methods using HPLC-MS/MS and GC-MS after
derivatization
WARNING — The use of this document can involve hazardous materials, operations and
equipment. This document does not purport to address all the safety problems associated with
its use. It is the responsibility of the user of this document to take appropriate measures for
ensuring the safety and health of the personnel prior to application of this document and to
fulfil statutory requirements for this purpose.
1 Scope
This document specifies methods for the determination of acrylamide in coffee and coffee products by
extraction with water, clean-up by solid-phase extraction and determination by HPLC-MS/MS and GC-
MS. It was validated in a method validation study on roasted coffee, soluble coffee, coffee substitutes
and coffee products with ranges from 53 μg/kg to 612,1 μg/kg.
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
No terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
4 Principle
The coffee sample is extracted with water or, in the case of soluble products, dissolved in water. A clean-
up by solid phase extraction is employed to remove interfering matrix compounds. Two alternative
methods can be used for the determination: high-performance liquid chromatography with mass
spectrometric detection (HPLC-MS/MS) or, after a bromination of the acrylamide, gas chromatography
with mass spectrometric detection (GC-MS). In both cases, isotopic labelled internal standard solutions
are used.
5 Reagents
WARNING — In view of health risks when working with acrylamide, appropriate preventive
and protection measures shall be taken, such as using a fume cupboard, aspirating acrylamide-
containing solutions only with a pipette, and avoiding skin and eye contact or inhalation of
acrylamide-containing vapour.
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If available, reagents of “residue analysis grade” or “analytical reagent grade” shall be used. The level
of impurities in the reagents that contribute to the blank should be negligibly small. The blank shall be
checked regularly.
5.1 Water, of grade 1 according to ISO 3696, MS-grade is recommended.
5.2 Operating gases of high purity, suitable for GC and mass spectrometry according to the
instructions of the manufacturer of the apparatus.
5.3 Solvents, such as methanol, ethyl acetate, acetonitrile, n-hexane, MS-grade is recommended.
5.4 Acrylamide, C H NO, purity >98 %, reference substance.
3 5
5.4.1 Acrylamide stock solution, mass concentration ρ = 1 000 μg/ml.
Weigh (0,10 ± 0,001) g of acrylamide into a 100 ml one-mark volumetric flask and swirl it in 30 ml
of water in order to dissolve the acrylamide. Fill up to the mark with water and mix well. The stock
solution is stable for at least 3 months when stored protected from light at a maximum of 6 °C.
Alternatively, a commercially available solution with a mass concentration of ρ = 1 000 µg/ml may be
used. The information of the manufacturer regarding the stability of the solution shall be observed.
5.4.2 Acrylamide calibration solution, ρ = 10 μg/ml.
Using a pipette, transfer (1,0 ± 0,001) ml of the acrylamide stock solution (5.4.1) into a 100 ml one-mark
volumetric flask and fill up to the mark with water. This solution shall be stored protected from light at
a maximum of 6 °C and shall be freshly prepared every working day. Depending on the working range,
more dilution steps might be necessary.
5.5 D3-acrylamide (acrylamide-2,3,3-d3) internal standard solution, C H D NO, purity >98 %,
3 2 3
reference substance.
5.5.1 D3-acrylamide stock solution (internal standard solution).
Weigh (0,10 ± 0,001) g of D3-acrylamide into a 100 ml one-mark volumetric flask and swirl it in 30 ml
of water in order to dissolve the D3-acrylamide. Fill up to the mark with water and mix well. The stock
solution is stable for at least 3 months when stored protected from light at a maximum of 6 °C.
Alternatively, a commercially available solution with a mass concentration of ρ = 1 000 µg/ml may be
used. The information of the manufacturer regarding the stability of the solution shall be observed.
5.5.2 D3-acrylamide internal standard solution.
Using a pipette, transfer (1,0 ± 0,001) ml of the D3-acrylamide stock solution (5.5.1) into a 100 ml one-
mark volumetric flask and fill up to the mark with water. This solution shall be stored protected from
light at a maximum of 6 °C and shall be freshly prepared every working day. Depending on the working
range, more dilution steps might be necessary.
NOTE 1 For HPLC-MS/MS, the solutions according to 5.4.1 to 5.5.2 can be prepared using the HPLC eluent as a
solvent. The stability of these solutions depends on the mobile phase used and has to be validated.
When using GC-MS, all standard solutions according to 5.4.2 and 5.5.2 shall be subjected to the
derivatization step according to 8.5.1.
13
NOTE 2 Instead of D3-acrylamide, it is also possible to use C acrylamide for the preparation of the internal
3
standard solution. However, in the following clauses, the procedure and calculation are described for D3-
acrylamide only.
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5.6 Saturated bromine water.
Saturate distilled water with bromine in a 100 ml one-mark volumetric flask (with a glass stopper)
until a phase of bromine is formed at the bottom of the flask (around 3,5 % of bromine at 4 °C). Acidify
the bromine water to a pH of about 1 using concentrated hydrobromic acid, (HBr, with a specific gravity
3
of 1,48 g/cm ).
If stored at 4 °C and protected from light, the solution can be used for about 4 weeks.
5.7 Potassium bromide, KBr.
5.8 Sodium thiosulfate (pentahydrate), Na S O · 5 H O.
2 2 3 2
5.9 Triethylamine, (C H ) N.
2 5 3
5.10 Sodium sulfate (anhydrous, granular), Na SO .
2 4
5.11 Carrez solution I.
Dissolve 10,6 g of potassium hexacyanoferrate trihydrate (II) K [Fe(CN) ] · 3 H O in 100 ml of water. If
4 6 2
stored at 4 °C and protected from light, the solution is stable for 6 months.
5.12 Carrez solution II.
Dissolve 21,9 g of zinc acetate dihydrate Zn(CH COO) · 2 H O in 100 ml of water. If stored at 4 °C and
3 2 2
protected from light, the solution is stable for 6 months.
5.13 Borate buffer, pH 8,6.
Mix 68 ml of a 0,1 molar sodium borate solution (20,12 g Na B O per litre of water) and 32 ml of
2 4 7
0,1 molar hydrochloric acid, c(HCl) = 0,1 mol/l, in a 100 ml one-mark volumetric flask.
6 Apparatus
Usual laboratory apparatus and, in particular, apparatus according to 6.1 to 6.14 are required.
Apparatus and parts of the apparatus which come into contact with the sample and extract shall be free
of residues which can cause blank values. Preferably glassware or equipment made of stainless steel or
PTFE (polytetrafluoroethylene) shall be used.
6.1 Analytical balance, capable of weighing to an accuracy of 0,1 mg.
6.2 Coffee mill, suitable for grinding roasted coffee beans.
6.3 Glassware, for collecting and storing the extracts, preferably made of amber glass, as sample vials
for manual or automatic use, equipped with an inert seal (e.g. vials with PTFE coated septum).
6.4 Ultrasonic bath, capable of being maintained at 40 °C.
6.5 Laboratory centrifuge, suitable for 15 ml and 50 ml centrifugal tubes and with a minimum g-force
of 2 000 g.
6.6 Centrifuge tubes, of 15 ml and 50 ml.
6.7 One-mark volumetric flask, of 20 ml and 100 ml.
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6.8 Pipettes, glass or automatic, suitable for measuring volume ranges of standard solutions and
sample extract dilutions.
6.9 Glass or polypropylene cartridges, with sorbents for the solid phase extraction (SPE), and for
the clean-up of extracts in 8.3.2 and 8.5.1 (examples are given in Table B.1).
6.10 High performance liquid chromatograph (for the test procedure according to 8.4), equipped
with ESI and mass spectrometric detector (HPLC-MS/MS); gas supply as specified by the manufacturer.
6.11 HPLC column (for the test procedure according to 8.4), suitable for acrylamide chromatography
(examples are given in Table C.1).
6.12 Gas chromatograph (for the test procedure according to 8.5) with mass spectrometric detector
(GC-MS) and operating gas supply (5.2) as specified by the manufacturer.
6.13 GC column, (for the test procedure according to 8.5) capillary column, suitable for acrylamide
chromatography (examples are given in Table C.2).
6.14 Membrane filter units, syringe filter (e.g. cellulose acetate filters 0,45 µm pore size)
suitable for filtration of sample eluate obtained by solid phase extraction before injection into the
chromatographic system.
7 Sampling
Sampling is not part of the method specified in this document. The sampling procedure shall be subject
to agreement by the interested parties. A representative, thoroughly mixed sample shall be used, which
has not been damaged or adulterated during transport or storage.
In order to exclude changes in the acrylamide levels, the analysis shall be performed shortly after
reception of the sample. The samples shall be stored under cool conditions below 6 °C at a maximum of
6 months, under the exclusion of light and they shall be exposed to room temperature only for analysis.
The date of receipt of the sample, as well as the date of roasting or the best-before date, shall be
documented along with the date of analysis.
8 Procedure
8.1 General
To avoid losses of the analyte, it is necessary that the samples are protected from light during extraction
and further preparation. For this reason, amber glassware shall always be used. Otherwise, the content
of the vessels and flasks shall be protected from incident light using aluminium foil.
8.2 Preparation of the sample extract
If necessary, grind the sample in a coffee mill (6.2) and homogenize thoroughly.
Weigh 2 g of the homogenized sample of roasted coffee, soluble coffee or coffee substitute or 5 g of
liquid coffee beverage to the nearest 1 mg using an analytical balance (6.1) and transfer it into a 50 ml
centrifuge tube (6.6).
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Add 2 ml of n-hexane to the test sample and shake briefly. Then spike the test sample with D3-acrylamide
as the internal standard solution in a concentration corresponding to the expected acrylamide level of
the sample.
EXAMPLE Weigh 2 g of coffee and add 100 µl internal standard solution (ρ = 10 µg/ml), which is equivalent
to an acrylamide mass fraction of 500 µg/kg in the coffee sample.
Add 20 ml of distilled water, shake briefly but vigorously, and sonicate (6.4) for 15 min at
approximately 40 °C.
Allow a few minutes for precipitation and in the case of non-sedimenting samples centrifuge (6.5) for
15 min at 2 000 g to separate suspended solids. Before liquid chromatography (8.4) or derivatization
and gas chromatographic separation (8.5), take 10 ml from the lower aqueous phase and use it for a
further clean-up according to 8.3. Take the lower aqueous phase through the upper hexane phase using
a pipette without removing the hexane phase. If necessary, the hexane phase may also be removed
cautiously using a Pasteur pipette.
8.3 Clean-up of the extracts
8.3.1 Carrez precipitation
Clean-up the sample extract prepared according to 8.2 by Carrez precipitation. Add 1 000 µl of Carrez
solution I (5.11) and shake. Add 1 000 µl of Carrez solution II (5.12) and shake again. After a short
exposure time, centrifuge for 4 min at 2 000 g. Decant the supernatant, wash the residue with 2 ml
to 3 ml of water, centrifuge and decant again. Combine both aqueous solutions.
8.3.2 Solid phase extraction
Clean-up the sample extract after Carrez precipitation (8.3.1) by solid phase extraction (SPE) using
two sequential cartridges with adsorber material (examples are given in Table B.1). The first cartridge
contains 500 mg of C18 material, the second cartridge 500 mg of ion exchanger. The cartridges can be
used in a serial alignment. If appropriate, a combined cartridge can be used.
Condition both SPE columns according to the manufacturer’s instructions successively with methanol
and distilled water. Place the complete sample extract (8.3.1) on top of the upper (first) SPE column,
allow to soak and add 2 ml to 3 ml of water. Collect the eluate until the cartridge is dry. Place the eluate
on top of the second or lower conditioned ion exchange column, add 2 ml to 3 ml of water and collect
the eluate. A complete elution can be achieved by using a light vacuum or pressure. Collect the eluate
including washing water in a 20 ml one-mark volumetric flask and fill up to 20 ml with water.
8.4 HPLC-MS/MS measurement
8.4.1 High-performance liquid chromatography (HPLC)
Prior to the HPLC-MS/MS analysis, add organic solvent to the cleaned-up extract (8.3.2) in order to
make up the desired eluent composition and filter through a membrane filter (6.14) before injecting a
suitable volume (e.g. 10 µl to 100 µl depending on the column used) onto the HPLC column.
Optimize the device parameters of the HPLC system in accordance with the manufacturer’s instructions.
The chromatographic conditions shall be adjusted to suit the selected column (examples are given in
Table C.1).
The clean-up stages according to 8.3.1 and 8.3.2 are essential for the chromatographic separation of the
analyte peaks from the interfering peaks. An example of chromatogram is given in Figures C.1 and C.2.
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8.4.2 Identification and quantification by mass spectrometry (HPLC-MS/MS)
Detect acrylamide using MS/MS in the positive ionization mode (electrospray ionization, ESI).
For identification, use the mass transition m/z = 72 → 55. Acrylamide is identified as present if a signal
at the mass track of the daughter ion (m/z 55) appears in the MS/MS chromatogram and the deviation
of the retention time from that of an authentic reference substance, analysed under the same HPLC
conditions, is less than 5 %.
Possible transitions for acrylamide and D3-acrylamide are given in Table 1.
Quantify the analyte by comparing the abundance of the parent-daughter ions of acrylamide with the
isotope-labelled internal standard solution using the mass transitions 72 → 55 (acrylamide) and 75 →
58 (D3-acrylamide).
A third mass transition 72 → 54 may be used for further confirmation of the results. The evaluation of
this transition was not part of the interlaboratory study (Annex A).
Table 1 — Mass spectrometric transitions used for the identification and quantification of
acrylamide
Reference Selected transitions for the identification and
substance quantification of acrylamide using MS/MS modus
m/z
Acrylamide 72 → 55 Identification and quantification
72 → 44 Identification (informative, qualifier)
D3-acrylamide 75 → 58 Identification and quantification
75 → 44 Identification (informative, qualifier)
8.5 Measurement with GC-MS
8.5.1 Derivatization and sample preparation for gas chromatography
Add 3,5 g of potassium bromide (5.7) to the aqueous sample solution (8.3.2). Add 2,5 ml of saturated
bromine water (5.6) and allow to react for 2 h at room temperature in the dark. Add a few drops of
sodium thiosulfate solution (5.8) to eliminate excessive bromine. When using a one molar solution,
a few drops are enough to eliminate the brown colour caused by bromine. A significant overdose of
sodium thiosulfate should be avoided.
Apply the extract quantitatively to a polymer resin cartridge (content 500 mg) with polar and strong
cationic exchange properties (for examples, see Table B.2), which has previously been conditioned
according to the manufacturer’s instructions. During this process and in the subsequent rinsing steps,
it is necessary to ensure that the column is not running dry. Wash the column subsequently with 3 ml
of water, 1 ml of borate buffer of pH 8,6 (5.13) and again with 0,5 ml of water. The column is then to be
sucked dry, followed by elution of the analyte with 2 ml of ethyl acetate with a contact time of 1 min to
2 min on the column.
NOTE 1 It can be advantageous to elute with more ethyl acetate to impro
...

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