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 - Dosage de l'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.

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Status
Published
Publication Date
29-Oct-2019
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
30-Oct-2019
Completion Date
30-Oct-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

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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.

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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
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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.

NOTE 2 Instead of D3-acrylamide, it is also possible to use C acrylamide for the preparation of the internal

standard solution. However, in the following clauses, the procedure and calculation are described for D3-

acrylamide only.
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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

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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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).
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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
...

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