EN ISO 18862:2025
(Main)Coffee and coffee products - Determination of acrylamide - Methods using high-performance liquid chromatography with tandem mass spectrometric detection (HPLC-MS/MS) and gas chromatography with mass spectrometric detection (GC-MS) after derivatization (ISO 18862:2025)
Coffee and coffee products - Determination of acrylamide - Methods using high-performance liquid chromatography with tandem mass spectrometric detection (HPLC-MS/MS) and gas chromatography with mass spectrometric detection (GC-MS) after derivatization (ISO 18862:2025)
This document specifies methods for the determination of acrylamide in coffee and coffee products by extraction with water, clean-up by solid-phase extraction (SPE) and determination by high-performance liquid chromatography with tandem mass spectrometric detection (HPLC-MS/MS) and gas chromatography with mass spectrometric detection (GC-MS) after derivatization. The methods were validated in a validation study for 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 Hochleistungs-Flüssigchromatographie mit massenspektrometrischer Detektion (HPLC-MS/MS) und Gaschromatographie mit massenspektrometrischer Detektion (GC-MS) nach Derivatisierung (ISO 18862:2025)
Dieses Dokument legt Verfahren zur Acrylamidbestimmung in Kaffee und Kaffee-Erzeugnissen durch Wasserextraktion, Aufreinigung durch Festphasen-Extraktion und Bestimmung mittels Hochleistungs-Flüssigchromatographie mit massenspektrometrischer Detektion (HPLC-MS/MS) und Gaschromatographie mit massenspektrometrischer Detektion (GC-MS) fest. Es wurde in einer Verfahrensvalidierungsstudie für Röstkaffee, löslichen Kaffee, Kaffeesurrogate und Kaffee-Erzeugnisse mit Bereichen von 53 µg/kg bis 612,1 µg/kg validiert.
Café et dérivés du café - Dosage de l’acrylamide - Méthodes par chromatographie liquide à haute performance avec détection par spectrométrie de masse en tandem (CLHP–SM/SM) et chromatographie en phase gazeuse avec détection par spectrométrie de masse (CG–SM) après dérivation (ISO 18862:2025)
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 (SPE) et dosage par chromatographie liquide à haute performance avec détection par spectrométrie de masse en tandem (CLHP-SM/SM) et chromatographie en phase gazeuse avec détection par spectrométrie de masse (CG-SM) après dérivation. Les méthodes ont été validées au cours d’une étude 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 - Metoda z uporabo tekočinske kromatografije visoke ločljivosti z masno spektrometrijo (HPLC-MS/MS) in plinske kromatografije z masno spektrometrijo (GC-MS) po derivatizaciji (ISO/FDIS 18862:2025)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN ISO 18862:2024
01-oktober-2024
Kava in proizvodi iz kave - Določevanje akrilamida - Metode z uporabo HPLC-
MS/MS in GC-MS po derivatizaciji (ISO/DIS 18862:2024)
Coffee and coffee products - Determination of acrylamide - Methods using HPLC-MS/MS
and GC-MS after derivatization (ISO/DIS 18862:2024)
Kaffee und Kaffee-Erzeugnisse- Bestimmung von Acrylamid- Verfahren mittels HPLC-
MS/MS und mittels GC-MS nach Derivatisierung (ISO/DIS 18862:2024)
Café et dérivés du café - Dosage de l'acrylamide - Méthodes par CLHP-SM/SM et CG-
SM après dérivation (ISO/DIS 18862:2024)
Ta slovenski standard je istoveten z: prEN ISO 18862
ICS:
67.140.20 Kava in kavni nadomestki Coffee and coffee substitutes
oSIST prEN ISO 18862:2024 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
oSIST prEN ISO 18862:2024
oSIST prEN ISO 18862:2024
DRAFT
International
Standard
ISO/DIS 18862
ISO/TC 34/SC 15
Coffee and coffee products —
Secretariat: ICONTEC
Determination of acrylamide —
Voting begins on:
Methods using HPLC-MS/MS and GC-
2024-08-16
MS after derivatization
Voting terminates on:
2024-11-08
Café et dérivés du café — Dosage de l'acrylamide — Méthodes
par CLHP-SM/SM et CG-SM après dérivation
ICS: 67.140.20
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
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Reference number
ISO/DIS 18862:2024(en)
oSIST prEN ISO 18862:2024
DRAFT
ISO/DIS 18862:2024(en)
International
Standard
ISO/DIS 18862
ISO/TC 34/SC 15
Coffee and coffee products —
Secretariat: ICONTEC
Determination of acrylamide —
Voting begins on:
Methods using HPLC-MS/MS and GC-
MS after derivatization
Voting terminates on:
Café et dérivés du café — Dosage de l'acrylamide — Méthodes
par CLHP-SM/SM et CG-SM après dérivation
ICS: 67.140.20
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
© ISO 2024
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
STANDARDS MAY ON OCCASION HAVE TO
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Published in Switzerland Reference number
ISO/DIS 18862:2024(en)
ii
oSIST prEN ISO 18862:2024
ISO/DIS 18862:2024(en)
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) .5
8.5 Measurement with GC-MS .6
8.5.1 Derivatization and sample preparation for gas chromatography .6
8.5.2 Gas chromatography .6
8.5.3 Identification and quantification by mass spectrometry .6
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 .7
10 Evaluation . 7
10.1 Criteria for identification .7
10.2 Calculation and final results .8
11 Precision data . 8
11.1 General .8
11.2 Repeatability .8
11.3 Reproducibility .8
11.4 Recovery .9
12 Measurement uncertainty . 9
13 Test report . 9
Annex A (informative) Performance characteristics . 10
Annex B (informative) Examples of absorber materials .11
Annex C (informative) Examples of columns and analysis conditions.12
Annex D (Informative) Examples for sample preparation and chromatographic conditions
using LC-MS/MS .18
Bibliography .25
iii
oSIST prEN ISO 18862:2024
ISO/DIS 18862:2024(en)
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
oSIST prEN ISO 18862:2024
ISO/DIS 18862:2024(en)
Introduction
When applying this document, all existing safety regulations have to be followed.
v
oSIST prEN ISO 18862:2024
oSIST prEN ISO 18862:2024
DRAFT International Standard ISO/DIS 18862:2024(en)
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 https:// www .electropedia .org/
— ISO Online browsing platform: available at https:// 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.
oSIST prEN ISO 18862:2024
ISO/DIS 18862:2024(en)
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.
oSIST prEN ISO 18862:2024
ISO/DIS 18862:2024(en)
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 0,1 molar
2 4 7
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.
oSIST prEN ISO 18862:2024
ISO/DIS 18862:2024(en)
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).
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.
oSIST prEN ISO 18862:2024
ISO/DIS 18862:2024(en)
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 app
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