Coffee and coffee products -- Determination of acrylamide -- Methods using HPLC-MS/MS and GC-MS after derivatization

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.

Café et dérivés du café -- Dosage de l'acrylamide -- Méthodes par CLHP-SM/SM et CG-SM après dérivation

Le présent document spécifie des méthodes de dosage de l'acrylamide dans le café et les dérivés du café par extraction ŕ 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 ŕ 612,1 μg/kg.

General Information

Status
Published
Publication Date
10-Jul-2016
Technical Committee
Drafting Committee
Current Stage
9020 - International Standard under periodical review
Start Date
15-Jul-2021
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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
---------------------- Page: 1 ----------------------
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: 2 ----------------------
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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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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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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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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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.
2 © ISO 2016 – All rights reserved
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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.
© ISO 2016 – All rights reserved 3
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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).
4 © ISO 2016 – All rights reserved
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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 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.

© ISO 2016 – All rights reserved 5
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ISO 18862:2016(E)
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 improve the recovery. However, this can

amplify the impact of interferences.

Dry the extract by adding the lowest possible amount of sodium sulfate (5.10) to ensure a water free

extract and then add 50 µl of triethylamine (5.9). This solution can be used directly for GC analysis.

NOTE 2 To check the completeness of the conversion reaction, methacrylamide (e.g. 100 µl of an aqueous

0,5 µg/ml solution) can be added to the sample solution prior to the bromination step. If the recovery for the

methacrylamide derivative is satisfactory, it is assumed that acrylamide was completely brominated.

6 © ISO 2016 – All rights reserved
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ISO 18862:2016(E)

NOTE 3 By adding triethylamine, the unstable dibromopropionamide is quantitatively converted to the stable

monobromopropenamide, which is detected by GC-MS. Without this conversion, uncontrolled HBr-elimination

can occur in the further course of analysis (e.g. at active sites in the injector), giving results that are not

reproducible.
8.5.2 Gas chromatography

Dilute the purified extract (8.5.1) with ethyl acetate (5.3) according to the expected analyte

concentration and, if necessary, filter using membrane filter (6.14) prior to GC/MS analysis.

The gas chromatograph (6.12) shall be set up in accordance with the manufacturer’s instructions and

the device par
...

DRAFT INTERNATIONAL STANDARD
ISO/DIS 18862
ISO/TC 34/SC 15 Secretariat: ABNT
Voting begins on: Voting terminates on:
2015-10-06 2016-01-06
Analysis of coffee and coffee products — Determination of
acrylamide — Methods using HPLC-MS/MS and GC-MS after
derivatisation

Analyse du café et de ses dérivés — Dosage de l’acrylamide — Méthodes utilisant CLHP-MS/MS et CG-MS

après dérivation
ICS: 67.140.20
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 18862:2015(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. ISO 2015
---------------------- Page: 1 ----------------------
ISO/DIS 18862:2015(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2015, 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 2015 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/DIS 18862
Contents Page

Foreword ............................................................................................................................................................. v

Introduction ........................................................................................................................................................ vi

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

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

3 Principle.................................................................................................................................................. 1

4 Reagents................................................................................................................................................. 1

5 Apparatus ............................................................................................................................................... 3

6 Sampling................................................................................................................................................. 4

7 Procedure ............................................................................................................................................... 4

7.1 General ................................................................................................................................................... 4

7.2 Preparation of the extract ..................................................................................................................... 4

7.3 Clean-up of the extracts ........................................................................................................................ 4

7.3.1 Carrez precipitation ............................................................................................................................... 4

7.3.2 Solid phase extraction .......................................................................................................................... 4

7.4 HPLC-MS/MS measurement ................................................................................................................. 5

7.4.1 High performance liquid chromatography (HPLC) ............................................................................ 5

7.4.2 Identification and quantification by mass spectrometry (HPLC-MS/MS) ........................................ 5

7.5 Measurement with GC-MS .................................................................................................................... 6

7.5.1 Derivatisation and sample preparation for gas chromatography .................................................... 6

7.5.2 Gas chromatography ............................................................................................................................ 6

7.5.3 Identification and quantification by mass spectrometry ................................................................... 6

8 Calibration .............................................................................................................................................. 7

8.1 General advice ....................................................................................................................................... 7

8.2 Determination of linearity and definition of the working range ........................................................ 7

8.3 Calibration with internal standard ....................................................................................................... 7

8.4 Determination of the laboratory specific recovery ............................................................................ 7

9 Evaluation............................................................................................................................................... 7

9.1 Criteria for identification ....................................................................................................................... 7

9.2 Calculation and final results................................................................................................................. 7

10 Procedure characteristics .................................................................................................................... 8

10.1 General ................................................................................................................................................... 8

10.2 Repeatability, r ....................................................................................................................................... 8

10.3 Reproducibility, R .................................................................................................................................. 8

10.4 Recovery................................................................................................................................................. 9

11 Measurement uncertainty ..................................................................................................................... 9

12 Test report .............................................................................................................................................. 9

Annex A (informative) Performance characteristics ................................................................................... 10

Annex B (informative) Examples of absorber materials ............................................................................. 11

Annex C (informative) Examples for recommended columns and analysis conditions .......................... 12

C.1 Generally suitable chromatographic columns ................................................................................. 12

C.2 Example method and approximate retention time for HPLC-MS/MS ............................................. 13

C.3 Example of suitable conditions and approximate retention time for GC-MS analysis ................ 13

C.4 Example chromatograms for HPLC-MS/MS ........................................................................................ 1

C.5 Example of chromatogram for GC-MS ................................................................................................ 1

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ISO/DIS 18862

Bibliography ........................................................................................................................................................3

iv © ISO 2015 – All rights reserved
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ISO/DIS 18862
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.

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.

The main task of technical committees is to prepare International Standards. Draft International Standards

adopted by the technical committees are circulated to the member bodies for voting. Publication as an

International Standard requires approval by at least 75 % of the member bodies casting a vote.

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.

ISO 18862 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 15, Coffee.

© ISO 2015 – All rights reserved v
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ISO/DIS 18862
Introduction

This Standard was set up by the German Institute for Standardization (DIN), Agricultural products,

subcommittee Food Analysis – vertical methods, subcommittee coffee

This standard can only be applied by experts. It is necessary to stick to existing safety regulations. Annexes A

to C are informative.

Warning — Users of this standard must be familiar with practical work in laboratories. This standard does not

point out all risks which might occur during the use of this method. It is the responsibility of the user to make

sure that all safety precautions necessary are taken and that all national safety regulations are obeyed.

vi © ISO 2015 – All rights reserved
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ISO/DIS 18862
Analysis of coffee and coffee products — Determination of
acrylamide — Methods using HPLC-MS/MS and GC-MS after
derivatisation
1 Scope

This standard specifies methods for the determination of the acrylamide content in coffee and coffee products

by means of extraction, clean-up by solid-phase extraction and determination by HPLC-MS/MS and GC-MS. It

is applicable to roasted coffee, soluble coffee, coffee substitutes and coffee products.

2 Normative references

The following referenced documents are indispensable for the application 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 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, by gas chromatography with mass spectrometric

detection (GC-MS). In both cases isotopic labelled internal standards are used.
4 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.

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.
4.1 Water, of grade 1 according to ISO 3696, MS-grade is recommended.

4.2 Operating gases of high purity, suitable for GC and mass spectrometry according to the instructions

of the manufacturer of the analytical instruments.

4.3 Solvents, such as methanol, ethyl acetate, acetonitrile, n-hexane, MS-grade is recommended.

4.4 Acrylamide, C H NO, purity > 98 %, reference compound.
3 5
4.4.1 Acrylamide stock standard solution, mass concentration  = 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 ( = 1 000 μg/ml) and mix well. The stock

solution is stable for at least three months when stored protected from light at a maximum of 6 °C.

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ISO/DIS 18862

Alternatively a commercially available solution with a mass concentration  = 1 000 µg/ml may be used. The

information of the manufacturer regarding the stability of the solution shall be observed.

4.4.2 Acrylamide working solution for calibration,  = 10 μg/ml

Using a pipette, transfer (1,0 ± 0,001) ml of the acrylamide stock standard solution (4.4.1) 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.

4.5 D3-acrylamide (acrylamide-2,3,3-d3) internal standard, C H D NO, purity > 98 %, reference

3 2 3
compound.

4.5.1 D3-acrylamide stock standard solution (internal standard), the D3-acrylamide stock standard

solution (internal standard) shall be prepared analogously to the acrylamide stock standard solution in 4.4.1.

Alternatively a commercially available solution with a mass concentration, , of 1 000 µg/ml can be used. The

information of the manufacturer regarding the stability of the solution shall be observed.

4.5.2 D3-acrylamide working solution (internal standard), the working solution of the internal standard is

prepared analogously to 4.4.2 from commercially available or self-prepared stock solutions (4.5.1).

NOTE 1 For HPLC-MS/MS the solutions according to 4.4.1 to 4.5.2 can be prepared using the HPLC eluent as a

solvent. The stability of these solutions depends on the liquid phase used and has to be validated.

When using GC-MS all standards according to 4.4.2 and 4.5.2 shall be subjected to the derivatization step

according to 7.5.1.

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.

4.6 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.

4.7 Potassium bromide, KBr, p.a.
4.8 Sodium thiosulfate (pentahydrate), Na S O · 5 H O, p.a.
2 2 3 2
4.9 Triethylamine, (C H ) N, p.a.
2 5 3
4.10 Sodium sulfate (anhydrous, granular), Na SO , p.a.
2 4
4.11 Carrez solution I

Dissolve 10,6 g of potassium hexacyanoferrate trihydrate (II) K [Fe(CN) ] · 3 H O p.a. in 100 ml of water.

4 6 2
If stored at 4 °C and protected from light, the solution is stable for 6 months.
4.12 Carrez solution II

Dissolve 21,9 g of zinc acetate dihydrate Zn(CH COO) · 2 H O p.a. in 100 ml of water.

3 2 2
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ISO/DIS 18862
If stored at 4 °C and protected from light, the solution is stable for 6 months.
4.13 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.
5 Apparatus
5.1 General

Standard laboratory apparatus and, in particular, apparatus according to 5.2 to 5.15 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.
5.2 Analytical balance, capable of weighing to an accuracy of 0,1 mg.
5.3 Coffee mill, suitable for grinding roasted coffee beans.

5.4 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).

5.5 Ultrasonic bath, capable of being maintained at 40 °C.

5.6 Laboratory centrifuge, suitable for 15 ml and 50 ml centrifugal tubes and with a minimum g-force of

2 000 g.
5.7 Centrifuge tubes, nominal capacities of 15 ml and 50 ml.
5.8 One-mark volumetric flask, nominal capacities of 20 ml and 100 ml.

5.9 Pipettes, glass or automatic, suitable for measuring volume ranges of standard and sample extract

dilutions.

5.10 Glass- or polypropylene cartridges, with sorbents for the solid phase extraction (SPE), and for the

clean-up of extracts in 7.3.2 and/or 7.5.1 (examples are given in Table B.1).

5.11 High performance liquid chromatograph (for the test procedure according to 7.4), equipped with ESI

and mass spectrometric detector (HPLC-MS/MS); gas supply as specified by the manufacturer.

5.12 HPLC column (for the test procedure according to 7.4), suitable for acrylamide chromatography

(examples are given in Table C.1).

5.13 Gas chromatograph (for the test procedure according to 7.5) with mass spectrometric detector (GC-

MS) and operating gas supply (4.2) as specified by the manufacturer.

5.14 GC column, (for the test procedure according to 7.5) capillary column, suitable for acrylamide

chromatography (examples are given in Table C.2).

5.15 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.

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ISO/DIS 18862
6 Sampling

Sampling is not part of the method specified in this standard. 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 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.
7 Procedure
7.1 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.
7.2 Preparation of the sample extract
If necessary, grind the sample in a coffee mill (5.3) and homogenise thoroughly.

Weigh 2 g of the homogenised 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 (5.2) and transfer it into a 50 ml centrifuge

tube.

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 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 ( = 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 (5.5) for 15 min at approximately 40 °C.

Allow a few minutes for precipitation and in the case of non-sedimenting samples centrifuge (5.6) for 15 min at

2000 g to separate suspended solids. Before liquid chromatography (7.4) or derivatization and gas

chromatographic separation (7.5), take 10 ml from the lower aqueous phase and use it for a further clean-up

according to 7.3. 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.
7.3 Clean-up of the extracts
7.3.1 Carrez precipitation

Clean-up the sample prepared according to 7.2 by Carrez precipitation. Add 1 000 µl Carrez solution I (4.11)

and shake. Add 1 000 µl Carrez solution II (4.12) and shake again. After a short exposure time centrifuge for 4

min at 2000 g. Decant the supernatant, wash the residue with 2 ml to 3 ml of water, centrifuge and decant

again. Combine both aqueous solutions.
7.3.2 Solid phase extraction

Clean-up the sample extract from the Carrez precipitation (7.3.1) by solid phase extraction (SPE) using two

sequential cartridges with adsorber material (examples are given in Table B.1).The first cartridge contains

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ISO/DIS 18862

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 (7.3.1) 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 again 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.
7.4 HPLC-MS/MS measurement
7.4.1 High performance liquid chromatography (HPLC)

Prior to the HPLC-MS/MS analysis, add organic solvent to the cleaned-up extract (7.3.2) in order to make up

the desired eluent composition and filter (5.15) through a membrane filter (5.15) 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 Tables C.1).

The clean-up stages according to 7.3.1 and 7.3.2 are essential for the chromatographic separation of the

analyte peaks from the interfering peaks. An example chromatogram is given in Figure C.1 and C.2.

7.4.2 Identification and quantification by mass spectrometry (HPLC-MS/MS)

Detect acrylamide using MS/MS in the positive ionization mode (electrospray ionisation, 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 compound, 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 using the mass transitions 72  55 (acrylamide) and 75  58 (D3-acrylamide).

NOTE 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 inter-laboratory study (Annex A).

Table 1 — Mass spectrometric transitions used for the identification and quantification of acrylamide

Reference Selected transitions for the identification and quantification of
compound 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)
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ISO/DIS 18862
7.5 Measurement with GC-MS
7.5.1 Derivatisation and sample preparation for gas chromatography

Add 3,5 g of potassium bromide (4.7) to the aqueous sample solution (7.3.2). Add 2,5 ml of saturated bromine

water (4.6) and allow to react for two hours at room temperature in the dark. Add a few drops of sodium

thiosulfate solution (4.8) 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 borate buffer of pH

8,6 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 improve the recovery. However this can amplify the

impact of interferences.

Dry the extract by adding the lowest possible amount of sodium sulphate (4.10) to ensure a water free extract

and then add 50 µl of triethylamine (4.9). This solution can be used directly for GC analysis.

NOTE 2 To check the completeness of the conversion reaction, methacrylamide (e. g. 100 µl of an aqueous 0,5 µg/ml

solution) can be added to the sample solution prior to the bromination step. If the recovery for the methacrylamide

derivative is satisfactory, it is assumed that acrylamide was completely brominated.

NOTE 3 By adding triethylamine the unstable dibromopropionamide is quantitatively converted to the stable

monobromopropenamide, which is detected by GC-MS. Without this conversion, uncontrolled HBr-elimination can occur in

the further course of analysis (e. g. at active sites in the injector), giving results that are not reproducible.

7.5.2 Gas chromatography

Dilute the purified extract (7.5.1) with ethyl acetate according to the expected analyte concentration and, if

necessary, filter using membrane filter units (5.15) prior to GC/MS analysis.

The gas chromatograph (5.13) shall be set up in accordance with the manufacturer’s instructions and the

device parameters shall be optimized. The chromatographic settings shall be adjusted to suit the selected

column. Examples are given in Tables C.2 and C.5 and example chromatograms are given in Figures C.3, C.4

and C.5.
7.5.3 Identification and quantification by mass spectrometry

The analytes are transferred to positive ions (molecular ions) and characteristic fragment ions by electron

impact ionization (EI). Use these ions for the quantification and identification.

Quantify by comparing the peak area of ions deriving from bromopropenamide with those from the isotope-

labelled internal standard (see Table 2).
Table 2 — Observed ions
Observed ions
Original compound
m/z
79 +
149: [C H BrON]
3 4 Identification and quantification
Bromopropenamide
79 +
Identification
106: [C H Br]
2 3
2 1 81 +
Bromopropenamide-D
153: [C H H BrON] Identification and quantification
3 2 2
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ISO/DIS 18862
2 1 81 +
Identification
110: [C H H Br]
2 2 1

NOTE Due to the natural isotope distribution of bromine, occurrences of ions with m/z 151

and m/z 108 are not capable of being evaluated.
8 Calibration
8.1 General advice

Before application of the GC method, all calibration solutions shall be subject to the derivatization procedure

according to 7.5.1.

To obtain an accurate calibration of the test procedure according to 7.4 it is necessary to determine the

retention time of acrylamide (LC-MS/MS) or, for procedures according to 7.5, to determine the retention time

of bromopropenamide (GC-MS). Identify the retention times using an aqueous so
...

NORME ISO
INTERNATIONALE 18862
Première édition
2016-07-15
Café et derivés du café — Dosage de
l'acrylamide — Méthodes par CLHP-
SM/SM et CG-SM après dérivation
Coffee and coffee products — Determination of acrylamide —
Methods using HPLC-MS/MS and GC-MS after derivatization
Numéro de référence
ISO 18862:2016(F)
ISO 2016
---------------------- Page: 1 ----------------------
ISO 18862:2016(F)
DOCUMENT PROTÉGÉ PAR COPYRIGHT
© ISO 2016

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publication ne peut être reproduite ni utilisée sous quelque forme que ce soit et par aucun procédé, électronique ou mécanique,

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Publié en Suisse
ii © ISO 2016 – Tous droits réservés
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ISO 18862:2016(F)
Sommaire Page

Avant-propos ..............................................................................................................................................................................................................................iv

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

1 Domaine d’application ................................................................................................................................................................................... 1

2 Références normatives ................................................................................................................................................................................... 1

3 Termes et définitions ....................................................................................................................................................................................... 1

4 Principe .......................................................................................................................................................................................................................... 1

5 Réactifs ........................................................................................................................................................................................................................... 1

6 Appareillage .............................................................................................................................................................................................................. 3

7 Échantillonnage ..................................................................................................................................................................................................... 4

8 Mode opératoire.................................................................................................................................................................................................... 5

8.1 Généralités .................................................................................................................................................................................................. 5

8.2 Préparation de l’extrait d’échantillon.................................................................................................................................. 5

8.3 Purification des extraits .................................................................................................................................................................. 5

8.3.1 Précipitation de Carrez ........................................................................................................................................... .... 5

8.3.2 Extraction en phase solide ...................................................................................................................................... 5

8.4 Analyse par CLHP-SM/SM ............................................................................................................................................................. 6

8.4.1 Chromatographie liquide à haute performance (CLHP) ................................................................ 6

8.4.2 Identification et quantification par spectrométrie de masse (CLHP-SM/SM) ............ 6

8.5 Analyse par CG-SM ............................................................................................................................................................................... 6

8.5.1 Dérivation et préparation de l’échantillon pour la chromatographie en

phase gazeuse ..................................................................................................................................................................... 6

8.5.2 Chromatographie en phase gazeuse ............................................................................................................... 7

8.5.3 Identification et quantification par spectrométrie de masse .................................................... 7

9 Étalonnage .................................................................................................................................................................................................................. 8

9.1 Conseil d’ordre général .................................................................................................................................................................... 8

9.2 Détermination de la linéarité et définition de la plage de travail ................................................................ 8

9.3 Étalonnage avec une solution d’étalon interne ........................................................................................................... 8

9.4 Détermination du taux de récupération spécifique du laboratoire ........................................................... 8

10 Évaluation .................................................................................................................................................................................................................... 8

10.1 Critères d’identification................................................................................................................................................................... 8

10.2 Calcul et résultats finaux ................................................................................................................................................................ 8

11 Données de fidélité ............................................................................................................................................................................................ 9

11.1 Généralités .................................................................................................................................................................................................. 9

11.2 Répétabilité ................................................................................................................................................................................................ 9

11.3 Reproductibilité ..................................................................................................................................................................................... 9

11.4 Taux de récupération ......................................................................................................................................................................10

12 Incertitude de mesure .................................................................................................................................................................................10

13 Rapport d’essai ....................................................................................................................................................................................................10

Annexe A (informative) Caractéristiques de performance.........................................................................................................11

Annexe B (informative) Exemples de matériaux absorbants ..................................................................................................12

Annexe C (informative) Exemples de colonnes et de conditions d’analyse ...............................................................13

Bibliographie ...........................................................................................................................................................................................................................19

© ISO 2016 – Tous droits réservés iii
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ISO 18862:2016(F)
Avant-propos

L’ISO (Organisation internationale de normalisation) est une fédération mondiale d’organismes

nationaux de normalisation (comités membres de l’ISO). L’élaboration des Normes internationales est

en général confiée aux comités techniques de l’ISO. Chaque comité membre intéressé par une étude

a le droit de faire partie du comité technique créé à cet effet. Les organisations internationales,

gouvernementales et non gouvernementales, en liaison avec l’ISO participent également aux travaux.

L’ISO collabore étroitement avec la Commission électrotechnique internationale (IEC) en ce qui

concerne la normalisation électrotechnique.

Les procédures utilisées pour élaborer le présent document et celles destinées à sa mise à jour sont

décrites dans les Directives ISO/IEC, Partie 1. Il convient, en particulier de prendre note des différents

critères d’approbation requis pour les différents types de documents ISO. Le présent document a été

rédigé conformément aux règles de rédaction données dans les Directives ISO/IEC, Partie 2 (voir www

.iso .org/directives).

L’attention est attirée sur le fait que certains des éléments du présent document peuvent faire l’objet de

droits de propriété intellectuelle ou de droits analogues. L’ISO ne saurait être tenue pour responsable

de ne pas avoir identifié de tels droits de propriété et averti de leur existence. Les détails concernant

les références aux droits de propriété intellectuelle ou autres droits analogues identifiés lors de

l’élaboration du document sont indiqués dans l’Introduction et/ou dans la liste des déclarations de

brevets reçues par l’ISO (voir www .iso .org/brevets).

Les appellations commerciales éventuellement mentionnées dans le présent document sont données

pour information, par souci de commodité, à l’intention des utilisateurs et ne sauraient constituer un

engagement.

Pour une explication de la signification des termes et expressions spécifiques de l’ISO liés à l’évaluation

de la conformité, ou pour toute information au sujet de l’adhésion de l’ISO aux principes de l’Organisation

mondiale du commerce (OMC) concernant les obstacles techniques au commerce (OTC), voir le lien

suivant: www .iso .org/iso/fr/foreword .html.

Le comité chargé de l’élaboration du présent document est l’ISO/TC 34, Produits alimentaires, sous-

comité SC 15, Café.
iv © ISO 2016 – Tous droits réservés
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ISO 18862:2016(F)
Introduction

Toutes les règles de sécurité existantes doivent être respectées lors de l’application du présent

document.
© ISO 2016 – Tous droits réservés v
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NORME INTERNATIONALE ISO 18862:2016(F)
Café et derivés du café — Dosage de l'acrylamide —
Méthodes par CLHP-SM/SM et CG-SM après dérivation

AVERTISSEMENT — Le présent document peut impliquer l’utilisation de produits et la mise

en œuvre de modes opératoires et d’appareillages à caractère dangereux. Il n’est pas destiné

à traiter de tous les problèmes de sécurité liés à son utilisation. Il incombe à l’utilisateur du

présent document de prendre les mesures appropriées pour assurer l’hygiène et la sécurité du

personnel avant l’application du présent document et de satisfaire aux exigences réglementaires

correspondantes.
1 Domaine d’application

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.
2 Références normatives

Les documents suivants cités dans le texte constituent, pour tout ou partie de leur contenu, des

exigences du présent document. Pour les références datées, seule l’édition citée s’applique. Pour les

références non datées, la dernière édition du document de référence s’applique (y compris les éventuels

amendements).

ISO 3696, Eau pour laboratoire à usage analytique — Spécification et méthodes d'essai

3 Termes et définitions
Aucun terme n’est défini dans le présent document.

L’ISO et l’IEC tiennent à jour des bases de données terminologiques destinées à être utilisées en

normalisation, consultables aux adresses suivantes:
— IEC Electropedia: disponible à l’adresse http: //www .electropedia .org/
— ISO Online browsing platform: disponible à l’adresse http: //www .iso .org/obp
4 Principe

L’échantillon de café est extrait à l’eau ou, dans le cas de produits solubles, est dissous dans l’eau. Une

purification par extraction en phase solide est utilisée pour éliminer les composés interférents de la

matrice. Deux méthodes alternatives peuvent être utilisées pour le dosage: la chromatographie liquide

à haute performance couplée à la spectrométrie de masse (CLHP-SM/SM) ou, après une bromation de

l’acrylamide, la chromatographie en phase gazeuse couplée à la spectrométrie de masse (CG-SM). Dans

les deux cas, des solutions d’étalons internes marqués par un isotope sont utilisées.

5 Réactifs

AVERTISSEMENT — Étant donné les risques pour la santé liés à l’utilisation de l’acrylamide,

des mesures préventives et de protection appropriées doivent être prises, comme l’utilisation

d’une hotte aspirante, l’aspiration des solutions contenant de l’acrylamide uniquement avec

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une pipette et le fait d’éviter tout contact avec la peau et les yeux ou toute inhalation de vapeur

contenant de l’acrylamide.

S’ils sont disponibles, des réactifs de « qualité pour analyse de résidus » ou de « qualité réactif

analytique » doivent être utilisés. Il convient que le niveau d’impuretés dans les réactifs contribuant au

blanc soit négligeable. Le blanc doit être contrôlé régulièrement.
5.1 Eau, de qualité 1 selon l’ISO 3696, la qualité SM étant recommandée.

5.2 Gaz opératoires de grande pureté, adaptés pour la CG et la spectrométrie de masse selon les

instructions du fabricant de l’appareil.

5.3 Solvants, tels que le méthanol, l’acétate d’éthyle, l’acétonitrile, le n-hexane, la qualité SM étant

recommandée.
5.4 Acrylamide, C H NO, d’une pureté > 98 %, substance de référence.
3 5
5.4.1 Solution mère d’acrylamide, concentration massique ρ = 1 000 μg/ml.

Peser (0,10 ± 0,001) g d’acrylamide dans une fiole jaugée de 100 ml et agiter dans 30 ml d’eau pour

dissoudre l’acrylamide. Compléter jusqu’au trait de jauge avec de l’eau et bien mélanger. La solution mère

est stable pendant au moins 3 mois lorsqu’elle est conservée à l’abri de la lumière à une température

maximale de 6 °C.

Alternativement, une solution disponible dans le commerce ayant une concentration massique

de ρ = 1 000 µg/ml peut être utilisée. Les informations fournies par le fabricant en ce qui concerne la

stabilité de la solution doivent être suivies.
5.4.2 Solution d’étalonnage d’acrylamide, ρ = 10 μg/ml.

À l’aide d’une pipette, transférer (1,0 ± 0,001) ml de la solution mère d’acrylamide (5.4.1) dans une fiole

jaugée de 100 ml et compléter jusqu’au trait de jauge avec de l’eau. Cette solution doit être conservée à

l’abri de la lumière à une température maximale de 6 °C et elle doit être renouvelée tous les jours. En

fonction de la plage de travail, des étapes de dilution supplémentaires peuvent être nécessaires.

5.5 Solution d’étalon interne de D3-acrylamide (acrylamide-2,3,3-d3), C H D NO, d’une

3 2 3
pureté > 98 %, substance de référence.
5.5.1 Solution mère de D3-acrylamide (solution d’étalon interne).

Peser (0,10 ± 0,001) g de D3-acrylamide dans une fiole jaugée de 100 ml et agiter dans 30 ml d’eau

pour dissoudre le D3-acrylamide. Compléter jusqu’au trait de jauge avec de l’eau et bien mélanger. La

solution mère est stable pendant au moins 3 mois lorsqu’elle est conservée à l’abri de la lumière à une

température maximale de 6 °C.

Alternativement, une solution disponible dans le commerce ayant une concentration massique

de ρ = 1 000 µg/ml peut être utilisée. Les informations fournies par le fabricant en ce qui concerne la

stabilité de la solution doivent être suivies.
5.5.2 Solution d’étalon interne de D3-acrylamide.

À l’aide d’une pipette, transférer (1,0 ± 0,001) ml de la solution mère de D3-acrylamide (5.5.1) dans une

fiole jaugée de 100 ml et compléter jusqu’au trait de jauge avec de l’eau. Cette solution doit être conservée

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à l’abri de la lumière à une température maximale de 6 °C et elle doit être renouvelée tous les jours. En

fonction de la plage de travail, des étapes de dilution supplémentaires peuvent être nécessaires.

NOTE 1 Dans le cas de la CLHP-SM/SM, les solutions selon 5.4.1 à 5.5.2 peuvent être préparées en utilisant la

phase mobile de CLHP comme solvant. La stabilité de ces solutions dépend de la phase mobile utilisée et doit être

validée.

En cas d’utilisation de la CG-SM, toutes les solutions étalons selon 5.4.2 et 5.5.2 doivent être soumises à

une étape de dérivation conformément à 8.5.1.

NOTE 2 À la place du D3-acrylamide, il est également possible d’utiliser de l’acrylamide C pour la

préparation de la solution d’étalon interne. Toutefois, dans les articles suivants, le mode opératoire et le calcul

décrits s’appliquent uniquement au D3-acrylamide.
5.6 Eau de brome saturée.

Saturer de l’eau distillée avec du brome dans une fiole jaugée de 100 ml (munie d’un bouchon en verre),

jusqu’à ce qu’une phase de brome se forme au fond de la fiole (environ 3,5 % de brome à 4 °C). Acidifier

l’eau de brome à un pH d’environ 1 en utilisant de l’acide bromhydrique concentré (HBr, avec un poids

spécifique de 1,48 g/cm ).

Conservée à 4 °C et à l’abri de la lumière, cette solution peut être utilisée pendant environ 4 semaines.

5.7 Bromure de potassium, KBr.
5.8 Thiosulfate de sodium (pentahydraté), Na S O · 5 H O.
2 2 3 2
5.9 Triéthylamine, (C H ) N.
2 5 3
5.10 Sulfate de sodium (anhydre, granulaire), Na SO .
2 4
5.11 Solution de Carrez I.

Dissoudre 10,6 g d’hexacyanoferrate de potassium trihydraté (II) K [Fe(CN) ] · 3 H O dans 100 ml

4 6 2

d’eau. Conservée à 4 °C et à l’abri de la lumière, cette solution est stable pendant 6 mois.

5.12 Solution de Carrez II.

Dissoudre 21,9 g d’acétate de zinc dihydraté Zn(CH COO) · 2 H O dans 100 ml d’eau. Conservée à 4 °C et

3 2 2
à l’abri de la lumière, cette solution est stable pendant 6 mois.
5.13 Tampon de borate, pH 8,6.

Mélanger 68 ml d’une solution de borate de sodium à 0,1 mol/l (20,12 g de Na B O par litre d’eau)

2 4 7

et 32 ml d’acide chlorhydrique à 0,1 mol/l, c(HCl) = 0,1 mol/l, dans une fiole jaugée de 100 ml.

6 Appareillage

Utiliser le matériel courant de laboratoire et, en particulier, les appareils décrits de 6.1 à 6.14.

Le matériel et les parties du matériel qui entrent en contact avec l’échantillon et l’extrait doivent être

exempts de résidus pouvant causer des valeurs à blanc. Il faut utiliser de préférence de la verrerie ou du

matériel en acier inoxydable ou en PTFE (polytétrafluoroéthylène).
6.1 Balance analytique, réalisant des pesées à 0,1 mg près.
6.2 Moulin à café, pouvant moudre des grains de café torréfié.
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6.3 Verrerie, pour recueillir et stocker les extraits, de préférence en verre brun, telle que des flacons

pour échantillons à usage manuel ou automatique munis d’un bouchon inerte (par exemple bouchon

avec septum revêtu de PTFE).
6.4 Bain à ultrasons, pouvant être maintenu à 40 °C.

6.5 Centrifugeuse de laboratoire, convenant pour des tubes à centrifuger de 15 ml et 50 ml et avec

une vitesse de centrifugation minimale de 2 000 g.
6.6 Tubes à centrifuger, de 15 ml et 50 ml.
6.7 Fioles jaugées, de 20 ml et 100 ml.

6.8 Pipettes, en verre ou automatiques, convenant pour mesurer les plages de volume des solutions

étalons et des dilutions d’extrait d’échantillon.

6.9 Cartouches en verre ou en polypropylène, avec des adsorbants pour l’extraction en phase solide

(SPE), et pour la purification des extraits en 8.3.2 et 8.5.1 (des exemples sont donnés dans le Tableau B.1).

6.10 Chromatographe liquide haute performance (pour le mode opératoire d’essai selon 8.4),

muni d’une source électrospray (ESI) et d’une détection par spectrométrie de masse (CLHP-SM/SM);

alimentation en gaz telle que spécifiée par le fabricant.

6.11 Colonne de CLHP (pour le mode opératoire d’essai selon 8.4), adaptée pour la chromatographie

de l’acrylamide (des exemples sont donnés dans le Tableau C.1).

6.12 Chromatographe en phase gazeuse (pour le mode opératoire d’essai selon 8.5) avec détection

par spectrométrie de masse (CG-SM) et alimentation en gaz opératoires (5.2) telle que spécifiée par le

fabricant.

6.13 Colonne de CG (pour le mode opératoire d’essai selon 8.5), colonne capillaire adaptée pour la

chromatographie de l’acrylamide (des exemples sont donnés dans le Tableau C.2).

6.14 Filtres à membrane, filtres à seringues (par exemple, filtres en acétate de cellulose à 0,45 µm de

taille de pore), adaptés pour la filtration de l’éluat d’échantillon issu de l’extraction en phase solide avant

l’injection dans le système chromatographique.
7 Échantillonnage

L’échantillonnage ne fait pas partie de la méthode spécifiée dans le présent document. Le mode

opératoire d’échantillonnage doit faire l’objet d’un accord entre les parties intéressées. Un échantillon

représentatif bien homogénéisé, qui n’a pas été endommagé ou altéré lors du transport ou du stockage,

doit être utilisé.

Afin d’éviter des changements de concentration en acrylamide, l’analyse doit être effectuée peu après la

réception de l’échantillon. Les échantillons doivent être conservés au frais, à une température inférieure

à 6 °C, pendant au maximum 6 mois, à l’abri de la lumière, et ils doivent être exposés à température

ambiante uniquement pour l’analyse.

La date de réception de l’échantillon, ainsi que la date de torréfaction ou la date limite de conservation

doivent être documentées avec la date d’analyse.
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8 Mode opératoire
8.1 Généralités

Pour éviter les pertes d’analyte, il est nécessaire que les échantillons soient protégés de la lumière

pendant l’extraction et les autres étapes de préparation. C’est pourquoi il faut toujours utiliser du verre

brun. Autrement, le contenu des récipients et des fioles doit être protégé de la lumière incidente par une

feuille d’aluminium.
8.2 Préparation de l’extrait d’échantillon

Si nécessaire, moudre l’échantillon dans un moulin à café (6.2) et bien homogénéiser.

Peser 2 g d’échantillon homogénéisé de café torréfié, de café soluble ou de substitut de café ou 5 g de

boisson à base de café liquide à 1 mg près à l’aide d’une balance analytique (6.1) et transférer dans un

tube à centrifuger (6.6) de 50 ml.

Ajouter 2 ml de n-hexane dans le tube à centrifuger et agiter brièvement. Doper ensuite l’échantillon

avec une solution d’étalon interne de D3-acrylamide à une concentration correspondant au niveau

d’acrylamide attendu dans l’échantillon.

EXEMPLE Peser 2 g de café et ajouter 100 µl de solution d’étalon interne (ρ = 10 µg/ml), ce qui équivaut à

une concentration massique d’acrylamide de 500 µg/kg dans l’échantillon de café.

Ajouter 20 ml d’eau distillée, agiter brièvement mais vigoureusement, puis soumettre aux ultrasons (6.4)

pendant 15 min à environ 40 °C.

Attendre quelques minutes pour que la précipitation se produise et en cas de non-sédimentation des

échantillons, centrifuger (6.5) pendant 15 min à 2 000 g pour séparer les matières solides en suspension.

Avant la chromatographie liquide (8.4) ou la dérivation et la séparation par chromatographie en

phase gazeuse (8.5), prélever 10 ml de la phase aqueuse inférieure et la soumettre à une purification

supplémentaire selon 8.3. Prélever la phase aqueuse inférieure à l’aide d’une pipette en faisant passer

cette dernière dans la phase supérieure d’hexane sans retirer la phase d’hexane. Si nécessaire, la phase

d’hexane peut également être retirée avec précaution à l’aide d’une pipette Pasteur.

8.3 Purification des extraits
8.3.1 Précipitation de Carrez

Purifier l’extrait d’échantillon selon 8.2 par précipitation de Carrez. Ajouter 1 000 µl de solution de

Carrez I (5.11) et agiter. Ajouter 1 000 µl de solution de Carrez II (5.12) et agiter à nouveau. Après un

court temps d’exposition, centrifuger pendant 4 min à 2 000 g. Décanter le surnageant, prélever la

phase aqueuse, laver le résidu avec 2 ml à 3 ml d’eau. Centrifuger et décanter à nouveau. Combiner les

deux solutions aqueuses.
8.3.2 Extraction en phase solide

Purifier l’extrait d’échantillon après précipitation de Carrez (8.3.1) par extraction en phase solide (SPE)

en utilisant successivement deux cartouches d’adsorbants différents (des exemples sont donnés dans

le Tableau B.1). La première cartouche contient 500 mg de phase C18 et la seconde 500 mg de phase

échangeuse d’ions. Les cartouches peuvent être utilisées en série. Si appropriée, une cartouche mixte

peut être utilisée.

Conditionner les deux colonnes SPE selon les instructions du fabricant, successivement avec du

méthanol et de l’eau distillée. Placer la totalité de l’extrait d’échantillon (8.3.1) au sommet de la colonne

SPE supérieure (première colonne), laisser pénétrer et ajouter 2 ml à 3 ml d’eau. Recueillir l’éluat

jusqu’à assèchement de la cartouche. Placer l’éluat au sommet de la seconde colonne échangeuse d’ions

conditionnée (colonne inférieure), ajouter 2 ml à 3 ml d’eau et recueillir l’éluat. Une élution complète

© ISO 2016 – Tous droits réservés 5
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peut être obtenue en appliquant un léger vide ou une légère pression. Recueillir l’éluat, y compris l’eau

de lavage, dans une fiole jaugée de 20 ml et compléter jusqu’au trait de jauge avec 20 ml d’eau.

8.4 Analyse par CLHP-SM/SM
8.4.1 Chromatographie liquide à haute performance (CLHP)

Avant l’analyse par CLHP-SM/SM, ajouter du solvant organique dans l’extrait purifié (8.3.2) afin

d’obtenir la composition de phase mobile souhaitée et filtrer sur un filtre à membrane (6.14) avant

l’injection d’un volume approprié (par exemple 10 µl à 100 µl en fonction de la colonne utilisée) dans la

colonne de CLHP.

Optimiser les paramètres du système CLHP selon les instructions du fabricant. Les conditions

chromatographiques doivent être ajustées en fonction de la colonne choisie (des exemples sont donnés

dans le Tableau C.1).

Les étapes de purification selon 8.3.1 et 8.3.2 sont essentielles pour la séparation chromatographique

des pics d’analyte et des pics d’interférences. Un exemple de chromatogramme est donné

aux Figures C.1 et C.2.
8.4.2 Identification et quantification par spectrométrie de masse (CLHP-SM/SM)

Détecter l’acrylamide par SM/SM en mode d’ionisation positive (ionisation par électronébulisation, ESI).

Pour l’identification, utiliser la transition MRM m/z = 72 → 55. L’acrylamide est identifié comme présent

si le signal de l’ion fils (m/z 55) est détecté sur le chromatogramme SM/SM et si l’écart du temps de

rétention, par rapport à celui de la substance de référence analysée dans les mêmes conditions de CLHP,

est inférieur à 5 %.

Les transitions possibles pour l’acrylamide et le D3-acrylamide sont données dans le Tableau 1.

Quantifier l’analyte en comparant l’abondance des ions parents/fils de l’acrylamide avec celle de

la solution d’étalon interne marquée par un isotope en utilisant les transitions de masse 72 → 55

(acrylamide) et 75 → 58 (D3-acrylamide).

Une troisième transition de masse 72 → 54 peut être utilisée pour confirmer les résultats. L’évaluation

de cette transition ne faisait pas partie de l’étude interlaboratoires (Annexe A).

Tableau 1 — Transitions spectrométriques de masse utilisées pour l’identification et la

quantification de l’acrylamide
Substance Transitions choisies pour l’identification et la
de référence quantification de l’acrylamide par SM/SM
m/z
Acrylamide 72 → 55 Identification et quantification
72 → 44 Identification (informative, qualifiante)
D3-acrylamide 75 → 58 Identification et quantification
75 → 44 Identification (informative, qualifiante)
8.5 Analyse par CG-SM
8.5.1 Dérivation et préparation de l’échantillon pour la chromatographie en ph
...

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