Foodstuffs - Determination of acrylamide in food and coffee by gas chromatography-mass spectrometry (GC-MS)

This Technical Specification specifies a method for the determination of acrylamide in cereal-based products, potato-based products and coffee by gas-chromatography mass spectrometry (GC-MS).
The method has been single-laboratory validated via the analysis of spiked samples (French fries (uncooked), bread, water biscuit, infant cereal, biscuit, green coffee, roast coffee and instant coffee), ranging from 30 μg/kg to 1 500 μg/kg acrylamide.
The results from the single laboratory validation were obtained by a laboratory with significant experience in acrylamide analysis. In addition, this method has also been studied by inter laboratory trial via the analysis of samples containing incurred acrylamide, ranging from approximately 200 μg/kg to 2 000 μg/kg. Critical points of the method are identified in 7.5 and Clause 8.

Lebensmittel - Bestimmung von Acrylamid in Lebensmitteln und Kaffee mit Gaschromatographie-Massenspektrometrie (GC-MS)

Diese Technische Spezifikation legt ein Verfahren zur Bestimmung von Acrylamid in Getreideerzeugnissen, Kartoffelerzeugnissen und Kaffee durch Gaschromatographie gekoppelt mit Massen¬spektrometrie (GC MS) fest.
Dieses Verfahren wurde in einer Einzellaborvalidierung durch die Analyse von aufgestockten Proben (Pommes frites (ungegart), Brot, Kräcker, Getreidebeikost, Kekse, Rohkaffee, Röstkaffee und löslicher Kaffee) im Bereich von 30 μg/kg bis 1 500 μg/kg Acrylamid untersucht.
Die Ergebnisse der Einzellaborvalidierung wurden von einem Labor mit umfangreicher Erfahrung in der Acrylamid Analytik erzielt. Außerdem wurde dieses Verfahren auch in einem Ringversuch durch Untersuchung von Proben mit einem Anteil an entstandenem Acrylamid von etwa 200 μg/kg bis 2 000 μg/kg untersucht. Kritische Punkte des Verfahrens werden in 7.5 und Abschnitt 8 benannt.

Produits alimentaires - Dosage par CG-SM des produits alimentaires et café avec GC-MS

Živila - Določevanje akrilamida v živilih in kavi s plinsko kromatografijo/masno spektrometrijo (GC-MS)

Ta tehnična specifikacija opredeljuje metodo za določanje akrilamida v žitnih proizvodih, izdelkih iz krompirja in kavi s plinsko kromatografijo/masno spektrometrijo (GC-MS).
Metoda je bila validirana v laboratoriju z analizo primešanih vzorcev (krompirček, (nekuhan), kruh, krekerji, žita za dojenčke, piškoti, surova kava, pražena in instant kava) v razponu 30–1500 μg/kg akrilamida.
Rezultati validacije laboratorija so bili pridobljeni v laboratoriju z bogatimi izkušnjami z analizo akrilamida. Poleg tega je bila ta metoda raziskana tudi z medlaboratorijskim preskusom z analizo vzorcev, ki so vsebovali akrilamid v razponu približno 200–2000 μg/kg. Kritične točke metode so navedene v točkah 7.5 in 8.

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SLOVENSKI STANDARD
SIST-TS CEN/TS 17083:2017
01-september-2017

äLYLOD'RORþHYDQMHDNULODPLGDYåLYLOLKLQNDYLVSOLQVNRNURPDWRJUDILMRPDVQR

VSHNWURPHWULMR *&06

Foodstuffs - Determination of acrylamide in food and coffee by gas chromatography-

mass spectrometry (GC-MS)
Lebensmittel - Bestimmung von Acrylamid in Lebensmitteln und Kaffee mit
Gaschromatographie-Massenspektrometrie (GC-MS)

Produits alimentaires - Dosage par CG-SM des produits alimentaires et café avec GC-

Ta slovenski standard je istoveten z: CEN/TS 17083:2017
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
SIST-TS CEN/TS 17083:2017 en,fr,de

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

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SIST-TS CEN/TS 17083:2017
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SIST-TS CEN/TS 17083:2017
CEN/TS 17083
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
June 2017
TECHNISCHE SPEZIFIKATION
ICS 67.050; 67.060; 67.140.20; 67.080.20
English Version
Foodstuffs - Determination of acrylamide in food and
coffee by gas chromatography-mass spectrometry (GC-MS)

Produits alimentaires - Dosage par CG-SM des produits Lebensmittel - Bestimmung von Acrylamid in

alimentaires et café avec GC-MS Lebensmitteln und Kaffee mit Gaschromatographie-
Massenspektrometrie (GC-MS)

This Technical Specification (CEN/TS) was approved by CEN on 14 May 2017 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to

submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS

available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in

parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

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

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

Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels

© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 17083:2017 E

worldwide for CEN national Members.
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Contents Page

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

1 Scope .................................................................................................................................................................... 4

2 Normative references .................................................................................................................................... 4

3 Principle ............................................................................................................................................................. 4

4 Reagents ............................................................................................................................................................. 4

5 Standard solution preparation .................................................................................................................. 6

6 Apparatus ........................................................................................................................................................... 7

7 Procedure........................................................................................................................................................... 8

8 GC-MS analysis ............................................................................................................................................... 10

9 Calculating and reporting ......................................................................................................................... 12

10 Test report ...................................................................................................................................................... 12

11 Precision data ................................................................................................................................................ 13

Annex A (informative) Typical chromatograms ............................................................................................ 14

Annex B (informative) GC-MS troubleshooting .............................................................................................. 16

Annex C (informative) Single laboratory validation .................................................................................... 18

Annex D (informative) Interlaboratory trial precision data ..................................................................... 20

Bibliography ................................................................................................................................................................. 22

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European foreword

This document (CEN/TS 17083:2017) has been prepared by Technical Committee CEN/TC 275 “Food

analysis - Horizontal methods”, the secretariat of which is held by DIN.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

This document has been prepared under a mandate given to CEN by the European Commission and the

European Free Trade Association.
Annexes A, B, C and D are informative.

WARNING 1 — The use of this Technical Specification 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 establish

appropriate safety and health practices and determine the applicability of regulatory limitations

prior to use.

WARNING 2 — Some precaution is required when using polyacrylamide-based plastics because

acrylamide may leach from these materials.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the

following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,

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

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

Turkey and the United Kingdom.
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1 Scope

This Technical Specification specifies a method for the determination of acrylamide in cereal-based

products, potato-based products and coffee by gas-chromatography mass spectrometry (GC-MS).

The method has been single-laboratory validated via the analysis of spiked samples (French fries

(uncooked), bread, water biscuit, infant cereal, biscuit, green coffee, roast coffee and instant coffee),

ranging from 30 μg/kg to 1 500 μg/kg acrylamide.

The results from the single laboratory validation were obtained by a laboratory with significant

experience in acrylamide analysis. In addition, this method has also been studied by inter laboratory

trial via the analysis of samples containing incurred acrylamide, ranging from approximately 200 μg/kg

to 2 000 μg/kg. Critical points of the method are identified in 7.5 and Clause 8.

2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are

indispensable for its application. For dated references, only the edition cited applies. For undated

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

EN ISO 3696:1995, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987)

3 Principle

The test portion is extracted with hot water and isotopically labelled acrylamide is added as internal

standard. High-fat samples are defatted with hexane, cleared with Carrez solution and centrifuged.

Sample extracts are brominated and extracted with ethyl acetate. Following removal of the ethyl acetate

by evaporation, triethylamine is added to partially debrominate, after which a portion of the sample

extract is injected onto a GC-MS system for quantification. The chromatographic separation is obtained

on a mid-polarity capillary GC column. The acrylamide derivative is ionized at 70 eV and recorded in

selected ion monitoring (SIM) mode, and quantified by comparison with the stable isotopically labelled

analogue.
4 Reagents

Use only reagents of recognized analytical grade and water complying with grade 1 of

EN ISO 3696:1995 (electrical conductivity below 0,1 µS/cm at 25 °C), unless specified otherwise.

Standard solutions are preferably prepared gravimetrically. An analytical balance (6.1) is used for the

preparation of both native and stable isotope labelled acrylamide.

WARNING — Acrylamide has been classified by the International Agency for Research on Cancer (IARC)

as probably carcinogenic to humans (see [2]). Bromine is very toxic and corrosive and hydrobromic

acid is corrosive and hazardous. The prepared brominating solution (see 4.17) shall be considered as

very toxic and corrosive.

Protective equipment such as laboratory coat, disposable gloves and safety glasses shall be used. All

handlings of acrylamide, bromine, hydrobromic acid, brominating solution and organic solvents shall be

performed in a fume cupboard with adequate air flow.

IMPORTANT — Dispose chemical waste according to applicable environmental rules and regulations.

Bromine is very toxic to aquatic organisms hence discharge into the environment shall be avoided.

Waste shall be disposed of appropriately.
4.1 Acrylamide, purity ≥ 99 %.
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13 13 13

4.2 [1,2,3- C ]-acrylamide, (acrylamide- C ) isotopic C purity 99 %, supplied as 1 000 µg/ml

3 3
solution

It is permissible to use deuterated acrylamide (acrylamide-d ) as an alternative internal standard. In

the following sections of the procedure, MS detection and calculation are prescribed for acrylamide- C

only.
4.3 Bromine, purity ≥ 99 %.
4.4 Ethyl acetate, MS grade.
4.5 Helium purified compressed gas, (purity equivalent to 99,995 % or better).
4.6 n-Hexane, MS grade.
4.7 Hydrobromic acid, 48 % in water, assay ≥ 99,9 %.
4.8 Ice, crushed.
4.9 Magnesium sulfate, anhydrous powder.
4.10 Potassium bromide, purity ≥ 98,5 %.

4.11 Potassium hexacyanoferrate trihydrate (II), K Fe(CN) .3H O, purity ≥ 98,5 %.

4 6 2
4.12 Sodium chloride.
4.13 Sodium thiosulfate pentahydrate, Na S O ·5 H O, purity ≥ 99,5 %.
2 2 3 2

4.14 Sodium thiosulfate solution, substance concentration c = 1 mol/l. Dissolve 24,82 g of sodium

thiosulfate pentahydrate in 100 ml of water.
H ) , purity ≥ 99 %.
4.15 Triethylamine, N(C2 5 3
4.16 Zinc acetate dihydrate, Zn(CH COO) •2 H O, purity ≥ 98,5 %.
3 2 2
4.17 Brominating solution.

Wear gloves and carry out the following steps in a fume cupboard. Prepare fresh reagent every three

months.

For preparation of bromine water (saturated), place a 1 l glass bottle containing ca. 400 ml of water in a

crushed ice bath and stir with a magnetic stirrer for ca. 10 min. Using a measuring cylinder, add

15 ml ± 1 ml of bromine (4.3), stopper the flask and continue stirring for at least 60 min. Cap securely

and store at 4 °C to 6 °C.

Weigh 400 g ± 2 g of potassium bromide (4.10) into a 2 l screw-capped glass bottle and add

1 000 ml ± 5 ml of water. Shake or stir to dissolve then add 20 ml ± 1 ml of hydrobromic acid (4.7) using

a measuring cylinder.

This is an example of a suitable product available commercially, manufactured by Cambridge Isotope Laboratories

Inc, Andover MA, USA. CLM-813-1.2 or similar. This information is given for the convenience of users of this Technical

Specification and does not constitute an endorsement of these products by CEN. Equivalent products may be used if they

provide similar results.
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Using measuring cylinders, add 320 ml ± 5 ml of saturated bromine water followed by 660 ml ± 5 ml of

water to the glass bottle (6.3) and mix well. Stopper and store at 4 °C to 6 °C. Discard after 3 months. To

dispose of excess saturated bromine water, add solid sodium thiosulfate pentahydrate (4.13) and swirl

until the brown colour disappears.
4.18 Carrez solution I.

Dissolve 10,6 g of potassium hexacyanoferrate trihydrate (II) (4.11) in 100 ml of water. Discard after

6 months.
4.19 Carrez solution II.

Dissolve 21,9 g of zinc acetate dihydrate (4.16) in 100 ml of water. Discard after 6 months.

5 Standard solution preparation
5.1 General

Prepare all standard solutions preferably gravimetrically. The tare masses of all recipients and the

masses after each preparation step are recorded and used for calculation of the standard

concentrations. Volumetric standard preparation may also be applied provided that the volumetric

glassware used complies with EN ISO 1042 (see [1]).

5.2 Acrylamide stock standard solution, mass concentration ρ approximately 1 000 µg/ml.

Weigh 0,1 g ± 0,001 g of acrylamide into a 100 ml volumetric flask. Add 30 ml of water, swirl to dissolve

and dilute to the mark with water and mix well. The stock solution is stable for 1 month when stored in

a refrigerator (4 °C to 6 °C) and protected from light.

Alternatively, commercially-available certified standard solutions may be used if available.

5.3 Acrylamide working standard solution, ρ approximately 1 µg/ml.

Pipette 100 µl of acrylamide stock standard solution (5.2) into a 100 ml volumetric flask, dilute to

100 ml with water and mix well. The solution is stable for 2 weeks when stored in a refrigerator (4 °C to

6 °C) and protected from light.
5.4 Acrylamide spiking solution, ρ approximately 100 µg/ml.

Pipette 10 ml of acrylamide stock solution (5.2) into a 100 ml volumetric flask, dilute to 100 ml with

water and mix well. The solution is stable for 2 weeks when stored in a refrigerator (4 °C to 6 °C) and

protected from light.
5.5 C -Acrylamide internal standard solution, ρ approximately 20 µg/ml.

Pipette 200 µl of C -acrylamide stock standard solution (4.2) into a 10 ml volumetric flask dilute to

10 ml with water. The solution is stable for 2 weeks when stored in a refrigerator (4 °C to 6 °C) and

protected from light.
5.6 Calibration standards

Prepare calibration standards of approximately 0 µg/l, 1 µg/l, 3 µg/l, 5 µg/l, 10 µg/l, 20 µg/l and

100 μg/l according to the following scheme. Pipette 15 ml of water into each of seven 40 ml screw-cap

vials and add acrylamide working standard solution (5.3) and C -acrylamide internal standard

solution (5.5) to each vial as detailed in Table 1. Carry out the bromination step (7.4) on each

calibration standard.
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It should be noted that a little bias caused by the different volumes of the calibration standard solutions

when calculated as given in 8.8 and 9.1 is assumed to be negligible. Alternatively, to avoid this bias, the

calibration standards may be prepared as follows.

The volume of water may be adjusted to give the same concentration of internal standard in each vial

(i.e. by using a 15 ml volumetric flask which will give a nominal internal standard concentration of

20 µg/l). The concentration (i.e. linear working) range of the calibration standards is for guidance only.

Other standard concentrations may be prepared if required but shall take into account expected

acrylamide levels.
Table 1 — Calibration standards (nominal values)
Water Working Internal Concentration Concentration Ratio of A:B
(ml) standard standard acrylamide internal
solution (5.3) solution (5.5) (A) standard (B)
(µl) (µl) (µg/l) (µg/l)
15 0 15 0 19,98 0
15 15 15 1,00 19,96 0,05
15 45 15 2,99 19,92 0,15
15 75 15 4,97 19,88 0,25
15 150 15 9,89 19,78 0,50
15 300 15 19,59 19,59 1,00
15 1 500 15 90,83 18,17 5,00
6 Apparatus

WARNING — All glassware shall be meticulously cleaned (except disposable glassware).

Usual laboratory glassware and equipment and, in particular, the following:
6.1 Analytical balance, capable of weighing to an accuracy of ± 0,0001 g.
6.2 Bottle, glass or plastic (polypropylene), ca. 175 ml to 250 ml capacity.
6.3 Bottle, glass 1 l and 2 l capacity.
6.4 Centrifuge, capable of ≥ 3 000 g, suitable for 50 ml centrifuge tubes.
6.5 Centrifuge tubes, 50 ml capacity.

6.6 Gas chromatography mass spectrometry (GC-MS) apparatus, comprising the following:

6.6.1 Injection system, split-splitless injector, suitable for temperatures up to 200 °C.

A programmed temperature vaporizing (PTV) injector may be used as an alternative to a split/splitless

injector.

6.6.2 GC oven, suitable for temperatures up to 300 °C and capable of temperature programming.

6.6.3 Sample carousel, suitable for use with vials and caps (6.13).
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6.6.4 GC capillary column, DB 35 (35 %-Phenyl)-dimethylpolysiloxane), length of 30 m, internal

diameter of 0,25 mm, d = 0,25 μm, or any column with comparable separation characteristics.

The use of a suitable guard column is highly recommended e.g. deactivated silica 5 m.

6.6.5 An interface to a mass spectrometer, with a temperature control device, suitable for

temperatures up to 250 °C.
6.6.6 A mass spectrometer with the following characteristics:
— electron ionization source;
— ionization energy of 70 eV;
— mass resolution of at least 1;

— temperature control devices for the ion source (280 °C), the GC-MS interface (280 °C). Optionally a

temperature control device for the quadrupole (150 °C);

— tuning stability of at least 48 h (allowing for the analysis of a sequence of samples and standards);

— response linearity range of at least two orders of magnitude.

6.7 Heater, dry-block or similar with nitrogen stream, thermostatically controlled at 40 °C ± 1 °C.

6.8 Pipettes, calibrated positive displacement 15 µl to 1 500 µl capacity.
6.9 Pipettes, glass or calibrated air displacement 4 ml to 10 ml capacity.
6.10 Shaker, orbital type.
6.11 Syringe filters, polytetrafluoroethylene (PTFE) 0,45 µm, 13 mm diameter.
6.12 Vials, glass screw cap 4 ml and 40 ml capacity.
6.13 Vials, glass for use with GC injection apparatus (6.6.3).
7 Procedure
7.1 General

Residues of acrylamide have occasionally been detected in laboratory wares e.g. filters. Acrylamide may

also be formed as an artefact in certain analytical procedures e.g. during extraction or in the GC injector

port. Reagent blank samples shall therefore be included as controls with each batch (series) of samples

to ensure that there is no significant background interference.

It has been proven that acrylamide is extracted sufficiently from various types of food by shaking with

water provided that the sample particle size is sufficiently small. To ensure that the particle size

is < 1 mm before extraction, the use of mechanical homogenization is strongly advised.

This is an example of a suitable product available commercially. This information is given for the convenience of the

users of this Technical Specification and does not constitute an endorsement by CEN of the product named. Equivalent

products may be used if they provide similar results.
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7.2 Sample treatment

If necessary, the sample is milled, ground or homogenized by suitable means. Allow frozen samples to

defrost fully by storing in a refrigerator (4 °C to 6 °C) overnight and then allow them to reach room

temperature before carrying out the following steps. Heat until boiling a sufficient amount of water to

carry out step 7.3 for all samples.
7.3 Test sample preparation

Weigh 5 g of homogeneous food sample, or 1 g of coffee (roasted coffee, soluble coffee or substitute

coffee) to the nearest 0,01 g into a bottle (6.2) and add 100 µl of internal standard soluti

...

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