oSIST prEN 16987:2016
(Main)Foodstuffs - Determination of acrylamide in coffee and coffee products by HPLC-MS/MS and GC-MS
Foodstuffs - Determination of acrylamide in coffee and coffee products by HPLC-MS/MS and GC-MS
This standard 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.
Lebensmittel - Bestimmung von Acrylamid in Kaffee und Kaffee-Erzeugnissen mit HPLC-MS/MS und GC-MS
Diese Norm legt Verfahren zur Bestimmung von Acrylamid in Kaffee und Kaffee-Erzeugnissen durch Extraktion mit Wasser, Aufreinigung mit Festphasenextraktion und Bestimmung mittels HPLC-MS/MS bzw. GC-MS fest. Es wurde in einem Ringversuch für Röstkaffee, löslichen Kaffee, Kaffeesurrogate und Kaffee-Erzeugnisse in Bereichen von 53 μg/kg bis 612,1 μg/kg validiert.
Produits alimentaires - Détermination de la teneur en acrylamide dans le café et les produits du café par CLHP-SM/SM et CG-SM
La présente norme spécifie des méthodes de détermination de la teneur en acrylamide dans le café et les produits du café par extraction à l’eau, puis purification par extraction en phase solide et détermination par CLHP-SM/SM et CG-SM. Elle a été validée lors d’une étude de validation de méthode sur le café torréfié, le café soluble, les succédanés de café et les produits du café dans des gammes allant de 53 μg/kg à 612,1 μg/kg.
Živila - Določevanje akrilamida v kavi in proizvodih iz kave s HPLC-MS/MS in GC-MS
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN 16987:2016
01-junij-2016
äLYLOD'RORþHYDQMHDNULODPLGDYNDYLLQSURL]YRGLKL]NDYHV+3/&0606LQ*&
06
Foodstuffs - Determination of acrylamide in coffee and coffee products by HPLC-MS/MS
and GC-MS
Lebensmittel - Bestimmung von Acrylamid in Kaffee und Kaffee-Erzeugnissen mit HPLC-
MS/MS und GC-MS
Produits alimentaires - Détermination de la teneur en acrylamide dans le café et les
produits du café par CLHP-SM/SM et CG-SM
Ta slovenski standard je istoveten z: prEN 16987
ICS:
67.140.20 Kava in kavni nadomestki Coffee and coffee substitutes
oSIST prEN 16987:2016 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
oSIST prEN 16987:2016
---------------------- Page: 2 ----------------------
oSIST prEN 16987:2016
DRAFT
EUROPEAN STANDARD
prEN 16987
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2016
ICS 67.140.20
English Version
Foodstuffs - Determination of acrylamide in coffee and
coffee products by HPLC-MS/MS and GC-MS
Lebensmittel - Bestimmung von Acrylamid in Kaffee
und Kaffee-Erzeugnissen mit HPLC-MS/MS und GC-MS
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 275.
If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.
This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
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.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2016 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 16987:2016 E
worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
oSIST prEN 16987:2016
prEN 16987:2016 (E)
Contents Page
Introduction . 3
European foreword . 3
1 Scope . 5
2 Normative references . 5
3 Principle . 5
4 Reagents . 5
5 Apparatus . 7
6 Procedure. 8
6.1 General . 8
6.2 Preparation of the sample extract . 8
6.3 Clean-up of the extracts . 8
6.3.1 Carrez precipitation . 8
6.3.2 Solid phase extraction . 8
6.4 HPLC-MS/MS measurement . 9
6.4.1 High performance liquid chromatography (HPLC). 9
6.4.2 Identification and quantification by mass spectrometry (HPLC-MS/MS) . 9
6.5 Measurement with GC-MS . 10
6.5.1 Derivatisation and sample preparation for gas chromatography . 10
6.5.2 Gas chromatography . 10
6.5.3 Identification and quantification by mass spectrometry . 10
7 Calibration . 11
7.1 General advice . 11
7.2 Determination of linearity and definition of the working range . 11
7.3 Calibration with internal standard . 11
7.4 Determination of the laboratory specific recovery . 11
8 Evaluation . 12
8.1 Criteria for identification . 12
8.2 Calculation and final results. 12
9 Precision data . 12
9.1 General . 12
9.2 Repeatability . 12
9.3 Reproducibility . 13
9.4 Recovery . 13
10 Measurement uncertainty . 13
11 Test report . 13
Annex A (informative) Performance characteristics . 14
Annex B (informative) Examples of absorber materials. 15
Annex C (informative) Examples for recommended columns and analysis conditions . 16
Bibliography . 23
2
---------------------- Page: 4 ----------------------
oSIST prEN 16987:2016
prEN 16987:2016 (E)
European foreword
This document (prEN 16987:2016) has been prepared by Technical Committee CEN/TC 275 “Food
analysis - horizontal methods”, the secretariat of which is held by DIN.
This document is currently submitted to the CEN Enquiry.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
3
---------------------- Page: 5 ----------------------
oSIST prEN 16987:2016
prEN 16987:2016 (E)
Introduction
When applying this European Standard, all existing safety regulations should be followed.
Annexes A to C are informative.
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 establish appropriate safety and
health practices and determine the applicability of regulatory limitations prior to use.
4
---------------------- Page: 6 ----------------------
oSIST prEN 16987:2016
prEN 16987:2016 (E)
1 Scope
This standard 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, 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, Water for analytical laboratory use — Specification and test methods (ISO 3696)
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, 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 EN 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 apparatus.
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 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 three 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
---------------------- Page: 7 ----------------------
oSIST prEN 16987:2016
prEN 16987:2016 (E)
4.4.2 Acrylamide calibration solution, ρ = 10 μg/ml.
Using a pipette, transfer (1,0 ± 0,001) ml of the acrylamide stock 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 solution (internal standard),
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 three 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.
4.5.2 D3-acrylamide internal standard solution.
Using a pipette, transfer (1,0 ± 0,001) ml of the D3-acrylamide stock solution (4.5.1) 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.
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 solvent 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 6.5.1.
13
Instead of D3-acrylamide, it is also possible to use C acrylamide for the preparation of the internal
3
standard solution. However in the following clauses the procedure and calculation are described for D3-
acrylamide only.
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
3
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.
4.8 Sodium thiosulfate (pentahydrate), Na S O · 5 H O.
2 2 3 2
4.9 Triethylamine, (C H ) N.
2 5 3
4.10 Sodium sulfate (anhydrous, granular), Na SO
2 4.
4.11 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.
6
---------------------- Page: 8 ----------------------
oSIST prEN 16987:2016
prEN 16987:2016 (E)
4.12 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.
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
2 4 7
0,1 molar 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, of 15 ml and 50 ml.
5.8 One-mark volumetric flask, 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 6.3.2 and/or 6.5.1 (examples are given in Table B.1).
5.11 High performance liquid chromatograph (for the test procedure according to 6.4), 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 6.5) 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 6.5) capillary column, suitable for acrylamide
chromatography (examples are given in Table C.2).
7
---------------------- Page: 9 ----------------------
oSIST prEN 16987:2016
prEN 16987:2016 (E)
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.
6 Procedure
6.1 General
To avoid losses of the analyte, it is necessary that the samples are protected from light during extraction
and further preparation. Therefore, amber glassware should always be used. Otherwise, the content of
the vessels and flasks shall be protected from incident light using aluminium foil.
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.
6.2 Preparation of the sample extract
If necessary, grind the sample in a coffee mill (5.3) 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 (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 (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 (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 (6.4) or derivatization
and gas chromatographic separation (6.5), take 10 ml from the lower aqueous phase and use it for a
further clean-up according to 6.3. 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.
6.3 Clean-up of the extracts
6.3.1 Carrez precipitation
Clean-up the sample extract prepared according to 6.2 by Carrez precipitation. Add 1 000 µl of Carrez
solution I (4.11) and shake. Add 1 000 µl of 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 again and decant. Combine both aqueous solutions.
6.3.2 Solid phase extraction
Clean-up the sample extract after Carrez precipitation (6.3.1) by solid phase extraction (SPE) using two
sequential cartridges with adsorber material (examples are given in Table B.1). The first cartridge
8
---------------------- Page: 10 ----------------------
oSIST prEN 16987:2016
prEN 16987:2016 (E)
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 (6.3.1) on top of the upper (first) SPE column,
allow t
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.