EN 16618:2015

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittelanalytik - Bestimmung von Acrylamid in Lebensmitteln mit Flüssigkeitschromatographie und Tandem-Massenspektrometrie (LC-ESI-MS/MS)Analyse des produits alimentaires - Dosage de l'acrylamide dans les produits alimentaires par chromatographie en phase liquide couplée à la spectrométrie de masse en tandem (CL-ESI-SM-SM)Food analysis - Determination of acrylamide in food by liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS)67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 16618:2015SIST EN 16618:2015en,fr,de01-julij-2015SIST EN 16618:2015SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16618
April 2015 ICS 67.050; 67.060; 67.080.20; 67.140.20 English Version
Food analysis - Determination of acrylamide in food by liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS)
Analyse des produits alimentaires - Dosage de l'acrylamide dans les produits alimentaires par chromatographie en phase liquide couplée à la spectrométrie de masse en tandem (CL-ESI-SM-SM)
Lebensmittelanalytik - Bestimmung von Acrylamid in Lebensmitteln mit Flüssigchromatographie und Tandem-Massenspektrometrie (LC-ESI-MS/MS) This European Standard was approved by CEN on 7 February 2015.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, 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.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2015 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16618:2015 ESIST EN 16618:2015

Typical chromatograms . 18 Annex B (informative)
Precision data . 20 Bibliography . 24
Figure 1 — Acrylamide WARNING — Acrylamide has been classified by the International Agency for Research on Cancer (IARC) as probably carcinogenic to humans. Protective equipment as laboratory coat, disposable gloves and safety glasses shall be used. All handlings of acrylamide and organic solvents shall be performed in a fume cupboard with adequate air flow. 4.2 Deuterium-labelled acrylamide – acrylamide-2,3,3-D3 (CAS 122775-19-3). The chemical structure is:
Figure 2 — Deuterium-labelled acrylamide Alternatively, 13C-labelled acrylamide (acrylamide-13C3, CAS 287399-26-2) may be used. 4.3 Methanol (CAS 67-56-1). 4.4 Glacial acetic acid (CAS 64-19-7). 4.5 n-Hexane (CAS 110-54-3). Alternatively, cyclohexane (CAS 110-82-7) may be used. 4.6 Eluent for SPE column 2 (5.2.3) Mix 60 parts per volume of methanol (4.3) with 40 parts per volume of water. 4.7 HPLC mobile phase Mix 1 part per volume of glacial acetic acid (4.4) with 1 000 parts per volume of water. 4.8 Stock solutions of acrylamide and acrylamide-2,3,3-D3, mass concentration
= 1 000
Weigh, to the nearest 0,05 mg, approximately 100 mg of acrylamide and acrylamide-2,3,3-D3 respectively into separate 100 ml volumetric flasks, dissolve in water and dilute to 100 ml. Solutions can be stored at 4 °C for at least 3 months. 4.9 Internal standard solution 1,
= 10
Transfer 1 000 -2,3,3-D3 (4.8) to a 100 ml volumetric flask and dilute to the mark with water. SIST EN 16618:2015

= 1 000 ng/ml. Transfer 5 000
internal standard solution 1 (4.9) to a 50 ml volumetric flask and dilute to the mark with water. 4.11 Acrylamide standard solution 1,
= 100
Transfer 5 000 4.8) to a 50 ml volumetric flask and dilute to the mark with water. 4.12 Acrylamide standard solution 2,
= 10
Transfer 5 000
1 (4.11) to a 50 ml volumetric flask and dilute to the mark with water. 4.13 Acrylamide standard solution 3,
= 100 ng/ml. Transfer 1 000
2 (4.12) to a 100 ml volumetric flask and dilute to the mark with water. 4.14 LC-MS calibration solutions Dilute aliquots from standard solutions (4.9), (4.11), (4.12) and (4.13) with water to give calibration solutions of e.g. 0 ng/ml, 5 ng/ml, 10 ng/ml, 20 ng/ml, 50 ng/ml, 100 ng/ml, 250 ng/ml, 500 ng/ml, 1 000 ng/ml, 2 000 ng/ml, 5 000 ng/ml and 10 000 ng/ml respectively of acrylamide, all containing 400 ng/ml of acrylamide-2,3,3-D3. Examples for the preparation of calibration solutions are given in Table 1. Table 2 indicates the relation between calibration solution concentrations and acrylamide contents of food samples. Calibration shall be performed on at least six concentration levels distributed properly over the working range. The analysis of an even higher number of calibration solutions should be analysed if such a broad range of concentrations (0
Table 1 — Preparation of LC-MS calibration solutions Calibration solution ng/ml Volumetric flask ml Internal standard solution (4.9)
Acrylamide standard solution
0 100 4 000 0 5 100 4 000 5 000 of (4.13) 10 100 4 000 10 000 of (4.13) 20 100 4 000 200 of (4.12) 50 100 4 000 500 of (4.12) 100 100 4 000 1 000 of (4.12) 250 100 4 000 2 500 of (4.12) 500 100 4 000 5 000 of (4.12) 1 000 100 4 000 1 000 of (4.11) 2 000 100 4 000 2 000 of (4.11) 5 000 100 4 000 5 000 of (4.11) 10 000 50 4 000 5 000 of (4.11) SIST EN 16618:2015

Roasted coffee
10 10 50 5 Apparatus Usual laboratory glassware and equipment and, in particular, the following: 5.1 LC-MS/MS system 5.1.1 HPLC apparatus, comprising the following: 5.1.1.1 Thermostated column compartment. 5.1.1.2 Injection system, capable of injecting 10
5.1.1.3 Mobile phase pump, capable of maintaining a mobile phase flow of 0,4 ml/min. 5.1.2 HPLC column The stationary phase of the column is graphitized carbon1a), particle size 5
mm x 2,1 mm with a guard column1a), particle size 5
mm x 2 mm. Alternative columns/stationary phases may be applied provided that similar performance to the graphitized carbon column can be demonstrated. 5.1.3 Mass spectrometer Triple quadrupole mass spectrometer operating in positive electrospray and, selected reaction monitoring mode (SRM), set to obtain unit resolution. 5.1.4 Data acquisition and analysis system Suitable data collection and evaluation software. 5.1.5 Divert valve (optional) HPLC valve installed between HPLC column and mass spectrometer in order to direct the HPLC effluent either to waste or to the mass spectrometer, see 7.1.1. 5.2 Solid phase extraction system 5.2.1 Vacuum manifold for solid phase extraction
1) a) Hypercarb™ column, Thermo Hypersil-Keystone® column, b) ISOLUTE ® Multimode SPE column and c
...