1、June 2015Translation by DIN-Sprachendienst.English price group 13No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 67
2、.050; 67.060; 67.080.20; 67.140.20!%CZa“2325562www.din.deDDIN EN 16618Food analysis Determination of acrylamide in food by liquid chromatography tandemmass spectrometry (LC-ESI-MS/MS);English version EN 16618:2015,English translation of DIN EN 16618:2015-06Lebensmittelanalytik Bestimmung von Acrylam
3、id in Lebensmitteln mit Flssigchromatographie undTandem-Massenspektrometrie (LC-ESI-MS/MS);Englische Fassung EN 16618:2015,Englische bersetzung von DIN EN 16618:2015-06Analyse des produits alimentaires Dosage de lacrylamide dans les produits alimentaires par chromatographie en phaseliquide couple la
4、 spectromtrie de masse en tandem (CL-ESI-SM-SM);Version anglaise EN 16618:2015,Traduction anglaise de DIN EN 16618:2015-06www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Document comprises 26 pages06.15 DIN EN 16618:2015-06 2 A comma is used as the decima
5、l marker. National foreword This document (EN 16618:2015) has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). The responsible German body involved in its preparation was the DIN-Normenausschuss Lebensmittel und landwirtschaftliche Produ
6、kte (DIN Standards Committee Food and Agricultural Products), Working Committee NA 057-01-14 AA Prozesskontaminanten. 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
7、-ESI-MS/MS) Analyse des produits alimentaires - Dosage de lacrylamide dans les produits alimentaires par chromatographie en phase liquide couple la spectromtrie de masse en tandem (CL-ESI-SM-SM) Lebensmittelanalytik - Bestimmung von Acrylamid in Lebensmitteln mit Flssigchromatographie und Tandem-Mas
8、senspektrometrie (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-da
9、te 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
10、 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, For
11、mer 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 andUnited Kingdom. CEN-CENELEC Management Centre: Avenue Marnix
12、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 EEUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGE
13、N 16618:2015 (E) 2 Contents Page Foreword 3 1 Scope 4 2 Normative references 4 3 Principle 4 4 Reagents .4 5 Apparatus .7 6 Sample preparation .8 7 Measurement . 10 8 Determination of concentrations 13 9 Precision 15 10 Test report . 17 Annex A (informative) Typical chromatograms 18 Annex B (informa
14、tive) Precision data . 20 Bibliography . 24 DIN EN 16618:2015-06EN 16618:2015 (E) 3 Foreword This document (EN 16618:2015) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This European Standard shall be given the stat
15、us of a national standard, either by publication of an identical text or by endorsement, at the latest by October 2015 and conflicting national standards shall be withdrawn at the latest by October 2015. Attention is drawn to the possibility that some of the elements of this document may be the subj
16、ect of patent rights. CEN and/or CENELEC 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. WARNING The use of this document can involve hazardou
17、s 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
18、 to use. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, Fr
19、ance, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. DIN EN 16618:2015-06EN 16618:2015 (E) 4 1 Scope This European Standard specifi
20、es a method for the determination of acrylamide in bakery ware such as bread, toasted bread, crisp bread, butter cookies, and biscuits, as well as potato products such as potato chips, potato crisps, and potato pan cake and roasted coffee, by liquid chromatography in combination with electrospray io
21、nization and tandem mass spectrometry (LC-ESI-MS/MS). This method has been validated in an interlaboratory study via the analysis of both naturally contaminated and spiked samples, ranging from 14,3 g/kg to 9 083 g/kg. It was developed at the Swedish National Food Administration and validated in a s
22、tudy organized by the Directorate General Joint Research Centre (DG JRC), Swedish National Food Administration and the Nordic Committee on Food Analysis (NMKL), see 1 and 2. The limit of quantification (LOQ) depends on the type of instrument used and on the actual performance of the instrument. The
23、majority of the laboratories participating in the validation study were able to determine acrylamide in a butter cookie sample at a level of 14,3 g/kg. Thus, the validation by interlaboratory study showed that LOQ can be expected to be in the range between below 15 g/kg and 30 g/kg. 2 Normative refe
24、rences 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.
25、EN ISO 1042:1999, Laboratory glassware - One-mark volumetric flasks (ISO 1042:1998) EN ISO 3696:1995, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987) 3 Principle Acrylamide is extracted with water and isotopic labelled acrylamide is added. The extract is centrifu
26、ged and the supernatant is cleaned up with two solid phase extraction (SPE) columns. The first SPE column contains silica based C18 groups as well as anion and cation exchangers, and since acrylamide is not retained by the column, the extract is just passed and collected. The reason for using this c
27、olumn is to retain as many matrix components as possible (non-polar compounds as well as anions and cations) without retaining acrylamide, i.e. this first SPE column is used as a chemical filter. The second SPE column contains a polymer based phase with a relatively high capacity to bind acrylamide.
28、 The extract is loaded onto the column, the column is washed with water and finally eluted with a mixture of 60 parts per volume of methanol and 40 parts per volume of water. The purpose of this step, apart from further cleaning of the extract, is to concentrate the extract and to obtain low limits
29、of quantification. After evaporation of the methanol, the extract is analysed by LC-MS/MS. For this purpose an HPLC column with graphitized carbon as stationary phase is used, since the retention factor (k) is relatively high (k = 4 when no organic solvent is added in the mobile phase) compared to o
30、ther commercially available columns. 4 Reagents Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696:1995, unless otherwise specified. Solvents shall be of quality for HPLC analysis. 4.1 Acrylamide (CAS 79-06-1), purity not less than 99,9 % mass fraction.
31、DIN EN 16618:2015-06EN 16618:2015 (E) 5 The chemical structure is: 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 sh
32、all 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
33、 (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 volu
34、me 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 g/ml. Weigh, to the nearest 0,05 mg, approximately 100 mg of acrylamide and acrylamide-2,3
35、,3-D3respectively 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 g/ml. Transfer 1 000 l of the stock solution of acrylamide-2,3,3-D3(4.8) to a 100 ml volumetric flask and dil
36、ute to the mark with water. DIN EN 16618:2015-06EN 16618:2015 (E) 6 4.10 Internal standard solution 2, = 1 000 ng/ml. Transfer 5 000 l of the 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 g/ml. Transfer 5
37、000 l of the stock solution of acrylamide (4.8) to a 50 ml volumetric flask and dilute to the mark with water. 4.12 Acrylamide standard solution 2, = 10 g/ml. Transfer 5 000 l of the acrylamide standard solution 1 (4.11) to a 50 ml volumetric flask and dilute to the mark with water. 4.13 Acrylamide
38、standard solution 3, = 100 ng/ml. Transfer 1 000 l of the acrylamide standard solution 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 sol
39、utions 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
40、 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 shou
41、ld be analysed if such a broad range of concentrations (0 g/kg to 10 000 g/kg) shall be covered. Table 1 Preparation of LC-MS calibration solutions Calibration solution ng/ml Volumetric flask ml Internal standard solution (4.9) l Acrylamide standard solution l 0 100 4 000 0 5 100 4 000 5 000 of (4.1
42、3) 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
43、.11) DIN EN 16618:2015-06EN 16618:2015 (E) 7 Table 2 Relation between acrylamide contents of calibration solutions and contents in food Calibration solution ng/ml Bakery and potato products g/kg Roasted coffee g/kg 10 10 50 5 Apparatus Usual laboratory glassware and equipment and, in particular, the
44、 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 l of sample. 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 stationar
45、y phase of the column is graphitized carbon1a), particle size 5 m, 50 mm x 2,1 mm with a guard column1a), particle size 5 m, 10 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 spectrome
46、ter 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 betw
47、een 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 colu
48、mn and c) ISOLUTE ENV+ SPE column from Biotage are examples of suitable products available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of these products. Equivalent products may be used if they can be sh
49、own to lead to the same results. DIN EN 16618:2015-06EN 16618:2015 (E) 8 5.2.2 SPE column 1 Multimode SPE column comprising non-polar, strong anion exchange, and strong cation exchange properties1b), 1 000 mg/6 ml. 5.2.3 SPE column 2 Crosslinked polystyrene-divinylbenzene copolymer for the extraction of polar analytes from aqueous samples1c), 500 mg/6 ml. 5.3 Analytical balance, accuracy to the nearest 0,01 mg. 5.4 Laboratory
