1、BSI Standards PublicationBS EN 16618:2015Food analysis Determinationof acrylamide in food by liquidchromatography tandem massspectrometry (LC-ESI-MS/MS)BS EN 16618:2015 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 16618:2015.The UK participation in its prepar
2、ation was entrusted to TechnicalCommittee AW/275, Food analysis - Horizontal methods.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its
3、correctapplication. The British Standards Institution 2015. Published by BSI StandardsLimited 2015ISBN 978 0 580 80855 5ICS 67.050; 67.060; 67.080.20; 67.140.20Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of t
4、heStandards Policy and Strategy Committee on 30 April 2015.Amendments issued since publicationDate Text affectedBS EN 16618:2015EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16618 April 2015 ICS 67.050; 67.060; 67.080.20; 67.140.20 English Version Food analysis - Determination of acrylamide i
5、n food by liquid chromatography tandem mass spectrometry (LC-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 Acrylam
6、id in Lebensmitteln mit Flssigchromatographie 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 st
7、atus 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,
8、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, Cro
9、atia, 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
10、United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR 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.
11、 EN 16618:2015 EBS EN 16618:2015EN 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
12、chromatograms 18 Annex B (informative) Precision data . 20 Bibliography . 24 BS EN 16618:2015EN 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 Sta
13、ndard shall be given the status 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
14、this document may be the subject 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
15、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
16、 regulatory limitations prior 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 Yugos
17、lav 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 the United Kingdom. BS EN 16618:2015EN 16618:2015 (E) 4 1 Scope This
18、European Standard specifies 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 combina
19、tion with electrospray ionization 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 Administra
20、tion and validated in a study 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 performanc
21、e of the instrument. The 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
22、30 g/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
23、any amendments) applies. 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
24、. The extract is centrifuged 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. Th
25、e reason for using this column 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 cap
26、acity to bind acrylamide. 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
27、and to obtain low limits 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 mo
28、bile phase) compared to other 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 th
29、an 99,9 % mass fraction. BS EN 16618:2015EN 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
30、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, 13
31、C-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) w
32、ith 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 g/ml. Weigh, to the nearest 0,05 mg, approximately 100 mg of acrylami
33、de and acrylamide-2,3,3-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 vol
34、umetric flask and dilute to the mark with water. BS EN 16618:2015EN 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
35、g/ml. Transfer 5 000 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.
36、 4.13 Acrylamide 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 giv
37、e 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 a
38、re 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 calibrati
39、on solutions should 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
40、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
41、4 000 5 000 of (4.11) BS EN 16618:2015EN 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 pa
42、rticular, 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 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
43、The stationary 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 M
44、ass 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 i
45、nstalled 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 Multi
46、mode SPE column 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 t
47、hey can be shown to lead to the same results. BS EN 16618:2015EN 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
48、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 balance, accuracy to the nearest 0,01 g. 5.5 Calibrated precision microlitre pipettes, of 200 l to 1 000 l, and of 1 000 l to 5 000 l capacity. 5.6 Centrifuge tu
49、bes, volume of 50 ml, polypropylene, disposable. 5.7 Mechanical shaker, e.g. wrist arm shaker, allowing well mixing of different phases, capable of holding 50 ml centrifuge tubes. 5.8 Vortex mixer. 5.9 Cooled centrifuge, capable of a centrifugal force of 3 600 g for 50 ml centrifuge tubes. 5.10 Volumetric flasks, volume of 50 ml, 100 ml, etc. according to EN ISO 1042:1999. 5.11 Glass vials, volume of at least 4 ml, suitable for the evaporation equipment. 5.12 Amber glass autosampler vials, suitable for the HPLC
