1、BS ISO 18862:2016Coffee and coffee products Determination of acrylamide Methods using HPLC-MS/MSand GC-MS after derivatizationBSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06BS ISO 18862:2016 BRITISH STANDARDNational forewordThis British Standard is the UK implementa
2、tion of ISO 18862:2016.The UK participation in its preparation was entrusted to TechnicalCommittee AW/15, Coffee.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. Us
3、ers are responsible for its correctapplication. The British Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 83831 6ICS 67.140.20Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of
4、theStandards Policy and Strategy Committee on 31 July 2016.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS ISO 18862:2016 ISO 2016Coffee and coffee products Determination of acrylamide Methods using HPLC-MS/MS and GC-MS after derivatizationCaf et de ses drivs Dosage de l
5、acrylamide Mthodes utilisant CLHP-MS/MS et CG-MS aprs drivationINTERNATIONAL STANDARDISO18862First edition2016-07-15Reference numberISO 18862:2016(E)BS ISO 18862:2016ISO 18862:2016(E)ii ISO 2016 All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2016, Published in SwitzerlandAll rights reserved. Un
6、less otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the
7、 address below or ISOs member body in the country of the requester.ISO copyright officeCh. de Blandonnet 8 CP 401CH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.iso.orgBS ISO 18862:2016ISO 18862:2016(E)Foreword ivIntroduction v1 Scope . 12 Normative r
8、eferences 13 Terms and definitions . 14 Principle 15 Reagents 16 Apparatus . 37 Sampling 48 Procedure. 48.1 General . 48.2 Preparation of the sample extract 48.3 Clean-up of the extracts . 58.3.1 Carrez precipitation 58.3.2 Solid phase extraction . 58.4 HPLC-MS/MS measurement 58.4.1 High-performance
9、 liquid chromatography (HPLC) . 58.4.2 Identification and quantification by mass spectrometry (HPLC-MS/MS) . 68.5 Measurement with GC-MS . 68.5.1 Derivatization and sample preparation for gas chromatography . 68.5.2 Gas chromatography . 78.5.3 Identification and quantification by mass spectrometry 7
10、9 Calibration 79.1 General advice . 79.2 Determination of linearity and definition of the working range . 79.3 Calibration with internal standard solution 79.4 Determination of the laboratory specific recovery 810 Evaluation 810.1 Criteria for identification . 810.2 Calculation and final results 811
11、 Precision data 911.1 General . 911.2 Repeatability . 911.3 Reproducibility . 911.4 Recovery . 912 Measurement uncertainty 913 Test report 10Annex A (informative) Performance characteristics 11Annex B (informative) Examples of absorber materials .12Annex C (informative) Examples of columns and analy
12、sis conditions .13Bibliography .19 ISO 2016 All rights reserved iiiContents PageBS ISO 18862:2016ISO 18862:2016(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards
13、 is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take p
14、art in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part 1. In partic
15、ular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).Attention is drawn to the possibility that some of the elements of thi
16、s document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO list of patent declarations received (see www.
17、iso.org/patents).Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to the
18、World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 15, Coffee.iv ISO 2016 All rights reservedBS ISO 18862:2016ISO 18862:2016(E
19、)IntroductionWhen applying this document, all existing safety regulations have to be followed. ISO 2016 All rights reserved vBS ISO 18862:2016BS ISO 18862:2016Coffee and coffee products Determination of acrylamide Methods using HPLC-MS/MS and GC-MS after derivatizationWARNING The use of this documen
20、t 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 take appropriate measures for ensuring the safety and health of the personnel prior to appl
21、ication of this document and to fulfil statutory requirements for this purpose.1 ScopeThis document 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 valid
22、ated in a method validation study on roasted coffee, soluble coffee, coffee substitutes and coffee products with ranges from 53 g/kg to 612,1 g/kg.2 Normative referencesThe following documents are referred to in the text in such a way that some or all of their content constitutes requirements of thi
23、s document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.ISO 3696, Water for analytical laboratory use Specification and test methods3 Terms and definitionsNo terms and definitions are l
24、isted in this document.ISO and IEC maintain terminological databases for use in standardization at the following addresses: IEC Electropedia: available at http:/www.electropedia.org/ ISO Online browsing platform: available at http:/www.iso.org/obp4 PrincipleThe coffee sample is extracted with water
25、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
26、 a bromination of the acrylamide, gas chromatography with mass spectrometric detection (GC-MS). In both cases, isotopic labelled internal standard solutions are used.5 ReagentsWARNING In view of health risks when working with acrylamide, appropriate preventive and protection measures shall be taken,
27、 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.INTERNATIONAL STANDARD ISO 18862:2016(E) ISO 2016 All rights reserved 1BS ISO 18862:2016ISO 18862:2016(E)If available, reage
28、nts 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.5.1 Water, of grade 1 according to ISO 3696, MS-grade is recommended.5.2 Operating gases
29、of high purity, suitable for GC and mass spectrometry according to the instructions of the manufacturer of the apparatus.5.3 Solvents, such as methanol, ethyl acetate, acetonitrile, n-hexane, MS-grade is recommended.5.4 Acrylamide, C3H5NO, purity 98 %, reference substance.5.4.1 Acrylamide stock solu
30、tion, mass concentration = 1 000 g/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 3 months when stored protected f
31、rom light at a maximum of 6 C.Alternatively, a commercially available solution with a mass concentration of = 1 000 g/ml may be used. The information of the manufacturer regarding the stability of the solution shall be observed.5.4.2 Acrylamide calibration solution, = 10 g/ml.Using a pipette, transf
32、er (1,0 0,001) ml of the acrylamide stock solution (5.4.1) 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
33、 steps might be necessary.5.5 D3-acrylamide (acrylamide-2,3,3-d3) internal standard solution, C3H2D3NO, purity 98 %, reference substance.5.5.1 D3-acrylamide stock solution (internal standard solution).Weigh (0,10 0,001) g of D3-acrylamide into a 100 ml one-mark volumetric flask and swirl it in 30 ml
34、 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 3 months when stored protected from light at a maximum of 6 C.Alternatively, a commercially available solution with a mass concentration of = 1 000 g/ml may be use
35、d. The information of the manufacturer regarding the stability of the solution shall be observed.5.5.2 D3-acrylamide internal standard solution.Using a pipette, transfer (1,0 0,001) ml of the D3-acrylamide stock solution (5.5.1) into a 100 ml one-mark volumetric flask and fill up to the mark with wa
36、ter. 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.NOTE 1 For HPLC-MS/MS, the solutions according to 5.4.1 to 5.5.2 can be prepared using the HPLC eluent a
37、s a solvent. The stability of these solutions depends on the mobile phase used and has to be validated.When using GC-MS, all standard solutions according to 5.4.2 and 5.5.2 shall be subjected to the derivatization step according to 8.5.1.NOTE 2 Instead of D3-acrylamide, it is also possible to use 13
38、C3acrylamide for the preparation of the internal standard solution. However, in the following clauses, the procedure and calculation are described for D3-acrylamide only.2 ISO 2016 All rights reservedBS ISO 18862:2016ISO 18862:2016(E)5.6 Saturated bromine water.Saturate distilled water with bromine
39、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 1,48 g/cm3).If stored at 4 C
40、 and protected from light, the solution can be used for about 4 weeks.5.7 Potassium bromide, KBr.5.8 Sodium thiosulfate (pentahydrate), Na2S2O3 5 H2O.5.9 Triethylamine, (C2H5)3N.5.10 Sodium sulfate (anhydrous, granular), Na2SO4.5.11 Carrez solution I.Dissolve 10,6 g of potassium hexacyanoferrate tri
41、hydrate (II) K4Fe(CN)6 3 H2O in 100 ml of water. If stored at 4 C and protected from light, the solution is stable for 6 months.5.12 Carrez solution II.Dissolve 21,9 g of zinc acetate dihydrate Zn(CH3COO)2 2 H2O in 100 ml of water. If stored at 4 C and protected from light, the solution is stable fo
42、r 6 months.5.13 Borate buffer, pH 8,6.Mix 68 ml of a 0,1 molar sodium borate solution (20,12 g Na2B4O7per litre of water) and 32 ml of 0,1 molar hydrochloric acid, c(HCl) = 0,1 mol/l, in a 100 ml one-mark volumetric flask.6 ApparatusUsual laboratory apparatus and, in particular, apparatus according
43、to 6.1 to 6.14 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.6.1 Analytical balanc
44、e, capable of weighing to an accuracy of 0,1 mg.6.2 Coffee mill, suitable for grinding roasted coffee beans.6.3 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 se
45、ptum).6.4 Ultrasonic bath, capable of being maintained at 40 C.6.5 Laboratory centrifuge, suitable for 15 ml and 50 ml centrifugal tubes and with a minimum g-force of 2 000 g.6.6 Centrifuge tubes, of 15 ml and 50 ml.6.7 One-mark volumetric flask, of 20 ml and 100 ml. ISO 2016 All rights reserved 3BS
46、 ISO 18862:2016ISO 18862:2016(E)6.8 Pipettes, glass or automatic, suitable for measuring volume ranges of standard solutions and sample extract dilutions.6.9 Glass or polypropylene cartridges, with sorbents for the solid phase extraction (SPE), and for the clean-up of extracts in 8.3.2 and 8.5.1 (ex
47、amples are given in Table B.1).6.10 High performance liquid chromatograph (for the test procedure according to 8.4), equipped with ESI and mass spectrometric detector (HPLC-MS/MS); gas supply as specified by the manufacturer.6.11 HPLC column (for the test procedure according to 8.4), suitable for ac
48、rylamide chromatography (examples are given in Table C.1).6.12 Gas chromatograph (for the test procedure according to 8.5) with mass spectrometric detector (GC-MS) and operating gas supply (5.2) as specified by the manufacturer.6.13 GC column, (for the test procedure according to 8.5) capillary colu
49、mn, suitable for acrylamide chromatography (examples are given in Table C.2).6.14 Membrane filter units, syringe filter (e.g. cellulose acetate filters 0,45 m pore size) suitable for filtration of sample eluate obtained by solid phase extraction before injection into the chromatographic system.7 SamplingSampling is not part of the method specified in this document. The sampling procedure shall be subject to agreement by the interested parties. A representative, thoroughly mixed sample shall b
