1、BS EN ISO 18465:2017Microbiology of the foodchain Quantitativedetermination of emetic toxin(cereulide) using LC-MS/MS(ISO 18465:2017)BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06BS EN ISO 18465:2017 BRITISH STANDARDNational forewordThis British Standard is the UK
2、implementation of EN ISO 18465:2017. The UK participation in its preparation was entrusted to Technical Committee AW/9, Microbiology.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provis
3、ions of a contract. Users are responsible for its correct application. The British Standards Institution 2017.Published by BSI Standards Limited 2017ISBN 978 0 580 84390 7 ICS 07.100.30 Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was publish
4、ed under the authority of the Standards Policy and Strategy Committee on 28 February 2017.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 18465 February 2017 ICS 07.100.30 English Version Microbiology of the food chai
5、n - Quantitative determination of emetic toxin (cereulide) using LC-MS/MS (ISO 18465:2017) Microbiologie de la chane alimentaire - Dtermination quantitative de la toxine mtique (creulide) par CL-SM/SM (ISO 18465:2016) Mikrobiologie der Lebensmittelkette - Quantitative Bestimmung von emetischem Toxin
6、 (Cereulid) mittels LC-MS/MS (ISO 18465:2017) This European Standard was approved by CEN on 28 December 2016. 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 alter
7、ation. 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
8、 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, Estoni
9、a, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FO
10、R STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 18465:2017 EBS EN ISO 18465:2
11、017EN ISO 18465:2017 (E) 3 European Foreword This document (EN ISO 18465:2017) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN, in collaboration with Technical Committee ISO/TC 34 “Food products“. This European Standar
12、d shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by August 2017 and conflicting national standards shall be withdrawn at the latest by August 2017. Attention is drawn to the possibility that some of the elements of this d
13、ocument 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. According to the CEN-CENELEC I
14、nternal 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, France, Germany, Greece, Hungary, Iceland
15、, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 18465:2017 has been approved by CEN as EN ISO 18465:2017 without any modif
16、ication. BS EN ISO 18465:2017ISO 18465:2017(E)Foreword ivIntroduction v1 Scope . 12 Normative references 13 Terms and definitions . 14 General principle . 15 Reagents 16 Apparatus and equipment 37 Procedure. 47.1 Sample preparation 47.2 Standard preparation 47.3 LC-MS analysis . 57.3.1 LC conditions
17、 . 57.3.2 MS conditions and tuning parameters 57.3.3 Transitions (multiple reaction monitoring, MRM) . 68 Calculation 69 Quality controls . 710 Precision . 810.1 General . 810.2 Repeatability . 810.3 Reproducibility . 9Annex A (informative) Results of the interlaboratory study 10Annex B (informative
18、) Possible transitions of cereulide in MS3.12Bibliography .14 ISO 2017 All rights reserved iiiContents PageBS EN ISO 18465:2017ISO 18465:2017(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of pre
19、paring International Standards 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
20、 liaison with ISO, also take part 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
21、 Directives, Part 1. In particular 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
22、 that some of the elements of this 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 paten
23、t declarations received (see www .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 info
24、rmation about ISOs adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: www .iso .org/ iso/ foreword .html.This document was prepared by the European Committee for Standardization (CEN) Technical Committee CEN/TC 275, Food Analysi
25、s Horizontal methods, in collaboration with ISO Technical Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology, in accordance with the agreement on technical cooperation between ISO and CEN (Vienna Agreement).iv ISO 2017 All rights reservedBS EN ISO 18465:2017ISO 18465:2017(E)Introduc
26、tionCereulide, the emetic toxin produced in foods by certain strains of Bacillus cereus, is a heat and acid stable toxin that causes nausea and vomiting when ingested. In very rare cases, people can die after ingestion of the toxin. Due to its stability, the toxin may still be present even when B. c
27、ereus can no longer be detected. The presence of cereulide seems to be linked to starch-rich foods like rice (dishes) and pasta (dishes). However, recent data suggest that the occurrence of food borne outbreaks due to cereulide is more common to foods in general9. The toxin has a cyclic structure an
28、d consists of in total 12 monomers as a repeat of (D-O-Leucine-D-Alanine-L-O-Valine-L-Valine). Several methods have been developed for the detection and/or quantification of the toxin. Some of these methods are nonspecific bio-assays3, 4and other methods are specifically based on the chemical analys
29、is using liquid chromatography with mass spectrometry (LC-MS/MS) for the detection and quantification of the toxin5, 6, 7, 8. The chemical methods are more specific for cereulide and have, therefore, been chosen as the starting point for standardization of a method for the quantification of cereulid
30、e. Recently, research has been done for the chemodiversity of cereulide. At least 18 cereulide variants were detected by UHPLC-TOFMS and ion-trap MSnsequencing, among which the previously unknown isocereulides AG10. ISO 2017 All rights reserved vBS EN ISO 18465:2017BS EN ISO 18465:2017Microbiology o
31、f the food chain Quantitative determination of emetic toxin (cereulide) using LC-MS/MS1 ScopeThis document describes the quantitative analysis of the emetic toxin cereulide using high performance liquid chromatography (HPLC) or ultra performance liquid chromatography (UHPLC) connected to a tandem ma
32、ss spectrometer (LC-MS/MS).This document is applicable to the analysis of the toxin in products intended for human consumption.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 this document. For date
33、d 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 methodsISO 1042, Laboratory glassware One-mark volumetric flasks3 Terms and defi
34、nitionsFor the purposes of this document, the following terms and definitions apply.ISO and IEC maintain terminological databases for use in standardization at the following addresses: IEC Electropedia: available at h t t p :/ www .electropedia .org/ ISO Online browsing platform: available at h t t
35、p :/ www .iso .org/ obp3.1cereulidetoxin cycloD-O-Leucine-D-Alanine-L-O-Valine-L-Valine3produced by certain strains of the species of B. cereus4 General principleCereulide is extracted from the food matrix by shaking the sample with acetonitrile. 13C6-Cereulide is used as an internal standard. The c
36、omponents in the solution are separated using HPLC or UHPLC and subsequently detected using tandem mass spectrometry (LC-MS/MS). For MS, the electro spray ionization technique (ESI) is used, using the positive mode. The level of emetic toxin (cereulide) is expressed as g cereulide/kg product.5 Reage
37、ntsUse only reagents of recognized analytical grade, unless otherwise specified.5.1 Water, according to ISO 3696.5.2 Acetonitrile, LC-MS grade.INTERNATIONAL STANDARD ISO 18465:2017(E) ISO 2017 All rights reserved 1BS EN ISO 18465:2017ISO 18465:2017(E)5.3 Methanol, LC-MS grade.5.4 Formic acid, 98 % t
38、o 100 % pro analyse grade.5.5 Synthetic 13C6-Cereulide.1)5.6 Synthetic Cereulide.1)5.7 Ammonium formate, pro analyse grade.5.8 Mobile phase A, consisting of 10 mmol/l ammonium formate (5.7) with 0,1 % (v/v) formic acid (5.4) in water (5.1).5.9 Mobile phase B, consisting of 0,1 % (v/v) formic acid (5
39、.4) in acetonitrile (5.2).5.10 13C6-Cereulidestock solution IS-A, = 100 000 ng/ml (in methanol).Weigh 10 mg to the nearest 0,01 mg 13C6-Cereulide (5.5) into a glass volumetric flask (6.11) of 100 ml and dissolve, make up to the mark with methanol (5.3). This solution is not corrected for the purity
40、of the compound. Store the solution in the freezer (6.15).NOTE Cereulide (labelled and non-labelled) stock and standard solutions are extremely stable, meaning over three years when stored in a freezer (6.15)5.11 13C6-Cereulidestandard solution IS-B, = 1 000 ng/ml (in methanol).Pipette (6.10) 1 000
41、l 13C6-Cereulide stock solution IS-A (5.10) in a glass volumetric flask (6.11) of 100 ml, make up to the mark with methanol (5.3) and mix the solution. Store the solution in the freezer (6.15).5.12 13C6-Cereulidestandard solution IS-C, = 100 ng/ml (in acetonitrile).Pipette (6.10) 500 l 13C6-Cereulid
42、e stock solution IS-A (5.10) in a glass volumetric flask (6.11) of 500 ml, make up to the mark with acetonitrile (5.2) and mix. Store the solution in the freezer (6.15).5.13 13C6- Cereulidestandard solution IS-D, = 10 ng/ml (in acetonitrile).Pipette (6.10) 1 000 l 13C6-Cereulide standard solution IS
43、-B (5.11) in a glass volumetric flask (6.11) of 100 ml, make up to the mark with acetonitrile (5.2) and mix. Store the solution in the freezer (6.15).5.14 Cereulidestock solution Cer-A, = 100 000 ng/ml (in methanol).Weigh 5 mg to the nearest 0,01 mg synthetic cereulide (5.6) into a glass volumetric
44、flask (6.11) of 50 ml and dissolve, make up to the mark with methanol (5.3). This solution is not corrected for the purity of the compound. Store the solution in the freezer (6.15).5.15 Cereulidestandard solution Cer-B, = 100 ng/ml (in acetonitrile).Pipette (6.10) 500 l cereulide stock solution A (5
45、.14) in a glass volumetric flask (6.11) of 500 ml, make up to the mark with acetonitrile (5.2) and mix the solution. Store the solution in the freezer (6.15).1) Chiralix is an example of a suitable product available commercially. This information is given for the convenience of users of this documen
46、t and does not constitute an endorsement by ISO of this product.2 ISO 2017 All rights reservedBS EN ISO 18465:2017ISO 18465:2017(E)5.16 Cereulidestock solution Cer-C, = 10 ng/ml (in acetonitrile).Pipette (6.10) 10 ml cereulide standard solution B (5.15) in a glass volumetric flask (6.11) of 100 ml,
47、make up to the mark with acetonitrile (5.2) and mix the solution. Store the solution in the freezer (6.15).5.17 Positive control sample or spiked sample (level approximately 10 ng/g).6 Apparatus and equipment6.1 Tandem mass spectrometer, equipped with ESI interface (in positive mode) and multiple re
48、action monitoring (MRM) mode.LC system, pump system (HPLC or UHPLC), degasser, autosampler, column oven.LC-MS software, suitable of data collection, integration.6.2 LC column (C-18)For HPLC, Supelco Discovery2)C-18, 100 mm 2,1 mm, 5 m or equivalent.For UHPLC, Waters BEH C-18,3)100 mm or 50 mm 2,1 mm
49、, 1,7 m or equivalent.6.3 Centrifuge, capable of a centrifugal force of 1 000g to 1 500g for 50 ml tubes.6.4 Centrifuge, capable of a centrifugal force of 10 000g to 12 000g for 2 ml tubes.6.5 Centrifuge tubes, (plastic) with closing cap, 2 ml disposable.6.6 Centrifuge tubes, (glass) with leakage free screw cap 50 ml.6.7 Horizontal mechanical shaker, capable of holding 50 ml centrifuge tubes.6.8 Analytical balance, accuracy to the nearest 0,01 mg.6.9 Grinder, e.g. mixer, blender, cryogenic mixer.6.
