1、May 2017 English price group 12No 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 07.100.30!%e)s“2660680www.din.deDIN
2、EN ISO 18465Microbiology of the food chain Quantitative determination of emetic toxin (cereulide) using LCMS/MS (ISO 18465:2017);English version EN ISO 18465:2017,English translation of DIN EN ISO 18465:2017-05Mikrobiologie der Lebensmittelkette Quantitative Bestimmung von emetischem Toxin (Cereulid
3、) mittels LCMS/MS (ISO 18465:2017);Englische Fassung EN ISO 18465:2017,Englische bersetzung von DIN EN ISO 18465:2017-05Microbiologie de la chane alimentaire Dtermination quantitative de la toxine mtique (creulide) par CLSM/SM (ISO 18465:2017);Version anglaise EN ISO 18465:2017,Traduction anglaise d
4、e DIN EN ISO 18465:2017-05www.beuth.deDocument comprises 21 pagesDTranslation by DIN-Sprachendienst.In case of doubt, the German-language original shall be considered authoritative.05.17 DIN EN ISO 18465:2017-05 2 A comma is used as the decimal marker. National foreword This document (EN ISO 18465:2
5、017) has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany), in collaboration with Technical Committee ISO/TC 34 “Food products”. The responsible German body involved in its preparation was DIN-Normenausschuss Lebensmittel und landwirtschaf
6、tliche Produkte (DIN Standards Committee Food and Agricultural Products), Working Group NA 057-01-06-02 AK “Spore-forming bacteria”. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 1042 DIN EN ISO 1042 ISO 3696 DIN ISO 3696 ISO 5725-2 D
7、IN ISO 5725-2 National Annex NA (informative) Bibliography DIN EN ISO 1042, Laboratory glassware One-mark volumetric flasks DIN ISO 3696, Water for analytical laboratory use Specification and test methods DIN ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results Part 2: Ba
8、sic method for the determination of repeatability and reproducibility of a standard measurement method EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 18465 February 2017 ICS 07.100.30 English Version Microbiology of the food chain - Quantitative determination of emetic toxin (cereulide) us
9、ing LC-MS/MS (ISO 18465:2017) Microbiologie de la chane alimentaire - Dtermination quantitative de la toxine mtique (creulide) parCL-SM/SM (ISO 18465:2017) Mikrobiologie der Lebensmittelkette - Quantitative Bestimmung von emetischem Toxin (Cereulid) mittels LC-MS/MS (ISO 18465:2017) This European St
10、andard 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 alteration. Up-to-date lists and bibliographical references concern
11、ing 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
12、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, Ger
13、many, 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 FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES K
14、OMITEE 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 EForeword .4Introduction . 51 Scope . 62 Normative references 63 Terms and defin
15、itions . 64 General principle . 65 Reagents 66 Apparatus and equipment 87 Procedure. 97.1 Sample preparation 97.2 Standard preparation 97.3 LC-MS analysis 107.3.1 LC conditions 107.3.2 MS conditions and tuning parameters .107.3.3 Transitions (multiple reaction monitoring, MRM) 118 Calculation .119 Q
16、uality controls 1210 Precision 1310.1 General 1310.2 Repeatability 1310.3 Reproducibility 14Annex A (informative) Results of the interlaboratory study 15Annex B (informative) Possible transitions of cereulide in MS3.17Bibliography .19Contents Page2EN ISO 18465:2017 (E) DIN EN ISO 18465:2017-05 Europ
17、ean foreword .3European 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 Standard shall be g
18、iven 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 document may
19、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 Internal Regu
20、lations, 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, Ireland, I
21、taly, 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 modification. 3EN
22、 ISO 18465:2017 (E) DIN EN ISO 18465:2017-05 ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each membe
23、r 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 part in the work. ISO collaborates closely with the International Elec
24、trotechnical 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 particular the different approval criteria needed for the different types o
25、f 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 this document may be the subject of patent rights. ISO shall not be h
26、eld 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 .iso .org/ patents).Any trade name used in this document is inform
27、ation 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 World Trade Organization (WTO) principles in the Technical Barr
28、iers 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 Analysis Horizontal methods, in collaboration with ISO Technical Committee ISO/TC 34, Food products, Subco
29、mmittee SC 9, Microbiology, in accordance with the agreement on technical cooperation between ISO and CEN (Vienna Agreement).4EN ISO 18465:2017 (E) DIN EN ISO 18465:2017-05 IntroductionCereulide, the emetic toxin produced in foods by certain strains of Bacillus cereus, is a heat and acid stable toxi
30、n 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. cereus can no longer be detected. The presence of cereulide seems to be linked to starch-rich foods like rice (dishes) a
31、nd 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 and consists of in total 12 monomers as a repeat of (D-O-Leucine-D-Alanine-L-O-Valine-L-Valine). Several methods have bee
32、n 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 analysis using liquid chromatography with mass spectrometry (LC-MS/MS) for the detection and quantification of the toxin5, 6,
33、 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 cereulide. Recently, research has been done for the chemodiversity of cereulide. At least 18 cereulide variants were detected b
34、y UHPLC-TOFMS and ion-trap MSnsequencing, among which the previously unknown isocereulides AG10.5EN ISO 18465:2017 (E) DIN EN ISO 18465:2017-05 1 ScopeThis document describes the quantitative analysis of the emetic toxin cereulide using high performance liquid chromatography (HPLC) or ultra performa
35、nce liquid chromatography (UHPLC) connected to a tandem mass 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 c
36、ontent constitutes requirements of this 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 methodsISO 1042, Labor
37、atory glassware One-mark volumetric flasks3 Terms and definitionsFor 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 .electroped
38、ia .org/ ISO Online browsing platform: available at h t t 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 acetonitr
39、ile. 13C6-Cereulide is used as an internal standard. The components 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
40、(cereulide) is expressed as g cereulide/kg product.5 ReagentsUse only reagents of recognized analytical grade, unless otherwise specified.5.1 Water, according to ISO 3696.5.2 Acetonitrile, LC-MS grade.6EN ISO 18465:2017 (E) DIN EN ISO 18465:2017-05 5.3 Methanol, LC-MS grade.5.4 Formic acid, 98 % to
41、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 (5.4
42、) 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 of
43、 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 l
44、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-Cereulide
45、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-B
46、 (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 fl
47、ask (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 (5.1
48、4) 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 document
49、and does not constitute an endorsement by ISO of this product.7EN ISO 18465:2017 (E) DIN EN ISO 18465:2017-05 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, 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
