1、Foodstuffs Determination of zearalenone in edible vegetable oils by LC-FLD or LC-MS/MSBS EN 16924:2017BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16924 May 2017 ICS 67.200.10 English Version Foodstuffs - Determ
2、ination of zearalenone in edible vegetable oils by LC-FLD or LC-MS/MS Produits alimentaires - Dosage de la zaralnone dans les huiles vgtales alimentaires par CL-FLD ou CL-SM/SM Lebensmittel - Bestimmung von Zearalenon in pflanzlichen Speiselen mit LC-FLD oder LC-MS/MS This European Standard was appr
3、oved by CEN on 27 February 2017. 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 concerning such nation
4、al 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 its own languag
5、e 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, Germany, Greece, H
6、ungary, 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 KOMITEE FR NORMU
7、NG 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 16924:2017 ENational forewordThis British Standard is the UK implementation of EN 16924:2017.The UK participat
8、ion in its preparation was entrusted to Technical Committee AW/275, Food analysis - Horizontal methods.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 provisions of a contract. Users are
9、responsible for its correct application. The British Standards Institution 2017 Published by BSI Standards Limited 2017ISBN 978 0 580 90470 7ICS 67.200.10Compliance with a British Standard cannot confer immunity from legal obligations. This British Standard was published under the authority of the S
10、tandards Policy and Strategy Committee on 30 June 2017.Amendments/corrigenda issued since publicationDate Text affectedBRITISH STANDARDBS EN 16924:2017EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16924 May 2017 ICS 67.200.10 English Version Foodstuffs - Determination of zearalenone in edible
11、 vegetable oils by LC-FLD or LC-MS/MS Produits alimentaires - Dosage de la zaralnone dans les huiles vgtales alimentaires par CL-FLD ou CL-SM/SM Lebensmittel - Bestimmung von Zearalenon in pflanzlichen Speiselen mit LC-FLD oder LC-MS/MS This European Standard was approved by CEN on 27 February 2017.
12、 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 concerning such national standards may be obtained on
13、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 its own language and notified to the CEN-CENELE
14、C 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, Germany, Greece, Hungary, Iceland, Ireland, Italy,
15、 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 KOMITEE FR NORMUNG CEN-CENELEC Management Centre
16、: 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 16924:2017 EBS EN 16924:2017EN 16924:2017 (E) 2 Contents Page European foreword . 3 Introduction 4 1 Scope 5 2 Normative references 5 3 Princi
17、ple . 5 4 Reagents . 5 5 Apparatus and equipment . 8 6 Procedure 10 7 Calculation 12 8 Precision 14 9 Test report 15 Annex A (informative) Typical chromatograms 16 Annex B (informative) Example conditions for suitable LC-MS/MS systems 22 Annex C (informative) Precision data 27 Bibliography . 30 BS E
18、N 16924:2017EN 16924:2017 (E) 3 European foreword This document (EN 16924:2017) 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 status of a national standard, either by public
19、ation of an identical text or by endorsement, at the latest by November 2017, and conflicting national standards shall be withdrawn at the latest by November 2017. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN shall not be h
20、eld 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 Regulations, the national standards organizations of the following cou
21、ntries 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, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, P
22、oland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. BS EN 16924:2017EN 16924:2017 (E) 4 Introduction The mycotoxin zearalenone is a resorcylic acid derivative, which is produced by several species of the fungi genus Fusarium, in particular
23、 by Fusarium roseum var. graminearum. Especially cereals like maize and wheat are affected, so that zearalenone can also be detected in the oils produced from them. WARNING 1 Suitable precaution and protection measures need to be taken when carrying out working steps with harmful chemicals. The haza
24、rdous substances ordinance, Regulation (EC) No 1907/2006 3, should be taken into account as well as appropriate National statements e.g. such as in 4. WARNING 2 The use of this document can involve hazardous materials, operations and equipment. This document does not purport to address all the safet
25、y 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 to use. WARNING 3 Zearalenone is known to have strong oestrogenic effects. BS EN 16924:2017EN
26、 16924:2017 (E) 5 1 Scope This European Standard describes a procedure for the determination of the zearalenone content in edible vegetable oils specifically maize germ oil by either of the following techniques: High performance liquid chromatography with fluorescence detection (LC-FLD) or high perf
27、ormance liquid chromatography with tandem mass spectrometry (LC-MS/MS) after basic extraction of the diluted oil. The method has been validated for zearalenone in naturally contaminated maize germ oil at levels of 61,2 g/kg to 515 g/kg 5. Laboratory experiences 6 have shown that this method is also
28、applicable to other vegetable oils such as wheat germ oil (n = 4), sunflower oil (n = 5), pumpkin seed oil (n = 1), soybean oil (n = 5), hemp seed oil (n = 5), rape seed oil (n = 11), and mixed oils including maize germ oil (n = 3). However occasionally, samples can result in interferences in the FL
29、D-chromatograms. In this case, the detection with MS/MS is recommended. 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 refere
30、nces, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696, Water for analytical laboratory use Specification and test methods (ISO 3696) 3 Principle After diluting the edible vegetable oil, zearalenone is extracted by shaking with an alkaline methanol - ammo
31、nium hydrogen carbonate mixture. For the determination by LC-FLD, an aliquot of the centrifuged methanolic-alkaline extract is evaporated to dryness, then the residue is diluted in acidified LC-eluent and the zearalenone content is determined by LC-FLD. For the determination by LC-MS/MS, an aliquot
32、of the centrifuged methanolic-alkaline extract is used directly for analysis. 4 Reagents Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696, unless otherwise specified. Solvents shall be of quality for LC analysis, unless otherwise specified. 4.1 Methano
33、l, p. a. (pro analysis) for extraction. 4.2 Dilution solvent, (defatting solvent), n-hexane or, alternatively, n-heptane, p. a. n-Heptane may be used instead of n-hexane, however, only n-hexane was used in the interlaboratory test. 4.3 Acetonitrile, LC quality. 4.4 Ammonium hydrogen carbonate (NH4HC
34、O3). 4.5 Ammonium hydrogen carbonate solution, mass concentration = 10 g/l. Weigh in 1 g of ammonium hydrogen carbonate (4.4) into a 100 ml volumetric flask and fill up to the mark with water. Prepare a fresh solution each day of analysis. BS EN 16924:2017EN 16924:2017 (E) 6 4.6 Alkaline methanol ex
35、traction solution. Mix 9 volumes of methanol (4.1) with 1 volume of ammonium hydrogen carbonate solution (4.5). Prepare a fresh alkaline methanol extraction solution each day of analysis. 4.7 Zearalenone, e.g. crystalline, purity greater than 98 % mass fraction, or as certified standard solution wit
36、h 100 g/ml. 4.8 Stock solution of zearalenone, mass concentration = 100 g/ml. Weigh in 10 mg of crystalline zearalenone (4.7) to the nearest 0,1 mg into a 100 ml volumetric flask and fill up to the mark with acetonitrile (4.3). The stock solution is stable for at least 4 weeks to 12 weeks if stored
37、at 18 C 7. This step can be omitted when using the certified standard solution. The certified standard solution then serves as stock solution. 4.9 Standard solution of zearalenone, = 10 g/ml. Pipette 1 ml of the stock solution of zearalenone (4.8) into a 10 ml volumetric flask and fill up to the mar
38、k with acetonitrile (4.3). Determine the exact concentration of zearalenone in this standard solution spectrometrically. For this purpose, record the absorption curve of the standard solution from 190 nm to 350 nm against acetonitrile. Calculate the mass concentration of zearalenone in the standard
39、solution stin g/ml according to Formula (1): max100stEM=(1) where Emaxis the maximum extinction value determined from the absorption curve (here: 274 nm); M is the molar mass of zearalenone = 318,4 g/mol; is the layer thickness of the cuvette in cm; is the molar extinction coefficient in acetonitril
40、e: 1262 m2/mol. 4.10 Reagents, mobile phases and calibration solutions for LC-FLD analysis: 4.10.1 Glacial acetic acid for LC-FLD, volume fraction (CH3COOH) approximately 99 %. 4.10.2 Mobile phase A for LC-FLD: Acetonitrile/water/glacial acetic acid (47+52+1, v+v+v). Mix 47 volumes of acetonitrile (
41、4.3) with 52 volumes of water and 1 volume of glacial acetic acid (4.10.1). For the stability of the chromatographic separation it is necessary to acidify the mobile phase. 4.10.3 Mobile phase B for LC-FLD: Acetonitrile (100 %) (4.3). 4.10.4 Calibration solutions of zearalenone for LC-FLD. Prepare a
42、 series of calibration solutions from the standard solution (4.9) of zearalenone. According to the pipetting scheme in Table 1, pipette the corresponding volumes of standard solution of zearalenone into volumetric flasks, evaporate the solvent with nitrogen at approximately 40 C to dryness and disso
43、lve the residue in mobile phase A (4.10.2). Dissolve by means of laboratory shaker (5.4) or ultrasonic bath (5.3) at room temperature. Then fill up to the mark. The calibration range shall be BS EN 16924:2017EN 16924:2017 (E) 7 adapted to the desired working range. The calibration solutions can be u
44、sed for approximately 1 month if stored in the refrigerator at 4 C. Table 1 Examples of suitable calibration solutions for LC-FLD Calibration solution Volume of volumetric flask ml Volume of standard solution (4.9) l Mass concentration of calibration solution ng/ml 1 10 25 25 2 10 50 50 3 10 100 100
45、 4 10 150 150 5 10 200 200 6 10 250 250 4.11 Reagents, mobile phases, and additional standards and standard solutions for LC-MS/MS analysis: 4.11.1 Methanol, LC quality. 4.11.2 Ammonium hydrogen carbonate (NH4HCO3), for LC-MS, molecular weight: 79 g/mol. 4.11.3 Mobile phase A for LC-MS/MS: 1 mmol/l
46、NH4HCO3in water/methanol (95+5, v+v). Dissolve 79 mg of ammonium hydrogen carbonate (4.11.2) in 950 ml of water and mix with 50 ml of methanol (4.11.1). 4.11.4 Mobile phase B for LC-MS/MS: 1 mmol/l NH4HCO3in water/methanol (5+95, v+v). Dissolve 79 mg of ammonium hydrogen carbonate (4.11.2) in 50 ml
47、of water and mix with 950 ml of methanol (4.11.1). 4.11.5 Working solution of zearalenone for calibration, = 200 ng/ml. Transfer 0,5 ml of the standard solution (4.9) into a 25 ml volumetric flask and fill up to the calibration mark with methanol (4.11.1). 4.11.6 Zearalanone, internal standard (ISTD
48、 1) for LC-MS/MS, e.g. crystalline or as certified standard solution with 10 g/ml. 4.11.7 Zearalenone, isotopic labelled internal Standard (ISTD 2) for LC-MS/MS, as optional internal standard, e.g. zearalenone 13C18-labelled. 4.11.8 Stock solution of zearalanone (ISTD 1), = 100 g/ml. Weigh in 10 mg
49、of zearalanone (ISTD 1) (4.11.6) to the nearest 0,1 mg into a 100 ml volumetric flask and fill up to the mark with acetonitrile (4.3). The ISTD 1 stock solution is stable for at least 4 weeks to 12 weeks if stored at 18 C 7. If the certified standard solution is used, the preparation of this stock solution of zearalanone (ISTD 1) is not necessary. BS EN 16924:2017EN 16924:2017 (E) 8 4.11.9 Internal standard solution of zearalanone (ISTD 1), = 10 g/ml. Transfer 1 ml of the stock solution of zearalano
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