SIST EN 15136:2006
(Main)Materials and articles in contact with foodstuffs - Certain epoxy derivatives subject to limitation - Determination of BADGE, BFDGE and their hydroxy and chlorinated derivatives in food simulants
Materials and articles in contact with foodstuffs - Certain epoxy derivatives subject to limitation - Determination of BADGE, BFDGE and their hydroxy and chlorinated derivatives in food simulants
This European standard describes a method for the determination of BADGE, BFDGE and their reaction products in food simulants: distilled water, 3 % w/v aqueous acetic acid, 10 % v/v aqueous ethanol solution and olive oil or sunflower oil.
A high performance liquid chromatography (HPLC) method is employed based on reversed phase HPLC and fluorescence detection.
The method is capable of determining BADGE and its derivatives at a minimum level of 0,05 µg/ml food simulant.
BFDGE and its derivatives can be determined at a minimum level of 0,1 µg/ml food simulant.
Direct HPLC analysis of the migration solutions may result in chromatograms difficult to interpret, due to interference from other components or the instability of the monomers resulting in a complex mixture of derivatives and/or reaction products. By forced hydrolysis of all epoxy groups and their reaction products, the quantification of the relevant substances is simplified and in addition the identities of the substances are indicatively confirmed.
NOTE In this European standard the term “BADGE, BFDGE and their derivatives” refers to the substances listed in Directive 2002/16/EC. These substances are listed in 4.1.
Werkstoffe und Gegenstände in Kontakt mit Lebensmitteln - Bestimmte Epoxyderivate, die Beschränkungen unterliegen - Bestimmung von BADGE, BFDGE und deren Hydroxy- und Chlorderivaten in Prüflebensmitteln
Diese Europäische Norm beschreibt ein Verfahren zur Bestimmung von BADGE, BFDGE und deren Reaktionsprodukten in den Prüflebensmitteln destilliertes Wasser, 3%ige Essigsäure (m/V) in wässriger Lösung, 10%iges Ethanol (Volumenanteil) in wässriger Lösung und Olivenöl oder Sonnenblumenöl.
Angewendet wird ein Hochleistungs-Flüssigkeitschromatographie-Verfahren (HPLC), das auf Umkehrphasen-HPLC und Fluoreszenzdetektion beruht.
Das Verfahren ist für die Bestimmung von BADGE und dessen Derivaten bei einem Mindestgehalt von 0,05 mg/ml Prüflebensmittel geeignet.
BFDGE und seine Derivate können bei einem Mindestgehalt von 0,1 µg/ml Prüflebensmittel bestimmt werden.
Die direkte HPLC-Analyse der Migrationslösungen kann zu Chromatogrammen führen, die auf Grund von Störungen durch andere Komponenten oder der Instabilität der Monomere, die zu einer komplexen Mischung von Derivaten und/oder Reaktionsprodukten führt, schwer auszuwerten sind. Durch beschleunigte Hydrolyse aller Epoxygruppen und von deren Reaktionsprodukten wird die quantitative Bestimmung der betreffenden Substanzen vereinfacht und zusätzlich werden die Identitäten der Substanzen nachweislich bestätigt.
ANMERKUNG In diesem Dokument bezieht sich der Ausdruck "BADGE, BFDGE und deren Derivate" auf Substanzen, die in der Richtlinie 2002/16/EG aufgeführt sind. Diese Substanzen sind in 4.1 angegeben.
Matériaux et objets en contact avec les denrées alimentaires - Dérivés époxy soumis a des limitations - Détermination du BADGE, du BFDGE et de leurs dérivés hydroxylés et chlorés dans les simulants d'aliments
La présente Norme européenne décrit une méthode de détermination du BADGE, du BFDGE et de leurs
produits de réaction dans les simulants d'aliments : eau distillée, solution aqueuse d'acide acétique a
3 % (m/v), solution aqueuse d'éthanol a 10 % (v/v) et huile d'olive ou de tournesol.
La détermination est effectuée par chromatographie liquide haute performance (CLHP) en phase inverse et
détection par fluorescence.
Cette méthode permet de déterminer le BADGE ainsi que ses dérivés a une concentration minimale de
0,05 µg/ml de simulant d’aliments.
Le BFDGE ainsi que ses dérivés peuvent etre déterminés a une concentration minimale de 0,1 µg/ml de
simulant d’aliments.
Une analyse CLHP directe des solutions de migration peut générer des chromatogrammes difficiles a
interpréter, en raison d'une interférence avec d'autres composants ou de l'instabilité des monomeres
engendrant un mélange complexe de dérivés et/ou de produits de réaction. L'hydrolyse forcée de tous les
groupes époxy et de leurs produits de réaction permet de simplifier la quantification des substances
appropriées et de confirmer l'identité des substances.
NOTE Dans le présent document, l'expression « BADGE, BFDGE et leurs dérivés » se rapporte aux substances
citées dans la Directive 2002/16/CE [1] et son amendement, Directive 2004/13/CE [2]. Ces substances sont répertoriées
en 4.1.
Materiali in predmeti v stiku z živili – Nekateri epoksi derivati, katerih koncentracija je omejena – Določevanje BADGE, BFDGE in njihovih hidroksi in kloriranih derivatov v modelnih raztopinah za živila
General Information
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Materials and articles in contact with foodstuffs - Certain epoxy derivatives subject to limitation - Determination of BADGE, BFDGE and their hydroxy and chlorinated derivatives in food simulantsULUDQLKMatériaux et objets en contact avec les denrées alimentaires - Dérivés époxy soumis a des limitations - Détermination du BADGE, du BFDGE et de leurs dérivés hydroxylés et chlorés dans les simulants d'alimentsWerkstoffe und Gegenstände in Kontakt mit Lebensmitteln - Bestimmte Epoxyderivate, die Beschränkungen unterliegen - Bestimmung von BADGE, BFDGE und deren Hydroxy- und Chlorderivaten in PrüflebensmittelnTa slovenski standard je istoveten z:EN 15136:2006SIST EN 15136:2006en,fr,de67.250ICS:SLOVENSKI
STANDARDSIST EN 15136:200601-september-2006
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 15136March 2006ICS 67.250 English VersionMaterials and articles in contact with foodstuffs - Certain epoxyderivatives subject to limitation - Determination of BADGE,BFDGE and their hydroxy and chlorinated derivatives in foodsimulantsMatériaux et objets en contact avec les denréesalimentaires - Dérivés époxy soumis à des limitations -Détermination du BADGE, du BFDGE et de leurs dérivéshydroxylés et chlorés dans les simulants d'alimentsWerkstoffe und Gegenstände in Kontakt mit Lebensmitteln- Bestimmte Epoxyderivate, die Beschränkungenunterliegen - Bestimmung von BADGE, BFDGE und derenHydroxy- und Chlorderivaten in PrüflebensmittelnThis European Standard was approved by CEN on 23 January 2006.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the Central Secretariat or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania,Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2006 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15136:2006: E
EN 15136:2006 (E) 2 Contents Page Foreword.3 Introduction.4 1 Scope.5 2 Normative references.5 3 Principle.5 4 Reagents.6 5 Apparatus.9 6 Samples.11 7 Procedure.13 8 Confirmation.17 9 Precision.17 10 Test report.18 Annex A (informative)
Structures of the main compounds cited in this standard.20 Annex B (informative)
Principle of the determination of BADGE, BFDGE and their hydroxyl and chlorinated derivatives in food simulants.24 Annex C (informative)
Typical chromatograms of BADGE, BFDGE and some of their derivatives.25 Annex D (informative)
Determination of BADGE and its hydrolysis and hydroxychlorinated products in foodstuffs.29 Annex ZA (informative)
Relationship between this
European
Standard and the Essential Requirements
of EU Directives.30 Bibliography.31
EN 15136:2006 (E) 3 Foreword This document (EN 15136:2006) has been prepared by Technical Committee CEN/TC 194 “Utensils in contact with food”, the secretariat of which is held by BSI. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by September 2006, and conflicting national standards shall be withdrawn at the latest by September 2006. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association, and supports essential requirements of EU Directive(s). For relationship with EU Directive(s), see informative Annex ZA, which is an integral part of this document. This document should be read in conjunction with EN 13130-1. WARNING:
All chemicals are hazardous to health to a greater or lesser extent.
It is beyond the scope of this European standard to give instructions for the safe handling of all chemicals, that meet, in full, the legal obligations in all countries in which this European standard may be followed.
Therefore, specific warnings are not given and users of this European standard should ensure that they meet all the necessary safety requirements in their own country. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EN 15136:2006 (E) 4 Introduction 2,2-Bis(4-hydroxyphenyl)propane bis(2,3-epoxypropyl)ether (BADGE) and bis(hydroxyphenyl)methane bis(2,3-epoxypropyl)ether (BFDGE) are monomers used in the manufacture of certain polymeric food contact materials and articles.
The main application of these monomers is in epoxy coatings for cans and ends. The substances may also be used in organosol coatings. After the manufacture, residues of the substances or the reaction products can remain in the finished product and might migrate into foodstuffs coming into contact with that product.
The analytical method described allows for the determination of BADGE, BFDGE and their reaction products in aqueous and fatty food simulants.
EN 15136:2006 (E) 5
1 Scope
This European standard describes a method for the determination of BADGE, BFDGE and their reaction products in food simulants: distilled water, 3 % w/v aqueous acetic acid, 10 % v/v aqueous ethanol solution and olive oil or sunflower oil. A high performance liquid chromatography (HPLC) method is employed based on reversed phase HPLC and fluorescence detection.
The method is capable of determining BADGE and its derivatives at a minimum level of 0,05 µg/ml food simulant.
BFDGE and its derivatives can be determined at a minimum level of 0,1 µg/ml food simulant. Direct HPLC analysis of the migration solutions may result in chromatograms difficult to interpret, due to interference from other components or the instability of the monomers resulting in a complex mixture of derivatives and/or reaction products. By forced hydrolysis of all epoxy groups and their reaction products, the quantification of the relevant substances is simplified and in addition the identities of the substances are indicatively confirmed.
NOTE In this European standard the term “BADGE, BFDGE and their derivatives” refers to the substances listed in Directive 2002/16/EC [1] and its amendment, Directive 2004/13/EC [2]. These substances are listed in 4.1. 2 Normative references The following referenced documents are indispensable for the application of this European standard. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN 13130-1:2004, Materials and articles in contact with foodstuffs – Plastics substances subject to limitation – Part 1: Guide to the test methods for the specific migration of substances from plastics to food and food simulants and the determination of substances in plastics and the selection of conditions of exposure to food simulants ISO 648, Laboratory glassware – One-mark pipettes
3 Principle
3.1 Determination of BADGE, BFDGE and their derivatives in food simulants Proper quantification of the sum of BADGE, BFDGE and/or their derivatives is obtained by analysing the simulants twice: a first analysis of the simulant as obtained from the migration is performed and, if necessary, the substances are fully hydrolysed and the hydrolysed substances are determined in a second HPLC analysis.
This second analysis is used for confirmation and final quantification of the sum of BADGE or BFDGE and their derivatives as the bis(diol) derivatives. In the first instance, after the migration period, samples from aqueous food simulants are directly injected into a reverse phase HPLC column and the substances are separated using a gradient elution profile. For fatty food simulants, the substances are extracted with acetonitrile followed by HPLC
EN 15136:2006 (E) 6 analysis. Detection is performed by means of fluorescence detection. Identification is based on retention time and comparison with reference substances, fluorescence and UV detection. If it appears from this first analysis that the summed migration level of BADGE/BFDGE and/or their derivatives is >0.5 mg/kg, confirmation and final quantification of BADGE, BFDGE and their derivatives is performed by forced hydrolysis of the epoxy components and the chlorinated components. If the substance of interest contains either an epoxy group or a HCl adduct, then this component will hydrolyse to the bis(diol) components. Compared to the HPLC chromatogram prior to hydrolysis a simpler HPLC chromatogram is obtained after hydrolysis, containing fewer peaks because the peaks of BADGE, BFDGE and their adducts disappear; if the peak(s) remain(s) then this substance should be considered an interfering substance originating from the matrix. Forced hydrolysis is obtained by mixing the sample solution with buffer and hydrolysis at 100 °C. Then a second HPLC analysis is performed to determine the concentration of monomer.2H2O. 3.2 Hydrolysis BADGE, BFGDE and their partly hydrolysed adducts hydrolyse in aqueous neutral and acid conditions. The chlorinated adducts are stable in acid conditions and hydrolyse only slowly in neutral aqueous media. However, in slightly alkaline conditions all adducts (excepting ether derivatives) hydrolyse to the bis(diol) component. To force complete hydrolysis of the epoxy and HCl adducts, the sample solutions are buffered at pH 8.5 and subsequently stored for a minimum of 20 h at 100 °C. After that period the bis(diol) substances are determined. NOTE The structures of BADGE, BFDGE and their derivatives are included in Annex A as well as some derivatives that may be formed during contact with simulants.
A flow chart showing the principle of the determination of BADGE, BFDGE and their derivatives in food simulants is given in Annex B. 4 Reagents
4.1 Analytes NOTE See Annex A for molecular structures. 4.1.1 BADGE
CAS no. 1675-54-3
4.1.2 BADGE.2HCl
CAS no. 4809-35-2
4.1.3 BADGE.2H2O
CAS no. 5581-32-8
4.1.4 BADGE.H2O
CAS no. 76002-91-0
4.1.5 BADGE.HCl
CAS no. 13836-48-1
4.1.6 BADGE.H2O.HCl
CAS no. 227947-06-0
4.1.7 BFDGE
CAS no. 2095-03-6
4.1.8 BFDGE.2HCl
4.1.9 BFDGE.2H2O
CAS no. 72406-26-9
4.1.10 BFDGE.HCl, prepared as follows: Dissolve approximately 50 mg BFDGE (4.1.7) in 25 ml dioxane, add 10 µl concentrated hydrochloric acid and reflux for 10 min.
Using a rotavapor evaporate the dioxane and dissolve the residue in 25 ml acetonitrile.
The mixture should contain the isomers of each of three substances BFDGE, BFDGE.HCl and BFDGE.2 HCl. This mixture is only used for reference purposes.
EN 15136:2006 (E) 7 4.1.11 BFDGE.H2O, prepared as follows: Dissolve 50 mg BFDGE in 25 ml dioxane, add 500 µl water and reflux for 10 min. Using a rotavapor evaporate the dioxane and dissolve the residue in acetonitrile. The mixture should contain the isomers of each of three substances BFDGE, BFDGE.H2O and BFDGE.2H2O. This mixture is only used for reference purposes. NOTE BFDGE and its derivatives consists of a mixture of isomers (see annex A). The ratio of the various isomers may differ depending on the supply source.
4.2 Reagents NOTE During the analysis, unless otherwise stated, only reagents of recognized analytical grade and distilled water of equivalent purity should be used.
4.2.1 Acetonitrile, HPLC grade
4.2.2 Boric acid 4.2.3 Ethanol 100 %, distilled 4.2.4 Methanol, HPLC grade 4.2.5 Sodium hydroxide 4.2.6 Water deionised, HPLC grade 4.3 Solutions 4.3.1 Borate buffer: 0,6 M
Dissolve 9,28 g of boric acid in 220 ml of water. Add 4,5 M sodium hydroxide solution to a pH of 8,5. Top up the volume to 250 ml with water. 4.3.2 Stock solutions of pure reference substances of BADGE, BFDGE and their derivatives in acetonitrile (500 µg/ml) NOTE If the derivatives of BADGE and BFDGE only are used for establishing their retention times before hydrolysis, qualitative solutions may be prepared. If the solutions are used to quantify each of the derivatives in order to avoid the hydrolysis procedure (see scheme in Annex B), quantitative solutions should be prepared of each of the derivatives. However, quantitative solutions containing only BADGE and BFDGE should always be prepared. Weigh to the nearest 0,1 mg approximately 50 mg of each of the relevant substances listed in 4.1 in a series of 100 ml volumetric flasks and add 80 ml acetonitrile (4.2.1). Place the flask for 5 min in an ultrasonic water-bath to dissolve the substances. Cool the solution to room temperature and fill the volumetric flask up to the mark with acetonitrile and mix carefully. Calculate the actual concentration of the substance in micrograms per millilitre of solution. To dissolve the bis(diol) derivatives add first 20 ml of water and then make up to the mark with acetonitrile. Repeat the procedure to obtain a second standard stock solution. Check the two primary stock solutions of analyte against one another. Check that the response factor, i.e. detector response divided by concentration of analyte solution, of the two primary stock solutions (or dilutions of that) does not differ by more than 5 %. If there is agreement within 5 %, make
EN 15136:2006 (E) 8 subsequent diluted standard solutions from only one of the primary stock solutions. If the levels of the two independently prepared stock solutions do not correspond to within ± 5 %, discard both stock solutions and prepare new solutions. NOTE 1 Solubility of bis(diol) derivatives in pure acetonitrile is limited. When stored in a refrigerator the substances may crystallize. Addition of water to the acetonitrile as described will improve the solubility.
NOTE 2 The solutions may be stored for up to 6 months in a refrigerator.
4.3.3 Intermediate standard solution of BADGE, BFDGE and their derivatives in acetonitrile (10 µg/ml)
Into a series of 50 ml volumetric flask pipette 1,0 ml of each of the stock solutions (4.3.2) and fill the volumetric flask up to the mark with acetonitrile to give approximately 10 µg/ml of the relevant substance. Calculate the actual concentration of the substances in µg/ml solution. NOTE Either a mixture of standard substances or individual standard solution may be prepared by adding each of the stock solutions (4.3.2) to one and the same flask or to individual flasks. However, at least standards containing only BADGE and BFDGE shall be prepared for quantification purposes of the substances after hydrolysis. 4.3.4 Sodium hydroxide solution in water 4.5M Dissolve 18 g of NaOH in 100 ml of water.
EN 15136:2006 (E) 9 5 Apparatus 5.1 HPLC vials. 5.2 Headspace vials of suitable volume, with inert gas-tight closure capable of sustaining the build-up of pressure during the heating period. 5.3 Micro pipette. 5.4 Mixer of vortex type. 5.5 Oven, capable of being maintained at 100 ºC ± 5 °C. 5.6 pH meter, accurate to within ± pH 0,1.
5.7 Pipettes, of 5 ml and 50 ml capacity, conforming to ISO 648. 5.8 Reaction-therm heating module with nitrogen gas flow supply and the temperature set to between 40 °C to 50 °C. 5.9 C18 solid phase extraction cartridges (360 mg size) with Luer connection to a 5 ml syringe.
5.10 HPLC apparatus 5.10.1 High performance liquid chromatograph preferably with 20 µl injection loop, a UV detector at 225 nm, and a variable fluorescence detector, set to λex =275 nm and λem =305 nm; detectors should be connected to an integrator.
NOTE For the determination of BFDGE and its derivatives the use of the prescribed fluorescence wavelengths is essential. Alternative wavelengths may provide better sensitivity but the response factor for the various isomers is different, making correct quantification impossible.
5.10.2 HPLC column, packed with C18 coated silica gel, capable of producing a symmetric peak of BADGE, and capable to separate from each other: BADGE, BFDGE and the hydrolysis and chlorohydrin reaction products of BADGE, BFDGE, as well as from peaks originating from simulants and/or solvents used. For the determination of BADGE, BFDGE and their adducts a reverse phase HPLC procedure with a gradient profile is applied, using an analytical column and an optional pre-column.
NOTE For guidance, the parameters which are found suitable for the analysis using the column selected are given below. Other columns and dimensions have also been found to be suitable.
HPLC SYSTEM
Analytical Column
: Stainless steel 250 mm x 4,6 mm, Spherisorb ODS2, partical size 5 µm Pre Column
: Stainless steel, 30 mm x 4,6 mm, Hypersil ODS, partical size 5 µm
Column temperature : 30 °C
Flow
: 1,1 ml/min Eluent gradient
: The following gradient profile, using a linear gradient in each step, is applied.
EN 15136:2006 (E) 10 Table 1 Time (min)% water% acetonitrile0 802010 653525 505045 505060 010075 010080 802085 8020 Depending on the composition of the samples injected, the final step(s) (from 60 min) in the gradient can be adapted or shortened. In case of strongly contaminated solutions it is advised to rinse the HPLC column properly with pure acetonitrile in between successive injections. If mainly clean samples are injected then the pre column may not be required. This will influence the retention times presented below. Injection volume:
20 µl
Detection:
See 5.10.1 Under the conditions given above the following retention times were obtained from standard solutions. The retention times should be seen as indicative values as retention times may shift from one run to another. Typical chromatograms, containing the majority of the substances listed, are depicted in Annex C. Table 2 Component Retention time (min) Component Retention time (min) p,p-BFDGE.2H2O 10,2 p,p-BFDGE.2HCl 28,9 o,p-BFDGE.2H2O 11,4 o,p-BFDGE.2HCl 30,2 o,o-BFDGE.2H2O 12,5 o,o-BFDGE.2HCl + p,p-BFDGE 31,6 BADGE.2H2O 13,4 o,p-BFDGE 32,7 p,p-BFDGE.1H2O 17,9 o,o-BFDGE 34,0 o,p-BFDGE.1H2O 18,7 BADGE.2HCl_isom 35,8 o,o-BFDGE.1H2O 19,5 BADGE.2HCl 36,6 BADGE.1H2O. 1ether
21,5 BADGE.1HCl
38,7 BADGE.1HCl.1H2O. 22,7 BADGE 41,0 BADGE.1H2O
23,1
Bisphenol A and p,p-Bisphenol F may appear at retention time 20,1 min and 15,2 min respectively. Use of methanol as modifier in the elution solvent may cause a different order of elution.
Injection of acetonitrile solutions may influence the separation efficiency of short retention time substances.
Dilution of the acetonitrile with water or buffer will avoid that problem.
EN 15136:2006 (E) 11 6 Samples
6.1 General Laboratory samples of the food simulant to be analyzed shall be obtained as described in EN 13130-1.
NOTE Blank samples of simulants of the same type as those to be analyzed are also required for calibration purposes. When using olive oil as a fatty food simulant, blank olive oil shall be extracted and analysed before starting the migration experiments as components originating from olive oil, which interfere in the HPLC analysis, may be present in the extract with acetonitrile (6.2.4).
NOTE 1 It has been found that olive oil is sensitive to interferences.
NOTE 2 The use of refined sunflower oil is advisable as that oil usually results in “clean” chromatograms.
NOTE 3 During the migration period with 10 % (v/v) aqueous ethanol, the epoxy groups may form ethanol ethers which cannot be hydrolysed.
It has been found that after 10 d at 40 °C approximately 15 % of the initial amount of BADGE was transformed into the mono ethanol derivative.
6.2 Preparation of test samples from food simulants 6.2.1 General Forced hydrolysis of BADGE, BFDGE and/or their derivatives is performed in case: HPLC chromatogram of the simulant (prior to hydrolysis (6.2.3) is complex and substances can not clearly be identified based on their retention time, sum of the migration level of BADGE, BFDGE and their derivatives in the simulant (prior to hydrolysis) is > 0,5 mg/kg. 6.2.2 Aqueous food simulants
After the migration period cool the solutions to room temperature and fill a HPLC vial with the sample solution. Continue as described in 7.3. 6.2.3 Forced hydrolysis of aqueous food simulants
Pipette 1,0 ml of the aqueous simulant, obtained after the migration period, in a headspace vial (5.2).
Add 1,0 ml borate buffer pH 8.5 (4.3.1) and homogenise the buffered solution by vortex mixing. Close the headspace vial hermetically and store the solution thus obtained at 100 °C (± 5 °C) for a period of 20 h (-0h +2h) to force hydrolysis of BADGE, BFDGE and their derivatives to the bis(diol) derivatives. After that period, cool the headspace vial to room temperature and transfer the solution into a HPLC vial.
Continue as described in 7.3. For 3 % acetic acid simulant, prior to hydrolysis, neutralize the simulant as follows. Pipette 1,0 ml of the simulant in a headspace vial (5.2), add 110 µl 4,5 M sodium hydroxide solution and mix carefully. Add 1,0 ml borate buffer pH 8.5 and continue as described above. 6.2.4 Fatty food simulant After the migration period, cool the simulant to room temperature. Weigh 10 g ± 0,01 g fat simulant into a 40 ml vial. Add 10,0 ml acetonitrile and shake vigorously for 1 min.
Allow the layers to separate for about 10 min. Transfer 4 ml ± 0,5 ml of the upper acetonitrile layer, using a Pasteur pipette, into a 5 ml syringe fitted to a C18 solid phase extraction cartridge (5.9). Pump the solution through the
EN 15136:2006 (E) 12 cartridge, collecting the eluent in a suitable vial. Transfer, from the solution thus obtained, an aliquot into a HPLC vial.
Continue as described in 7.3. 6.2.5 Forced hydrolysis of fatty food simulant extracts From the acetonitrile solution obtained after extraction of the fatty food simulant (6.2.4) pipette 2,0 ml into a headspace vial (5.2) and evaporate to dryness in a reaction therm heating module (5.8) under a nitrogen stream and heating.
Re-dissolve the residue in 400 µl acetonitrile, add 1600 µl borate buffer pH 8,5 (4.3.1) and homogenise the buffered solution by vortex mixing. Close the headspace vial hermetically and store the remaining solution at 100 °C (± 5 °C) for a period of 20 h (-0h +2h) to force hydrolysis. After this period cool the headspace vial to room temperature and transfer the solution in a HPLC vial. Continue as described in 7.3. 6.2.6 Preparation of blank food simulant samples Treat food simulants, which have not been in contact with coating material in the same way as described in 6.2.2 to 6.2.5. 6.3 Preparation of standards 6.3.1 General Prepare working standards of BADGE, BFDGE and/or their derivatives in food simulants in the first instance according to 6.3.2 and/or 6.3.3.
In case of forced hydrolysis of the test samples (6.2.3 and 6.2.5), prepare working standards of totally hydrolysed BADGE (BADGE.2H2O) and/or BFDGE (BFDGE.2H2O) according to 6.2.4 or 6.2.5.
NOTE Construction of calibration curves for all individual substances may be omitted (see 7.4.2). Quantification of individual substances may also be derived from a calibration curve of BADGE or BFDGE assuming an equal response factor for each substance. However a conversion for differences in molecular weight may be needed.
6.3.2 Working standards for the determination of BADGE, BFDGE and their derivatives in aqueous food simulants (range of 0,05 to 2 µµµµg/ml)
Transfer suitable volumes of the solution, prepared in 4.3.3, into a series of 25 ml volumetric flasks to obtain a calibration curve covering the range of 0,05 µg/ml up to 2 µg/ml for BADGE, BFDGE and their derivatives.
Make up to the mark with the relevant simulant and mix thoroughly.
Calculate the actual concentrations in the simulant expressed in µg/ml. Continue as described in 7.3. NOTE 1 The solutions in aqueous food simulants should be prepared freshly before analysis. NOTE 2 This working standard solution(s) are intended for reference purposes in respect to retention time and response factor of the individual substances. The results may be used to decide on the requirement set out in 7.4.2. NOTE 3 The working standard solution containing only BADGE and/or BFDGE should be used for quantitative purposes according to 6.2.4. 6.3.3 Working standards for the determination of BADGE, BFDGE and their derivatives in fatty food simulant (range of 0,05 µµµµg/g to 2 µµµµg/g) Weigh 10 g ± 0,01 g blank fatty food simulant into a series of 40 ml vials. Add suitable volumes of the stock solution, prepared as described in 4.3.2, to obtain a calibration curve covering the range of 0,05 µg/g up to 2 µg/g for BADGE, BFDGE and their derivatives. Alternatively the intermediate standard
EN 15136:2006 (E) 13 (4.3.3) can be added, but then the final volume of 10,0 ml acetonitrile should be taken into account as described below.
Calculate the actual concentration in the oil expressed in µg/g. Add acetonitrile to a final volume of 10,0 ml to the oil and shake vigorously for 1 min. Allow the layers to separate for about 10 min. Transfer 4 ml ± 0,5 ml of the upper acetonitrile layer, using a Pasteur pipette, into a 5 ml syringe fitted to a C18 solid phase extraction cartridge (5.9). Pump the solution through the cartridge, collecting the eluent in a suitable vial. Transfer, from the solution thus obtained, an aliquot into a HPLC vial. Continue as described in 7.3. NOTE 1 The solutions in acetonitrile are stable for at least several weeks when stored at room temperature with the exclusion of light. NOTE 2 These working standard solution(s) are intended for reference purposes in respect to retention time and response factor of the individual substances. The results may be used to decide on the requirement set out in 7.4.2. NOTE 3 The working standard solution containing only BADGE and/or BFDGE should be used for quantitative purposes according to 6.3.5. 6.3.4 Preparation of totally hydrolysed BADGE, BFDGE calibration solutions in the aqueous food simulants Mix 1,0 ml of the calibration solutions containing only BADGE and/or BFDGE as prepared in 6.3.2 with 1,0 ml borate buffer (4.3.1) in a headspace vial (5.2). Close the headspace vial hermetically and store the solution thus obtained at 100 °C ± 5 °C for a period of 20 h (-0h +2h) to force hydrolysis of BADGE, BFDGE and their derivatives to the bis(diol) derivatives. After that period, cool the headspace vial to room temperature and transfer the solution into a HPLC vial. Continue as described in 7.3. Prior to hydrolysis, neutralize the standard solutions prepared in 3% acetic acid as follows. Add to 1,0 ml calibration solution in 3 % acetic acid 110 µl 4,5 M sodium hydroxide solution (4.3.4) and mix carefully. Add 1,0 ml borate buffer (4.3.1) and continue as described above.
6.3.5 Preparation of totally hydrolysed BADGE and BFDGE calibration solutions in fatty food simulant From the acetonitrile solution obtained after extraction of the fatty fo
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