SIST-TS CEN/TS 13130-18:2005
(Main)Materials and articles in contact with foodstuffs - Plastics substances subject to limitation - Part 18: Determination of 1,2-dihydroxybenzene, 1,3-dihydroxybenzene, 1,4-dihydroxybenzene, 4,4'-dihydroxybenzophenone and 4,4'dihydroxybiphenyl in food simulants
Materials and articles in contact with foodstuffs - Plastics substances subject to limitation - Part 18: Determination of 1,2-dihydroxybenzene, 1,3-dihydroxybenzene, 1,4-dihydroxybenzene, 4,4'-dihydroxybenzophenone and 4,4'dihydroxybiphenyl in food simulants
This document, part of EN 13130, specifies two methods as follows.
Method A is an analytical procedure for the determination of the monomers 1,2-DHB,
1,3-DHB and 1,4-DHB in the food simulants water, 3 % w/v aqueous acetic acid, 15 % v/v aqueous ethanol and olive oil. The levels of monomer determined are expressed in milligrams of monomer per kilogram of food simulant. The method is appropriate for the quantitative determination of 1,2-DHB and 1,3-DHB at a minimum level of 0,15 mg/kg in food simulants and, of 1,4-DHB at a minimum level of 0,10 mg/kg in food simulants.
Method B is an analytical procedure for the determination of DHBphen and DHBiph in the food simulants water, 3 % w/v aqueous acetic acid, 15 % v/v aqueous ethanol and olive oil. The method is appropriate for the quantitative determination of DHBphen or DHBiph at minimum levels of 0,2 mg/kg and 0,25 mg/kg, respectively, in food simulants.
NOTE The methods should also be applicable to other aqueous food simulants as well as to the other fatty food simulants e.g. sunflower oil, corn oil or a mixture of synthetic triglycerides.
Werkstoffe und Gegenstände in Kontakt mit Lebensmitteln - Substanzen in Kunststoffen, die Beschränkungen unterliegen - Teil 18: Bestimmung von 1,2-Dihydroxybenzol, 1,3-Dihydroxybenzol, 1,4-Dihydroxybenzol, 4,4'-Dihydroxy-benzophenon und 4,4'-Dihydroxybiphenyl in Prüflebensmitteln
Dieses Dokument, Teil der EN 13130, legt folgende zwei Verfahren fest.
Verfahren A ist ein Analyseverfahren zur Bestimmung von monomerem 1,2-DHB, 1,3-DHB und 1,4-DHB in
den Prüflebensmitteln Wasser, 3%ige Essigsäure (m/V) in wässriger Lösung, 15%iges Ethanol (Volumenan-
teil) in wässriger Lösung und Olivenöl. Der ermittelte Gehalt an Monomeren wird in Milligramm Monomer je
Kilogramm Prüflebensmittel angegeben. Das Verfahren eignet sich zur quantitativen Bestimmung von
1,2-DHB und 1,3-DHB bei einem Mindestgehalt von 0,15 mg/kg Prüflebensmittel und von 1,4-DHB bei einem
Mindestgehalt von 0,10 mg/kg Prüflebensmittel.
Verfahren B ist ein Analyseverfahren zur Bestimmung von DHBphen und DHBiph in den Prüflebensmitteln
Wasser, 3%ige Essigsäure (m/V) in wässriger Lösung, 15%iges Ethanol (Volumenanteil) in wässriger Lösung
und Olivenöl. Das Verfahren eignet sich zur quantitativen Bestimmung von DHBphen oder DHBiph bei einem
Mindestgehalt von 0,2 mg/kg bzw. 0,25 mg/kg Prüflebensmittel.
ANMERKUNG Die Verfahren sollten auch auf andere wässrige Prüflebensmittel sowie weitere fettige Prüflebensmittel,
wie z. B. Sonnenblumenöl, Maiskeimöl oder eine Mischung synthetischer Triglyceride, anwendbar sein.
Matériaux et objets en contact avec les denrées alimentaires - Substances dans les matieres plastiques soumises a des limitations - Partie 18 : Détermination des 1,2-dihydroxybenzene, 1,3-dihydroxybenzene, 1,4-dihydroxybenzene, 4,4'-dihydroxybenzophénone et 4,4'-dihydroxybiphénil dans les simulants d'aliments
Le présent document qui fait partie de l'EN 13130, spécifie deux méthodes d'essai.
La méthode A est une procédure d'analyse permettant de déterminer le 1,2-DHB, le 1,3-DHB et le 1,4-DHB dans les simulant d'aliments suivants : eau, solution aqueuse d'acide acétique a 3 % (m/v), solution aqueuse d'éthanol a 15 % (v/v) et huile d'olive. La teneur en monomere obtenue est exprimée en milligrammes de monomere par kilogrammes de denrée alimentaire. Cette méthode convient pour la détermination quantitative du 1,2-DHB et du 1,3-DHB, pour une concentration minimale de 0,15 mg par kilogramme de simulant d'aliments et du 1,4-DHB, pour une concentration minimale de 0,10 mg par kilogramme de simulant d'aliments.
La méthode B est une procédure d'analyse permettant de déterminer le DHBphen et le DHBiph dans les simulant d'aliments suivants : eau, solution aqueuse d'acide acétique a 3 % (m/v), solution aqueuse d'éthanol a 15 % (v/v) et huile d'olive. Cette méthode convient pour la détermination quantitative du DHBphen et du DHBiph, pour une concentration minimale de 0,2 mg/kg et de 0,25 mg/kg de simulants d'aliments, respectivement.
NOTE Il convient que ces méthodes soient également applicables a d'autres simulants aqueux ainsi qu'a d'autres simulants gras tels que l'huile de tournesol, l'huile de mais ou un mélange de triglycérides synthétiques.
Materiali in predmeti v stiku z živili - Snovi v polimernih materialih, katerih koncentracija je omejena - 18. del: Določevanje 1,2-dihidroksibenzena, 1,3- dihidroksibenzena, 1,4- dihidroksibenzena, 4,4'- dihidroksibenzofenona and 4,4' dihidroksibifenila v modelnih raztopinah za živila
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST-TS CEN/TS 13130-18:2005
01-april-2005
0DWHULDOLLQSUHGPHWLYVWLNX]åLYLOL6QRYLYSROLPHUQLKPDWHULDOLKNDWHULK
NRQFHQWUDFLMDMHRPHMHQDGHO 'RORþHYDQMHGLKLGURNVLEHQ]HQD
GLKLGURNVLEHQ]HQDGLKLGURNVLEHQ]HQD
GLKLGURNVLEHQ]RIHQRQDDQG
GLKLGURNVLELIHQLODYPRGHOQLKUD]WRSLQDK]DåLYLOD
Materials and articles in contact with foodstuffs - Plastics substances subject to limitation
- Part 18: Determination of 1,2-dihydroxybenzene, 1,3-dihydroxybenzene, 1,4-
dihydroxybenzene, 4,4'-dihydroxybenzophenone and 4,4'dihydroxybiphenyl in food
simulants
Werkstoffe und Gegenstände in Kontakt mit Lebensmitteln - Substanzen in Kunststoffen,
die Beschränkungen unterliegen - Teil 18: Bestimmung von 1,2-Dihydroxybenzol, 1,3-
Dihydroxybenzol, 1,4-Dihydroxybenzol, 4,4'-Dihydroxy-benzophenon und 4,4'-
Dihydroxybiphenyl in Prüflebensmitteln
Matériaux et objets en contact avec les denrées alimentaires - Substances dans les
matieres plastiques soumises a des limitations - Partie 18 : Détermination des 1,2-
dihydroxybenzene, 1,3-dihydroxybenzene, 1,4-dihydroxybenzene, 4,4'-
dihydroxybenzophénone et 4,4'-dihydroxybiphénil dans les simulants d'aliments
Ta slovenski standard je istoveten z: CEN/TS 13130-18:2005
ICS:
67.250 Materiali in predmeti v stiku z Materials and articles in
živili contact with foodstuffs
SIST-TS CEN/TS 13130-18:2005 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST-TS CEN/TS 13130-18:2005
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SIST-TS CEN/TS 13130-18:2005
TECHNICAL SPECIFICATION
CEN/TS 13130-18
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
February 2005
ICS 67.250
English version
Materials and articles in contact with foodstuffs - Plastics
substances subject to limitation - Part 18: Determination of 1,2-
dihydroxybenzene, 1,3-dihydroxybenzene, 1,4-
dihydroxybenzene, 4,4'-dihydroxybenzophenone and
4,4'dihydroxybiphenyl in food simulants
Matériaux et objets en contact avec les denrées Werkstoffe und Gegenstände in Kontakt mit Lebensmitteln
alimentaires - Substances dans les matières plastiques - Substanzen in Kunststoffen, die Beschränkungen
soumises à des limitations - Partie 18 : Détermination des unterliegen - Teil 18: Bestimmung von 1,2-
1,2-dihydroxybenzène, 1,3-dihydroxybenzène, 1,4- Dihydroxybenzol, 1,3-Dihydroxybenzol, 1,4-
dihydroxybenzène, 4,4'-dihydroxybenzophénone et 4,4'- Dihydroxybenzol, 4,4'-Dihydroxy-benzophenon und 4,4'-
dihydroxybiphénil dans les simulants d'aliments Dihydroxybiphenyl in Prüflebensmitteln
This Technical Specification (CEN/TS) was approved by CEN on 16 December 2004 for provisional application.
The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their
comments, particularly on the question whether the CEN/TS can be converted into a European Standard.
CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available
promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)
until the final decision about the possible conversion of the CEN/TS into an EN is reached.
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, Slovakia,
Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels
© 2005 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 13130-18:2005: E
worldwide for CEN national Members.
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Contents
page
Foreword .3
Introduction.5
1 Scope.6
2 Normative references.7
3 Method A – Determination of 1,2-dihydroxybenzene, 1,3-dihydroxybenzene and/or 1,4-
dihydroxybenzene in food simulants.7
4 Method B – Determination of 4,4’-dihydroxybenzophenone and 4,4’dihydroxybiphenyl in
food simulants.16
Bibliography.24
2
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Foreword
This document (CEN/TS 13130-18:2005) has been prepared by Technical Committee CEN/TC 194 “Utensils
in contact with food”, the secretariat of which is held by BSI.
This part of EN 13130 has been prepared within the Standards, Measurement and Testing project,
MAT1-CT92-0006, “Development of Methods of Analysis for Monomers” and has been prepared by
Subcommittee (SC 1) of TC 194 "Utensils in contact with food" as one of a series of test methods for plastics
materials and articles in contact with foodstuffs.
This standard is intended to support Directives 2002/72/EC [1], 89/109/EEC [2], 82/711/EEC [3] and its
amendments 93/8/EEC [4] and 97/48/EC [5], and 85/572/EEC [6].
At the time of preparation and publication of this part of EN 13130 the European Union legislation relating to
plastics materials and articles intended to come into contact with foodstuffs is incomplete. Further Directives
and amendments to existing Directives are expected which could change the legislative requirements which
this standard supports. It is therefore strongly recommended that users of this standard refer to the latest
relevant published Directive(s) before commencement of a test or tests described in this standard.
This part of EN 13130 should be read in conjunction with EN 13130-1.
Further parts of EN 13130, under the general title Materials and articles in contact with foodstuffs - Plastics
substances subject to limitation, have been prepared, and others are in preparation, concerned with the
determination of specific migration from plastics materials into foodstuffs and food simulants and the
determination of specific monomers and additives in plastics. The parts of EN 13130 are as follows.
Part 1: Guide to test methods for the specific migration of substances from plastics to foods and food
simulants and the determination of substances in plastics and the selection of conditions of exposure to food
simulants
Part 2: Determination of terephthalic acid in food simulants
Part 3: Determination of acrylonitrile in food and food simulants
Part 4: Determination of 1,3-butadiene in plastics
Part 5: Determination of vinylidene chloride in food simulants
Part 6: Determination of vinylidene chloride in plastics
Part 7: Determination of monoethylene glycol and diethylene glycol in food simulants
Part 8: Determination of isocyanates in plastics
Part 9: Determination of acetic acid, vinyl ester in food simulants
Part 10: Determination of acrylamide in food simulants
Part 11: Determination of 11-aminoundecanoic acid in food simulants
Part 12: Determination of 1,3-benzenedimethanamine in food simulants
Part 13: Determination of 2,2-bis(4-hydroxyphenyl)propane (Bisphenol A) in food simulants
3
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Part 14: Determination of 3,3-bis(3-methyl-4-hydroxyphenyl)-2-indoline in food simulants
Part 15: Determination of 1,3-butadiene in food simulants
Part 16: Determination of caprolactam and caprolactam salt in food simulants
Part 17: Determination of carbonyl chloride in plastics
Part 18: Determination of 1,2-dihydroxybenzene, 1,3-dihydroxybenzene,
1,4-dihydroxybenzene, 4,4’-dihydroxybenzophenone and 4,4’dihydroxybiphenyl in food simulants
Part 19: Determination of dimethylaminoethanol in food simulants
Part 20: Determination of epichlorohydrin in plastics
Part 21: Determination of ethylenediamine and hexamethylenediamine in food simulants
Part 22: Determination of ethylene oxide and propylene oxide in plastics
Part 23: Determination of formaldehyde and hexamethylenetetramine in food simulants
Part 24: Determination of maleic acid and maleic anhydride in food simulants
Part 25: Determination of 4-methyl-pentene in food simulants
Part 26: Determination of 1-octene and tetrahydrofuran in food simulants
Part 27: Determination of 2,4,6-triamino-1,3,5-triazine in food simulants
Part 28: Determination of 1,1,1-trimethylolpropane in food simulants
Parts 1 to 8 are European Standards. Parts 9 to 28 are Technical Specifications.
WARNING All chemicals are hazardous to health to a greater or lesser extent. It is beyond the
scope of this Technical Specification to give instructions for the safe handling of all chemicals, that
meet, in full, the legal obligations in all countries in which this Technical Specification may be
followed. Therefore, specific warnings are not given and users of this Technical Specification 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 announce this CEN Technical Specification: Austria, Belgium, Cyprus, Czech Republic,
Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland
and United Kingdom.
4
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Introduction
1,2-dihydroxybenzene (1,2-DHB), (pyro)catechol, C H (OH), PM/Ref. No 15880,
6 4 4
1,3-dihydroxybenzene (1,3-DHB), resorcinol, C H (OH), PM/Ref. No 15910, 1,4-dihydroxybenzene
6 4 2
(1,4-DHB), hydroquinone, C H (OH) , PM/Ref-No. 15940, 4,4'-dihydroxybenzophenone (DHBphen), HOC H
6 4 2 6 4
COC H OH, PM/Ref. No 15970 and 4,4'-dihydroxybiphenyl (DHBiph), HOC H C H OH,
6 4 6 4 6 4
PM/Ref. No 16000, are monomers used in the manufacture of certain plastics materials and articles intended
to come into contact with foodstuffs. After manufacture, residual monomer(s) can remain in the polymer and
may migrate into foodstuffs coming into contact with the plastics material or article.
Method A describes the determination of 1,2-DHB, 1,3-DHB, 1,4-DHB in food simulants.
Method B describes the determination of DHBphen and DHBiph in food simulants. However, in cases where
migration occurs, pay attention to the clear identification of the analyte, since under the conditions described
in the method DHBphen and DHBiph have the same retention time.
NOTE The following should be taken into account when carrying out a migration test (see EN 13130-1). From
migration experiments carried out for 10 d at 40 °C it was recognized that 1,2-DHB, 1,3-DHB and 1,4-DHB are subject to
losses due to chemical decomposition. The loss observed for 1,2-DHB was up to 20 % in aqueous simulants, that for
1,3-DHB was 5 % to 12 % in olive oil and that for 1,4-DHB was 14 % to 68 %, in both aqueous food simulants and olive oil.
For DHBiph a small loss of 5 % to 12 % due to chemical decomposition was observed when using 15 % v/v aqueous
ethanol as food simulant.
The methods described have been pre-validated by collaborative trials with two laboratories.
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1 Scope
This document, part of EN 13130, specifies two methods as follows.
Method A is an analytical procedure for the determination of the monomers 1,2-DHB,
1,3-DHB and 1,4-DHB in the food simulants water, 3 % w/v aqueous acetic acid, 15 % v/v aqueous ethanol
and olive oil. The levels of monomer determined are expressed in milligrams of monomer per kilogram of food
simulant. The method is appropriate for the quantitative determination of 1,2-DHB and 1,3-DHB at a minimum
level of 0,15 mg/kg in food simulants and, of 1,4-DHB at a minimum level of 0,10 mg/kg in food simulants.
Method B is an analytical procedure for the determination of DHBphen and DHBiph in the food simulants
water, 3 % w/v aqueous acetic acid, 15 % v/v aqueous ethanol and olive oil. The method is appropriate for the
quantitative determination of DHBphen or DHBiph at minimum levels of 0,2 mg/kg and 0,25 mg/kg,
respectively, in food simulants.
NOTE The methods should also be applicable to other aqueous food simulants as well as to the other fatty food
simulants e.g. sunflower oil, corn oil or a mixture of synthetic triglycerides.
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2 Normative references
The following referenced documents are indispensable for the application 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.
EN 13130-1:2004, Materials and articles in contact with foodstuffs – Plastics substances subject to limitation –
Part 1: Guide to test methods for the specific migration of substances from plastics to foods and food
simulants and the determination of substances in plastics and the selection of conditions of exposure to food
simulants.
3 Method A – Determination of 1,2-dihydroxybenzene, 1,3-dihydroxybenzene and/or
1,4-dihydroxybenzene in food simulants
3.1 Principle
The aqueous simulant test samples are analyzed directly by high performance liquid chromatography (HPLC)
with ultra violet (UV) detection. The olive oil test samples are extracted with distilled water and the resultant
aqueous solution analyzed by HPLC. Quantification is achieved using 2-methyl-1,3-dihydroxybenzene as an
internal standard with calibration against relevant food simulant samples fortified with known amounts of
1,2-DHB, 1,3-DHB or 1,4-DHB. Confirmation of 1,2-DHB, 1,3-DHB or 1,4-DHB is carried out by diode array
detection.
3.2 Reagents
NOTE All reagents should be of recognized analytical quality unless otherwise stated.
3.2.1 Analytes
3.2.1.1 1,2-dihydroxybenzene (1,2-DHB), C H (OH) , purity greater than 99 %.
6 4 2
3.2.1.2 1,3-dihydroxybenzene (1,3-DHB), C H (OH) , purity greater than 99 %.
6 4 2
3.2.1.3 1,4-dihydroxybenzene (1,4-DHB), C H (OH) , purity greater than 99 %.
6 4 2
3.2.1.4 2-methyl-1,3-dihydroxybenzene (2-methylresorcinol) (2-MR), CH C H (OH) , internal standard,
3 6 3 2
purity greater than 98 %.
3.2.2 Chemicals
3.2.2.1 Hexane, glass distilled grade.
3.2.2.2 Methanol, glass distilled grade.
3.2.2.3 Water, glass distilled grade.
3.2.2.4 Acetonitrile, HPLC grade.
3.2.2.5 Water, distilled, HPLC grade.
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3.2.2.6 Sodium dihydrogen orthophosphate, NaH PO .2H O.
2 4 2
3.2.2.7 Glacial acetic acid.
3.2.2.8 Mobile phase for high performance liquid chromatography, prepared as follows.
Dissolve 7,5 g of sodium dihydrogen orthophosphate (3.2.2.6) in 800 ml of water (3.2.2.5), add 150 ml
acetonitrile (3.2.2.4) using a measuring cylinder and adjust to pH (3,6 ± 0,2) with glacial acetic acid (3.2.2.7)
(approximately 5 ml). Make up to 1 l with water (3.2.2.5).
NOTE Degassing the mobile phase may be necessary with some HPLC equipment.
3.2.3 Solutions
NOTE The solutions prepared in 3.2.3.1 to 3.2.3.7 can be stored in the dark at – 20 °C for up to 6 weeks.
3.2.3.1 Standard stock solution 1,2-DHB and/or 1,4-DHB in methanol at a defined concentration of
approximately 0,5 g/l
Weigh 50 mg ± 2 mg of 1,2-DHB, or 1,4-DHB into a 100 ml volumetric flask to an accuracy of 0,2 mg and
dilute to the mark with methanol (3.2.2.2). Close and mix thoroughly.
Calculate the exact concentration of 1,2-DHB and/or 1,4-DHB in milligrams per litre.
Repeat the procedure to provide a second standard stock solution.
3.2.3.2 Standard stock solution 1,3-DHB in methanol at a defined concentration of approximately
0,125 g/l
Weigh 12,5 mg ± 2 mg of 1,3-DHB into a 100 ml volumetric flask to an accuracy of 0,2 mg and dilute to the
mark with methanol (3.2.2.2). Close and mix thoroughly.
Calculate the exact concentration of 1,3-DHB in milligrams per litre.
Repeat the procedure to provide a second standard stock solution.
3.2.3.3 Stock internal standard solution of 2-MR in methanol at a defined concentration of
approximately 1 g/l
Weigh 100 mg ± 2 mg of 2-MR into a 100 ml volumetric flask to an accuracy of 0,2 mg and dilute to the mark
with methanol (3.2.2.2). Close and mix thoroughly.
Calculate the exact concentration of 2-MR in milligrams per litre.
3.2.3.4 Intermediate standard solutions of 1,2-DHB and 2-MR in methanol
Into six 25 ml volumetric flasks, pipette 0 ml, 2,0 ml, 4,0 ml, 8,0 ml, 16,0 ml and 20,0 ml of the 1,2-DHB
standard stock solution (3.2.3.1). Add 5,0 ml of the 2-MR stock internal standard solution (3.2.3.3) and dilute
to the mark with methanol. The resulting intermediate standards contain approximately 0 mg/l, 40 mg/l,
80 mg/l, 160 mg/l, 320 mg/l and 400 mg/l 1,2-DHB and 200 mg/l 2-MR.
Calculate the exact concentrations of 1,2-DHB and 2-MR in milligrams per litre.
Repeat the procedure using the second solution prepared in 3.2.3.1 to provide a second set of intermediate
standards.
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3.2.3.5 Intermediate standard solutions of 1,3-DHB and 2-MR in methanol
Into six 25 ml volumetric flasks, pipette 0 ml, 2,0 ml, 4,0 ml, 8,0 ml, 16,0 ml and 20,0 ml of the 1,3-DHB
standard stock solution (3.2.3.2). Add 5,0 ml of the 2-MR stock internal standard solution (3.2.3.3) and dilute
to the mark with methanol. The resulting intermediate standards contain approximately 0 mg/l, 10 mg/l,
20 mg/l, 40 mg/l, 80 mg/l and 100 mg/l 1,3-DHB and 200 mg/l 2-MR.
Calculate the exact concentrations of 1,3-DHB and 2-MR in milligrams per litre.
Repeat the procedure using the second solution prepared in 3.2.3.2 to provide a second set of intermediate
standards.
3.2.3.6 Intermediate standard solutions of 1,4-DHB and 2-MR in methanol
Into a 100 ml volumetric flask, pipette 10 ml of the 1,4-DHB standard stock solution (3.2.3.1) and dilute to the
mark with methanol thus obtaining a diluted stock solution of 1,4-DHB of approximately 50 mg/l.
Into six 25 ml volumetric flasks, pipette 0 ml, 1,5 ml, 5,0 ml, 7,5 ml, 10,0 ml and 15,0 ml of this diluted 1,4-DHB
stock solution. Add 5,0 ml of the 2-MR stock internal standard solution (3.2.3.3) and dilute to the mark with
methanol. These standards contain approximately 0 mg/l, 3 mg/l, 10 mg/l, 15 mg/l, 20 mg/l and 30 mg/l
1,4-DHB and 200 mg/l 2-MR.
Calculate the exact concentrations of 1,4-DHB and 2-MR in milligrams per litre of solution.
Repeat the procedure using the second stock solution prepared in 3.2.3.1 to provide a second set of
intermediate standards.
3.2.3.7 Intermediate internal standard solution:
Pipette 5 ml of the 2-MR stock internal standard solution (3.2.3.3) into a 25 ml volumetric flask and dilute to
the mark with methanol. This solution contains approximately 200 mg/l 2-MR.
Calculate the exact concentration of 2-MR in milligrams per litre of solution. The calculated concentration shall
be the same as in 3.2.3.4, 3.2.3.5 and 3.2.3.6.
3.3 Apparatus
NOTE An instrument or item of apparatus is listed only where it is special or made to a particular specification, the
usual laboratory glassware and equipment being assumed to be available.
3.3.1 High performance liquid chromatograph, with a 20 µl injection loop, and a variable wavelength UV
detector, set to 280 nm, connected to a strip chart recorder or integrator.
3.3.2 HPLC column, capable of fully resolving 1,2-DHB, 1,3 DHB and/or 1,4-DHB from 2-MR, such that the
peaks of 1,2-DHB, 1,3-DHB and/or 1,4-DHB and 2-MR do not overlap by more than 1 % peak area with each
other and with interferences arising from the injection media.
Appropriate operating conditions shall be established for the specific equipment used for the determination.
NOTE The following column has been found to be suitable:
Column 250 mm x 4,6 mm I.D. octadecylsilane, 10 % carbon loading with Hypersil 5 µm
packing
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The following operating conditions were found to be suitable for this column:
Injector : 20 µl total loop fill
Mobile phase flow rate : 1 ml/min
Detector (UV) : 280 nm
3.4 Samples
3.4.1 General
Laboratory samples of the food simulant to be analyzed shall be obtained as described in EN 13130-1.
Samples shall be kept refrigerated at 4 °C in closed containers with the exclusion of light for up to four weeks.
Analyte-free samples of relevant food simulants of the same type as those to be analyzed shall also be
prepared for calibration purposes.
Take into account the possible loss of analyte due to chemical decomposition in aqueous food simulants (see
NOTE in the Introduction).
3.4.2 Preparation of test samples
3.4.2.1 Aqueous food simulants
Pipette 1 ml of the intermediate internal standard solution (3.2.3.7) into a dry 25 ml volumetric flask and dilute
to the mark with the food simulant obtained from the migration experiment (see EN 13130-1), mixing
thoroughly. If the aqueous simulant sample contains suspended solids, filter using a 0,2 µm disposable HPLC
membrane filter. Repeat the procedure to provide a duplicate sample.
3.4.2.2 Olive oil
Weigh 25 g ± 0,1g of the olive oil food simulant obtained from the migration experiment (see EN 13130-1) into
a dry beaker. Pour the olive oil into a 100 ml separating funnel. Pipette into the separating funnel 1 ml of the
intermediate internal standard solution (3.2.3.7) and mix thoroughly. Rinse the beaker with 25 ml ± 1 ml of
hexane and transfer to the separating funnel. Mix and add 10 ml ± 1 ml of distilled water using a measuring
cylinder.
Shake for 1 min and allow 5 min for the phases to separate. Collect the aqueous phase and re-extract the
olive oil with a further 10 ml ± 1 ml of distilled water. Allow the phases to separate and run off the aqueous
phase. Combine the aqueous extracts in a 25 ml volumetric flask and make up to the mark with distilled water.
Pass the aqueous solution through a glass funnel, provided with a small plug of cotton wool to retain any olive
oil.
Repeat the procedure to provide a duplicate sample.
3.4.3 Preparation of blank samples
3.4.3.1 Prepare a sample as in 3.4.2.1 and 3.4.2.2, but omit the addition of the internal standard,
replacing it with 1 ml of methanol (3.2.2.2).
3.4.3.2 Obtain blank samples of simulant (i.e. free of 1,2-DHB, 1,3-DHB or 1,4-DHB and 2-MR).
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SIST-TS CEN/TS 13130-18:2005
CEN/TS 13130-18:2005 (E)
3.4.4 Preparation of calibration samples
3.4.4.1 Working standards for aqueous food simulants
For the determination of 1,2-DHB, pipette 1 ml of each intermediate standard (3.2.3.4) into six 25 ml
volumetric flasks, and dilute to the mark with the appropriate analyte-free aqueous food simulant (3.4.3.2) to
give approximately 0 mg/kg, 1,6 mg/kg, 3,2 mg/kg, 6,4 mg/kg, 12,8 mg/kg and 16 mg/kg 1,2-DHB and
8 mg/kg 2-MR. Close and mix thoroughly.
For at least one food simulant, repeat the procedure using the second set of intermediate standards (3.2.3.4).
Calculate the exact concentration of 1,2-DHB in milligrams per kilogram in the working standards.
For the determination of 1,3-DHB, pipette 1 ml of each intermediate standard (3.2.3.5) into six 25 ml
volumetric flasks, and dilute to the mark with the appropriate analyte-free aqueous food simulant (3.4.3.2) to
give approximately 0 mg/kg, 0,4 mg/kg, 0,8 mg/kg, 1,6 mg/kg, 3,2 mg/kg and 4,0 mg/kg 1,3-DHB and 8 mg/kg
2-MR. Close and mix thoroughly.
For at least for one food simulant, repeat the procedure using the second set of intermediate standards
(3.2.3.5).
Calculate the exact concentration of 1,3-DHB in milligrams per kilogram in the working standards.
For the determination of 1,4-DHB, pipette 1 ml of each intermediate standard (3.2.3.6) into six 25 ml
volumetric flasks, and dilute to the mark with the appropriate analyte-free aqueous food simulant (3.4.3.2) to
give approximately 0 mg/kg, 0,12 mg/kg, 0,40 mg/kg, 0,60 mg/kg, 0,80 mg/kg and 1,20 mg/kg 1,4-DHB and
8 mg/kg 2-MR. Close and mix thoroughly.
For at least for one food simulant, repeat the procedure using the second set of intermediate standards
(3.2.3.6).
Correct the calculated concentrations by multiplying each by a factor 1,04 to allow for the dilution of the food
simulant with methanol. The corrected values shall be the relevant concentrations of the calibration samples.
3.4.4.2 Working standards for olive oil simulant
Using a beaker, weigh 25 g ± 0,1g of analyte-free olive oil (3.4.3.2) into a 100 ml separating funnel. Add by
pipette, 1 ml of the first intermediate standard (3.2.3.4 or 3.2.3.5 or 3.2.3.6), mix well and add 25 ml ± 1 ml of
hexane, rinsing the above beaker in the process.
Extract with distilled water following the procedure described in 3.4.1.2.
Repeat this procedure for each intermediate standard, and for the second set of intermediate standards.
Calculate the exact concentration of 1,2-DHB, 1,3-DHB or 1,4-DHB in milligrams per kilogram in the olive oil
calibration samples.
3.5 Procedure
3.5.1 HPLC analysis
The test samples, blanks and calibration samples prepared in 3.4.1 to 3.4.3 shall be analyzed as they are
without any further sample treatment. Identify the 1,2-DHB, 1,3-DHB and/or 1,4-DHB and
2-MR peaks on the basis of the retention times and measure the peak area.
Examine the baseline stability and response linearity of the detector before starting measurements.
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SIST-TS CEN/TS 13130-18:2005
CEN/TS 13130-18:2005 (E)
Maintain the same operating conditions throughout the measurement of all samples and calibration solutions.
Inject each solution at least in duplicate.
NOTE Under the conditions given in 3.3.2 the retention times for 1,2-DHB, 1,3-DHB, 1,4-DHB and 2-MR were
9,3 min, 6,4 min, 4,4 min and 7,6 min, respectively.
Inject the samples (3.4.1.1 and 3.4.1.2) applying duplicate injections together with the blank samples (3.4.2.1)
under the conditions in 3.3.2.
3.5.2 Calibration
Applying duplicate injections, analyze the calibration samples (3.4.3.1 and 3.4.3.2) under the conditions
described in 3.3.2. Measure the areas of the 1,2-DHB, 1,3-DHB, 1,4-DHB and 2-MR peaks. Construct
calibration curves plotting peak area ratio (PAR) of 1,2-DHB/2-MR 1,3-DHB/2-MR, 1,4-DHB/2-MR values
against the corrected concentration values of 1,2-DHB, 1,3-DHB, 1,4-DHB in milligrams per kilogram of food
simulant.
NOTE Commission Directive 90/128/EEC [1] states that the specific gravity of all simulants should conventionally be
assumed to be ‘1’. Milligrams of substance released per litre of simulant will thus correspond numerically to milligrams of
substance released per kilogram of simulant and, taking into account of the provisions laid down in Directive 82/711//EEC
[3], to milligrams of substance released per kilogram of foodstuff.
The calibration curves shall be rectilinear and the correlation coefficient shall be 0,996 or better.
The two sets of calibrant solutions made from independently prepared stock solutions shall be cross-checked
by generating two calibration curves which on the basis of peak area measurement shall agree to within ± 5 %
of one another.
3.5.3 Evaluation of data
3.5.3.1 HPLC interferences
NOTE So far no interferences have been observed when following this method.
If the blank sample with 2-MR omitted shows an interference in the 2-MR region of the chromatogram
exceeding 10 % of the area of 2-MR in the calibration samples (3.4.3.1 and 3.4.3.2), and if the analysis of
replicate blank samples reveals that this interference varies by more than ± 20 % in absolute size, external
calibration shall be used.
If the analysis of the zero point calibration sample shows peaks in the 1,2-DHB, 1,3-DHB and/or 1,4-DHB
region corresponding to less than 0,6 mg/kg, 0,25 mg/kg and/or 0,1 mg/kg, respectively, when calculated
according to 3.5.3.2 and the absolute area of duplicates does not vary by more than 10 %, the PAR of the
zero point calibration sample shall be subtracted from the ratios of the test sample and the calibration samples
and the data plotted as in 3.5.2. If the interference corresponds to more than 0,6 mg/kg, 0,25 mg/kg and/or
0,1 mg/kg, respectively, the m
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