oSIST prEN 16466-1:2023
(Main)Food authenticity - Isotopic analysis of acetic acid and water in vinegar - Part 1: 2H-NMR analysis of acetic acid
Food authenticity - Isotopic analysis of acetic acid and water in vinegar - Part 1: 2H-NMR analysis of acetic acid
This document specifies an isotopic method to control the authenticity of vinegar and food containing vinegar as an ingredient (for example Aceto Balsamico di Modena), with a density below 1,28 g/cm3. This method is applicable on acetic acid of vinegar (from wine, cider, agricultural alcohol, etc.) in order to characterize the botanical origin of acetic acid and to detect adulterations of vinegar using synthetic acetic acid or acetic acid from a non-allowed origin (together with the method described in EN 16466-2).
The isotopic analysis of the extracted acetic acid by 2H-NMR is based on a similar method already normalized for wine analysis [10].
The application of this document can involve the use of hazardous substances, operations and equipment. This document does not claim to address all associated safety issues. It is the responsibility of the user of this document to take appropriate measures for the safety and health protection of personnel before use, and to check the applicability of existing national and European rules and regulations.
Lebensmittelauthentizität - Isotopenanalyse von Essigsäure und Wasser in Essig - Teil 1: 2H-NMR-Analyse von Essigsäure
Dieses Dokument legt ein isotopenanalytisches Verfahren zur Überprüfung der Authentizität von Essig und von Lebensmitteln, die Essig als Zutat enthalten, (zum Beispiel Aceto Balsamico di Modena) mit einer Dichte von weniger als 1,28 g/cm3 fest. Dieses Verfahren ist anwendbar auf Essigsäure aus Essig (aus Wein, Apfelwein, Agraralkohol usw.), um den botanischen Ursprung der Essigsäure zu charakterisieren und um Verfälschungen von Essig durch Verwendung synthetischer Essigsäure oder Essigsäure nicht zulässigen Ursprungs nachzuweisen (in Kombination mit dem in EN 16466-2 beschriebenen Verfahren).
Die Isotopenanalyse der extrahierten Essigsäure durch 2H NMR-Spektroskopie beruht auf einem ähnlichen Verfahren, das bereits für die Weinanalyse genormt wurde [10].
Die Anwendung dieses Dokuments kann die Verwendung gefährlicher Stoffe, Vorgänge und Prüfeinrichtungen vorsehen. Dieses Dokument erhebt nicht den Anspruch, alle damit verbundenen Sicherheitsprobleme zu behandeln. Es liegt in der Verantwortung des Anwenders dieses Dokuments, vor der Anwendung geeignete Maßnahmen für die Sicherheit und den Gesundheitsschutz des Personals zu ergreifen und die Anwendbarkeit bestehender nationaler und europäischer Regeln und Vorschriften (Regelwerke) zu prüfen.
Authenticité des aliments - Analyse isotopique de l'acide acétique et de l'eau dans le vinaigre - Partie 1 : Analyse RMN-2H de l'acide acétique
Le présent document spécifie une méthode isotopique permettant de contrôler l’authenticité du vinaigre et des aliments contenant du vinaigre en tant qu’ingrédient (par exemple l’« Aceto Balsamico di Modena »), dont la densité est inférieure à 1,28 g/cm3. La présente méthode est applicable à l’acide acétique du vinaigre (de vin, de cidre, d’alcool agricole, etc.) afin de caractériser l’origine botanique de l’acide acétique et de détecter des adultérations du vinaigre utilisant de l’acide acétique synthétique ou de l’acide acétique d’une origine non autorisée (en complément de la méthode décrite dans l’EN 16466-2).
L’analyse isotopique par RMN-2H de l’acide acétique extrait est fondée sur une méthode similaire à celle de l’analyse du vin, déjà normalisée [10].
L’application du présent document peut impliquer l’utilisation de substances, d’opérations et d’équipement dangereux. Le présent document ne prétend pas traiter toutes les questions de sécurité associées. Il est de la responsabilité de l’utilisateur du présent document de prendre préalablement les mesures appropriées pour la sécurité et la protection de la santé du personnel et de vérifier l’applicabilité de règles et règlements nationaux et européens existants.
Pristnost živil - Izotopska analiza ocetne kisline in vode v kisu - 1. del: 2H-NMR-analiza ocetne kisline
General Information
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Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN 16466-1:2023
01-julij-2023
Pristnost živil - Izotopska analiza ocetne kisline in vode v kisu - 1. del: 2H-NMR-
analiza ocetne kisline
Food authenticity - Isotopic analysis of acetic acid and water in vinegar - Part 1: 2H-NMR
analysis of acetic acid
Lebensmittelauthentizität - Isotopenanalyse von Essigsäure und Wasser in Essig - Teil 1:
2H-NMR-Analyse von Essigsäure
Authenticité des aliments - Analyse isotopique de l'acide acétique et de l'eau dans le
vinaigre - Partie 1 : Analyse RMN-2H de l'acide acétique
Ta slovenski standard je istoveten z: prEN 16466-1
ICS:
67.220.20 Dodatki jedem Food additives
oSIST prEN 16466-1:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 16466-1:2023
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oSIST prEN 16466-1:2023
DRAFT
EUROPEAN STANDARD
prEN 16466-1
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2023
ICS 67.220.10 Will supersede EN 16466-1:2013
English Version
Food authenticity - Isotopic analysis of acetic acid and
water in vinegar - Part 1: 2H-NMR analysis of acetic acid
Authenticité des aliments - Analyse isotopique de Lebensmittelauthentizität - Isotopenanalyse von
l'acide acétique et de l'eau dans le vinaigre - Partie 1 : Essigsäure und Wasser - Teil 1: 2H-NMR-Analyse von
Analyse RMN-2H de l'acide acétique Essigsäure
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 460.
If this draft becomes a European Standard, 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.
This draft European Standard was established by CEN 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-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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 16466-1:2023 E
worldwide for CEN national Members.
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oSIST prEN 16466-1:2023
prEN 16466-1:2023 (E)
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 5
5 Reagents . 5
6 Apparatus . 6
7 Procedure . 7
7.1 Extraction of acetic acid from vinegar . 7
7.1.1 General. 7
7.1.2 Liquid-liquid extraction . 7
7.1.3 Purification of the extract . 8
7.2 Composite NMR determination of acetic acid from vinegar . 10
7.2.1 General. 10
7.2.2 NMR preparation. 10
7.2.3 Acquisition of composite NMR spectra . 10
7.2.4 Calculations and expression of the result . 11
7.2.5 Quality control of the analysis . 12
8 Precision . 12
8.1 General. 12
8.2 Repeatability . 12
8.3 Reproducibility . 12
Annex A (informative) Results of the collaborative study (2020) . 13
Bibliography . 15
2
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oSIST prEN 16466-1:2023
prEN 16466-1:2023 (E)
European foreword
This document (prEN 16466-1:2023) has been prepared by Technical Committee CEN/TC 460 “Food
authenticity”, the secretariat of which is held by DIN.
This document is currently submitted to the CEN Enquiry.
This document will supersede EN 16466-1:2013.
The main changes compared to the previous edition are listed below:
a) extension to matrices other than wine vinegar;
b) modification of the NMR sequence;
c) standard editorially revised.
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prEN 16466-1:2023 (E)
Introduction
Vinegar is defined in EN 13188 [3] as the acetic acid solution resulting from a double fermentation:
a) transformation of sugars to ethanol; and
b) transformation of ethanol to acetic acid.
Conversely, EN 13189 [4] defines acetic acid as “Product made from materials of non-agricultural
origin”.
Wine vinegar is defined by the European Regulations 1308/2013 [5] as the product obtained
exclusively from the acetous fermentation of wine, which is in turn defined as the product exclusively
obtained from the alcoholic fermentation of fresh grapes, whether crushed or not, or of grape must.
Similarly, cider vinegar is defined as the product obtained from the acetous fermentation of apple. Thus,
the acetic acid part of these vinegars cannot be obtained from the fermentation of exogenous sugars
(e.g. from beet or cane).
Regulation EC no 813/2000 [6] defines Aceto Balsamico Tradizionale di Modena and Aceto Balsamico
Tradizionale di Reggio Emilia, as PDOs and Regulation EC no. 583/2009 [7] defines Aceto Balsamico di
Modena as PGI.
In all types of vinegar, both the ethanol and the acetic acid should be obtained by a biotechnological
process, and the use of acetic acids obtained from either petroleum derivatives or the pyrolysis of wood
is not permitted according to the above definitions.
2 13
The isotopic analysis of acetic acid extracted from vinegar by H-SNIF-NMR and C-IRMS enables the
distinction of grape origin from other sources, such as beet, cane, malt, apple and synthesis [8]. Isotopic
methods have been recognized by the European Committee for Standardization (CEN) and in part by
the Organization Internationale de la Vigne et du Vin (OIV) as a means of detecting the non-permitted
presence of exogenous acetic acid and water in vinegar (CEN) and specifically wine vinegar (OIV). The
2
methods used are EN 16466-1 for D/H in the methyl site of acetic acid [(D/H) ] using H-SNIF-NMR
CH3
(Site Specific Natural Isotope Fractionation-Nuclear Magnetic Resonance), EN 16466-2 and
13 12
OIV 510/2013 for analysis of C/ C in acetic acid using IRMS (Isotope Ratio Mass Spectrometry), and
18 16
EN 16466-3 and OIV 511/2013 for analysis of O/ O in water using IRMS. Recently, it was
experimentally proven that OIV and CEN methods are also applicable to the analysis of acetic acid
13
extracted from Aceto Balsamico di Modena (D/H, C) [9].
This document provides the base for the analytical methods. The setup of the required apparatus
depends to a large extent on its design principles, and the specific recommendations of the
manufacturers should be followed. It is intended to serve as a frame in which the analyst can define his
own analytical work in accordance with the standard procedure.
This document has been based on an international collaborative study of the methods published in
Molecules 2020, 25, 2932 [1]; and is an update and extension of the previous version published in 2013
after a first an international collaborative study published in Analytica Chimica Acta 649 (2009) 98-105
[2], and organized under the auspices of the Permanent International Vinegar Committee (CPIV,
Brussels).
4
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oSIST prEN 16466-1:2023
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1 Scope
This document specifies an isotopic method to control the authenticity of vinegar and food containing
3
vinegar as an ingredient (for example Aceto Balsamico di Modena), with a density below 1,28 g/cm .
This method is applicable on acetic acid of vinegar (from wine, cider, agricultural alcohol, etc.) in order
to characterize the botanical origin of acetic acid and to detect adulterations of vinegar using synthetic
acetic acid or acetic acid from a non-allowed origin (together with the method described in
EN 16466-2).
2
The isotopic analysis of the extracted acetic acid by H-NMR is based on a similar method already
normalized for wine analysis [10].
The application of this document can involve the use of hazardous substances, operations and
equipment. This document does not claim to address all associated safety issues. It is the responsibility
of the user of this document to take appropriate measures for the safety and health protection of
personnel before use, and to check the applicability of existing national and European rules and
regulations.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp/
— IEC Electropedia: available at https://www.electropedia.org/
4 Principle
The acetic acid from vinegar is first extracted with diethyl ether (or alternatively another solvent with
similar properties such as tert-butyl methyl ether), using a liquid-liquid extractor, during at least 5 h.
The solvent is then eliminated by distillation. The acetic acid extracted from the sample is then analysed
1 2
by H-NMR and H-SNIF-NMR using a composite NMR experiment [10], [11]: the weight ratio between
1
tetramethylurea (TMU) and acetic acid is determined by H-NMR, and the isotopic ratio of hydrogen
2
, is determined by H-SNIF-NMR (Nuclear Magnetic
atoms at the methyl site of acetic acid, (D/H)
CH3
Resonance analysis of the Deuterium), on the same NMR tube.
5 Reagents
All reagents and consumables used shall meet stated requirements of the used method/apparatus (as
specified by the manufacturer). However, all reagents and consumables can be replaced by items with
similar performance.
5.1 Diethyl ether, for analysis. Purity ≥ 99 %.
5.2 Standard N,N-tetramethylurea (TMU), standard TMU with a calibrated isotope ratio D/H.
5
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5.3 Hexafluorobenzene (C6F6), used as field-frequency stabilization substance (lock). Alternatively,
trifluoroacetic acid (TFA, CAS: 76-05-1) or trifluoroacetic anhydride (TFAA, CAS: 407-25-0), or a mix of
them (e.g. 10 mL C F + 1 mL TFA).
6 6
6 Apparatus
All materials listed below are commercially available and used in food control laboratories:
6.1 For the extraction of acetic acid from vinegar
6.1.1 Liquid-liquid extractor of 400 ml or 800 ml.
6.1.2 Cadiot type (with spinning band) or Vigreux distillation column.
6.1.3 Round bottom flask of 500 ml.
6.1.4 Erlenmeyer of 250 ml.
6.1.5 Condenser.
6.1.6 Heating mantle.
6.1.7 Pumices stones.
6.1.8 Micropipettes enabling to take from 0,1 to 5 ml.
6.1.9 Fume hood.
6.2 For composite SNIF-NMR analysis of acetic acid from vinegar
6.2.1 Filter 0,45 μm.
6.2.2 NMR spectrometer fitted with a specific deuterium probe tuned to a frequency v0,
characteristic of the field B0 (e.g. B0 = 7,05 T, v0 = 46,05 MHz and for B0 = 9,4 T, v0 = 61,4 MHz) with a
decoupling channel (B2) and a field-frequency stabilization channel (lock) at the fluorine frequency.
The second channel, B2, is also used for the 1H-NMR experiment.
The resolution measured on the spectrum for 2H experiment, transformed without exponential
multiplication (i.e. LB = 0) and expressed as the width at the half-height of the methyl signals of acetic
acid and the methyl signal of TMU shall be less than 0,7 Hz.
The sensitivity (signal-to-noise ratio), measured with an exponential multiplying factor LB equal to 2 Hz
shall be greater than or equal to 150 for the methyl signal of acetic acid containing less than 25 % of
water.
For example, using a NMR spectrometer of field B0 = 7,05 T, 200 scans are necessary to reach this value
(depending on the noise of the instrument).
6.2.3 Automatic sample changer (optional).
6.2.4 Data-processing software enabling lorentzian integration.
6.2.5 10 mm sample tubes of sufficient quality for NMR spectrometer 400 MHz.
6.2.6 Fume hood.
6
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7 Procedure
7.1 Extraction of acetic acid from vinegar
7.1.1 General
3
This extraction procedure is only applicable to sample with a density below 1,25g/cm . For samples
wit
...
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