SIST EN 16466-1:2025

SLOVENSKI STANDARD
01-januar-2025
Nadomešča:
SIST EN 16466-1:2013
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: EN 16466-1:2024
ICS:
67.220.20 Dodatki jedem Food additives
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 16466-1
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2024
EUROPÄISCHE NORM
ICS 67.220.10 Supersedes EN 16466-1:2013
English Version
Food authenticity - Isotopic analysis of acetic acid and
water in vinegar - Part 1: H-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 in Essig - Teil 1: H-NMR-
analyse RMN- H de l'acide acétique Analyse von Essigsäure
This European Standard was approved by CEN on 24 June 2024.

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 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-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.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATIO N

EUROPÄISCHES KOMITEE FÜR NORMUN G

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 16466-1:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 5
5 Reagents . 6
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 . 14
European foreword
This document (EN 16466-1:2024) has been prepared by Technical Committee CEN/TC 460 “Food
authenticity”, the secretariat of which is held by DIN.
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 May 2025, and conflicting national standards shall be
withdrawn at the latest by May 2025.
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 held responsible for identifying any or all such patent rights.
This document supersedes EN 16466-1:2013.
EN 16466-1:2013:
a) extension to matrices other than wine vinegar;
b) modification of the NMR sequence;
c) standard editorially revised.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: 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 the
United Kingdom.
Introduction
[3]
Vinegar is defined in EN 13188 as the acetic acid solution resulting from a double fermentation:
a) transformation of sugars to ethanol; and
b) transformation of ethanol to acetic acid.
[4]
Conversely, EN 13189 defines acetic acid as “Product made from materials of non-agricultural origin”.
[5]
Wine vinegar is defined by the European Regulations 1308/2013 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).
Council Regulation (EC) No 813/2000 of 17 April 2000 supplementing the Annex to Commission
Regulation (EC) No 1107/96 on the registration of geographical indications and designations of origin
[6]
under the procedure laid down in Article 17 of Regulation (EEC) No 2081/92 defines Aceto
Balsamico Tradizionale di Modena and Commission Regulation (EC) No 583/2009 of 3 July 2009
entering a name in the register of protected designations of origin and protected geographical
[7]
indications [Aceto Balsamico di Modena (PGI)] 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-NMR and C-IRMS enables the
distinction of vinegar origin from other sources, such as beet, cane, malt, apple and synthesis compared
[8]
to grapes . 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 methods used are EN 16466-1 for D/H in the methyl site of acetic acid [(D/H) ]
CH
using H-NMR (Site Specific Natural Isotope Fractionation-Nuclear Magnetic Resonance),
[16] [18] 13 12
EN 16466-2 and OIV 510/2013 for analysis of C/ C in acetic acid using IRMS (Isotope Ratio
[17] [19] 18 16
Mass Spectrometry), and 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
13 [9]
analysis of acetic acid extracted from Aceto Balsamico di Modena (D/H, C) .
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 analysts can define their
own analytical work in accordance with the standard procedure.
[1]
This document has been based on an international collaborative study ; and is an update and
[2]
extension of the previous version published in 2013 after a first international collaborative study ,
and organized under the auspices of the Permanent International Vinegar Committee (CPIV, Brussels).
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.
1 Scope
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/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).
The isotopic analysis of the extracted acetic acid by H-NMR is based on a similar method already
[10]
normalized for wine analysis .
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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/
3.1
ppm
part per million
pseudo-unit for the chemical shift (δ) of a resonance signal
Note 1 to entry: The frequency difference of a signal’s resonance f from a reference compound’s resonance
signal
freference (both frequencies in Hz, cycles per second) is divided by the frequency of the reference compound’s
resonance and multiplied with 10 . This yields a normalized shift measure that is independent of the
spectrometer’s field strength. See the following formula for explanation:
ff −
signal reference
δ  ×10
signal
f
reference
−6 −2
Note 2 to entry: ppm is not a (SI) unit but a symbol for an order of magnitude (10 ), like the % symbol (10 ).
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 extract
...