EN ISO 9233-2:2013

SLOVENSKI STANDARD
01-september-2013
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Cheese, cheese rind and processed cheese - Determination of natamycin content - Part
2: High-performance liquid chromatographic method for cheese, cheese rind and
processed cheese (ISO 9233-2:2007 including Amd 1:2012)
Käse, Käserinde und Schmelzkäse - Bestimmung des Natamycingehalts - Teil 2:
Verfahren mit Hochleistungs-Flüssigchromatographie für Käse, Käserinde und
Schmelzkäse (ISO 9233-2:2007 + Amd 1:2012)
Fromage, croûte de fromage et fromages fondus - Détermination de la teneur en
natamycine - Partie 2: Méthode par chromatographie liquide à haute performance pour
fromage, croûte de fromage et fromages fondus (ISO 9233-2:2007, Amd 1:2012 inclus)
Ta slovenski standard je istoveten z: EN ISO 9233-2:2013
ICS:
67.100.30 Sir Cheese
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 9233-2
NORME EUROPÉENNE
EUROPÄISCHE NORM
May 2013
ICS 67.100.30
English Version
Cheese, cheese rind and processed cheese - Determination of
natamycin content - Part 2: High-performance liquid
chromatographic method for cheese, cheese rind and processed
cheese (ISO 9233-2:2007 including Amd 1:2012)
Fromage, croûte de fromage et fromages fondus - Käse, Käserinde und Schmelzkäse - Bestimmung des
Détermination de la teneur en natamycine - Partie 2: Natamycingehalts - Teil 2: Verfahren mit Hochleistungs-
Méthode par chromatographie liquide à haute performance Flüssigchromatographie für Käse, Käserinde und
pour fromage, croûte de fromage et fromages fondus (ISO Schmelzkäse (ISO 9233-2:2007 einschließlich Amd
9233-2:2007, Amd 1:2012 inclus) 1:2012)
This European Standard was approved by CEN on 16 May 2013.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2013 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 9233-2:2013: E
worldwide for CEN national Members.

Contents Page
Foreword .3

Foreword
The text of ISO 9233-2:2007 including Amd 1:2012 has been prepared by Technical Committee ISO/TC 34
“Food products” of the International Organization for Standardization (ISO) and has been taken over as EN
ISO 9233-2:2013 by Technical Committee CEN/TC 302 “Milk and milk products - Methods of sampling and
analysis” the secretariat of which is held by NEN.
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 November 2013, and conflicting national standards shall be withdrawn
at the latest by November 2013.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech
Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 9233-2:2007 including Amd 1:2012 has been approved by CEN as EN ISO 9233-2:2013
without any modification.
INTERNATIONAL ISO
STANDARD 9233-2
IDF
140-2
First edition
2007-12-15
Cheese, cheese rind and processed
cheese — Determination of natamycin
content —
Part 2:
High-performance liquid
chromatographic method for cheese,
cheese rind and processed cheese
Fromage, croûte de fromage et fromages fondus — Détermination de la
teneur en natamycine —
Partie 2: Méthode par chromatographie liquide à haute performance
pour fromage, croûte de fromage et fromages fondus

Reference numbers
ISO 9233-2:2007(E)
IDF 140-2:2007(E)
©
ISO and IDF 2007
ISO 9233-2:2007(E)
IDF 140-2:2007(E)
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Published in Switzerland
ii © ISO and IDF 2007 – All rights reserved

ISO 9233-2:2007(E)
IDF 140-2:2007(E)
Contents Page
Foreword. iv
Foreword. v
1 Scope . 1
2 Terms and definitions. 1
3 Principle. 1
4 Reagents. 2
5 Apparatus . 2
6 Sampling. 3
7 Preparation of test sample. 3
7.1 Cheese rind . 3
7.2 Cheese interior and processed cheese. 4
8 Procedure . 4
8.1 Test portion . 4
8.2 Preparation of test solution . 4
8.3 Determination. 5
9 Calculation and expression of results. 6
9.1 Calculation of natamycin mass fraction. 6
9.2 Calculation of surface-area-related natamycin mass . 7
9.3 Correction of results. 7
9.4 Expression of results . 7
10 Precision. 7
10.1 Interlaboratory tests . 7
10.2 Repeatability. 7
10.3 Reproducibility. 7
11 Test report . 8
Annex A (informative) Examples . 9
Annex B (informative) Results of interlaboratory trial. 11
Bibliography . 12

ISO 9233-2:2007(E)
IDF 140-2:2007(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
ISO technical committees. Each member body interested in a subject for which a technical committee has
been established has the right to be represented on that committee. International organizations, governmental
and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 9233-2|IDF 140-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5,
Milk and milk products and the International Dairy Federation (IDF) and is being published jointly by ISO and
IDF.
This first edition of ISO 9233-2|IDF 140-2, together with ISO 9233-1|IDF 140-1, cancels and replaces the first
edition of ISO 9233:1991, which has been technically revised.
ISO 9233|IDF 140 consists of the following parts, under the general title Cheese, cheese rind and processed
cheese — Determination of natamycin content:
⎯ Part 1: Molecular absorption spectrometric method for cheese rind
⎯ Part 2: High-performance liquid chromatographic method for cheese, cheese rind and processed cheese

iv © ISO and IDF 2007 – All rights reserved

ISO 9233-2:2007(E)
IDF 140-2:2007(E)
Foreword
IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National
Committee in every member country. Every National Committee has the right to be represented on the IDF
Standing Committees carrying out the technical work. IDF collaborates with ISO in the development of
standard methods of analysis and sampling for milk and milk products.
Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the
National Committees for voting. Publication as an International Standard requires approval by at least 50% of
IDF National Committees casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. IDF shall not be held responsible for identifying any or all such patent rights.
ISO 9233-2|IDF 140-2 was prepared by the International Dairy Federation (IDF) and Technical Committee
ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products and is being published jointly by IDF
and ISO.
All work was carried out by the Joint ISO-IDF Action Team on Selected food additives and vitamins of the
Standing Committee on Analytical methods for additives and contaminants under the aegis of its project
leader, Mr. M. Carl (DE).
This first edition of ISO 9233-2|IDF 140-2, together with ISO 9233-1|IDF 140-1, cancels and replaces the first
edition of IDF 140A:1992, which has been technically revised.
ISO 9233|IDF 140 consists of the following parts, under the general title Cheese, cheese rind and processed
cheese — Determination of natamycin content:
⎯ Part 1: Molecular absorption spectrometric method for cheese rind
⎯ Part 2: High-performance liquid chromatographic method for cheese, cheese rind and processed cheese

ISO 9233-2:2007(E)
INTERNATIONAL STANDARD
IDF 140-2:2007(E)
Cheese, cheese rind and processed cheese — Determination
of natamycin content —
Part 2:
High-performance liquid chromatographic method for cheese,
cheese rind and processed cheese
1 Scope
This part of ISO 9233|IDF 140 specifies a method for the determination of natamycin mass fraction in cheese,
cheese rind and processed cheese of above 0,5 mg/kg and of the surface-area-related natamycin mass in
cheese rind of above 0,03 mg/dm .
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
natamycin content
mass fraction of substances determined by the procedure specified in this part of ISO 9233|IDF 140
NOTE The natamycin content is expressed in milligrams per kilogram.
2.2
surface-area-related natamycin mass in cheese rind
surface-area-related mass of substances determined by the procedure specified in this part of
ISO 9233|IDF 140
NOTE The surface-area-related natamycin mass is expressed in milligrams of natamycin per square decimetre of
cheese rind.
2.3
cheese rind
outer layer of the cheese of thickness 5 mm, excluding the coating layer, if present.
3 Principle
A known quantity of sample is extracted with methanol. The extract is diluted with water followed by cooling to
between −15 °C and −20 °C to precipitate most of the fat, followed by filtration. The natamycin content or
surface-area-related natamycin mass is determined in the filtrate (after concentration, if necessary) by high-
performance liquid chromatography (HPLC).
ISO 9233-2:2007(E)
IDF 140-2:2007(E)
4 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified, and only distilled or
demineralized water or water of equivalent purity.
4.1 Methanol (CH OH).
4.2 Methanol, aqueous solution.
Mix 2 volumes of methanol (4.1) with 1 volume of water.
4.3 Natamycin standard solutions.
4.3.1 Natamycin standard stock solution, of concentration 500 mg/l.
Immediately before use, dissolve in methanol (4.1) a quantity of a natamycin preparation of known natamycin
content, corresponding to 50 mg of pure natamycin (C H NO ), in a 100 ml one-mark volumetric flask (5.1).
33 47 13
Make up to the mark with water and mix.
4.3.2 Natamycin standard working solution, of concentration 5 mg/l.
Pipette 5,0 ml of the natamycin standard stock solution (4.3.1) in a 50 ml one-mark volumetric flask (5.1).
Dilute to the mark with aqueous methanol (4.2) and mix.
Pipette 5,0 ml of the thus diluted solution into another 50 ml one-mark volumetric flask (5.1). Dilute to the mark
with aqueous methanol (4.2) and mix. The concentration of this natamycin standard working solution is
5 µg/ml.
This concentration shall be close to that of the test solution measured in 8.3.3. Adjust the standard working
dilution by pipetting and diluting another quantity, if required.
4.4 Acetic acid (CH CO H), glacial.
3 2
5 Apparatus
Usual laboratory equipment and, in particular, the following.
5.1 One-mark volumetric flasks, of capacities 50 ml and 100 ml.
5.2 Slicer or similar apparatus, capable of cutting cheese portions of thickness 5 mm and of width about
30 mm (Figure A.1 shows an example).
5.3 Fine slicer, capable of cutting thin cheese slices of maximum thickness 1 mm (Figure A.2 shows an
example).
5.4 Grinder or blender.
5.5 Sharp knife, capable of cutting cheese slices into small pieces.
5.6 Magnetic stirrer or shaking machine.
5.7 Conical flasks, of capacities 100 ml and 200 ml, made of coloured glass and fitted with ground-glass
stoppers.
5.8 Syringes, disposable, of capacity 10 ml.
2 © ISO and IDF 2007 – All rights reserved

ISO 9233-2:2007(E)
IDF 140-2:2007(E)
5.9 Membrane microfilters, of pore size 0,20 µm and 0,45 µm, resistant to attack by alcoholic solutions.
1)
5.10 Folded paper filters, fast speed, of diameter 150 mm. [e.g. S and S, No. 595 1/2 ].
5.11 Funnel, of diameter approximately 70 mm.
5.12 Freezer, capable of freezing at a temperature of between −15 °C and −20 °C.
1)
5.13 Extraction cartridges, to concentrate the filtered extract, if necessary [e.g. Sep-pack C18 or Waters
1)
No. 51910 ].
5.14 Liquid chromatograph, with UV detector, capable of measuring at 303 nm and equipped with a
recorder and/or integrator.
5.15 Analytical column, of length 150 mm, of internal diameter 4,6 mm, type C8, having a particle size of
1)
5 µm [e.g.
...