Milk, milk products, infant formula and adult nutritionals - Determination of fatty acids composition - Capillary gas chromatographic method (ISO 16958:2015)

ISO 16958:2015 specifies a method for the quantification of individual and/or all fatty acids in the profile of milk, milk products, infant formula and adult nutritional formula, containing milk fat and/or vegetable oils, supplemented or not supplemented with oils rich in long chain polyunsaturated fatty acids (LC-PUFA). This also includes groups of fatty acids often labelled [i.e. trans fatty acids (TFA), saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), omega-3, omega-6 and omega-9 fatty acids] and/or individual fatty acids [i.e. linoleic acid (LA), α-linolenic acid (ALA), arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)].
The determination is performed by direct transesterification in food matrices, without prior fat extraction, and consequently it is applicable to liquid samples or reconstituted powder samples with water having total fat ≥ 1,5 % m/m.
The fat extracted from products containing less than 1,5 % m/m fat can be analysed with the same method after a preliminary fat extraction using methods referenced in Clause 2. Dairy products, like soft or hard cheeses with acidity level ≤ 1 mmol/100 g of fat, can be analysed after a preliminary fat extraction using methods referenced in Clause 2. For products supplemented or enriched with PUFA with fish oil or algae origins, the evaporation of solvents should be performed at the lowest possible temperature (e.g. max. 40 °C) to recover these sensitive fatty acids.

Milch, Milcherzeugnisse, Säuglingsnahrung und Nahrungsergänzungsmittel für Erwachsene - Bestimmung der Fettsäurenzusammensetzung - Verfahren mit Kapillargaschromatographie (ISO 16958:2015)

Diese Internationale Norm legt ein Verfahren zur Quantifizierung von einzelnen und/oder allen Fettsäuren im Profil von Milch, Milcherzeugnissen, Säuglingsnahrung und Nahrungsergänzungsmitteln für Erwachsene fest, die Milchfett und/oder pflanzliche Öle enthalten, gegebenenfalls ergänzt um Öle, die reich an langkettigen mehrfach ungesättigten Fettsäuren (LC-PUFA, en: long chain polyunsaturated fatty acids) sind. Dazu gehören auch Gruppen von Fettsäuren, die häufig auf dem Etikett angegeben werden (d. h. trans-Fettsäuren [TFA, en: trans fatty acids], gesättigte Fettsäuren [SFA, en: saturated fatty acids], einfach ungesättigte Fettsäuren [MUFA, en: monounsaturated fatty acids], mehrfach ungesättigte Fettsäuren [PUFA, en: polyunsaturated fatty acids], Omega-3-, Omega-6- und Omega-9-Fettsäuren), und/oder einzelne Fettsäuren (d. h. Linolsäure [LA, en: linoleic acid], α-Linolensäure [ALA, en: α-linolenic acid], Arachidonsäure [ARA, en: arachidonic acid], Eicosapentaensäure [EPA, en: eicosapentaenoic acid] und Docosahexaensäure [DHA, en: docosahexaenoic acid]).
Die Bestimmung erfolgt durch direkte Umesterung in Lebensmittelmatrices ohne vorherige Fettextraktion und ist daher auf flüssige Proben oder auf mit Wasser wiederaufbereitete Pulverproben mit einem Gesamtfettgehalt ≥ 1,5 % m/m anwendbar.
Nach einer vorab erfolgten Fettextraktion unter Anwendung der in Abschnitt 2 angegebenen Verfahren kann das aus Produkten mit einem Fettgehalt von weniger als 1,5 % m/m extrahierte Fett nach demselben Verfahren analysiert werden. Milcherzeugnisse, wie z. B. Weich- oder Hartkäse mit einem Säuregrad ≤ 1 mmol/100 g Fett, können nach einer vorab erfolgten Fettextraktion unter Anwendung der in Abschnitt 2 angegebenen Verfahren analysiert werden. Im Falle von Produkten, die um aus Fischöl oder Algen stammende PUFA ergänzt oder damit angereichert wurden, sollte die Verdampfung von Lösemitteln bei der niedrigstmöglichen Temperatur (z. B. höchstens 40 °C) erfolgen, um diese empfindlichen Fettsäuren zurückzugewinnen.

Lait, produits laitiers, formules infantiles et produits nutrionnels pour adultes - Détermination de la composition en acides gras - Méthode de chromatographie en phase gazeuse sur colonne capillaire (ISO 16958:2015)

ISO 16958:2015 spécifie une méthode de quantification des acides gras individuels et/ou de tous les acides gras dans le profil du lait, des produits laitiers, des formules infantiles et des préparations nutritionnelles pour adultes contenant de la matière grasse de lait et/ou des huiles végétales, supplémentées ou non supplémentées avec des huiles riches en acides gras polyinsaturés à chaîne longue (AGPI-CL). Cela inclut également les groupes d'acides gras souvent déclarés [c'est-à-dire, les acides gras trans (AGT), les acides gras saturés (AGS), les acides gras monoinsaturés (AGMI), les acides gras polyinsaturés (AGPI), les acides gras oméga-3, oméga-6 et oméga-9] et/ou les acides gras individuels [c'est-à-dire l'acide linoléique (AL), l'acide α-linolénique (AAL), l'acide arachidonique (ARA), l'acide éicosapentaénoïque (AEP), l'acide docosahexaénoïque (ADH)].
La détermination est effectuée par transestérification directe dans les matrices d'aliments, sans extraction préalable de la matière grasse. Elle s'applique donc aux échantillons liquides ou aux échantillons pulvérulents reconstitués avec de l'eau et ayant une teneur totale en matière grasse supérieure ou égale à 1,5 % (m/m).
La matière grasse extraite de produits contenant moins de 1,5 % (m/m) de matière grasse peut être analysée avec la même méthode après une extraction préalable de la matière grasse en utilisant les méthodes référencées dans l'Article 2 Les produits laitiers tels que les fromages à pâte molle ou à pâte dure ayant un niveau d'acidité inférieur ou égal à 1 mmol/100 g de matière grasse peuvent être analysés après une extraction préalable de la matière grasse en utilisant les méthodes référencées dans l'Article 2. Pour les produits supplémentés ou enrichis en AGPI extrait d'huile de poisson ou d'algues, il convient que l'évaporation de solvants soit effectuée à la plus faible température possible (par exemple, 40 °C maximum) pour récupérer ces acides gras sensibles.

Mleko, mlečni proizvodi, hrana za dojenčke in prehranska dopolnila za odrasle - Določevanje sestave maščobnih kislin - Kapilarna plinska kromatografija (ISO 16958:2015)

General Information

Status
Published
Public Enquiry End Date
19-Mar-2020
Publication Date
18-Jun-2020
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
11-Jun-2020
Due Date
16-Aug-2020
Completion Date
19-Jun-2020

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SLOVENSKI STANDARD
SIST EN ISO 16958:2020
01-julij-2020
Mleko, mlečni proizvodi, hrana za dojenčke in prehranska dopolnila za odrasle -
Določevanje sestave maščobnih kislin - Kapilarna plinska kromatografija (ISO
16958:2015)

Milk, milk products, infant formula and adult nutritionals - Determination of fatty acids

composition - Capillary gas chromatographic method (ISO 16958:2015)
Milch, Milcherzeugnisse, Säuglingsnahrung und Nahrungsergänzungsmittel für
Erwachsene - Bestimmung der Fettsäurenzusammensetzung - Verfahren mit
Kapillargaschromatographie (ISO 16958:2015)

Lait, produits laitiers, formules infantiles et produits nutrionnels pour adultes -

Détermination de la composition en acides gras - Méthode de chromatographie en
phase gazeuse sur colonne capillaire (ISO 16958:2015)
Ta slovenski standard je istoveten z: EN ISO 16958:2020
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
67.100.10 Mleko in predelani mlečni Milk and processed milk
proizvodi products
SIST EN ISO 16958:2020 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 16958:2020
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SIST EN ISO 16958:2020
EN ISO 16958
EUROPEAN STANDARD
NORME EUROPÉENNE
June 2020
EUROPÄISCHE NORM
ICS 67.100.10
English Version
Milk, milk products, infant formula and adult nutritionals -
Determination of fatty acids composition - Capillary gas
chromatographic method (ISO 16958:2015)

Lait, produits laitiers, formules infantiles et produits Milch, Milcherzeugnisse, Säuglingsnahrung und

nutrionnels pour adultes - Détermination de la Nahrungsergänzungsmittel für Erwachsene -

composition en acides gras - Méthode de Bestimmung der Fettsäurenzusammensetzung -

chromatographie en phase gazeuse sur colonne Verfahren mit Kapillargaschromatographie (ISO

capillaire (ISO 16958:2015) 16958:2015)
This European Standard was approved by CEN on 10 May 2020.

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, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16958:2020 E

worldwide for CEN national Members.
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SIST EN ISO 16958:2020
EN ISO 16958:2020 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 16958:2020
EN ISO 16958:2020 (E)
European foreword

The text of ISO 16958:2015 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 16958:2020

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 December 2020, and conflicting national standards

shall be withdrawn at the latest by December 2020.

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.

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, 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, Turkey and the

United Kingdom.
Endorsement notice

The text of ISO 16958:2015 has been approved by CEN as EN ISO 16958:2020 without any modification.

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SIST EN ISO 16958:2020
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SIST EN ISO 16958:2020
INTERNATIONAL ISO
STANDARD 16958
IDF
231
First edition
2015-11-01
Milk, milk products, infant
formula and adult nutritionals —
Determination of fatty acids
composition — Capillary gas
chromatographic method
Lait, produits laitiers, formules infantiles et produits nutritionnels
pour adultes — Détermination de la composition en acides gras —
Méthode de chromatographie en phase gazeuse sur colonne capillaire
Reference numbers
ISO 16958:2015(E)
IDF 231:2015(E)
ISO and IDF 2015
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SIST EN ISO 16958:2020
ISO 16958:2015(E)
IDF 231:2015(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO and IDF 2015, Published in Switzerland

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form

or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior

written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of

the requester.
ISO copyright office International Dairy Federation

Ch. de Blandonnet 8 • CP 401 Silver Building • Bd Auguste Reyers 70/B • B-1030 Brussels

CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11 Tel. + 32 2 325 67 40
Fax +41 22 749 09 47 Fax + 32 2 325 67 41
copyright@iso.org info@fil-idf.org
www.iso.org www.fil-idf.org
ii © ISO and IDF 2015 – All rights reserved
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SIST EN ISO 16958:2020
ISO 16958:2015(E)
IDF 231:2015(E)
Contents Page

Forewords .....................................................................................................................................................................................................................................iv

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

5 Reagents ........................................................................................................................................................................................................................ 2

6 Apparatus ..................................................................................................................................................................................................................... 6

7 Sampling ........................................................................................................................................................................................................................ 8

8 Preparation of test sample ......................................................................................................................................................................... 9

8.1 Liquid and powder milk and infant formula with a fat content ≥ 1,5 % m/m ................................. 9

8.2 Liquid and powder milk and infant formula with a fat content < 1,5 % m/m ................................. 9

8.3 Cheese ............................................................................................................................................................................................................. 9

9 Procedure..................................................................................................................................................................................................................... 9

9.1 Test portion ................................................................................................................................................................................................ 9

9.2 Quantitative determination ......................................................................................................................................................10

9.2.1 Determination of response factors................................................................................................................10

9.2.2 Determination of the test portion ..................................................................................................................10

9.2.3 Fatty acid identification ..........................................................................................................................................10

10 Calculation and expression of results ..........................................................................................................................................12

10.1 Calculation ...............................................................................................................................................................................................12

10.1.1 Calculation of response factor ...........................................................................................................................12

10.1.2 Fatty acids on the product ....................................................................................................................................12

10.1.3 Fatty acids on the total fat .....................................................................................................................................13

10.1.4 Sum of class or group of fatty acids in 100 g product ...................................................................13

10.1.5 Sum of class or group of fatty acids in 100 g fat ................................................................................13

10.1.6 Performance of the transesterification......................................................................................................13

10.2 Expression of results .......................................................................................................................................................................14

11 Precision ....................................................................................................................................................................................................................14

11.1 Interlaboratory test..........................................................................................................................................................................14

11.2 Repeatability ..........................................................................................................................................................................................14

11.3 Reproducibility ....................................................................................................................................................................................15

11.4 Limit of detection ...............................................................................................................................................................................15

11.5 Limit of quantitation .......................................................................................................................................................................15

12 Test report ................................................................................................................................................................................................................15

Annex A (normative) Groups or classes of fatty acids and individual fatty acids ...............................................16

Annex B (informative) Examples of the gas–liquid chromatographic analysis .....................................................20

Annex C (informative) Results of an interlaboratory trial ..........................................................................................................30

Bibliography .............................................................................................................................................................................................................................45

© ISO and IDF 2015 – All rights reserved iii
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SIST EN ISO 16958:2020
ISO 16958:2015(E)
IDF 231:2015(E)
Forewords

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.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).

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. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www.iso.org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation on the meaning of ISO specific terms and expressions related to conformity

assessment, as well as information about ISO’s adherence to the WTO principles in the Technical

Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information

The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 5, Milk and

milk products and the International Dairy Federation (IDF), in collaboration with AOAC INTERNATIONAL.

It is being published jointly by ISO and IDF and separately by AOAC INTERNATIONAL. The method

described in this International Standard is equivalent to the AOAC Official Method 2012.13:

Determination of labeled fatty acids content in milk products and infant formula.

iv © ISO and IDF 2015 – All rights reserved
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SIST EN ISO 16958:2020
ISO 16958:2015(E)
IDF 231:2015(E)

IDF (the International Dairy Federation) is a non-profit private sector organization representing the

interests of various stakeholders in dairying at the global level. IDF members are organized in National

Committees, which are national associations composed of representatives of dairy-related national

interest groups including dairy farmers, dairy processing industry, dairy suppliers, academics and

governments/food control authorities.

ISO and IDF collaborate closely on all matters of standardization relating to methods of analysis

and sampling for milk and milk products. Since 2001, ISO and IDF jointly publish their International

Standards using the logos and reference numbers of both organizations.

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. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www.iso.org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

ISO 16958 | IDF 231 was prepared by the IDF Standing Committee on Analytical Methods for Composition

and the ISO Technical Committee ISO/TC 34, Food products, Subcommittee 5 on Milk and milk products

(ISO/TC 34/SC 5), in collaboration with AOAC INTERNATIONAL. It is being published jointly by ISO and

IDF, and separately by AOAC INTERNATIONAL. The method described in this International Standard

is equivalent to the AOAC Official Method 2012.13: Determination of labeled fatty acids content in milk

products and infant formula

All work was carried out by the ISO-IDF Project Group C11 of the Standing Committee on Analytical

Methods for Composition under the aegis of its project leader, Mr Pierre-Alain Golay (CH).

© ISO and IDF 2015 – All rights reserved v
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SIST EN ISO 16958:2020
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SIST EN ISO 16958:2020
ISO 16958:2015(E)
INTERNATIONAL STANDARD
IDF 231:2015(E)
Milk, milk products, infant formula and adult
nutritionals — Determination of fatty acids composition —
Capillary gas chromatographic method
1 Scope

This International Standard specifies a method for the quantification of individual and/or all fatty acids

in the profile of milk, milk products, infant formula and adult nutritional formula, containing milk fat

and/or vegetable oils, supplemented or not supplemented with oils rich in long chain polyunsaturated

fatty acids (LC-PUFA). This also includes groups of fatty acids often labelled [i.e. trans fatty acids (TFA),

saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA),

omega-3, omega-6 and omega-9 fatty acids] and/or individual fatty acids [i.e. linoleic acid (LA), α-linolenic

acid (ALA), arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)].

The determination is performed by direct transesterification in food matrices, without prior fat

extraction, and consequently it is applicable to liquid samples or reconstituted powder samples with

water having total fat ≥ 1,5 % m/m.

The fat extracted from products containing less than 1,5 % m/m fat can be analysed with the same

method after a preliminary fat extraction using methods referenced in Clause 2. Dairy products, like

soft or hard cheeses with acidity level ≤ 1 mmol/100 g of fat, can be analysed after a preliminary fat

extraction using methods referenced in Clause 2. For products supplemented or enriched with PUFA

with fish oil or algae origins, the evaporation of solvents should be performed at the lowest possible

temperature (e.g. max. 40 °C) to recover these sensitive fatty acids.
2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are

indispensable for its application. For dated references, only the edition cited applies. For undated

references, the latest edition of the referenced document (including any amendments) applies.

ISO 1042, Laboratory glassware — One-mark volumetric flasks

ISO 1735 | IDF 5, Cheese and processed cheese products — Determination of fat content — Gravimetric

method (Reference method)

ISO 1740 | IDF 6, Milk fat products and butter — Determination of fat acidity (Reference method)

ISO 14156 | IDF 172, Milk and milk products — Extraction methods for lipids and liposoluble compounds

3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
fatty acids content

mass fraction of individual or groups of substances determined by the procedure specified in this

International Standard
Note 1 to entry: See Table A.1.

Note 2 to entry: The fatty acid content is expressed as a mass fraction in grams (or in milligrams) of the fatty

acids per 100 g of product (see Table A.1). Fatty acid results can be converted into other results expression

formats (see 10.2).
© ISO and IDF 2015 – All rights reserved 1
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SIST EN ISO 16958:2020
ISO 16958:2015(E)
IDF 231:2015(E)
4 Principle

Addition of the internal standard solution to the sample, preparation of fatty acid methyl esters (FAMEs)

by direct transesterification with methanolic sodium methoxide for liquid samples; dissolution (i.e.

reconstitution) in water for powder sample and direct transesterification with methanolic sodium

methoxide. The same transesterification procedure is applied to fat extracted from various foods (e.g.

low fat products, cheeses).

Separation of FAMEs using capillary gas-liquid chromatography. Identification of FAMEs by comparison

with the retention time of pure standards and quantification as fatty acids by reference to an internal

standard (C11:0 FAME) and instrument response factors. Verification of the transesterification

performance using a second internal standard (C13:0 TAG).
5 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.
5.1 n-Hexane, [CH (CH ) CH ], chromatography grade.
3 2 4 3
5.2 Methanol, [CH OH], chromatography grade.
5.3 Water, HPLC grade or equivalent purity quality.

5.4 Sodium methoxide solution, [CH ONa], dissolved in methanol 30 % m/v, or 25 % m/v, depending

on local availability.

5.5 Transesterification solution, (sodium methoxide solution 5 % m/v in methanol).

Into a 300 ml volumetric flask, pipette 50 ml (or 60 ml) of sodium methoxide solution 30 % m/v (or

25 % m/v) and mix gently with 250 ml of methanol using a magnetic stirrer. Remove the magnetic

stirrer, then cool to room temperature and make up to the mark with methanol.

Stored in the dark at 4 °C, this solution is stable for one week. Allow the solution to come to room

temperature before use. This solution volume is sufficient to analyse approximately 40 samples. In case

of a smaller number of analyses, the reagent volume can be adapted accordingly.

Perform the transesterification reaction at ambient temperature (between 20 °C and 25 °C).

NOTE Value indicated in brackets () corresponds to sodium methoxide solution with 25 % m/v concentration

5.6 di-Sodium hydrogen citrate sesquihydrate, [HOC(COOH)(CH COONa) .1,5 H O].
2 2 2
5.7 Sodium chloride, [NaCl].

5.8 Neutralization solution, (di-sodium hydrogen citrate sesquihydrate 10 % m/v, sodium chloride

15 % m/v in water).

Weigh 50,0 g of di-sodium hydrogen citrate sesquihydrate and 75,0 g of sodium chloride in a 500 ml

volumetric flask. Dissolve in 450 ml of water using a magnetic stirrer. Remove the magnetic stirrer,

then make up to the mark with water.

Stored in the dark at 4 °C, this solution is stable for one month. Presence of salt crystals may appear in

the solution during storage, but disappear after shaking.

Allow the solution to come to room temperature before use. This solution volume is sufficient to analyse

approximately 40 samples or more. In case of a smaller number of analyses (or single analysis), the

mass and volume of solution can be adapted accordingly.
2 © ISO and IDF 2015 – All rights reserved
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SIST EN ISO 16958:2020
ISO 16958:2015(E)
IDF 231:2015(E)
5.9 Tert-butyl methyl ether (MTBE), chromatography grade.
5.10 Methyl undecanoate (C11:0 FAME), of purity ≥ 99 % mass fraction.
5.11 Tritridecanoin (C13:0 TAG), of purity ≥ 99 % mass fraction.
5.12 C11:0 FAME/C13:0 TAG standard solution.

Into a 250 ml volumetric flask, weigh to the nearest 0,1 mg about 500 mg of tritridecanoin and 500 mg

of methyl undecanoate. Dissolve and make up to the mark with MTBE.

Stored in the dark at 4 °C, this solution is stable for one week. Allow the solution to come to room

temperature before use.

This solution volume is sufficient to analyse approximately 40 samples or more. In case of a smaller

number of analyses, standard mass and volume of solvent can be adapted accordingly.

5.13 Octadecenoic acid methyl esters, cis and trans isomers mixture of C18:1 with trans-4 to trans-16

octadecenoic (all isomers) and principal cis isomers. Concentration 2,5 mg/ml in methylene chloride.

NOTE This standard is commercially available from Supelco Inc, brand of Sigma-Aldrich (Cat. 40495-U) .

5.14 Linoleic acid methyl esters, cis and trans isomers mixture of C18:2 with trans-9,trans-12-

octadecadienoic acid (approximately 50 %), cis-9,trans-12-octadecadienoic acid (approximately

20 %), trans-9,cis-12-octadecadienoic acid (approximately 20 %) and cis-9,cis-12-octadecadienoic acid

(approximately 10 %). Concentration 10 mg/ml in methylene chloride.

NOTE This standard is commercially available from Supelco Inc, brand of Sigma-Aldrich (Cat. 47791) .

5.15 Linolenic acid methyl esters, cis and trans isomers mixture of C18:3 with

— cis-9,cis-12,cis-15-octadecatrienoic acid methyl ester (approximately 3 % m/m),

— cis-9,cis-12,trans-15-octadecatrienoic acid methyl ester (approximately 7 % m/m),

— cis-9,trans-12,cis-15-octadecatrienoic acid methyl ester (approximately 7 % m/m),

— cis-9,trans-12,trans-15-octadecatrienoic acid methyl ester (approximately 15 % m/m),

— trans-9,cis-12,cis-15-octadecatrienoic acid methyl ester (approximately 7 % m/m),

— trans-9,cis-12,trans-15-octadecatrienoic acid methyl ester (approximately 15 % m/m),

— trans-9,trans-12,cis-15-octadecatrienoic acid methyl ester (approximately 15 % m/m), and

— trans-9,trans-12,trans-15-octadecatrienoic acid methyl ester (approximately 30 % m/m).

Concentration 10 mg/ml in methylene chloride.

NOTE This standard is commercially available from Supelco Inc, brand of Sigma Aldrich (Cat. 47792) . This

standard contains all trans isomers of C18:3 (eight in total) but their abundance and ratio are different to those

observed in refined/deodorized oils and fats.

5.16 Methyl octadecadienoate conjugated acids, mixture of C18:2 cis-9,trans-11 and cis-10,trans-12-

octadecadienoate conjugated acids, of purity ≥ 99 % mass fraction.

1) Supelco Inc., brand of Sigma Aldrich, is an example of suitable product available commercially. This information

is given for the convenience of users of this document and does not constitute an endorsement by either ISO or IDF

of the product named. Equivalent products may be used if they can be shown to lead to the same results.

© ISO and IDF 2015 – All rights reserved 3
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SIST EN ISO 16958:2020
ISO 16958:2015(E)
IDF 231:2015(E)

NOTE This standard is commercially available from Supelco Inc, brand of Sigma Aldrich (Cat. 05507) . This

standard contains the two principal CLA isomers, but isomer ratio may vary from lot to lot.

5.17 Qualitative cis and trans isomers standard mixture solution

For the retention time (RT) identification of cis and trans isomers (i.e. C18:1, C18:2, C18:3 and CLA),

prepare a qualitative standard solution with the standards listed in 5.13 to 5.16. All standards that are

commercially available could be used. Into a 50 ml volumetric flask, add each standard isomer solution

in equal proportion. Dissolve and make up to the mark with hexane. Dilute in accordance with the type

of injector used.
5.18 FAME standards calibration solution
5.18.1 Preparation with individual FAME standards
5.18.1.1 Individual FAME standards
Purchase individual FAME standards as follows (purity ≥ 99 %):

Butyric acid methyl ester (C4:0), caproic acid methyl ester (C6:0), caprylic acid methyl ester (C8:0),

capric acid methyl ester (C10:0), undecanoic acid methyl ester (C11:0), lauric acid methyl ester (C12:0),

tridecanoic acid methyl ester (C13:0), myristic acid methyl ester (C14:0), myristoleic acid methyl ester

(C14:1 cis-9 or n-5), pentadecanoic acid methyl ester (C15:0), cis-10-pentadecenoic acid methyl ester

(C15:1 cis-10 n-5), palmitic acid methyl ester (C16:0), palmitoleic acid methyl ester (C16:1 ci

...

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