EN ISO 16958:2020

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

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.

Contents Page
European foreword . 3

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.

INTERNATIONAL ISO
STANDARD 16958
IDF
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
ISO 16958:2015(E)
IDF 231:2015(E)
© 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

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 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

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 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 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

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.
1)
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.
1)
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.
1)
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 16958:2015(E)
IDF 231:2015(E)
1)
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 cis-9 or n-7),
heptadecanoic acid methyl ester (C17:0), cis-10-heptadecenoic acid methyl ester (C17:1 cis-10 or n-7),
stearic acid methyl ester (C18:0), elaidic acid methyl ester (C18:1 trans-9 or n-9), oleic acid methyl ester
(C18:1 cis-9 or n-9), linolelaidic acid methyl ester (C18:2 all trans-9,12 or n-6), linoleic acid methyl ester
(C18:2 all cis-9,12 or n-6), arachidic acid methyl ester (C20:0), gamma-linoleic acid methyl ester (C18:3
all cis-6,9,12 or n-6), cis-11-eicosenoic acid methyl ester (C20:1 cis-11 or n-9), linolenic acid methyl
ester (C18:3 all cis-9,12,15 or n-3), heneicosanoic acid methyl ester (C21:0), cis-11,14-eicosadienoic acid
methyl ester (C20:2 all cis-11,14 or n-6), behenic acid methyl ester (C22:0), cis-8,11,14-eicosatrienoic
acid methyl ester (C20:3 all cis-8,11,14 or n-6 cis), erucic acid methyl ester (C22:1 cis-13 or n-9), cis-
11,14,17-eicosatrienoic acid methyl ester (C20:3 all cis-11,14,17 or n-3), arachidonic acid methyl ester
(C20:4 all cis-5,8,11,14 or n-6), cis-13,16-docosadienoic acid methyl ester (C22:2 all cis-13,16 or n-6),
lignoceric acid methyl ester (C24:0), cis-5,8,11,14,17- eicosapentaenoic acid methyl ester (C20:5 all cis-
5,8,11,14,17 or n-3), nervonic acid methyl ester (C24:1 cis-15 or n-9), cis-4,7,10,13,16,19-docosahexaenoic
acid methyl ester (C22:6 all cis-4,7,10,13,16,19 or n-3).
NOTE Purchasing of individual FAME standards is more expensive than a single FAME standard mixture. In
addition, weighing each FAME standard individually could give imprecision and requires high precision of weighing.
5.18.1.2 Stock solution 1 – Saturated
Into a 100 ml volumetric flask, accurately weigh to the nearest 0,1 mg about 25 mg of Lignoceric acid
methyl ester (C24:0), 25 mg of behenic acid methyl ester (C22:0), 25 mg of heneicosanoic acid methyl
ester (C21:0), 25 mg of arachidic acid methyl ester (C20:0), 25 mg of sacid methyl ester (C18:0),
25 mg of heptadecanoic acid methyl ester (C17:0), 50 mg of palmitic acid methyl ester (C16:0), 25 mg
of pentadecanoic acid methyl ester (C15:0), 25 mg of myristic acid methyl ester (C14:0), 25 mg of
tridecanoic acid methyl ester (C13:0), 25 mg of lauric acid methyl ester (C12:0), 25 mg of undecanoic
acid methyl ester (C11:0), 25 mg of capric acid methyl ester (C10:0), 25 mg of caprylic acid methyl ester
(C8:0), 25 mg of caproic acid methyl ester (C6:0) and 25 mg butyric acid methyl ester (C4:0). Make up to
the mark with n-hexane.
Palmitic acid is weighed in double amount. Short chain fatty acid methyl esters (i.e. C4:0, C6:0 and C8:0)
are volatile and shall be weighed at the end of the procedure.
4 © ISO and IDF 2015 – All rights reserved

ISO 16958:2015(E)
IDF 231:2015(E)
5.18.1.3 Stock solution 2 – Monounsaturated
Into a 100 ml volumetric flask, accurately weigh to the nearest 0,1 mg about 25 mg of nervonic acid
methyl ester (C24:1 cis-15 or n-9), 25 mg of erucic acid methyl ester (C22:1 cis-13 or n-9), 25 mg of cis-
11-eicosenoic acid methyl ester (C20:1 cis-11 or n-9), 25 mg of oleic acid methyl ester (C18:1 cis-9 or
n-9), 25 mg of elaidic acid methyl ester (C18:1 trans-9 or n-9 trans), 25 mg of cis-10-heptadecenoic acid
methyl ester (C17:1 cis-10 or n-7), 25 mg of palmitoleic acid methyl ester (C16:1 cis-9 or n-7), 25 mg of
cis-10-pentadecenoic acid methyl ester (C15:1 cis-10 or n-5) and 25 mg of myristoleic acid methyl ester
(C14:1 cis-9 or n-5). Make up to the mark with n-hexane.
5.18.1.4 Stock solution 3 – Polyunsaturated
Into a 100 ml volumetric flask, accurately weigh to the nearest 0,1 mg about 25 mg of Linolelaidic acid
methyl ester (C18:2 all trans-9,12 or n-6 trans), 25 mg of linoleic acid methyl ester (C18:2 all cis-9,12
or n-6), 25 mg of gamma-linoleic acid methyl ester (C18:3 all cis-9,12 or n-6), 25 mg of linolenic acid
methyl ester (C18:3 all cis-12,15 or n-3), 25 mg of cis-11,14-eicosadienoic acid methyl ester (C20:2 all cis-
11,14 or n-6), 25 mg of cis-8,11,14-eicosatrienoic acid methyl ester (C20:3 all cis-8,11,14 or n-6), 25 mg
of cis-11,14,17-eicosatrienoic acid methyl ester (C20:3 all cis-11,14,17 or n-3), 25 mg of arachidonic acid
methyl ester (C20:4 all cis-5,8,11,14 or n-6), 25 mg of cis-13,16-docosadienoic acid methyl ester (C22:2
cis-13,16 or n-6), 25 mg of cis-5,8,11,14,17- eicosapentaenoic acid methyl ester (C20:5 all cis-5,8,11,14,17
or n-3) and 25 mg of cis-4,7,10,13,16,19-docosahexaenoic acid methyl ester (C22:6 cis-4,7,10,13,16,19 or
n-3). Make up to the mark with n-hexane.
5.18.1.5 Preparation of FAME standards calibration solution
Into a 100 ml volumetric flask, pipette 25,0 ml of the calibration standard stock solution 1 (5.18.1.2),
25,0 ml of the calibration standard stock solution 2 (5.18.1.3) and 25,0 ml of the calibration standard
stock solution 3 (5.18.1.4). Then make up to the mark with n-hexane. Dilute in accordance with the type
of injector used.
Stored in the dark at −20 °C, this solution is stable for about six months. To prevent contamination of the
standard solution, distribute the solution into different vials (ready to inject) and store them at −20 °C
before use. Use each vial once, then discard it.
5.18.2 Preparation from a quantitative FAME standard mixture
5.18.2.1 Quantitative FAME standard mixture
2)
Purchase a quantitative FAME standard mixture: Nu-Check-Prep, Cat. Number GLC- Nestle-36 .
The FAME calibration standard mixture is carefully prepared by mass by the supplier. The mass
percentage of each component is indicated in the accompanying certificate. Each ampoule contains
approximately 100 mg of the FAME calibration standard mix. All individual FAME standards are
distributed in equal proportions in the standard mixture, except for palmitic acid methyl ester (C16:0)
which is added in double amount.
5.18.2.2 Preparation of FAME standards calibration mixture
Before use, allow the ampoule to come to room temperature (maximum 25 °C) in the dark without
heating. Cut the ampoule with a glass knife, using a Pasteur pipette, rapidly transfer the content of the
ampoule into a 50 ml pre-tarred volumetric flask, weigh and make up to the mark with n-hexane. Dilute
in accordance with the type of injector used.

2) Nu-Check-Prep GLC-Nestle36 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 16958:2015(E)
IDF 231:2015(E)
Stored in the dark at −20 °C, this solution is stable for about six months. To prevent contamination of the
standard solution, distribute the solution into different vials (ready to inject) and store them at −20 °C
before use. Use each vial once, then discard it.
6 Apparatus
WARNING — Since the determination involves the use of volatile flammable solvents, all
electrical apparatus employed shall comply with legislation relating to the hazards in using
such solvents.
Usual laboratory equipment and, in particular, the following.
6.1 Analytical balance, capable of weighing to the nearest 1 mg, with a readability of 0,1 mg.
6.2 Volumetric flasks, of capacities 50 ml, 100 ml, 250 ml, 300 ml and 500 ml.
6.3 Volumetric pipettes, with one mark, of capacities 2 ml, 5 ml, 10 ml, 25 ml and 50 ml, class AS
(ISO 1042).
6.4 Volumetric pipettes, with two marks, of capacities 2 ml and 5 ml, class AS (ISO 1042).
6.5 Micropipette, of capacity 200 µl.
6.6 Dispensers, of capacities 2 ml, 5 ml and 10 ml.
6.7 Test tube, of diameter 26 mm, of length 100 mm, fitted with PTFE-lined screw cap.
6.8 Test tube mixer vortex-genie, or equivalent.
6.9 Laboratory centrifuge, equipped with adapters for test tubes with external diameter of 26 mm.
6.10 Gas-liquid chromatograph, equipped with flame ionization detector and split/splitless or on-
column injector. Auto-sampler and integration system preferably computerized.
Use of the cleanest possible glassware and caps is required to avoid impurities in the FAME
chromatogram.
6.10.1 Carrier gas, hydrogen or helium, purity ≥ 99,9997 %.
NOTE The use of hydrogen or helium as carrier gas affects principally the chromatography duration
(i.e. increase of time between 10 min to 15 min with helium) but does not have significant impact on the
chromatographic resolution with optimized conditions.
The other gases necessary for the detector (FID) should be free from organic impurities (i.e. CnHm of
below 1 ppm) and have purity at least ≥ 99,995 %. Synthetic air or compressed air can be used. The use
of gas generator is also possible.
6.10.2 Capillary column, bonded with cyanopropyl-polysiloxane phase or equivalent (100 m length,
0,25 mm internal diameter, 0,2 micron film thickness), that elutes the FAMEs primarily by carbon chain
length and secondarily by the number of double bonds.
Traces of oxygen and humidity will damage the polar phase of the column. If pure gas is not available,
use a gas purifying filter device.
6 © ISO and IDF 2015 – All rights reserved

ISO 16958:2015(E)
IDF 231:2015(E)
6.10.3 Flame ionization detector, capable of being heated to a temperature 50 °C above the final
temperature of the column oven.
6.10.4 Split/splitless injector, capable of being heated to a temperature 30 °C above the final
temperature of the column oven.
6.10.5 On-column injector, capable of being not heated (cold), or being heated to a temperature 30 °C
above the final temperature of the column oven.
NOTE The installation of one single injector (i.e. split/splitless or on-column) on the GC instrument is sufficient.
6.10.6 Injection syringe, capacity 10 μl.
6.10.7 Integration system.
6.11 Gas chromatographic conditions
The oven temperature and the carrier gas flow depend on the column selected, and on the carrier
gas used (i.e. hydrogen or helium). In any case, the selected conditions shall produce the separation
between cis and trans zone for C18:1, C18:2, C18:3 and conjugated linoleic acids (CLA), as shown in
Annex B, Figures B.1, B.2 and B.3.
The examples in 6.11.1 and 6.11.2 list applicable conditions for a correct separation/identification of
cis and trans.
6.11.1 Example 1 – Split injection mode
— Column: 100 m length, 0,25 mm internal diameter, 0,2 μm film thickness, fused silica capillary column.
— Stationary phase: cyanopropyl-polysiloxane.
— Carrier gas type: helium.
— Column head carrier gas pressure: 225 kPa (175 kPa – 225 kPa).
— Split flow: 25,5 ml/min.
— Split ratio: 10:1.
— Injector temperature: 250 °C.
— Detector temperature: 275 °C.
— Oven temperature programme: initial temperature of 60 °C, maintained for 5 min, raised at a rate of
−1
15 °C min up to 165 °C, maintained at this temperature for 1 min and then raised at a rate of 2 °C
−1
min up to 225 °C for 20 min.
— Amount of sample injected: 1,0 μl.
An example of the full GC profile obtained with these conditions is shown in Annex B, Figure B.4.
6.11.2 Example 2 – On-column injection mode
— Column: 100 m length, 0,25 mm internal diameter, 0,2 μm film thickness, fused silica capillary column.
— Stationary phase: cyanopropyl-polysiloxane.
— Carrier gas type: hydrogen.
— Column head carrier gas pressure: 210 kPa (175 kPa – 225 kPa).
ISO 16958:2015(E)
IDF 231:2015(E)
— Injector temperature: cold.
— Detector temperature: 275 °C.
— Oven temperature programme: initial temperature of 60 °C, maintained for 5 min, raised at a rate of
−1
15 °C min up to 165 °C, maintained at this temperature for 1 min and then raised at a rate of 2 °C
−1
min up to 225 °C for 17 min.
— Amount of sample injected: 1,0 μl.
An example of the full GC profile obtained with these conditions is shown in Annex B, Figure B.5.
6.12 Resolution between C18:1 cis and trans
For the accurate quantification of C18:1 TFA (level ≥ 0,5 g/100 g fat), a sufficient resolution between
C18:1 trans-13/14 and C18:1 cis-9 (oleic acid) is required. The resolution is determined with the injection
of the qualitative cis and trans C18:1 FAME isomers standard mixture solution (5.17).
Inject into the gas chromatograph 1,0 µl of the calibrating solution (5.13). Determine peak width at half
height and distance between the left of the chromatogram and the top of peak for C18:1 trans-13/14 and
C18:1 cis-9 (oleic acid methyl ester). The resolution criteria R is calculated by using Formula (1):
Rt=×11,(8 −+tW)/()W (1)
   
RR21
1 1
 
 
 
 1  2
 
 
 
 
2 2
   
where
t is the distance, in centimetres, between the left of the chromatogram and the top of peak
R1
1 (C18:1 trans-13/14);
t is the distance, in centimetres, between the left of the chromatogram and the top of peak
R2
2 (C18:1 cis-9);
is the peak width, in centimetres, at half height of peak 1 (C18:1 trans-13/14);
W
 



 1




2
is the peak width, in centimetres, at half height of peak 2 (C18:1 cis-9).
W
 
 
2



 
 
The resolution is sufficient when R criterion ≥ 1,00 ± 5 % (see Annex B, Figure B.3).
NOTE In case of insufficient resolution, but with R close to the target value, the fine tuning of chromatography
conditions (i.e. slight modification of carrier gas pressure/flow, or oven temperature programme) can give an
acceptable R value.
7 Sampling
It is important that the laboratory receives a sample that is representative and has not been damaged
or changed during transport or storage.
Sampling is not part of the method specified in this International Standard. A recommended sampling
method is given in ISO 707 | IDF 50.
8 © ISO and IDF 2015 – All rights reserved

ISO 16958:2015(E)
IDF 231:2015(E)
8 Preparation of test sample
8.1 Liquid and powder milk and infant formula with a fat content ≥ 1,5 % m/m
Bring the sample to room temperature and shake vigorously before use. Ensure that the sample is
homogeneous (i.e. mix well).
8.2 Liquid and powder milk and infant formula with a fat content < 1,5 % m/m
Bring the sample to room temperature and shake vigorously before use. Ensure that the sample is
homogeneous (i.e. mix well).
Extract the fat in accordance with ISO 14156 | IDF 172 taking care to evaporate completely the
extraction solvent(s) by heating to a temperature not higher than 40 °C to avoid the degradation of long
chain polyunsaturated fatty acids (LC-PUFA).
NOTE See also ISO 1211 | IDF 1, ISO 1737 | IDF 13, ISO 8381 | IDF 123 and ISO 8262-1 | IDF 124-1 for useful
guidance on fat extraction methods.
8.3 Cheese
Bring the sample to room temperature.
...