prEN ISO 16958

SLOVENSKI STANDARD
01-november-2025
Mleko, mlečni proizvodi, hrana za dojenčke in prehranska dopolnila za odrasle -
Določevanje sestave maščobnih kislin - Kapilarna plinska kromatografija
(ISO/FDIS 16958:2025)
Milk, milk products, infant formula and adult nutritionals - Determination of fatty acids
composition - Capillary gas chromatographic method (ISO/FDIS 16958:2025)
Milch, Milcherzeugnisse, Säuglingsnahrung und Nahrungsergänzungsmittel für
Erwachsene - Bestimmung der Fettsäurenzusammensetzung - Verfahren mit
Kapillargaschromatographie (ISO/FDIS 16958:2025)
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 (ISO/FDIS 16958:2025)
Ta slovenski standard je istoveten z: prEN ISO 16958
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.

FINAL DRAFT
International
Standard
ISO/FDIS 16958
IDF 231
ISO/TC 34/SC 5
Milk, milk products, infant
Secretariat: NEN
formula and adult nutritionals —
Voting begins on:
Determination of fatty acids
2025-09-12
composition — Capillary gas
chromatographic method Voting terminates on:
2025-12-05
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
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
ISO/CEN PARALLEL PROCESSING LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
Reference number
ISO/FDIS 16958:2025(en)
231:2025(en) © ISO and IDF 2025

FINAL DRAFT
ISO/FDIS 16958:2025(en)
International
Standard
ISO/FDIS 16958
IDF 231
ISO/TC 34/SC 5
Milk, milk products, infant
Secretariat: NEN
formula and adult nutritionals —
Voting begins on:
Determination of fatty acids
composition — Capillary gas
Voting terminates on:
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
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
© ISO and IDF 2025
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
IN ADDITION TO THEIR EVALUATION AS
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
ISO/CEN PARALLEL PROCESSING
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
or ISO’s member body in the country of the requester.
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
ISO copyright office International Dairy Federation TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
CP 401 • Ch. de Blandonnet 8 Silver Building • Bd Auguste Reyers 70/B
CH-1214 Vernier, Geneva B-1030 Brussels
Phone: +41 22 749 01 11 Phone: +32 2 325 67 40
Fax: +32 2 325 67 41
Email: copyright@iso.org Email: info@fil-idf.org
Website: www.iso.org Website: www.fil-idf.org
Reference number
Published in Switzerland
ISO/FDIS 16958:2025(en)
IDF 231:2025(en)
ii
ISO/FDIS 16958:2025(en)
IDF 231:2025(en)
Contents Page
Foreword .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 % (mass fraction) .9
8.2 Liquid and powder milk and infant formula with a fat content < 1,5 % (mass fraction) .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 (LOD) . 15
11.5 Limit of quantitation (LOQ) . 15
12 Test report .15
Annex A (normative) Groups or classes of fatty acids and individual fatty acids .16
Annex B (normative) Gas–liquid chromatographic analysis . 19
Annex C (informative) Results of an interlaboratory trial .29
Bibliography .45

iii
ISO/FDIS 16958:2025(en)
IDF 231:2025(en)
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.
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO’s adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and
milk products, and the International Dairy Federation (IDF), in collaboration with AOAC INTERNATIONAL,
and in collaboration with the European Committee for Standardization (CEN) Technical Committee CEN/
TC 302, Milk and milk products - Methods of sampling and analysis,in accordance with the Agreement on
technical cooperation between ISO and CEN (Vienna Agreement). 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.
This second edition cancels and replaces the first edition (ISO 16958 | IDF 231:2015), of which it constitutes
a minor revision.
The changes are as follows:
— references to other standards have been updated;
— information on standard solutions and chromatographic columns has been updated;
— the Bibliography has been expanded.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
ISO/FDIS 16958:2025(en)
IDF 231:2025(en)
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.
IDF draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). IDF takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, IDF had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. IDF shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
This document was prepared by the IDF Standing Committee on Analytical Methods for Composition
and ISO Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, 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).

v
ISO/FDIS 16958:2025(en)
FINAL DRAFT International Standard
IDF 231:2025(en)
Milk, milk products, infant formula and adult nutritionals —
Determination of fatty acids composition — Capillary gas
chromatographic method
1 Scope
This document specifies a method for the quantification of individual and/or all fatty acids content 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 % (mass fraction).
The fat extracted from products containing less than 1,5 % (mass fraction) fat can be analysed with the
same method after a preliminary fat extraction using methods referenced in Clause 2. Dairy products, such
as 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
is performed at the lowest possible temperature (e.g. max. 40 °C) to recover these sensitive fatty acids.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. 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 1740 | IDF 6, Milkfat products and butter — Determination of fat acidity (Reference method)
ISO 14156 | IDF 172, Milk and milk products — Extraction methods for lipids and liposoluble compounds
ISO 23319| IDF 250, Cheese and processed cheese products, caseins and caseinates — Determination of fat
content — Gravimetric method
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/

ISO/FDIS 16958:2025(en)
IDF 231:2025(en)
3.1
fatty acids content
mass fraction of individual or groups of substances
Note 1 to entry: The fatty acids content is determined by the procedure specified in this document. See Table A.1.
Note 2 to entry: The fatty acids content is expressed as a mass fraction in grams (or in milligrams) of the fatty acids
per 100 g of product. Fatty acid results can be converted into other results expression formats (see 10.2).
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 is done by using capillary gas-liquid chromatography. Identification of FAMEs is done
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 or n-heptane, [CH (CH ) CH ], chromatography grade.
3 2 4 3 3 2 5 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 250 ml volumetric flask, pipette 42 ml (or 50 ml) of sodium methoxide solution 30 % m/v (or 25 %
m/v) and mix gently with 200 ml of methanol using a magnetic stirrer. Remove the magnetic stirrer (i.e.
using a rigid rod with magnetic extremity), 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 cases 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].
ISO/FDIS 16958:2025(en)
IDF 231:2025(en)
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 cases of a smaller number of analyses (or single analysis), the mass
and volume of solution can be adapted accordingly.
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 cases 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 Sigma-Aldrich, brand of Merck (Cat. 40495-U), or Cayman
1)
Chemical (cat. 29363).
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 Sigma-Aldrich, brand of Merck (Cat. L8404) .
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 % (mass fraction));
— cis-9,cis-12,trans-15-octadecatrienoic acid methyl ester (approximately 7 % (mass fraction));
— cis-9,trans-12,cis-15-octadecatrienoic acid methyl ester (approximately 7 % (mass fraction));
— cis-9,trans-12,trans-15-octadecatrienoic acid methyl ester (approximately 15 % (mass fraction));
— trans-9,cis-12,cis-15-octadecatrienoic acid methyl ester (approximately 7 % (mass fraction));
1) Sigma-Aldrich, brand of Merck, and Cayman Chemical are examples 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 products named. Equivalent products may be used if they can be shown to lead to the same results.

ISO/FDIS 16958:2025(en)
IDF 231:2025(en)
— trans-9,cis-12,trans-15-octadecatrienoic acid methyl ester (approximately 15 % (mass fraction));
— trans-9,trans-12,cis-15-octadecatrienoic acid methyl ester (approximately 15 % (mass fraction));
— trans-9,trans-12,trans-15-octadecatrienoic acid methyl ester (approximately 30 % (mass fraction)).
Concentration 10 mg/ml in methylene chloride.
1)
NOTE This standard is commercially available from Sigma-Aldrich, brand of Merck (Cat. L6031) . 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)
NOTE This standard is commercially available from Sigma-Aldrich, brand of Merck (Cat. O5507) . This standard
contains the two principal CLA isomers, but the isomer ratio can 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 can be used. Into a 50 ml volumetric flask, add each standard isomer solution in
equal proportion. Dissolve and make up to the mark with n-hexane or n-heptane. 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 can 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 acid methyl ester (C18:0), 25 mg of heptadecanoic acid

ISO/FDIS 16958:2025(en)
IDF 231:2025(en)
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 or n-heptane.
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.
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 or n-heptane.
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 or n-heptane.
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 or n-heptane. 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.
NOTE A FAME standards calibration solution can be prepared by weighing each individual FAME standard
according to 5.18.1 or by using the FAME standard mixture according to 5.18.2.
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- 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
2) Nu-Check-Prep GLC-36 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/FDIS 16958:2025(en)
IDF 231:2025(en)
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 volumetric flask, weigh and make up to the mark with n-hexane or n-heptane. 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.
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 ≥ 99,999 7 %.
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.
ISO/FDIS 16958:2025(en)
IDF 231:2025(en)
3)
6.10.2 Capillary column, bonded with cyanopropyl-polysiloxane phase (e.g. CP-Sil 88 Agilent ). or
3)
equivalent, such as poly(cyanopropyl siloxane) phase (e.g. SP-2560 Sigma-Aldrich , brand of Merck), (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.
6.10.3 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.
6.10.8 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 Figures B.1, B.2 and B.3.
The following examples list applicable conditions for a correct separation/identification of cis and trans:
— 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 to 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
−1
of 15 °C min up to 165 °C, maintained at this temperature for 1 min and then raised at a rate of
−1
2 °C min up to 225 °C for 20 min.
— Amount of sample injected: 1,0 μl.
3) CP-Sil 88 Agilent and SP-2560 Sigma-Aldrich are examples of suitable products 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 products named. Equivalent products may be used if they can be shown to lead to the same results.

ISO/FDIS 16958:2025(en)
IDF 231:2025(en)
NOTE Results obtained during the collaborative study show that the use of different split ratio (i.e. from
1:10 to 1:1:100) is not affecting the results as peak response is also linked to injected FAME concentration
solution.
b) 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).
— Injector temperature: cold.
— Detector temperature: 275 °C.
— Oven temperature programme: initial temperature of 60 °C, maintained for 5 min, raised at a rate
−1
of 15 °C min up to 165 °C, maintained at this temperature for 1 min and then raised at a rate of
−1
2 °C min up to 225 °C for 17 min.
— Amount of sample injected: 1,0 μl.
Examples of the full GC profiles obtained with the conditions given in examples 1 and 2 are shown in
Figures B.4 and B.5, respectively.
6.10.9 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 1
R1
(C18:1 trans-13/14);
t is the distance, in centimetres, between the left of the chromatogram and the top of peak 2
R2
(C18:1 cis-9);
W is the peak width, in centimetres, at half height of peak 1 (C18:1 trans-13/14);
 
 
2
W is the peak width, in centimetres, at half height of peak 2 (C18:1 cis-9).
 
 
2
The resolution is sufficient when R criterion ≥ 1,00 ± 5 % (see Figure B.3).
NOTE In cases 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.

ISO/FDIS 16958:2025(en)
IDF 231:2025(en)
Sampling is not part of the method specified in this document. A recommended sampling method is given in
ISO 707 | IDF 50.
8 Preparation of test sample
8.1 Liquid and powder milk and infant formula with a fat content ≥ 1,5 % (mass fraction)
Bring the sample
...