prEN ISO 12966-4

SLOVENSKI STANDARD
01-julij-2025
Živalske in rastlinske maščobe in olja - Plinska kromatografija metilnih estrov
maščobnih kislin - 4. del: Določevanje s kapilarno plinsko kromatografsko metodo
(ISO/DIS 12966-4:2025)
Animal and vegetable fats and oils - Gas chromatography of fatty acid methyl esters -
Part 4: Determination by capillary gas chromatography (ISO/DIS 12966-4:2025)
Tierische und pflanzliche Fette und Öle - Gaschromatographie von
Fettsäuremethylestern - Teil 4: Bestimmung mittels Kapillargaschromatographie
(ISO/DIS 12966-4:2025)
Corps gras d’origines animale et végétale - Chromatographie en phase gazeuse des
esters méthyliques d’acides gras - Partie 4: Détermination par chromatographie capillaire
en phase gazeuse (ISO/DIS 12966-4:2025)
Ta slovenski standard je istoveten z: prEN ISO 12966-4
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
67.200.10 Rastlinske in živalske Animal and vegetable fats
maščobe in olja and oils
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

DRAFT
International
Standard
ISO/DIS 12966-4
ISO/TC 34/SC 11
Animal and vegetable fats and
Secretariat: BSI
oils — Gas chromatography of fatty
Voting begins on:
acid methyl esters —
2025-05-08
Part 4:
Voting terminates on:
2025-07-31
Determination by capillary gas
chromatography
Corps gras d'origines animale et végétale — Chromatographie en
phase gazeuse des esters méthyliques d'acides gras —
Partie 4: Détermination par chromatographie capillaire en phase
gazeuse
ICS: 67.200.10
THIS DOCUMENT IS A DRAFT CIRCULATED
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Reference number
ISO/DIS 12966-4:2025(en)
DRAFT
ISO/DIS 12966-4:2025(en)
International
Standard
ISO/DIS 12966-4
ISO/TC 34/SC 11
Animal and vegetable fats and
Secretariat: BSI
oils — Gas chromatography of fatty
Voting begins on:
acid methyl esters —
Part 4:
Voting terminates on:
Determination by capillary gas
chromatography
Corps gras d'origines animale et végétale — Chromatographie en
phase gazeuse des esters méthyliques d'acides gras —
Partie 4: Détermination par chromatographie capillaire en phase
gazeuse
ICS: 67.200.10
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
© ISO 2025
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
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Published in Switzerland Reference number
ISO/DIS 12966-4:2025(en)
ii
ISO/DIS 12966-4:2025(en)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents and materials . 2
5.1 Reference standards .2
5.1.1 Reference fatty acid methyl esters (FAMEs) .2
5.1.2 Fats and oils with certified fatty acid composition .3
5.1.3 Quantitative FAME standard mixture containing cis and trans fatty acid methyl
esters from C4:0 to C22:6 .3
5.1.4 Calibration FAME standard solution at 2 mg/ml for the calculation of the
correction factors .3
5.2 Internal standards .3
5.3 Iso-octane (2,2,4-trimethyl pentane) .4
5.4 Methyl tert-Butyl ether (MTBE) (2-Methoxy-2-methylpropane) .4
5.5 n-Hexane .4
5.6 n-Heptane .4
5.7 Qualitative cis and trans isomers standard mixture solution .4
5.8 Dichloromethane (Methylene chloride) .5
6 Apparatus . 5
7 Sampling . 5
8 Preparation of test sample . 5
9 Preparation of methyl esters from fats, oils, and fatty acids . 6
10 Procedure . 6
10.1 General .6
10.2 GC conditions .6
10.3 Performance check .6
11 Calculations . 7
11.1 Qualitative analysis and peak identification .7
11.2 Quantitative analysis .8
11.2.1 Fatty acid methyl esters quantification, by mass (g/100 g) .9
11.2.2 Fatty acid methyl esters quantification, by area % .9
11.2.3 Butyric acid and caproic acid methyl esters quantification (only), by mass
(g/100 g) in fat containing short chain fatty acids .9
11.2.4 Total trans fatty acid methyl esters quantification (only), by mass (g/100 g) .10
11.3 Expression of the results for food labelling .10
12 Precision .11
12.1 Results of interlaboratory test .11
12.2 Repeatability .11
12.3 Reproducibility .11
13 Test report .11
Annex A (informative) Theoretical flame ionization detector correction factor (TCF) for fatty
acid methyl esters (FAMEs) .12
Annex B (informative) Examples of chromatograms . 14
Annex C (informative) Elution zone of the trans fatty acid methyl esters (TFA) . 19

iii
ISO/DIS 12966-4:2025(en)
Annex D (informative) Stoichiometric factors (St(FA)) for converting fatty acid methyl esters
(FAME) to fatty acids (FA) .25
Annex E (informative) Determination of the composition of fatty acid methyl esters expressed
by area % in liquid vegetable oils .26
Annex F (informative) Results of an interlaboratory trial .28
Bibliography .35

iv
ISO/DIS 12966-4:2025(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 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).
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 11,
Animal and vegetable fats and oils.
The first edition of ISO 12966-4 cancelled and replaced ISO 5508:1990 and ISO 15304:2002. This second
edition cancels and replaces the first edition (ISO 12966-4:2015), which has been technically revised.
The main changes are as follows:
— extension of the scope for the separation of fatty acid methyl esters from C4 to C24,
— addition of ruminant fat in the scope,
— quantification by area (%) or by mass (g/100g) using internal standards and corrections factors
calculated with a quantitative fatty acid methyl esters standard mixture containing cis and trans fatty
acid methyl esters from C4:0 to C22:6,
— quantification of total trans fatty acid methyl esters by mass (g/100g),
— the use of 100 m, 0,25 mm ID, 0,20 µm film thickness columns are required to separate most C18:1 trans-
and cis-isomers,
— addition of a method for determination of the composition of fatty acid methyl esters expressed by area
% in liquid vegetable oils.
A list of all parts in the ISO 12966 series can be found on the ISO website.
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.

v
ISO/DIS 12966-4:2025(en)
Introduction
This part of ISO 12966 is one of a suite of four International Standards for the preparation and determination
of fatty acid methyl esters by gas chromatography in animal and vegetable fats and oils. ISO 12966 (all parts)
is applicable to crude, refined, partially hydrogenated, or fully hydrogenated fats, oils, and fatty acids derived
from animal and vegetable sources, and fats extracted from foodstuff.
ISO 12966 (all parts) is not suitable for milk and milk products (or fat coming from milk and milk products),
or products supplemented with conjugated linoleic acid (CLA). Furthermore, it is not intended to be applied
to polymerized and oxidized fats and oils.
This part of ISO 12966 gives the conditions for the analysis of fatty acid methyl esters by capillary gas
chromatography, while ISO 12966-2 and ISO 12966-3 cover the preparation of fatty acid methyl esters by
different methods. ISO 12966-1 is a guideline to the modern gas chromatography of fatty acid methyl esters.

vi
DRAFT International Standard ISO/DIS 12966-4:2025(en)
Animal and vegetable fats and oils — Gas chromatography of
fatty acid methyl esters —
Part 4:
Determination by capillary gas chromatography
1 Scope
This part of ISO 12966 specifies a method for the determination of fatty acid methyl esters (FAMEs) derived
by transesterification or esterification from fats, oils, and fatty acids by capillary gas chromatography
(GLC). Fatty acid methyl esters from C4 to C24 can be separated using this part of ISO 12966 including
saturated fatty acid methyl esters, cis- and trans-monounsaturated fatty acid methyl esters, and cis- and
trans-polyunsaturated fatty acid methyl esters.
The method is applicable to crude, refined, partially hydrogenated, or fully hydrogenated fats, oils, and fatty
acids derived from animal and vegetable sources, and fats extracted from foodstuff.
Milk and milk products (or fat coming from milk and milk products) are excluded from the scope of this part
of ISO 12966.
This part of ISO 12966 is not applicable to di-, tri-, polymerized, hydroxylated and oxidized fatty acids, and
fats and oils.
A method for the determination of the composition of fatty acid methyl esters expressed by area % in liquid
vegetable oils is proposed in Annex E.
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 661, Animal and vegetable fats and oils — Preparation of test sample
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 5555, Animal and vegetable fats and oils — Sampling
ISO 6353, — Reagents for chemical analysis
ISO 12966-1, Animal and vegetable fats and oils — Gas chromatography of fatty acid methyl esters — Part 1:
Guidelines on modern gas chromatography of fatty acid methyl esters
ISO 12966-2, Animal and vegetable fats and oils — Gas chromatography of fatty acid methyl esters — Part 2:
Preparation of methyl esters of fatty acids
ISO 12966-3, Animal and vegetable fats and oils — Gas chromatography of fatty acid methyl esters — Part 3:
Preparation of methyl esters using trimethylsulfonium hydroxide (TMSH)
3 Terms and definitions
No terms and definitions are listed in this document.

ISO/DIS 12966-4:2025(en)
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/
4 Principle
Using capillary gas chromatography, FAMEs are separated on a highly polar stationary phase with respect
to their chain length, degree of (un)saturation, and geometry and position of the double bonds. Peaks are
identified by comparison with the retention time of pure standards and quantified as fatty acids methyl
esters by reference to internal standards and instrument correction factors.
5 Reagents and materials
Unless otherwise stated, use only reagents as specified in ISO 6353 2 and ISO 6353 3 (if listed there). If not,
then use reagents of recognized analytical grade and water of at least grade 3, as defined in ISO 3696.
WARNING — Attention is drawn to the regulations which specify the handling of dangerous matter.
Technical, organizational, and personal safety measures shall be followed.
5.1 Reference standards
5.1.1 Reference fatty acid methyl esters (FAMEs)
Methyl esters of pure fatty acids, in particular, cis- and trans-isomers of octadecenoic (oleic), trans-isomers
of octadecadienoic (linoleic), and octadecatrienoic (α-linolenic) acids. Wide ranges of cis- and trans methyl
ester isomers are available on the market. The following are examples of suitable products available
commercially.
5.1.1.1 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 iso-octane (5.3), or MTBE
(5.4), or n-heptane (5.6), or n-hexane (5.5), or dichloromethane (5.8).
1)
NOTE This standard is commercially available from Supelco Inc, brand of Sigma-Aldrich (Cat. 40495-U) .
5.1.1.2 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 iso-octane (5.3), or MTBE (5.4), or n-heptane (5.6), or n-hexane (5.5), or
dichloromethane (5.8).
1)
NOTE This standard is commercially available from Supelco Inc, brand of Sigma-Aldrich (Cat. 47791) .
5.1.1.3 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),
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/DIS 12966-4:2025(en)
— 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 iso-octane (5.3), or MTBE (5.4), or n-heptane (5.6), or n-hexane (5.5), or
dichloromethane (5.8).
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.
5.1.1.4 Methyl octadecadienoate conjugated acids (CLA), 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 Supelco Inc, brand of Sigma Aldrich (Cat. O5507) . This
standard contains the two principal CLA isomers, but isomer ratio may vary from lot to lot.
5.1.2 Fats and oils with certified fatty acid composition
5.1.3 Quantitative FAME standard mixture containing cis and trans fatty acid methyl esters from
C4:0 to C22:6
2)
This type of FAME mixture is commercially available . It is also possible to prepare the FAME standard
mixture from individual and pure FAME standards, but the purchasing of individual FAME standards is
more expensive and the preparation is time consuming and requires high precision.
The amount of each FAME standard present in the mixture is necessary for determining the correction
factor (area/amount) for each FAME (Figure B.1).
5.1.4 Calibration FAME standard solution at 2 mg/ml for the calculation of the correction factors
Allow quantitative FAME standard mixture (5.1.3) to come to room temperature in the dark without heating.
Using a Pasteur pipet, rapidly transfer the content of the vial into a 50 ml volumetric flask, and dilute to the
mark with iso-octane (5.3), or MTBE (5.4), or n-heptane (5.6), or n-hexane (5.5). Dilute accordingly to the
type of injector used.
5.2 Internal standards
For the quantification of the fatty acids, in grams per 100 g, the use of a FAME as an internal standard (IS) is
mandatory.
If it is necessary to check the recovery and the effectiveness of the derivatization method, then either or
both a TAG and a FAME internal standard should be used. While the TAG-IS is added to the sample prior
to the FAME preparation, the FAME-IS is added before or after the FAME preparation. The FAME-IS is used
to calculate the recovery of the FAME from the TAG-IS and therefore, the efficiency of the derivatisation
procedure. In this case, a different chain length of the standards is required.
For the quantification of all fatty acids in vegetable oils, animal fats or extracted fats, C21:0 FAME or C19:0-
FAME are the recommended internal standards, depending on the risk of coelutions such as C21:0/C18:2
conjugated.
For the quantification of all fatty acids in fish oil, C23:0 FAME is the recommended internal standard.
For the quantification of butyric acid (C4:0) and caproic acid (C6:0) only, in fat containing short chain fatty
acids, C5:0 FAME is the recommended internal standard.
2) Examples of suitable products available commercially: Nu-Check-Prep, Cat. No. GLC 36 or GLC 37 (including C23:0
FAME). This information is given for the convenience of users of this document and does not constitute an endorsement
by ISO of these products. Equivalent products may be used if they can be shown to lead to the same results.

ISO/DIS 12966-4:2025(en)
For the quantification of trans fatty acids only, in vegetable oils, animal fats or extracted fats, C19:0-FAME
is the recommended internal standard. The concentration of the internal standard has to be adapted to the
expected content of trans fatty acids.
Depending on the capillary column and carrier gas used, the coelutions may be different. It is recommended
to carry out further analysis of the sample without the addition of the internal standard to check the natural
content of the fatty acid, which is used as the internal standard (C19:0 or C21:0 or C23:0). The content shall
be considered in the calculation.
The internal standard solutions are stable if precautions are taken to eliminate the loss of solvent and
therefore, a change in the concentration of the IS. For example, store the solution in a refrigerator in a well-
sealed amber bottle when not in use. Pure standards are available on the market. Purity of the IS shall be
confirmed by thin-layer chromatography, high-performance liquid chromatography, gas chromatography
analysis, or by any other appropriate technique.
The following are examples of suitable standard solutions:
Internal standard solution of C5:0 FAME - valeric acid methyl ester (purity ≥ 99 % mass fraction), mass
concentration 0,5 mg/ml in iso-octane (5.3), or MTBE (5.4), or n-heptane (5.6), or n-hexane (5.5) can be used
as the internal standard.
Internal standard solution of C19:0 FAME - nonadecanoic acid methyl ester (purity ≥ 99 % mass fraction),
mass concentration 5 mg/ml in iso-octane (5.3), or MTBE (5.4), or n-heptane (5.6), or n-hexane (5.5) can be
used as the internal standard.
Internal standard solution of C21:0 FAME - heneicosanoic acid methyl ester (purity ≥ 99 % mass fraction),
mass concentration 5 mg/ml in iso-octane (5.3), or MTBE (5.4), or n-heptane (5.6), or n-hexane (5.5) can be
used as the internal standard.
Internal standard solution of C23:0 FAME - tricosanoic acid methyl ester (purity ≥ 99 % mass fraction), mass
concentration 5 mg/ml in iso-octane (5.3), or MTBE (5.4), or n-heptane (5.6), or n-hexane (5.5) can be used
as the internal standard.
In order to check the recovery and the effectiveness of the derivatization method, the suitable standard
solution is the following:
Internal standard solution of C13:0 TAG - tritridecanoin (purity >99 % mass fraction), mass concentration
5 mg/ml in iso-octane (5.3), or MTBE (5.4), or n-heptane (5.6), or n-hexane (5.5) can be used as the internal
standard.
NOTE If the TAG-IS is hard to dissolve in the cold, a hot methylation procedure, as specified in ISO 12966 2, 4.3,
4.4, and 4.5, shall be used.
5.3 Iso-octane (2,2,4-trimethyl pentane)
5.4 Methyl tert-Butyl ether (MTBE) (2-Methoxy-2-methylpropane)
5.5 n-Hexane
5.6 n-Heptane
5.7 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 5.1.1.1 to 5.1.1.4. 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 iso-octane (5.3), or MTBE (5.4), or n-heptane (5.6),
or n-hexane (5.5). Dilute in accordance with the type of injector used.

ISO/DIS 12966-4:2025(en)
It is also possible to identify the retention times of cis and trans isomers using a fat or an oil with certified
fatty acid composition (5.1.2), for example, a reference hydrogenated oil samples.
5.8 Dichloromethane (Methylene chloride)
6 Apparatus
Usual laboratory equipment and, in particular, the following.
6.1 Gas chromatograph, equipped with flame ionization detector, split or splitless injector, and data
acquisition system.
NOTE The use of on-column and programmable temperature vaporizer (PTV) injectors are also possible.
6.2 Capillary column, fused silica capillary 100 m and 0,25 mm i.d. coated with cyanopropyl-polysiloxane
3)
stationary phase, such as SP-2560 or CP-Sil 88 to a thickness of 0,20 µm. Commercially prepared columns
are available from different suppliers.
The use of 100 m, 0,25 mm ID, 0,20 µm film thickness columns with SP-2560 or CP-Sil 88 as the stationary
phase are required as the separation capacity of these columns is sufficient to separate most C18:1 trans-
and cis-isomers and is in accordance with resolution specification indicated in 10.3. If this separation is not
required, a 50 m or 60 m column can also be used. However, some 50 m or 60 m long columns might also
achieve this separation mostly for vegetable oils. Other types of columns (BPX70, DB-23, HP-23, Rtx-2330,
SP-2330, SP-2380, etc.) are also possible, but a shift in the elution order is possible. For fast GC analysis, short
columns are also possible (10 m to 15 m), but with limited information which in certain cases, will not be a
problem.
6.3 Micro syringe, for gas chromatography, 10 μl delivery with a hardened needle.
6.4 Carrier gas, hydrogen (recommended), nitrogen or helium, 99,999 5 % pure or better, gas
chromatography quality, dried, oxygen removed by suitable filters (<0,1 mg/kg), free from organic
impurities.
WARNING — Hydrogen, which is used with capillary columns, can double the speed of the analysis
(in comparison with helium), but is hazardous. Hydrogen generators and safety devices are available
and it is essential that a suitable device be incorporated into the apparatus.
6.5 Flame gases, hydrogen and air, gas chromatography quality, free from organic impurities.
6.6 Make-up gas, nitrogen or helium, gas chromatography quality, free from organic impurities.
7 Sampling
A representative sample should be sent to the laboratory. It should not be damaged or changed during
transport or storage.
Sampling is not part of the method specified in this part of ISO 12966. A recommended sampling method is
given in ISO 5555.
8 Preparation of test sample
Prepare the test sample in accordance with ISO 661.
3) 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 ISO of these products. Equivalent products may be used if they can
be shown to lead to the same results.

ISO/DIS 12966-4:2025(en)
9 Preparation of methyl esters from fats, oils, and fatty acids
The fatty acid methyl esters shall be prepared in accordance with ISO 12966-2 or ISO 12966-3. The use
of ISO 12966-3 is not recommended for fatty acid methyl esters quantification by mass (g/100g) because
only 70 % to 80 % of free fatty acids are esterified and this preparation can lead to an isomerization of
unsaturated fatty acids such as methyl octadecadienoate conjugated acids (CLA).
Prior to methylation, the internal standard solution, is added to the reaction flask so that after the oil or fat
is added, the mass fraction is between 0,01 and 0,20 mg IS/mg oil or fat.
For example, 2 ml of internal standard solution (5.2) can be added to 100 mg test portion of fat prior to
methylation.
Dissolve the prepared FAMEs in iso-octane (5.3), or MTBE (5.4), or n-heptane (5.6), or n-hexane (5.5). The
mass concentration should be approximately 15 mg/ml to 20 mg/ml for split injection. For on-column
injection, the mass concentration should be adapted.
It is recommended to carry out further analysis of the sample without the addition of the internal standard
to check the natural content of the fatty acid which is used as the internal standard. This natural content
shall be considered in the calculation.
10 Procedure
WARNING — Due to the toxic character of some solvents, a ventilated hood shall be used.
10.1 General
The first sample in an analysis batch shall always be a blank FAME dissolution solvent. No peaks shall be
detected in this blank run.
10.2 GC conditions
Adapt the temperatures and GC conditions considering the type of fat, oil, or fatty acid analysed and the
apparatus used. The following conditions have been proven to be suitable for the separation of FAMEs (C4 to
C24) on 100 m columns. However, other conditions are also possible and can be used.
Injector temperature: 250°C
Detector temperature: 250°C
Oven temperature: 60°C (2 min) to 172°C with 30°C/min, hold 5 min at 172°C, 172°C to 210°C with
1°C/min, hold 22 min at 210°C
Carrier gas hydrogen: column head pressure, 150 kPa (constant pressure)
linear velocity; (30 to 40) cm/s, flow rate approx. 1,0 ml/min
split ratio, 1:25 or 1:100 depending on the dilution
Injection volume: 1 µl (equivalent to 15 µg to 20 μg FAME)
Examples of chromatograms are shown in Annex B.
NOTE For the analysis of animal fats, the complete elution of all FAMEs can be checked with certified reference
standards.
10.3 Performance check
Column performance is checked using a suitable mixture of fatty acid methyl esters covering the range of
fatty acids under investigation. Since commercial GC designs are different and the separation obtained is not

ISO/DIS 12966-4:2025(en)
identical to the example chromatograms, small changes in the sample size, sample concentration, or oven
temperature may be required. If so, adjust the sample size, sample concentration, or oven temperature until
the best separation results are obtained. If the column oven temperature needs to be adjusted, it should be
adjusted by small increments, preferably in steps of 1 °C.
Pay particular attention to the separation and identification of critical pair such as C16:1/C17:0, C18:3n-3/
C20:1, and C21:0/C18:2 conjugated.
For the accurate quantification of C18:1 TFA (level ≥ 0,3 g/100 g fat), a sufficient resolution between C18:1
trans-13/14 and C18:1 cis-9 (oleic acid) is required. Resolution is determined with the injection of the
qualitative cis and trans C18:1 FAME isomers standard mixture solution (5.1.1.1) or the injection of the
qualitative cis and trans isomers standard mixture solution (5.7).
Dilute in accordance with the type of injector used. Inject into the gas chromatograph the calibrating solution
(4.1.1.1 or 4.7). 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):
11, 8×−()tt
RR21
R = (1)
 
 
WW−
1 1
   
 
1 2
   
 2 2 
where
t is the distance, in centimeters, between the left of the chromatogram and the top of peak 1 (C18:1
R1
trans-13/14);
t is the distance, in centimeters, between the left of the chromatogram and the top of peak 2 (C18:1
R2
cis-9);
W is the peak width, in centimeters, at half-height, of peak 1 (C18:1 trans-13/14);
 
 
 
W is the peak width, in centimeters, at half-height, of peak 2 (C18:1 cis-9).
 
 
2
The resolution is sufficient when the R criterion is ≥ 1.00 ± 5 % (see Appendix C, Figure C.7).
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.
11 Calculations
11.1 Qualitative analysis and peak identification
The individual FAMEs are identified by their retention times and in comparison with FAME reference
standards, by using qualitative cis and trans isomers standard mixture solution (5.7) or by using a fat or an
oil with certified fatty acid composition (5.1.2).
When unknown peaks are observed, they should be identified using appropriate procedures such as GC-MS,
FTIR, silver-ion chromatography, and classical chemical methods. Peaks of unknown identity should not be
included in the summation of peak areas when calculating the fatty acid composition, unless they have been
confirmed to be fatty acids. It is also possible to summarize unknown peaks as such.
C18:1 trans fatty acids identification: identify and group all trans isomers of C18:1 eluted before the oleic
acid methyl ester (C18:1 n-9 cis) according to chromatograms in Annex C.

ISO/DIS 12966-4:2025(en)
C18:2 trans fatty acids identification: identify and group all trans isomers of C18:2 eluted before the linoleic
acid methyl ester (C18:2 n-6 cis) according to chromatograms in Annex C.
C18:3 trans fatty acids identification: identify and group all trans isomers of C18:3 eluted before the linolenic
acid methyl ester (C18:3 n-3 cis) according to chromatograms in Annex C.
11.2 Quantitative analysis
Areas are corrected with specific correction factors (F). These factors are determined for each single
instrument. For this purpose, a calibration FAME standard solution (5.1.4) containing certified fatty acid
composition, is injected three times.
These correction factors are not identical with theoretical FID correction factors, which are given in
Annex A, as they also include the performance of the injection system, etc. However, in the case of bigger
differences, the whole system shall be checked for performance.
For the calibration FAME standard solution (5.1.4), the mass fraction w , in grams per 100 g of FAME, i, is
i
given by Formula (2):
m
i
w = ×100 (2)
i
∑m
where
m is the mass of the FAME, i, in the calibration FAME standard solution;
i
Σm is the total of the masses of the various components, as FAMEs of the calibration FAME standard
solution.
From the chromatogram of the calibration FAME standard solution (5.1.4), calculate the percentage by area
for the FAME, i, as follows in Formula (3):
A
i
x = ×100 (3)
i
∑A
where
A is the area of the FAME, i, in the calibration FAME standard solution;
i
ΣA is the sum of all areas of all FAMEs of the calibration FAME standard solution.
The correction factor, F , is then determined as shown in Formula (4):
i
w mA×∑
i i
F == (4)
i
x Am×∑
i i
where
w mass fraction in grams per 100 g of FAME, i;
i
x percentage by area for the FAME, i;
i
A is the area the FAME, i;
i
ΣA is the sum of all areas of all FAMEs of the calibration FAME standard solution;
m is the mass of the FAME, i, in the calibration FAME standard solution;
i
Σm is the total of the masses of the various components, as FAMEs of the calibration FAME standard
solution.
ISO/DIS 12966-4:2025(en)
The mean of the three injections is used for each correction factor F . The variation between three injections
i
is optimal when coefficients of variation are less than 2 %.
The correction factor calculated for C18:2 cis-9,12 (or n-6) can be applied for C-18:2 CLA (cis-9,trans-11) and
that calculated for C18:3 cis-9,12,15 (n-3) can be applied for C18:3 trans isomers.
11.2.1 Fatty acid methyl esters quantification, by mass (g/100 g)
The mass fraction in grams per 100 g, of the fatty
...