ISO/PRF 12966-4

ISO/DIS PRF 12966-4:2025(en)
ISO/TC 34/SC 11
Secretariat: BSI
Date: 2025-11-052026-01-16
Animal and vegetable fats and oils — Gas chromatography of fatty
acid methyl esters —
Part 4:
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
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PROOF
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iii
Contents
Foreword . v
Introduction . vii
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
5.2 Internal standards. . 3
5.3 Iso-octane (2,2,4-trimethyl pentane). . 5
5.4 Methyl tert-Butyl ether (MTBE) (2-Methoxy-2-methylpropane). . 5
5.5 n-Hexane. . 5
5.6 n-Heptane. . 5
5.7 Qualitative cis and trans isomers standard mixture solution. . 5
5.8 Dichloromethane (methylene chloride). . 5
6 Apparatus . 5
7 Sampling . 6
8 Preparation of test sample . 6
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 . 7
11 Calculations . 8
11.1 Qualitative analysis and peak identification . 8
11.2 Quantitative analysis . 8
11.3 Expression of the results for food labelling . 11
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 for fatty acid
methyl esters . 13
Annex B (informative) Examples of chromatograms . 16
Annex C (informative) Elution zone of the trans fatty acid methyl esters . 23
Annex D (informative) Stoichiometric factors for converting fatty acid methyl esters to fatty
acids . 28
Annex E (informative) Determination of the composition of fatty acid methyl esters expressed
by area % in liquid vegetable oils . 30
Annex F (informative) Results of an interlaboratory trial . 31
Bibliography . 41
iv
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
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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
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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, in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 307, Oilseeds, vegetable and animal fats and oils and their by-products - Methods of
sampling and analysis, in accordance with the Agreement on technical cooperation between ISO and CEN
(Vienna Agreement).
This second edition cancels and replaces the first edition (ISO 12966-4:2015), which has been technically
revised.
The main changes are as follows:
— the Scope has been extended to the separation of fatty acid methyl esters from C4 to C24;
— ruminant fat has been added to the Scope,
— quantification by area (%) or by mass (g/100 g) 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; has been added
— quantification of total trans fatty acid methyl esters by mass (g/100 g) has been added;
— the use of 100 m, 0,25 mm ID, 0,20 µm film thickness columns are now required to separate most C18:1
trans- and cis-isomers;
— a method has been added for determination of the composition of fatty acid methyl esters expressed by
area % in liquid vegetable oils.
v
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.
vi
Introduction
This document is one part of a series of four International Standards for the preparation and determination of
fatty acid methyl esters (FAMEs) by gas chromatography in animal and vegetable fats and oils. The ISO 12966
series 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.
The ISO 12966 series 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 document gives the conditions for the analysis of FAMEs by capillary gas chromatography, while
ISO 12966-2 and ISO 12966-3 cover the preparation of FAMEs by different methods. ISO 12966-1 is a
guideline to the modern gas chromatography of FAMEs.
vii
Animal and vegetable fats and oils — Gas chromatography of fatty acid
methyl esters —
Part 4:
Determination by capillary gas chromatography
1 Scope
This document 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).
FAMEs from C4 to C24 can be separated using this document including saturated FAMEs, cis- and trans-
monounsaturated FAMEs, and cis- and trans-polyunsaturated FAMEs.
This document 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.
This document does not apply to milk and milk products (or fat coming from milk and milk products) or
products supplemented with conjugated linoleic acid (CLA).
This document does not apply to di-, tri-, polymerized, hydroxylated and oxidized fatty acids, and fats and oils.
A method for the determination of the composition of FAMEs 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 6353-2, Reagents for chemical analysis — Part 2: Specifications — First series
ISO 6353-3, Reagents for chemical analysis — Part 3: Specifications — Second series
ISO 12966-2:2017, 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 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 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 a mass fraction of 3 %);
— cis-9,cis-12,trans-15-octadecatrienoic acid methyl ester (approximately a mass fraction of 7 %;
— cis-9,trans-12,cis-15-octadecatrienoic acid methyl ester (approximately a mass fraction of 7 %;
— cis-9,trans-12,trans-15-octadecatrienoic acid methyl ester (approximately a mass fraction of 15 %;
— trans-9,cis-12,cis-15-octadecatrienoic acid methyl ester (approximately a mass fraction of 7 %;
— trans-9,cis-12,trans-15-octadecatrienoic acid methyl ester (approximately a mass fraction of 15 %;

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.

— trans-9,trans-12,cis-15-octadecatrienoic acid methyl ester (approximately a mass fraction of 15 %;
— trans-9,trans-12,trans-15-octadecatrienoic acid methyl ester (approximately a mass fraction of 30 %.
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 can vary from lot to lot.
5.1.2 Fats and oils with certified fatty acid composition.
Fats and oils with certified fatty acid composition, (e.g. External Reference Materialsexternal reference
materials or Certified Reference Material.certified reference material). This type of samplessample is
accompanied with a certificate that provides mean values and its associated uncertainty for each fatty acid.
These products are often, for example, commercially available from proficiency testing programsprogrammes
(BIPEA, AOCS, FAPAS, JRC, etc).
5.1.3 Quantitative FAME standard mixture containing cis and trans FAMEs 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 (see 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 triacylglycerol (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.

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.

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.
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 should be adapted to the
expected content of trans fatty acids.
Depending on the capillary column and carrier gas used, the coelutions can 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.
If the TAG-IS is hard to dissolve in the cold, a hot methylation procedure, as specified in ISO 12966-2:2017,
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 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 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.
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, reference hydrogenated oil samples.
5.8 Dichloromethane (methylene chloride).
6 Apparatus
The usual laboratory equipment and, in particular, the following shall be used.
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, it is possible that some 50 m or 60 m long columns
can 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.
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.

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 document. A recommended sampling method is given in
ISO 5555.
8 Preparation of test sample
Prepare the test sample in accordance with ISO 661.
9 Preparation of methyl esters from fats, oils, and fatty acids
The FAMEs shall be prepared in accordance with ISO 12966-2 or ISO 12966-3. The use of ISO 12966-3 is not
recommended for FAMEs quantification by mass (g/100 g) 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 approximately 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 FAMEs covering the range of fatty acids under
investigation. Since commercial GC designs are different and the separation obtained is not identical to the
example chromatograms, small changes in the sample size, sample concentration or oven temperature can 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
(5.1.1.1 or 5.7). 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):
1,18×(𝑡𝑡 −𝑡𝑡 )
𝑅𝑅2 𝑅𝑅1
𝑅𝑅 = (1)
(𝑊𝑊1 +𝑊𝑊1 )
( )1 ( )2
2 2
where
𝑡𝑡 is the distance, in minutes, between the left of the chromatogram and the top of peak 1 (C18:1
𝑅𝑅1
trans-13/14);
𝑡𝑡 is the distance, in minutes, between the left of the chromatogram and the top of peak 2 (C18:1
𝑅𝑅2
cis-9);
𝑊𝑊 1 is the peak width, in minutes, at half-height, of peak 1 (C18:1 trans-13/14);
( )1
𝑊𝑊 1 is the peak width, in minutes, at half-height, of peak 2 (C18:1 cis-9).
( )2
The resolution is sufficient when the R criterion is ≥ 1,00 ± 5 % (see Figure C.6).
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.
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
11.2.1 General
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 given
i
by Formula (2):
𝑚𝑚
𝑖𝑖
𝑤𝑤 = ×100 (2)
𝑖𝑖
∑𝑚𝑚
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):
𝐴𝐴
𝑖𝑖
𝑥𝑥 = ×100 (3)
𝑖𝑖
∑𝐴𝐴
where
Ai is the area of the FAME, i, in the calibration FAME standard solution;
ΣA is the sum of all areas of all FAMEs of the calibration FAME standard solution.
The correction factor, Fi, is then determined as shown in Formula (4):
𝑤𝑤 𝑚𝑚 ×∑𝐴𝐴
𝑖𝑖 𝑖𝑖
𝐹𝐹 = = (4)
𝑖𝑖
𝑥𝑥 𝐴𝐴 ×∑𝑚𝑚
𝑖𝑖 𝑖𝑖
where
w mass fraction in grams per 100 g of FAME, i;
i
xi percentage by area for the FAME, i;
A is the area of 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.
The mean of the three injections is used for each correction factor Fi. The variation between three injections
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 C18:2 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.
11.2.111.2.2 FAMEs quantification, by mass (g/100 g)
The mass fraction in grams per 100 g, of the fatty acid, i, expressed as methyl ester, is then given by
Formula (5):
𝑚𝑚 ×𝐹𝐹 ×𝐴𝐴
IS 𝑖𝑖 𝑖𝑖
𝑤𝑤 = ×100 (5)
𝑖𝑖
𝑚𝑚×𝐹𝐹 ×𝐴𝐴
IS IS
where
A is the area of the FAME, i;
i
AIS is the area of the internal standard;
F is the correction factor of the fatty acid, i, expressed as FAME;
i
FIS is the correction factor of the internal standard, expressed as FAME;
m is the mass of the test portion, in milligrams;
m is the mass of the internal standard, in milligrams, corrected by its purity (usually 0,99).
IS
The results are expressed to two decimal places.
11.2.211.2.3 FAMEs quantification, by area %
The content of each of the individual fatty acids from C4 to C24 present in the chromatogram, wi, expressed
as a percentage by area of methyl esters, is then given by Formula (6):
𝐴𝐴 ×𝐹𝐹
𝑖𝑖 𝑖𝑖
𝑤𝑤 = × 100 (6)
𝑖𝑖
∑(𝐴𝐴 ×𝐹𝐹 )
𝑖𝑖 𝑖𝑖
where
A is the area of the fatty acid, i, expressed as FAME;
i
Fi is the correction factor of the fatty acid, i, expressed as FAME.
The results are expressed to two decimal places.
11.2.311.2.4 Butyric acid and caproic acid methyl esters quantification (only), by mass (g/100 g) in
fat containing short chain fatty acids
When short chain fatty acids are present in the sample, the mass fraction of butyric acid (C4:0) and caproic
acid (C6:0) is calculated using the C5:0 FAME internal standard. Assuming that the response factors of C4:0
and C6:0 are equivalents to the response factor of the internal standard, area is not corrected by the correction
factor (F .
i)
The mass fraction in grams per 100 g, of butyric acid (C4:0) or caproic acid (C6:0), expressed as methyl ester,
wi, is then given by Formula (7):
𝑚𝑚 ×𝐴𝐴
IS 𝑖𝑖
𝑤𝑤 = ×100 (7)
𝑖𝑖
𝑚𝑚×𝐴𝐴
IS
where
Ai is the area of the butyric acid or the caproic acid, expressed as FAME (C4:0 FAME or C6:0
FAME);
AIS is the area of the internal standard (C5:0 FAME);
m is the mass of the test portion, in milligrams;
mIS is the mass of the internal standard (C5:0 FAME), in milligrams, corrected by its purity (usually
0,99).
The results are expressed to two decimal places.
11.2.411.2.5 Total trans FAMEs quantification (only), by mass (g/100 g)
For the quantification of total trans fatty acids (sum of trans C18:1, trans C18:2 and trans C18:3), in grams per
100 g, the use of an internal standard, such as C21:0-FAME, is necessary.
The mass fraction in grams per 100 g, of the fatty acid, i, expressed as methyl ester, W , is then given by
i
Formula (8):
𝑚𝑚 ×(𝐹𝐹 ×𝐴𝐴 +𝐹𝐹 ×𝐴𝐴 +𝐹𝐹 ×𝐴𝐴 )
IS 𝑡𝑡C18:1 𝑡𝑡C18:1 𝑡𝑡C18:2 𝑡𝑡C18:2 C18:3 𝑡𝑡C18:3
𝑤𝑤 = ×100 (8)
𝑖𝑖
𝑚𝑚×𝐹𝐹 ×𝐴𝐴
IS IS
where
F is the correction factor of the fatty acid, trans C18:1, expressed as FAME;
tC18:1
F is the correction factor of the fatty acid, trans C18:2, expressed as FAME;
tC18:2
F is the correction factor of the fatty acid, C18:3, expressed as FAME;
C18:3
FIS is the correction factor of the internal standard, expressed as FAME (C21:0 FAME);
A is the area of the FAME, trans C18:1;
tC18:1
AtC18:2 is the area of the FAME, trans C18:2;
A is the area of the FAME, trans C18:3;
tC18:3
A is the area of the internal standard (IS) (C21:0 FAME);
IS
m is the mass of the test portion, in milligrams;
m is the mass of the internal standard (C21:0 FAME), in milligrams, corrected by its purity
IS
(usually 0,99).
The results are expressed to two decimal places.
11.3 Expression of the results for food labelling
For food labelling, the results expressed as FAME can be converted into results expressed in fatty acid (FA)
using the corresponding stoichiometric factors St(FA) listed in Annex D.
Content of saturated fatty acids: sum of all saturated fatty acids (C4:0; C5:0; C6:0; C7:0; C8:0; C9:0; C10:0; iso-
C10:0; C11:0; C12:0; anteiso- and iso-C12:0; C13:0; anteiso- and iso-C13:0; C14:0; anteiso- and iso-C14:0;
C15:0; anteiso- and iso-C15:0; C16:0; anteiso- and iso-C16:0; C17:0; anteiso- and iso-C17:0; C18:0; anteiso- and
iso-C18:0; C19:0; anteiso-C19:0; C20:0; iso-C20:0; C21:0; iso-C21:0; C22:0; C24:0; C25:0 and C26:0).
Content of cis-monounsaturated fatty acids: sum of all cis-monounsaturated fatty acids (C10:1; C11:1; C12:1;
C14:1; C15:1; C16:1; C17:1; C18:1; C19:1; C20:1; C22:1; and C24:1).
Content of cis-polyunsaturated fatty acids: sum of all cis-polyunsaturated fatty acids (C16:2; C16:3; C16:4;
C18:2; C18:3; C18:4; C20:2; C20:3; C20:4; C20:5; C22:2; C22:3; C22:4; C22:5; and C22:6).
Content of cis-n-3 polyunsaturated fatty acids: sum of all n-3 PUFAs (9,12,15-C18:3; 6,9,12,15-C18:4;
8,11,14,17-C20:4; 5,8,11,14,17-C20:5; 7,10,13,16,19-C22:5 and 4,7,10,13,16,19-C22:6).
Content of cis-n-6 polyunsaturated fatty acids: sum of all n-6 PUFAs (9,12-C18:2; 6,9,12-C18:3; 8,11,14-C20:3;
5,8,11,14-C20:4; 7,10,13,16-C22:4; and 4,7,10,13,16-C22:5).
Content of trans-fatty acids: sum of all trans-fatty acids (trans-C18:1, trans-C18:2, trans-C18:3).
Content of conjugated linoleic acids: sum of C18:2 fatty acids containing conjugated double bonds in cis or
trans configuration, mainly cis-9, trans-11-C18:2 (CLA).
12 Precision
12.1 Results of interlaboratory test
Details of an interlaboratory test on the precision of the method are summarized in Annex F. The values
derived from this interlaboratory test might not be applicable to concentration ranges and matrices other than
those given.
12.2 Repeatability
The absolute difference between two independent single test results obtained using the same method on
identical test material in the same laboratory, by the same operator, using the same equipment within a short
interval of time, will, in not more than 5 % of cases, be greater than r given in Tables F.1 to F.12.
12.3 Reproducibility
The absolute difference between two single test results, obtained using the same method on identical test
material, in different laboratories, with different operators, using different equipment, will, in not more than
5 % of cases, be greater than R given in Tables F.1 to F.12.
13 Test report
The test report shall specify:
a) all information necessary for the complete identification of the sample;
b) the sampling method us
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