ISO/TS 17764-1:2002

Animal feeding stuffs — Determination of the content of fatty acids — Part 1: Preparation of methyl esters

ISO/TS 17764-1:2002 specifies two methods for preparing the methyl esters of fatty acids of animal and vegetable fats, oils and fatty acid mixtures for raw materials for compound animal feeds, and fatty acids originating from fat extracts of animal feeding stuffs, including fats and fatty acid mixtures containing butyric acid. The general method, the boron trifluoride (BF3) method is concerned with the preparation of methyl esters of fatty acids with six or more C atoms, originating from fats, oils and free fatty acids. The KOH/HCl method is concerned with the preparation of methyl esters of fatty acids with four or more C atoms. This method can also be used for the quantitative determination of fatty acids with a chain length shorter than 10 C atoms in free fatty acid mixtures. The methyl esters produced can be used for gas-liquid chromatography (GLC). Unsaponifiable materials are not removed and can, when present in considerable amounts, interfere with the chromatographic analysis. ISO/TS 17764-2:2002 describes the application of gas chromatography with capillary columns and flame ionization detection for the determination of the content of fatty acids in a fat by making use of the methyl esters of the fatty acids obtained in accordance with the methods specified in ISO/TS 17764-1:2002.

Aliments des animaux — Détermination de la teneur en acides gras — Partie 1: Préparation des esters méthyliques

General Information

• Status: Published
• Publication Date: 14-Nov-2002
• ICS: 65.120 - Animal feeding stuffs
• Technical Committee: ISO/TC 34/SC 10 - Animal feeding stuffs
• Drafting Committee: ISO/TC 34/SC 10 - Animal feeding stuffs
• Current Stage: 9060 - Close of review
• Start Date: 02-Dec-2026

Standards Content (Sample)

TECHNICAL ISO/TS
SPECIFICATION 17764-1
First edition
2002-11-01

Animal feeding stuffs — Determination of
the content of fatty acids —
Part 1:
Preparation of methyl esters
Aliments des animaux — Détermination de la teneur en acides gras —
Partie 1: Préparation des esters méthyliques




Reference number
ISO/TS 17764-1:2002(E)
©
ISO 2002

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ISO/TS 17764-1:2002(E)
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ISO/TS 17764-1:2002(E)
Foreword
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— an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical
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ISO/TS 17764-1 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 10,
Animal feeding stuffs.
ISO/TS 17764 consists of the following parts, under the general title Animal feeding stuffs — Determination of
the content of fatty acids:
 Part 1: Preparation of methyl esters
 Part 2: Gas chromatographic method

© ISO 2002 — All rights reserved iii

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TECHNICAL SPECIFICATION ISO/TS 17764-1:2002(E)

Animal feeding stuffs — Determination of the content of fatty
acids —
Part 1:
Preparation of methyl esters
1 Scope
ISO/TS 17764 specifies methods for the quantitative determination of individual fatty acids and of the sum of
the fatty acids (elutable fatty acids).
This part of ISO/TS 17764 specifies two methods for preparing the methyl esters of fatty acids of animal and
vegetable fats, oils and fatty acid mixtures for raw materials for compound animal feeds, and fatty acids
originating from fat extracts of animal feeding stuffs, including fats and fatty acid mixtures containing butyric
acid.
The general method, the boron trifluoride (BF ) method, is concerned with the preparation of methyl esters of
3
fatty acids with six or more C atoms, originating from fats, oils and free fatty acids.
The KOH/HCl method is concerned with the preparation of methyl esters of fatty acids with four or more C
atoms. This method can also be used for the quantitative determination of fatty acids with a chain length
shorter than ten C atoms in free fatty acid mixtures.
The methyl esters produced can be used for gas-liquid chromatography (GLC).
NOTE 1 Unsaponifiable materials are not removed and can, when present in considerable amounts, interfere with the
chromatographic analysis.
NOTE 2 ISO/TS 17764-2 describes the application of gas chromatography with capillary columns and flame ionization
detection for the determination of the content of fatty acids in a fat by making use of the methyl esters of the fatty acids
obtained in accordance with the methods specified in this part of ISO/TS 17764.
2 Normative references
The following referenced documents are indispensable for the application 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 661:1989, Animal and vegetable fats and oils  Preparation of test sample
ISO 3696:1987, Water for analytical laboratory use  Specification and test methods
ISO 6492:1999, Animal feeding stuffs  Determination of fat content
© ISO 2002 — All rights reserved 1

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ISO/TS 17764-1:2002(E)
3 Fat extraction
3.1 General
Extract the fat for determination of the content of the saponifiable fatty acids in an animal feeding stuff or
mixed feed in accordance with the category of the feeding stuff, as described in ISO 6492:1999, with the
following amendments.
3.2 Samples of category A
Perform a fat extraction in accordance with 9.5.1 of ISO 6492:1999.
Evaporate the solvent with a rotating evaporator in a water bath at a temperature of not more than 40 °C.
Then dry the residue for 2 h in a vacuum drying oven at 40 °C ± 2 °C.
3.3 Samples of category B
Extract the fat in two steps. For the first step, proceed in accordance with 9.5.1 of ISO 6492:1999, treating the
test portion as described in 9.3 of ISO 6492:1999 as a sample of category A.
Collect the fat extract in a dry flask. Allow the solvent to evaporate from the residue by exposing the thimble to
the air.
Perform a hydrolysis of the residue in accordance with 9.4 of ISO 6492:1999.
After hydrolysis, dry the residue in an extraction thimble for 60 min in a vacuum drying oven at 40 °C ± 2 °C.
Extract the residue according to 9.5.1 of ISO 6492:1999.
Add the fat extract to the first extract.
Evaporate the solvent with a rotating evaporator in a water bath at a temperature of not more than 40 °C and
dry the residue for 2 h in a vacuum drying oven at 40 °C ± 2 °C.
4 Preparation of test sample of fat or fat extract
If the fat sample or the fat extract is not completely molten, heat the sample to a temperature of not more than
10 °C above the melting temperature. See ISO 661.
5 Method for the preparation of methyl esters of fatty acids with six or more C
atoms (BF method)
3
5.1 Principle
The glycerides are saponified with methanolic sodium hydroxide. The soaps are converted to methyl esters by
reaction with a boron trifluoride/methanol complex.
5.2 Reagents
Use only reagents and solvents of recognized analytical grade.
5.2.1 Water, complying with at least grade 3 in accordance with ISO 3696:1987.
5.2.2 Heptadecanoic acid (internal standard), of purity at least 99 %.
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ISO/TS 17764-1:2002(E)
5.2.3 Sodium hydroxide, methanolic solution, c(NaOH) ≈ 0,5 mol/l.
Dissolve 2 g of sodium hydroxide in 100 ml of methanol containing not more than 0,5 % (mass fraction) of
water. If the solution has to be stored for a considerable time, a small amount of white precipitate of sodium
carbonate can form; this has no effect on the preparation of the methyl esters.
Instead of sodium hydroxide, a methanolic solution of potassium hydroxide with the same concentration may
be used.
5.2.4 Boron trifluoride (BF ), methanolic solution, 10 % to 15 % (mass fraction).
3
WARNING — Boron trifluoride is poisonous. For this reason, it is not recommended that the analyst
prepare the methanolic solution of boron trifluoride from methanol and boron trifluoride.
Solutions are available commercially.
In gas chromatographic analysis of the methyl esters, certain reagents can give rise to peaks which interfere
with the analysis. In particular, BF solutions can cause peaks in the area of the
...