ISO 24363:2023
(Main)Determination of fatty acid methyl esters (cis and trans) and squalene in olive oil and other vegetable oils by gas chromatography
Determination of fatty acid methyl esters (cis and trans) and squalene in olive oil and other vegetable oils by gas chromatography
This document specifies the determination of the fatty acid methyl esters (FAME) and squalene in olive oil and other vegetable oils by gas chromatography (GC). This document is applicable to the determination of FAME from C12 to C24, including saturated, cis- and trans-monounsaturated, cis- and trans-polyunsaturated FAME and squalene.
Détermination des esters méthyliques d'acides gras (cis et trans) et du squalène dans l'huile d'olive et d'autres huiles végétales par chromatographie en phase gazeuse
General Information
Standards Content (Sample)
INTERNATIONAL ISO
STANDARD 24363
First edition
2023-02
Determination of fatty acid methyl
esters (cis and trans) and squalene in
olive oil and other vegetable oils by
gas chromatography
Détermination des esters méthyliques d'acides gras (cis et trans)
et du squalène dans l'huile d'olive et d'autres huiles végétales par
chromatographie en phase gazeuse
Reference number
ISO 24363:2023(E)
© ISO 2023
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ISO 24363:2023(E)
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ISO 24363:2023(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents and materials . 2
6 Apparatus . 3
7 Sample . 3
7.1 Sampling . 3
7.2 Preparation of test sample . 4
8 Procedure .4
8.1 Preparation of FAME samples . 4
8.2 Gas chromatography . 5
8.2.1 General . 5
8.2.2 System suitability . 5
9 Expression of results . 5
9.1 Quantitative analysis . 5
9.1.1 FAME, by area % . 5
9.1.2 FAME, by mass (g/100 g) . 6
9.1.3 Squalene . 6
10 Precision of the method .6
10.1 Interlaboratory test . . 6
10.2 Repeatability . 7
10.3 Reproducibility . . 7
11 Test report . 7
Annex A (informative) Example chromatogram of FAME and squalene in an extra virgin
olive oil sample . 8
Annex B (informative) Quantitative analysis using correction factors — Calculation of
FAME using correction factors .9
Annex C (informative) Results of an interlaboratory test .11
Bibliography .22
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ISO 24363:2023(E)
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).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
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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.
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.
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INTERNATIONAL STANDARD ISO 24363:2023(E)
Determination of fatty acid methyl esters (cis and trans)
and squalene in olive oil and other vegetable oils by gas
chromatography
1 Scope
This document specifies the determination of the fatty acid methyl esters (FAME) and squalene in olive
oil and other vegetable oils by gas chromatography (GC).
This document is applicable to the determination of FAME from C12 to C24, including saturated, cis- and
trans-monounsaturated, cis- and trans-polyunsaturated FAME and squalene.
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
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/
3.1
squalene content
mass fraction of squalene in the sample
Note 1 to entry: As determined under the conditions specified in this document.
Note 2 to entry: The squalene content is expressed in milligrams per kilogram of oil, without a decimal place.
3.2
fatty acids methyl esters
FAME
percentage area of triglyceride fatty acids as well as free fatty acids that has been methylated in the
sample
Note 1 to entry: As determined under the conditions specified in this document.
Note 2 to entry: The FAME are expressed as percentage area of FAME (% area of individual fatty acid per 100 %
area of total FAME present in the sample taken).
4 Principle
FAME are formed by trans-esterification with methanolic solution of potassium hydroxide at room
temperature. FAME from C12 to C24, including saturated, cis- and trans- monounsaturated and cis- and
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ISO 24363:2023(E)
trans- polyunsaturated FAME are then determined by capillary GC. In the same method, the amount of
squalene is determined in mg/kg using a standard squalene calibration curve.
For oils with FFA > 2,0 %, a prior silica gel solid phase extraction (SPE) clean-up is recommended.
5 Reagents and materials
WARNING — Attention is drawn to the regulations which specify the handling of hazardous
substances. Technical, organizational and personal safety measures shall be followed.
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and
distilled water or water of equivalent purity.
5.1 Potassium hydroxide, (≥85 g/100 g potassium hydroxide).
5.2 Methanol, containing not more than 0,5 % mass fraction water.
5.3 Methanolic potassium hydroxide solution (2 M). Dissolve, with gentle heating, 13,1 g
potassium hydroxide (5.1) in 100 ml absolute methanol (5.2).
5.4 Heptane. Heptane may be replaced by iso-octane (2,2,4-trimethyl pentane, chromatography
grade).
5.5 Sodium sulfate, anhydrous, ≥ 99 %.
5.6 Internal standard solution. For the quantification of fatty acids, in g/100 g, the use of an internal
standard is necessary.
NOTE The use of an external calibration with reference mixtures of different fatty acids is also possible.
Prepare an internal standard solution of 5,0 mg/ml heneicosanoic acid methyl ester (C21:0 FAME) in
heptane (5.4). Use C21:0 FAME with a purity of not less than 99 %.
5.7 Reference standard
5.7.1 Fatty acid methyl esters
Use a suitable mixture of FAME and squalene covering the range of analytes expected in the sample
material.
For identification, use a mixture of 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, together with a reference chromatogram.
Use pairs of standards such as C18: 3/ C20: 1 and C23: 0/ squalene to ensure critical pair separation and
identification.
5.7.2 Squalene
5.7.2.1 For the quantification of squalene, a standard curve is necessary.
5.7.2.2 Standard stock solution: 10,0 mg/ml of squalene (>99 % purity) in heptane (5.4).
5.7.2.3 Standard solutions: Using 5.7.2.2 prepare a series of solutions containing 0,5 to 5,0 mg/ml
of squalene in heptane (5.4). Inject 1 μl of each standard solution (5.7.2.3) into the chromatograph and
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ISO 24363:2023(E)
record the resulting chromatograms. Construct a standard curve by plotting the content of squalene
(in μg/ml) against the peak area.
To check for loss of squalene during FAME preparation, a comparison of the results before and after
methylation is recommended.
5.8 Carrier gas. Inert gas (helium or hydrogen), thoroughly dried and with an oxygen content
<10 mg/kg.
5.9 Auxiliary gases:
a) Hydrogen (purity ≥ 99,9 %), free from organic impurities.
b) Air or oxygen, free from organic impurities.
c) Nitrogen (purity > 99 %).
6 Apparatus
The usual laboratory equipment and, in particular, the following shall be used.
6.1 Analytical balance, capable of weighing to the nearest 0,001 g
6.2 Graduated or automatic pipette, capacity 0,1 to 2 ml
6.3 Pipette tip, capacity 0,1 to 2 ml
6.4 Screw-top test tubes, capacity 10 ml with cap fitted with a PTFE liner.
6.5 Vortex.
6.6 GC vials and caps, capacity 2 ml.
6.7 Gas chromatograph for capillary column analysis, split injector, flame ionization detector
(FID) and suitable integration system.
1)
6.8 Capillary column, for GC. Length 100 m and 0,25 mm internal diameter coated with SP-2560
1)
or CP Sil 88 , 100 % cyanopropyl-silicone stationary phase with a film thickness of 0,25 μm. Other
columns of similar polarity and selectivity may also be used.
Condition the column by temperature programming the oven at 3 °C/min from ambient temperature to
a temperature 10 °C below the decomposition limit of the stationary phase. Maintain the oven at this
temperature for 1 h until stabilization of the baseline. Return it to 165 °C for use under the conditions
of the method.
7 Sample
7.1 Sampling
Sampling is not part of the method specified in this document. A recommended sampling method is
given in ISO 5555. It is important that the laboratory receives a sample which is representative and has
not been damaged or changed during transport or storage.
1) SP-2560 and CP Sil 88 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 ISO of these products.
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ISO 24363:2023(E)
7.2 Preparation of test sample
Prepare the test sample in accordance with ISO 661.
8 Procedure
8.1 Preparation of FAME samples
8.1.1 Weigh 0,1 g of sample into a 10 ml test tube (6.4). When testing for squalene or mass of fatty
acid methyl ester, record the mass.
For samples with free fatty acid levels > 2,0 %, prior clean-up to remove free fatty acids is recommended.
A suitable method is as follows:
— Rinse a 1 g silica gel cartridge (6 ml) in a vacuum elution apparatus with 6 ml of hexane without
vacuum.
— Load a sample of oil (approximately 0,12 g in 0,5 ml hexane) onto the column.
— Using the vacuum, load the column and then elute with a total of 10 ml of hexane/diethyl ether
(87:13 v/v).
— Combine the fractions and evaporate to dryness under reduced pressure at room temperature.
— Dissolve the residue in hexane and transfer into a tube and take to dryness under nitrogen.
— Use in 8.1.1.
8.1.2 Add 200 μl internal standard solution (5.6) into the same test tube (6.4) when testing for mass
of FAME.
The internal standard solution (5.6) is not necessary for squalene determination, but a squalene
standard curve is required.
8.1.3 Add 2 ml of heptane (5.4) to all samples and lightly shake to mix.
8.1.4 Add 0,2 ml of methanolic potassium hydroxide solution (5.3) to all samples.
8.1.5 Cap and vortex (6.5) for 30 s.
8.1.6 Leave to stratify until the upper solution becomes clear (approximately 30 min).
8.1.7 Transfer the upper layer containing the methyl esters and squalene into the GC vial (6.6).
Na SO may be added to dry the upper layer, if necessary. Sample transfer is not required if autosampler
2 4
needle is adjusted to stop above the Na SO layer
2 4
8.1.8 Analyse the samples by GC as soon as possible or keep the solution in the refrigerator for no
more than 12 h before analysis.
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ISO 24363:2023(E)
8.2 Gas chromatography
8.2.1 General
The following conditions have been proven to be suitable for the separation of FAME (C12 to C24) and
squalene:
— Injector temperature: 250 °C.
— Detector temperature: 250 °C.
— Oven temperature: The initial temperature is set at 165 °C for 30 min, then programmed to increase
at a rate of 2 °C/min to 200 °C and the final temperature is maintained for a further 12 min.
— Carrier gas hydrogen: column head pressure, 26 psi; 1,0 ml/min; constant flow.
— Split ratio: 1:100.
— Injection volume: 1 μl.
An example chromatogram is shown in Annex A.
8.2.2 System suitability
Using an injection size of 1 μl, check the performance of the column (6.8) using the reference standard
solution (5.7.1 and 5.7.2). Adjust the test portion size, test portion concentration or oven temperature
(in 1° increments) if necessary, to produce a chromatogram with optimal separation that matches
the chromatogram provided with the mixture of FAME and squalene used in the reference standard
solution. Pay particular attention to the separation and identification of critical pairs such as C18: 3/
C20: 1 and C23: 0/ squalene. Isomers of both C16:1c and C18:1c may be partially separated depending on
the column used and chromatographic conditions. However, for this analysis they should be integrated
as a single peak for C16:1c and a single peak for C18:1c.
9 Expression of results
9.1 Quantitative analysis
9.1.1 FAME, by area %
For the scope of this document, for samples in which significant amounts of components below C12:0
are absent, calculate the content of each of the individual fatty acids from C12:0 to C24:0, present in the
chromatogram, expressed as a percentage by area of methyl esters, as shown by Formula (1):
W = (A / ∑A) × 100 (1)
i i
where
W is the content individual fatty acids methyl ester;
i
A is the area under the peak of the individual FAME i;
i
∑A is the sum of the areas under all the peaks of all the individual FAME.
The results are expressed to two decimal places.
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ISO 24363:2023(E)
9.1.2 FAME, by mass (g/100 g)
If the quantification by mass of an individual fatty acid methyl ester (i) is needed, the calculation shown
by Formula (2) should be applied. For example, the calculation of linolenic acid content (in g/100 g)
using an internal standard:
L = [(A x m ) / (A × M)] × 0,1 (2)
i is is
where
L is the linolenic acid content, in g/100 g
A is the area under the peak of linolenic acid in the sample;
i
m is the mass of internal standard added, in mg;
is
A is the area under the peak of internal standard;
is
M is the mass of the sample taken for the determination, in g;
0,1 is used to convert the result to g/100 g.
Refer to Annex A for an example of a chromatogram.
Refer to Annex B for quantitative analysis using correction factors, in the presence of fatty acids methyl
esters with less than 12 carbon atoms.
9.1.3 Squalene
9.1.3.1 Calculation using a standard calibration curve
Calculate the content of squalene present in the sample, expressed as mg/kg oil, as shown by
Formula (3).
C = (C × V)/m (3)
s c
where
C is the concentration of squalene in mg/kg oil;
s
C is the content of squalene from the standard calibration curve, in μg/ml;
c
V is the final volume of the sample preparation, in ml;
m is the mass of the sample taken used in the sample preparation, in g (8.1.1).
Refer to Annex A for an example chromatogram.
To determine the ratio of fatty acid and squalene content both should be expressed on the sa
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