FprEN 18120

SLOVENSKI STANDARD
01-september-2025
Alge in izdelki iz alg - Določanje sestave maščobnih kislin
Algae and algae Products - Determination of the fatty acid composition
Algen und Algenprodukte - Bestimmung der Fettsäurezusammensetzung
Algues et produits à base d'algues - Détermination de la composition en acides gras
Ta slovenski standard je istoveten z: prEN 18210
ICS:
13.020.55 Biološki izdelki Biobased products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

DRAFT
EUROPEAN STANDARD
prEN 18210
NORME EUROPÉENNE
EUROPÄISCHE NORM
July 2025
ICS
English Version
Algae and algae Products - Determination of the fatty acid
composition
Algues et produits à base d'algues- Détermination de la Algen und Algenprodukte - Bestimmung der
composition en acides gras Fettsäurezusammensetzung
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 454.
If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.

This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2025 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 18210:2025 E
worldwide for CEN national Members.

prEN 18210:2025 (E)
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 7
5 Apparatus . 7
6 Reagents and materials . 7
7 Sampling and sample handling . 8
8 Procedure . 8
8.1 General. 8
8.2 Specific procedure description . 8
8.2.1 Indirect method . 8
8.2.2 Direct method . 11
9 Calculations . 13
10 Quality control . 15
10.1 General. 15
10.2 Interlaboratory reproducibility . 15
11 Test report . 15
Annex A (informative) Results of the interlaboratory trial for fatty acids . 16
Annex B (Informative) Results of interlaboratory study for fatty acids . 48
Bibliography . 82

prEN 18210:2023 (E)
European foreword
This document (prEN 18210:2025) has been prepared by Technical Committee CEN/TC 454 “Algae and
algae products”, the secretariat of which is held by NEN.
This document is currently submitted to the CEN Enquiry.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
prEN 18210:2025 (E)
Introduction
The European Commission (EC) has requested the European Committee for Standardization (CEN) to
draft European standards or European standardization deliverables to support the implementation of
Article 3 of Directive 2009/28/EC for algae and algae-based products or intermediates. This request,
presented as Mandate M/5471, also contributes to the Communication on “Innovating for Sustainable
Growth: A Bio Economy for Europe”.
The former working group CEN Technical Board Working Group 218 “Algae” was created in 2016 to
develop a work programme as part of this Mandate. The technical committee CEN/TC 454 'Algae and
algae products' was established to carry out the work programme that will prepare a series of standards.
The importance of algae and algae-based products or intermediates has increased significantly in Europe
as these products have been shown to be a valuable source, including but not limited to, of carbohydrates
(comprising, in particular, hydrocolloids with a large commercial importance, such as agar, alginate or
carrageenan), proteins, lipids, and several pigments. These materials are suitable for use in a wide range
of applications from food and feed purposes to other sectors, such as textiles, cosmetics, cosmeceuticals,
pharmaceuticals, biopolymers, biofuel, and fertilizer/biostimulants. Standardization of analytical
methods has been highlighted as having an important role in promoting the use of algae and algae
products.
The work of CEN/TC 454 should contribute to the reliability of the supply chain, thereby improving the
confidence of industry and consumers in algae, which include macroalgae, microalgae, cyanobacteria,
Labyrinthulomycetes, algae-based products or intermediates and should promote and support
commercialization of products of the European algae industry.
In this context, the fatty acid profile of algae and algal products is an important aspect for assessing the
potential nutritional quality and biological activity of algal biomass, thus paving the way for multiple
relevant and high added-value applications.
prEN 18210:2023 (E)
1 Scope
The document encompasses the determination of the fatty acid profile in algae and algae products,
thereby including micro- and macroalgae, according to the definitions adopted by CEN. This
determination shall enable that all fatty acids present at a significant level (> 1 % of the total fatty acids)
in the algal matrix are quantified in an accurate and reproducible way. The concentration of each fatty
acid shall be available in relative (in %) and, by means of an appropriate internal standard, absolute
(mg/g dw) terms. Moreover, the method described in the standard shall ensure a practical, quick, and
safe technical approach, whose protocol details and all related know-how shall be easily and
economically transferrable to all the sector stakeholders. The standard shall ensure this objective by a
comprehensive and fully detailed description of all technical steps from the sample itself (including its
state and form) to the gas chromatographic technique and the calculation of the fatty acid content. The
wording shall also avoid any risk of ambiguity or wrong interpretation. Finally, this methodological
standard will be informed by other equivalent standards applied to other matrices and will take into
account other standards concerning specific treatment or extractive procedure of the sample prior to the
fatty acid analysis itself.
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.
EN 17399, Algae and algae products - Terms and definitions
EN 17605:2022, Algae and algae products - Methods of sampling and analysis - Sample treatment
EN 17908, Algae and algae products — Methods of sampling and analysis — Determination of total lipids
content using the Ryckebosch-Foubert method
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 17399 and the following 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
total lipid content
all lipid substances extracted from the test portion under the operating conditions specified, expressed
in mg/g or g/kg relative to dry weight
3,2
transesterification
chemical reaction involving the conversion of fatty acid constituents regardless of their specific chemical
form (triacylglycerols, free fatty acids, phospholipids, etc.) into fatty acid methyl esters
3,3
direct transesterification approach
methodological approach consisting of the transesterification of all fatty acid constituents present in the
sample regardless of their specific chemical form (triacylglycerols, free fatty acids, phospholipids, etc.)
prEN 18210:2025 (E)
3,4
indirect transesterification approach
methodological approach consisting of the transesterification of all fatty acid constituents present in the
oil extracted from the sample by the Ryckebosch method regardless of their specific chemical form
(triacylglycerols, free fatty acids, phospholipids, etc.)
3,5
derivatization
first part of the process of preparing samples to be analysed by Gas Chromatography and comprising the
steps of sample preparation for transesterification and those corresponding to the transesterification
reaction itself
3,6
fatty acid methyl ester extraction
second part of the process of preparing samples to be analysed by Gas Chromatography and comprising
the steps of extracting the fatty acid methyl esters from the transesterification reaction mixture into an
organic phase and the purification of this phase
3,7
fatty acid methyl ester analysis
final part of the analytical method corresponding to the injection of the prepared fatty acid methyl esters
in a Gas Chromatography system for detection, identification, and quantification
3,8
internal standard
fatty acid that is chemically similar to the fatty acids in algae, but it is not present in the sample, being
added in the form of methyl ester in a constant known amount to every sample prior to the derivatization
process
3,9
fatty acid methyl ester peak area
area of a specific fatty acid methyl ester in a Gas Chromatography chromatogram
3,10
relative fatty acid methyl ester peak area
share in percentage of the area of a specific fatty acid methyl ester within the sum of the areas of all fatty
acid methyl esters in a Gas Chromatography chromatogram
3,11
total relative fatty acid content
sum of all identified and significant (> 1 % of the sum of the areas of all fatty acid methyl esters) relative
fatty acid methyl ester peak areas in a Gas Chromatography chromatogram
3,12
relative fatty acid content
share in percentage of a particular relative fatty acid methyl ester peak area in the total relative fatty acid
content in the test portion under the specified operating conditions
3,13
total absolute fatty acid content
concentration of all fatty acid constituents in the algal sample corresponding to the fatty acid methyl
esters identified by Gas Chromatography and expressed in mg of fatty acid methyl ester/g of sample or g
fatty acid methyl ester/kg of sample in dry weight terms
prEN 18210:2023 (E)
3,14
absolute fatty acid content
concentration of a specific fatty acid constituent in the algal sample corresponding to a fatty acid methyl
ester identified by Gas Chromatography and expressed in mg of fatty acid methyl ester/g of sample or g
fatty acid methyl ester/kg of sample in dry weight terms
4 Principle
The determination of the fatty acid composition (in relative and absolute terms) has two main phases: (i)
derivatization and extraction and (ii) chromatographic separation and quantification. For the first phase,
the fatty acids present within larger molecules (such as triacylglycerols and phospholipids) in the sample
are trans esterified, thereby forming fatty acid methyl esters (FAMEs). Afterwards, these FAMEs are
extracted with an adequate solvent (such as n-heptane), producing an organic solution. In the second
phase, this solution in injected and volatilized in a system of gas chromatography with a suitable detector.
The interaction of the FAMEs in the gas phase with the inner filling of a specific chromatographic column
enables separation of them. Finally, each separated FAME is detected and a detection signal proportional
to its amount is proportionately transformed into a chromatogram peak area, enabling quantification.
5 Apparatus
5,1 Analytical balance, with a readability of 0,1 mg and preferably 0,01 mg
5,2 Water bath, with temperature control and, preferably, high resistance to corrosion
5,3 Vortex, with velocity regulation
5,4 Centrifuge, with cooling and rotor for tubes
5,5 Gas chromatograph, with auto-sampler and an adequate detector presenting signal transduction
into a parameter enabling FAME quantification, such as flame ionization detector
6 Reagents and materials
6,1 Tube rack for holding 20 ml tubes or comparable glassware
6,2 Pyrex screw capped tubes, 20 ml (one unit per 1/test portion) or comparable glassware
6,3 Beaker with adequate volumetry for containing the transesterification reactant
6,4 Bowl or tray to hold ice slurry used to cool transesterification reactant
6,5 Measurement cylinder for measuring necessary amount of reactant
6,6 Micropipettes with the necessary volumetric ranges and respective tips
6,7 Pasteur pipettes, disposable, 2 ml (one unit per test portion)
6,8 Pipette tips, disposable, 5 ml (for cotton and sodium sulphate column preparation)
6,9 Glass vials for gas chromatography, preferably amber, 1,5-2,0 ml
6,10 Methanol (with a purity not less than a volume fraction of 99,0 %, v/v)
prEN 18210:2025 (E)
6,11 Catalyst, hydrochloric acid (formed in situ from acetyl chloride and methanol, mixed in a
volumetric ratio 1:19, respectively)
6,12 Water, demineralized
6,13 n-Heptane, gas chromatography grad
6,14 Hydrophilic cotton
6,15 Sodium sulphate, anhydrous
6,16 Internal standard, C21:0 (methyl ester form, 10 mg/ml concentration)
7 Sampling and sample handling
Sampling shall be performed according to the sampling standard EN 17605:2022, with the following
adaptations / additions:
— fine grinding shall be conducted as defined in 3,13 in EN 17605:2022;
— transport shall be performed on dry ice;
— storage shall be at −80 °C.
Extensive storage times of the test sample should be avoided. In case this could not be avoided, the test
sample should be freeze-dried again before usage.
Three test portions from the test sample shall be analysed as to be able to calculate the mean and standard
deviation.
8 Procedure
8.1 General
The starting point for all possible procedures described is a freeze-dried sample that has been prepared
according to in EN 17605:2022.
Determination of the total fatty acid content can be performed by two different approaches:
— The indirect (two step) method, by which the fatty acid profile can be determined with FAME
formation and extraction after having extracting the oil first
— The direct method, involving a direct transesterification of the fatty acids in the algal biomass [1]
8.2 Specific procedure description
The complete procedure, except for the weighing, should be conducted in the fume hood.
8.2.1 Indirect method
The indirect method shall be performed with hydrochloric acid (formed from acetyl chloride and
methanol) as catalyst [2].
prEN 18210:2023 (E)
8.2.1.1 Indirect method with acetyl chloride as catalyst generator (production of HCl In situ)
8.2.1.1.1 Objective and field of application
This method has as its main objective to describe the protocol used in the determination of the fatty acid
profile by acid catalysis (using hydrochloric acid formed from acetyl chloride and methanol as catalyst)
in algal matrices after oil extraction according to EN 17908.
8.2.1.1.2 Definition and process summary
The different fatty acids that compose many of the main lipid classes can be separated in saturated
(palmitic, stearic, arachidic acids, etc.), monounsaturated (palmitoleic, oleic acids, etc.) and
polyunsaturated fatty acids (linoleic, alpha-linolenic, eicosapentaenoic, docosahexaenoic acids, etc.).
Their separation and identification is possible by gaseous chromatography [3].
8.2.1.1.3 Methodological description
8.2.1.1.3.1 Reagents
All reagents must be of analytical or chromatographic grade:
a) Milli-Q water
b) Acetyl chloride
c) Methanol
d) n-Heptane p.a.
e) Anhydrous sodium sulphate
8.2.1.1.3.2 Equipment and tools
a) Glass tubes with screwcap of 15 ml
b) Graduated cylinder
c) Micropipette tips of 5 ml
d) Cotton
e) Glass rod
f) Pasteur pipette
g) 2 ml Vials
h) Vortex agitator
i) Water bath
j) Bench centrifuge SIGMA 2K12
k) Gas chromatograph (VARIAN, BRUKER or other)
prEN 18210:2025 (E)
8.2.1.1.3.3 Sample preparation
For the performance of the analysis and achieving the determination of the fatty acid profile, previous oil
extraction (by the Ryckebosch-Foubert method) of previously freeze-dried sample is needed.
8.2.1.1.3.4 Procedure for oil extraction
First apply the oil extraction method for algae and algae products (as described in EN 17908) to the
freeze-dried sample.
8.2.1.1.3.5 Procedure for esterification
Depending on the type of technique used for the FAME analysis (GC-FID vs GC-MS or cold-on-column vs
split-splitless), a different amount of internal standard and different quantities of chemicals may be
required for the analysis (Table 1). Dilution or concentration of the n-heptane phase might still be
necessary.
Table 1 — Amounts of internal standard and chemicals as a function of oil quantity available for
transesterification with hydrochloric acid as catalyst.
Amount of oil Internal Acetyl
(mg) standard (mg) chloride:methanol
(ml)
150 3,0 5
50 1,0 5
20 0,4 5
10 0,2 5
5 0,1 5
a) Weigh adequate amount of oil (extracted by the Ryckebosch-Foubert method) into 15 ml volume
glass tubes with screwcap in, at least, triplicate.
b) In the case of using internal standard (for the case of being required the calculation of absolute fatty
acid content), add an adequate internal standard (e.g. C21:0) in the form of methyl ester and in
concentration of 10 mg/ml (in an adequate solvent, such as n-heptane).
c) Add 5 ml of a mixture of acetyl chloride:methanol (1:19). This should be prepared at low temperature
(the container with the mixture should be immersed in an ice water slurry) and with dropwise
addition of the acetyl chloride to the methanol.
d) Stir the tubes in the vortex for 30 seconds (all samples must be thoroughly homogenized).
e) Put the tubes (closed with their screwcaps) in an 80 °C bath for 1 hour (no agitation needed), take
out and let cool at room temperature during 30 minutes.
f) Add 1 ml of ultra-pure water (milli-Q water) to each tube as well as 2 ml of n-heptane p.a. and stir
vigorously.
g) Let it rest for 10 minutes and centrifuge for 3 min at 3000 × g at room temperature.
h) Collect the organic phase (superior phase) for a 15 ml glass tube, taking care to filtrate through a
prepared column with cotton (1 cm thickness layer) and anhydrous sodium sulphate (1 cm thickness
layer).
prEN 18210:2023 (E)
i) In order to prepare the column: use a 5 ml micropipette tip. Put cotton with the help of a glass rod.
Then add the anhydrous sodium sulphate.
j) Wash the column thrice with 2 ml of n-heptane p.a. (total volume of 6 ml).
k) Concentrate the filtrated n-heptane solution to approximately 1 ml under a nitrogen stream at room
temperature and transfer with a Pasteur pipette to a 2 ml vial. Take care to wash the glass tube with
500 µl of n-heptane using the vortex.
l) Inject 2,0 µl of sample in the GC chromatograph or store at - 20 °C (for up to a month) until its
determination.
After injection in the chromatograph, verify the resultant chromatogram, taking attention to the retention
time of each fatty acid peak.
If the sample is too diluted (dependent on evaluation of chromatogram), it is necessary to concentrate it.
All solvent present in the vial is evaporated under nitrogen stream and 200 µl of n-heptane is added and
stirred, being afterwards transferred to a new vial with insert. Another chromatographic injection is then
performed.
8.2.2 Direct method
8.2.2.1 General
The direct method shall be performed with the direct transesterification method with acid catalysis [1].
The direct method shall be performed with hydrochloric acid (formed from acetyl chloride and methanol)
as catalyst [2].
8.2.2.2 Direct method with acetyl chloride as catalyst generator (production of HCl In situ)
8.2.2.2.1 Objective and field of application
This document has as its main objective to describe the protocol used in the determination of the fatty
acid profile by acid catalysis (using hydrochloric acid formed from acetyl chloride and methanol as
catalyst) in algal matrices directly.
8.2.2.2.2 Definition and process summary
The different fatty acids that compose many of the main lipid classes can be separated in saturated
(palmitic, stearic, arachidic acids, etc.), monounsaturated (palmitoleic, oleic acids, etc.) and
polyunsaturated fatty acids (linoleic, alpha-linolenic, eicosapentaenoic, docosahexaenoic acids, etc.).
Their separation and identification is possible by gaseous chromatography [1].
8.2.2.2.3 Methodological description
8.2.2.2.3.1 Reagents
All reagents must be of analytical or chromatographic grade.
a) Milli-Q water
b) Acetyl chloride
c) Methanol
d) n-Heptane p.a.
prEN 18210:2025 (E)
e) Anhydrous sodium sulphate
8.2.2.2.3.2 Equipment and tools
a) Usual laboratory material
b) Glass tubes with screwcap of 15 ml
c) Graduated cylinder Micropipette tips of 5 ml
d) Cotton
e) Glass rod
f) Pasteur pipette
g) 2 ml Vials
h) Vortex agitator
i) Water bath
j) Bench centrifuge SIGMA 2K12
k) Gas chromatograph (VARIAN, BRUKER or other)
8.2.2.2.3.3 Sample preparation
For the performance of the analysis and achieving the determination of the fatty acid profile, it is possible
to apply directly the method described below to the freeze-dried sample.
8.2.2.2.3.4 Procedure
Depending on the type of technique used for the FAME analysis (GC-FID vs GC-MS or cold-on-column vs
split-splitless), a different amount of starting material might be needed. A guideline concerning sample
weight and quantities of chemicals required for the analysis can be found in Table 2. Dilution or
concentration of the n-heptane phase might still be necessary.
Table 2 — Sample weight and related quantities of internal standard and chemicals as a function
of lipid content for the direct transesterification with hydrochloric acid as catalyst
Lipid content (%, Weight of sample Internal Acetyl
w/dw) (mg) standard (mg) chloride:methanol (ml)
>20 50 0,5 5
10-20 75 0,5 5
5-10 150 0,5 5
1-5 300 0,2 5
<1 500 0,1 5
a) Weigh the adequate amount of freeze-dried and ground sample into 15 ml volume glass tubes with
screwcap in, at least, triplicate.
b) In the case of using internal standard (for the case of being required the calculation of absolute fatty
acid content), add an adequate internal standard (e.g. C21:0) in the form of methyl ester and in the
prEN 18210:2023 (E)
concentration of 10 mg/ml (in an adequate solvent, such as n-heptane), taking the lipid content (%)
in the sample as a guide for choosing the amount of internal standard.
c) Add 5 ml of a mixture of acetyl chloride:methanol (1:19). This should be prepared at low temperature
(the container with the mixture should be immersed in an ice water slurry) and with dropwise
addition of the acetyl chloride to the methanol.
d) Stir the tubes in the vortex for 30 seconds (all samples must be thoroughly homogenized).
e) Put the tubes (closed with their screwcaps) in an 80 °C bath for 1 hour (no agitation needed), take
out and let cool at room temperature during 30 minutes.
f) Add 1 ml of ultra-pure water (milli-Q water) to each tube as well as 2 ml of n-heptane p.a. and stir
vigorously.
g) Let it rest for 10 minutes and centrifuge for 3 minutes at 3000 × g at room temperature.
h) Collect the organic phase (superior phase) for a 15 ml glass tube, taking care to filtrate through a
prepared column with cotton (1 cm thickness layer) and anhydrous sodium sulphate (1 cm thickness
layer).
i) In order to prepare the column- use a 5 ml micropipette tip. Put cotton with the help of a glass rod,
then add the anhydrous sodium sulphate.
j) Wash the column thrice with 2 ml of n-heptane p.a. (total volume of 6 ml).
k) Concentrate the filtrated n-heptane solution to approximately 1 ml under a nitrogen stream at room
temperature and transfer with a Pasteur pipette to a 2 ml vial. Take care to wash the glass tube with
500 µl of n-heptane using the vortex.
l) Inject 2,0 µl of sample in the GC chromatograph or store at - 20 °C (for up to a month) until its
determination.
After injection in the chromatograph, verify the resultant chromatogram, taking attention to the retention
time of each fatty acid peak.
If the sample is too diluted (dependent on evaluation of chromatogram), it is necessary to concentrate it.
All solvent present in the vial is evaporated under nitrogen stream and 200 µl of n-heptane is added and
stirred, being afterwards transferred to a new vial with insert. Another chromatographic injection is then
performed.
9 Calculations
The result calculations are different depending on whether a relative (percentual) or an absolute (mg/g
dw) fatty acid composition is aimed at.
For the percentual fatty acid composition, firstly each chromatographic peak’s area is divided by the sum
of all peaks’ areas and multiplied by 100 %. For instance, for the fatty acid j, FAj:
FA Area
( )
j

FA % = x1 00 (1)
( )
j

(∑FA Area)
i
prEN 18210:2025 (E)
where:
[FA ] is the concentration in percent of the fatty acid j, also termed as relative fatty acid
j
methyl ester peak area;
FA Area is the area of the chromatographic peak corresponding to fatty acid j;
j
Σ FA Area is the sum of the areas of the chromatographic peaks encompassing all identified fatty
i
acids.
For calculating the relative fatty acid composition, only those fatty acids present at a significant level (>
1 % of the total fatty acids) and identified are considered and summed, yielding the total relative fatty
acid content, which enables to calculate the relative fatty acid content:
FAj'%
[ ]( )
 
FA % of total id.sign. FA = x1 00 (2)
( )
j'
 
(∑ FAj' %)
[ ]( )
where:
[FA ’] is the relative fatty acid methyl ester peak area of an identified and
j
significant fatty acid;
Σ [FA ’] is the total relative fatty acid content;
j
[FA ’](% of total id.sign.FA) is the relative fatty acid content (expressed as percentage of the total
j
identified and significant fatty acids.
For the absolute fatty acid composition, calculation is based on the internal standard (fatty acid κ in the
form of methyl ester), being each chromatographic peak’s area divided by the area of the internal
standard, thus producing a proportionality factor, which, in turn, is multiplied by the amount (in mg) of
internal standard κ added to the analysed sample and divided by the sample weight (in g dw). For
instance, for the fatty acid j, FAj:
FA Area
mg mFA
j
κ

FA = x (3)
j 

g dw FA Area m
 κ sample
where:
[FA ] is the concentration in mg/g dw (per sample dry weight) of the fatty acid j in the form
j
of methyl ester
FA Area is the area of the chromatographic peak corresponding to fatty acid j
j
FA Area is the area of the chromatographic peak corresponding to the fatty acid operating as
κ
internal standard, κ
is the mass of the fatty acid methyl ester operating as internal standard, κ, added to the
mFAκ
analysed sample (in mg)
m is the mass of the analysed sample (in g dw)
sample
Moisture can be determined in the analysed sample (though freeze-dried, sample may contain residual
moisture) and the concentration of the fatty acids may be adjusted accordingly. In reporting results, it
shall be made clear whether fatty acid content is expressed per freeze-dried sample weight or in terms
of completely dry sample.
prEN 18210:2023 (E)
10 Quality control
10.1 General
All steps should be performed quickly, under convenient and very clean conditions, so that to prevent
any degradation of sensitive analytes, contamination or oxidation due to the influence of excessive
temperature, daylight, air or residues in the apparatus and glassware used, or from the samples prepared
previously or simultaneously. In particular, contamination from sample to sample should be prevented.
10.2 Interlaboratory reproducibility
A maximum relative standard deviation of 10% shall be obtained when analysing three test portions of
the same test sample.
During the statistical evaluation of the interlaboratory study (ILS) for the analysis method of the eight
fatty acids an acceptable maximal standard deviation of 25% of the consensus value was used.
Based on the results of the ILS (Annex B), the method has proven to provide acceptable results for the
micro algae species Nannochloropsis and macro algae species Codium.
Based on the results of the ILS for Nannochloropsis and Codium, and the variations found in the
concentrations in mg/g d.w. of the fatty acids analysed (from about 0,2 mg/g to about 33 mg/g d.w.) it
can be stated that method leads to reproducible results for the two very differing algae species. Therefore,
it is expected that the method will also be applicable for other fatty acids in other algae species.
Table 3 — Results of the ILS
Fatty acid Material A (Nannochloropsis)  Material B (Codium)

Direct Indirect Direct Indirect  Direct Indirect Direct Indirect

(%) (%) (mg/g (mg/g  (%) (%) (mg/g (mg/g

14:0  4,6±-0,5 4,4±0,7 4,6±0,5 2,8±0,9 5,9±0,7 5,3±1,2 0,64±0,0 0,35±0,1

16:0  19±0,30 19±1,5 19±1,2 12±3,7  44±2,1 43±6 5,0±0,28 3,2±0,68
18:0  0,30±0, 0,57±0, 0,31±0, 0,30±0, 1,5±0,5 3,0±1,4 0,16±0,0 0,2±0,1

16:1  27±1,0 26±2,3 27±2,9 16±4,4  7,4±1,1 6,1±1,9 0,86±0,0 0,42±0,1

18:1  5,2±0,1 5,6±0,3 5,2±-0,8 3,6±0,5 19±4,1 18±3,9 2,1±0,66 1,3±0,42

18:2  3,7±0,2 3,8±0,1 3,7±0,3 2,4±0,2 5,7±0,8 5,7±1,2 0,66±0,1 0,39±0,1

20:4  7,6±0,1 7,2±0,8 7,6±0,1 4,7±1,2  5,7±0,5 4,8±1,6 0,65±0,0 0,35±0,1

20:5  33±1,6 32±3,8 33±1,0 21±5,9  7,7±1,6 6,3±2,9 0,89±0,1 0,45±0,2

11 Test report
The test report shall contain the following information components as a minimum:
a) Identification of the laboratory performing the test and the date of the test;
b) Identification of the product (or sample) tested;
c) Reference to this document (prEN 18210:2025);
d) Results of the test in percentage and/or in dry weight basis expressed as mean value ± standard
deviation;
e) Any unusual features noted during the determination which might may affect the result.
prEN 18210:2025 (E)
Annex A
(informative)
Results of the interlaboratory trial for fatty acids
A.1 Introduction
In addition to the results already described in 10,2 an interlaboratory trial has been conducted to
optimize the draft standard. The main results are presented in this annex.
Statistical approach can be found in the report on the interlaboratory trial for algae and algae products
(2020-07) [4]. This implies all z-scores are calculated as follows:
xX−
z= (4)
σ
P
or as
xX−
z'= (5)
σ +u
P
where:
x result of participant
X consensus value
σp standard deviation for ILT (=25% of consensus value)
u uncertainty of the consensus value (in case u > 0,3 σp), this means the
variation in results is higher than expected.
z-scores between -2 and 2 (95% confidence level) are considered as correct. In graphs: orange lines |z-
score|≤2, red lines |z-score|≤3.
A.2 Results of the interlaboratory trial
A.2.1 General
From December 2023 to March 2024 an interlaboratory trial (ILT) for eight fatty acids in macro algae
species Codium (material A) and the micro algae species Nannochloropsis (material B) was organized by
Wageningen Food Safety Research, part of Wageningen University & Research, on behalf of CEN TC 454
‘Algae and algae products’. This ILT enabled participants to test the analytical performance according to
the draft protocol ‘Algae and algae products – Determination of the fatty acid composition’. During this
ILT laboratories were requested to report the following eight fatty acids:
— 14:0 (myristic acid)
— 16:0 (palmatic acid)
— 18:0 (octadecanoic acid)
— 16:1 (palmitoleic acid)
prEN 18210:2023 (E)
— 18:1 (oleic acid)
— 18:2 (linoleic acid)
— 20:4 (arachidonic acid)
— 20:5 (EPA/Eicosapentaenoic) acid.
All laboratories that participated were requested to analyse the algae samples according to the direct and
indirect method as described in the draft standard. From all laboratories 6 triplicate results were
expected to be reported for a specific fatty acid. Separate results had to be reported for both material A
and material B, and then the following quantities:
— % of the specific fatty acids for the direct and indirect method
— mg/g d.w. result for the specific fatty acid for the direct and indirect method
So, in principle, from every laboratory 288 results were obtained (8 fatty acids x 2 sample types (material
A and material B) x 2 methods (indirect and direct) x 2 measuring results (in % and in mg/g d.w.) x 3
triplicate results). Also the standard deviation was requested to report, and the following parameters:
— the internal standard used during the method and/or indirect method
— the amount of oil generated with the Ryckebosch-Foubert method (only for the indirect method)
— the type of injector used for the analytical part of the method.
Results in % for 14:0 (myristic acid)
A.2.2 Material A – Codium
A.2.2.1 Results in % for 14:0 (myristic acid) – direct method
Figure A.1 shows the results of the laboratories that participated. All results reported are in good
agreement with each other (i.e. within the 2z’-interval). The consensus value is 4,1 ± 0,87 %.

Figure A.1 — Graphical representation % results for 14:0 for the direct method in material A
prEN 18210:2025 (E)
A.2.2.2 Results in % for 14:0 (myristic acid) – indirect method
Figure A.2 shows the results of the laboratories that participated. All results reported are in good
agreement with each other (i.e. within the 2z’-interval). PT7853 did not report any results for the indirect
method. The consensus value is 3,1 ± 0,46 %.

Figure A.2 — Graphical representation % results for 4:0 for the indirect method in material A
A.2.2.3 Results in mg/g d.w. for 14:0 (myristic acid) – direct method
Figure A.3 shows the results of the laboratories that participated. Five laboratories reported data in good
agreement with each other (i.e. within the 2z’-interval). Two laboratories, PT7844 and PT7856 reported
data with z’-scores of 6,96 and 5,09, respectively. The consensus value is 0,72 ± 0,13 mg/g d.w.

Figure A.3 — Graphical representation mg/g results for 14:0 for the direct method in material A
A.2.2.4 Results in mg/g d.w. for 14:0 (myristic acid) – indirect method
Figure A.4 shows the results of the laboratories that participated. Five laboratories reported data in good
agreement with each other (i.e. within the 2z’-interval). PT7853 did not report any results for the indirect
method. One laboratory, PT7856, reported data with a z’-score of 8,88. The consensus value is 0,49 ±
0,097 mg/g d.w.
prEN 18210:2023 (E)
Figure A.4 — Graphical representation results for 14:0 for the indirect method in material A
A.2.2.5 Conclusion for data reported on 14:0 (myristic acid) in material A
Most of the results reported for fatty acid 14:0 are in good agreement between the laboratories that
participated to the ILT. All laboratories reported results in % for the direct and indirect method within
the 2z’-interval. The results in mg/g d.w. showed deviating z’-scores for one or two laboratories. Z’-scores
higher than 2 were obtained mainly by PT7844 and PT7856, ranging from z’-scores of 5,09 to 8,88.
A.2.3 Material B - Codium
A.2.3.1 Results in % for 14:0 (myristic acid) – direct method
Figure A.5 shows the results of the laboratories that participated. Five results reported are in good
agreement with each other (i.e. within the 2z’-interval). For two labs the results are the outside 2z’-
interval, being PT7853 with a z’-score of 3,64 and PT7858 with a z’-score of 2,80. The consensus value is
4,1 ± 0,79 %.
Figure A.5 — Graphical representation % results for 14:0 for the direct method in material B
A.2.3.2 Results in % for 14:0 (myristic acid) – indirect method
Figure A.6 shows the results of the laboratories that participated. All results reported are in good
agreement with each other (i.e. within the 2z’-interval). PT7853 did not report any results for the indirect
method. The consensus value is 4,4 ± 0,71 %.
prEN 18210:2025 (E)
Figure A.6 — Graphical representation % results for 14:0 for the indirect method in material B
A.2.3.3 Results in mg/g d.w. for 14:0 (myristic acid) – direct method
Figure A.7 shows the results of the laboratories that participated. Six laboratories reported data in good
agreement with each other (i.e. within the 2z’-interval). One laboratory, PT7856 reported data with a z’-
score of 8,77. The consensus value is 4,8 ± 1,0 mg/g d.w.

Figure A.7 — Graphical representation mg/g results for 14:0 for the direct method in material B
A.2.3.4 Results in mg/g d.w. for 14:0 (myristic acid) – indirect method
Figure A.8 shows the results of the laboratories that participated. Four laboratories reported data in good
agreement with each other (i.e. within the 2z’-interval). Two laboratories reported data outside the 2z’
interval: PT7844 reported results with a z’-score of -2,76, and PT7856 reported data with a z’-score of
9,48. PT7853 did not report any results for the indirect method. The consensus value is 3,4 ± 0,049 mg/g
d.w.
prEN 18210:2023 (E)
Figure A.8 — Graphical representation results for 14:0 for the indirect method in material B
A.2.3.5 Conclusion for data reported on 14:0 (myristic acid) in material B
This is applicable for all results in % for both the direct and indirect method. For the results in mg/g d.w.
in general for four or five laboratories the results are in good agreement. There are some deviating results
with high z’-scores (ranging from almost -3 up to almost 10). These deviations were mainly reported by
PT7844 and PT7856 for the direct method, and by PT7856 for the indirect method.
Most of the results reported for fatty acid 14:0 are in good agreement between the laboratories that
participated to the ILT. All results in % were in good agreement with each other for the indirect method.
For the direct method two laboratories obtained a z’-score outside the 2z’-interval. For the mg/g d.w.
results the pattern was different: for the direct method one laboratory with a z’-scores outside the 2z’-
interval was obtained, and for the indirect method two. Z’-scores higher than 2 were obtained mainly by
PT7844 and PT7856, ranging from z’-scores of -3,64 to 9,48.
A.2.4 General conclusion for data reported on 14:0 (myristic acid)
Most of the results reported for fatty acid 14:0 are in good agreement between the laboratories. This is
applicable for all results % and in mg/g d.w. for both the direct and indirect method. For the results in
mg/g d.w. for five laboratories the results are in good agreement. There are some deviating results with
high z’-scores, which need attention before going in the interlaboratory study. It should be noted that the
deviations are mainly caused by two laboratories, being PT7844 (2 deviating results on 8 results
reported) and PT7856 (5 deviating results on 8 results reported).
The results of the eight fatty acids (both in % and in mg/g d.w.) were considered that well, that the draft
protocol was considered ready for testing the fatty acid 14:0 during the interlaboratory
...