SIST EN 18210:2026

• Abstract:

  This 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 enables 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 will be available in relative (in %) and, by means of an appropriate internal standard, absolute (mg/g dw) terms. Moreover, the method described in this standard ensures a practical and safe technical approach, whose protocol details and all related know-how will be easily and economically transferrable to all the sector stakeholders. This document ensures 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 avoids 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.

• Status: Published
• Public Enquiry End Date: 29-Sep-2025
• Publication Date: 15-Jun-2026
• ICS: 13.020.55 - Biobased products
• Technical Committee: I13 - Imaginarni 13
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 02-Jun-2026
• Due Date: 07-Aug-2026
• Completion Date: 16-Jun-2026
• Mandate: M/547 - [C(2016)1582] Standardisation request to CEN on Algae and their products
• Ref Project: EN 18210:2026 - Algae and algae products - Determination of the fatty acid composition

SIST EN 18210:2026 is the Slovenian adoption of EN 18210:2026, a CEN European Standard for algae and algae products. It specifies a method for determining the fatty acid composition of algae, including microalgae and macroalgae, and for reporting results in relative terms and, when an internal standard is used, in absolute terms. The standard is aimed at laboratories and other sector users who need a reproducible gas chromatographic procedure for routine analysis, comparison, or documentation.

What does SIST EN 18210:2026 specify?

SIST EN 18210:2026 specifies how to determine the fatty acid profile of algae and algae products so that fatty acids present at a significant level are quantified in a reproducible way. It covers both relative results, expressed as percentages, and absolute results, expressed in mg/g dw when an appropriate internal standard is used.

The standard is organized into clauses for scope, references, terms, principle, apparatus, reagents, sampling and handling, procedure, calculations, quality control, and test reporting. It also includes two informative annexes with interlaboratory data and a bibliography.

Annex	What it covers
Annex A	Results of the interlaboratory trial for fatty acids
Annex B	Results of the interlaboratory study for fatty acids

The scope ties the method to algae and algae products, including micro- and macroalgae, and to fatty acids present above the document’s significance threshold. It also points readers toward related standards for vocabulary, sampling, sample treatment, and lipid extraction.

What are the key requirements of SIST EN 18210:2026?

Clause 4 sets out a two-phase analytical approach: first derivatization and extraction of fatty acid methyl esters, then gas chromatographic separation and quantification. In practice, this means the laboratory must convert fatty acids in the sample into a form suitable for GC, separate them, and measure peak areas for reporting.

Sample handling and preparation

Clause 7 requires sampling according to EN 17605:2022, with fine grinding for homogeneity, transport on dry ice, and storage at −80 °C. The standard also recommends avoiding long storage times and freeze-drying again if prolonged storage cannot be avoided. Three test portions are to be analysed so that a mean value and standard deviation can be calculated.

Two analytical routes

Clause 8 allows two approaches: an indirect method and a direct method. The indirect method first extracts oil with the Ryckebosch-Foubert method in EN 17908 and then transesterifies the oil; the direct method applies transesterification directly to the freeze-dried sample. This choice matters because laboratories can match the method to the sample type, available equipment, and the reporting format they need.

Reagents, equipment, and safety

Clauses 5 and 6 specify the core equipment and materials, including an analytical balance, water bath, vortex, centrifuge, gas chromatograph with an autosampler, and reagents such as methanol, acetyl chloride, n-heptane, sodium sulphate, and an internal standard. The method uses hydrochloric acid formed in situ from acetyl chloride and methanol, and the procedure is to be carried out in a fume hood except for weighing. In practice, that means the laboratory needs controlled handling conditions and suitable GC capability before it can run the method reliably.

Chromatographic analysis

Clause 8 gives GC operating guidance for both GC-FID and GC-MS, including injector mode, carrier gas, column type, and temperature program. The standard states that the prepared fatty acid methyl esters are injected, separated on a high-polarity column, and detected by a detector that converts signal into a quantitative chromatographic response. For users, this is the core of the measurement step and the point where peak identity and retention time must be checked.

Calculation of results

Clause 9 distinguishes between relative and absolute composition. Relative composition is calculated from chromatographic peak areas, while absolute composition is calculated against the internal standard and sample mass in dry-weight terms. The standard also notes that moisture can be considered and that the report must make clear whether results are expressed per freeze-dried sample weight or completely dry sample.

Quality control and reporting

Clause 10 requires clean, fast handling to avoid degradation, contamination, and oxidation, and it sets a reproducibility target: three test portions from the same sample should give a maximum relative standard deviation of 10 %. Clause 11 requires the test report to identify the laboratory, sample, standard, method used, results, and any deviations or unusual features. It also asks the laboratory to state how moisture correction and percentage normalization were handled, because those choices affect how results are interpreted.

What terms does SIST EN 18210:2026 define?

• Lipid content - all lipid substances extracted from the test portion under the specified operating conditions, expressed on a mass or percentage basis.
• Transesterification - the chemical conversion of fatty acid constituents, whatever their original form, into fatty acid methyl esters.
• Direct transesterification - a method in which all fatty acid constituents in the sample are transesterified directly.
• Indirect transesterification - a method in which fatty acids are transesterified after oil has first been extracted from the sample.
• Derivatization - the sample-preparation stage that covers preparation for transesterification and the transesterification reaction itself.
• Internal standard - a fatty acid methyl ester added in a known amount to each sample before derivatization so absolute content can be calculated.
• Absolute fatty acid content - the concentration of a specific fatty acid constituent in the sample, reported in mg/g dw or an equivalent dry-weight basis.

Who uses SIST EN 18210:2026?

SIST EN 18210:2026 is used by analytical laboratories that test algae and algae products for fatty acid composition. It is also relevant to producers, processors, and quality teams that need to compare batches, document product characteristics, or support specifications.

The introduction links the standard to the algae supply chain and to applications in food, feed, textiles, cosmetics, pharmaceuticals, biopolymers, biofuel, and fertilizer or biostimulants. That makes the standard useful wherever fatty acid profile is part of product assessment, research, or market documentation.

Which standards are used with SIST EN 18210:2026?

• EN 17399 - provides the vocabulary used for algae and algae products.
• EN 17605:2022 - gives the sampling and sample treatment framework used for sampling, fine grinding, transport, and storage.
• EN 17908 - gives the Ryckebosch-Foubert method for total lipids, which is used in the indirect route before transesterification.

What does the SIST EN 18210:2026 document contain?

The document contains a full analytical workflow from sample handling to final reporting. Clauses 4 to 9 describe the principle, apparatus, reagents, sampling, direct and indirect procedures, and the calculation of relative and absolute results.

Tables in the procedure section support method setup by linking sample or oil amount to internal standard and reagent quantities, and by giving GC-FID and GC-MS operating conditions. Annex A and Annex B summarize interlaboratory trial and study results, including the z-score approach used to evaluate laboratory performance.

The test report clause gives the minimum reporting items and the points that need clarification in practice, especially moisture correction and the basis used for percentage reporting. For users, this makes the standard not only a method description but also a reporting framework for consistent comparison of fatty acid profiles.

SLOVENSKI STANDARD
01-julij-2026
Alge in izdelki iz alg - Ugotavljanje 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: EN 18210:2026
ICS:
13.020.55 Biološki izdelki Biobased products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 18210
EUROPEAN STANDARD
NORME EUROPÉENNE
May 2026
EUROPÄISCHE NORM
ICS 13.020.55
English Version
Algae and algae products - Determination of the fatty acid
composition
Algues et produits à base d'algues - Détermination de Algen und Algenprodukte - Bestimmung der
la composition en acides gras Fettsäurezusammensetzung
This European Standard was approved by CEN on 13 April 2026.

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. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists 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.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2026 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 18210:2026 E
worldwide for CEN national Members.

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
9 Calculations . 14
10 Quality control . 16
11 Test report . 17
Annex A (informative) Results of the interlaboratory trial for fatty acids . 18
Annex B (informative) Results of the interlaboratory study for fatty acids . 25
Bibliography . 32

European foreword
This document (EN 18210:2026) has been prepared by Technical Committee CEN/TC 454 “Algae and
algae products”, the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by November 2026, and conflicting national standards shall
be withdrawn at the latest by November 2026.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document has been prepared under a standardization request addressed to CEN by the European
Commission. The Standing Committee of the EFTA States subsequently approves these requests for its
Member States.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: 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 the United
Kingdom.
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/547, 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.

1 Scope
This 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 enables 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
will be available in relative (in %) and, by means of an appropriate internal standard, absolute (mg/g dw)
terms. Moreover, the method described in this standard ensures a practical and safe technical approach,
whose protocol details and all related know-how will be easily and economically transferrable to all the
sector stakeholders. This document ensures 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 avoids 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 — Vocabulary
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
lipid content
all lipid substances extracted from the test portion under the operating conditions specified, expressed
in mg/g or g/kg or percentage 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
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.)
3.4
indirect transesterification
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
identified fatty acid methyl esters in a Gas Chromatography chromatogram
3.11
total relative fatty acid content
sum of all identified and significant (significant meaning > 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
Note 1 to entry: The significance threshold of 1 % is given as a recommendation according to the chromatographic
sensitivity. Relative fatty acid contents below 1 % can still be used but will entail a large error.
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
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 fatty acid methyl ester 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 reagent
6.4 Bowl or tray to hold ice slurry used to cool transesterification reagent
6.5 Measurement cylinder for measuring necessary amount of reagent
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)
6.11 Catalyst, hydrochloric acid in methanol (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 grade
6.14 Hydrophilic cotton
6.15 Sodium sulphate, anhydrous solvent
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 to achieve a homogeneous sample 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.
It is advisable that extensive storage times of the test sample are avoided. In case this could not be
avoided, it is advisable that the test sample is 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 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 Indirect method
8.2.1 General
The indirect method shall be performed with hydrochloric acid (formed from acetyl chloride and
methanol) as catalyst [2]. The complete procedure, except for the weighing, shall be conducted in the
fume hood.
8.2.2 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 as catalyst formed from acetyl chloride and methanol as
catalyst) in algal matrices after oil extraction according to EN 17908.
8.2.3 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].
8.2.4 Methodological description
8.2.4.1 Reagents
All reagents shall be of analytical or chromatographic grade:
a) Ultrapure water
b) Acetyl chloride
c) Methanol
d) n-Heptane p.a.
e) Anhydrous sodium sulphate
8.2.4.2 Equipment and tools
a) Glass tubes with screwcap of 15 ml
b) Graduated cylinder
c) Micropipette tips of 5 ml
d) Hydrophilic cotton
e) Glass rod
f) Pasteur pipette
g) 2 ml Vials
h) Vortex agitator
i) Water bath
j) Bench centrifuge
k) Gas chromatograph
8.2.4.3 Sample preparation
For the performance of the analysis and achieving the determination of the fatty acid profile, oil shall be
previously extracted by the Ryckebosch-Foubert method, EN 17908, from a previously freeze-dried
sample.
8.2.4.4 Procedure for oil extraction
First apply the oil extraction method for algae and algae products to the freeze-dried sample according
to EN 17908.
8.2.4.5 Procedure for esterification
Depending on the type of technique used for the fatty acid methyl ester analysis (GC-FID vs GC-MS or
cold-on-column vs split-splitless), a different amount of internal standard and different quantities of
chemicals can be required for the analysis (recommended amounts are shown in Table 1). Dilution or
concentration of the n-heptane phase might still be necessary. The specific chromatographic conditions
for performing GC-FID or GC-MS analysis are shown in Table 2.

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
Table 2 — Specific operational conditions and parameters for the determination of the fatty acid
composition through either GC-FID or GC-MS
Operational GC-FID GC-MS
conditions/parameters
Injection type Split/splitless Splitless
Injector temperature 250 °C 220 °C
Carrier gas Helium Helium
Type of column High polarity polyethilene High polarity polyethilene
glycol glycol
Column length 30 m 30 m
Column inner diameter 0,25 mm 0,32 mm
Column film thickness 0,25 μm 0,25 μm
Oven temperature program Starting at 180 °C and Starting at 58 °C for 2 min,
increasing to 200 °C at increasing to 160 °C at
4 °C/min, holding for 10 25 °C/min, to 210 °C at
min at 200 °C, heating to 2 °C/min, and to 225 °C at
210 °C at the same rate, 20 °C/min, holding at this
and holding at this temperature for 15 min
temperature for 14,5 min
Detector temperature 250 °C 230 °C
Electron impact ionization  70 eV

a) Weigh adequate amount of oil (extracted by the Ryckebosch-Foubert method, EN 17908) 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).
d) This shall be prepared at low temperature (the container with the mixture shall be immersed in
an ice water slurry) and with dropwise addition of the acetyl chloride to the methanol.
e) Stir the tubes in the vortex for 30 s (all samples shall be thoroughly homogenized).
f) Put the tubes (closed with their screwcaps) in an 80 °C bath for 1 h (no agitation needed), take
out and let cool at room temperature during 30 min.
g) Add 1 ml of ultra-pure water (ultrapure water) to each tube as well as 2 ml of n-heptane p.a. and
stir vigorously.
h) Let it rest for 10 min and centrifuge for 3 min at 3 000 × g at room temperature.
i) 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).
j) 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.
k) Wash the column thrice with 2 ml of n-heptane p.a. (total volume of 6 ml).
l) 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.
m) 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.
8.3 Direct method
8.3.1 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]. The complete procedure, except for the weighing, shall be conducted in the fume hood.
8.3.2 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 as catalyst formed from acetyl chloride and
methanol as catalyst) in algal matrices directly.
8.3.3 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].
8.3.4 Methodological description
8.3.4.1 Reagents
All reagents shall be of analytical or chromatographic grade.
a) Ultrapure water
b) Acetyl chloride
c) Methanol
d) n-Heptane p.a.
e) Anhydrous sodium sulphate
8.3.4.2 Equipment and tools
a) Usual laboratory material
b) Glass tubes with screwcap of 15 ml
c) Graduated cylinder
d) Micropipette tips of 5 ml
e) Hydrophilic cotton
f) Glass rod
g) Pasteur pipette
h) 2 ml Vials
i) Vortex agitator
j) Water bath
k) Bench centrifuge
l) Gas chromatograph
8.3.4.3 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.
8.3.4.4 Procedure
Depending on the type of technique used for the fatty acid methyl ester analysis (GC-FID vs GC-MS or
cold-on-column vs split-splitless), a different amount of starting material might be needed. A guideline
with recommendations concerning sample weight and quantities of chemicals required for the analysis
can be found in Table 3. Dilution or concentration of the n-heptane phase might still be necessary.
Table 3 — 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
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 shall be prepared at low temperature
(the container with the mixture shall 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 s (all samples shall be thoroughly homogenized).
e) Put the tubes (closed with their screwcaps) in an 80 °C bath for 1 h (no agitation needed), take out
and let cool at room temperature during 30 min.
f) Add 1 ml of ultra-pure water (ultrapure water) to each tube as well as 2 ml of n-heptane p.a. and stir
vigorously.
g) Let it rest for 10 min and centrifuge for 3 min at 3 000 × 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
...