oSIST prEN 18206:2025

SLOVENSKI STANDARD
01-september-2025
Alge in izdelki iz alg - Metode vzorčenja in analize - Določevanje vsebnosti beta
karotena, fukoksantina in luteina v mikroalgah
Algae and algae products - Methods of sampling and analysis - Determination of beta
carotene, fucoxanthin and lutein content in microalgae
Algen und Algenprodukte - Verfahren zur Probenentnahme und Analyse - Bestimmung
des Gehalts an Fucoxanthin, Beta-Carotin und Lutein in Mikroalgen
Algues et produits d’algues - Méthodes d’échantillonnage et d’analyse - Détermination
de la teneur en fucoxanthine, bêta-carotène, et lutéine dans les microalgues
Ta slovenski standard je istoveten z: prEN 18206
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
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2025
ICS
English Version
Algae and algae products - Methods of sampling and
analysis - Determination of fucoxanthin, beta carotene and
lutein content in microalgae
Algues et produits à base d'algues - Détermination de Algen und Algenprodukte - Bestimmung des
la teneur en caroténoïdes des algues Carotinoidgehalts in Algen
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 18206:2025 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 . 5
5 Apparatus . 6
6 Reagents and materials . 6
7 Sampling and preparation of the sample . 7
8 Procedure . 8
9 Calculation of carotenoid concentration . 12
10 Test Report . 13
Annex A (informative) Carotenoid standards and standard curves for calibration . 14
Annex B (informative) Results of Interlaboratory Study for the determination of beta
carotene, fucoxanthin and lutein content in micro and macro algae . 15
B.1 Introduction . 15
B.2 Results of the Interlaboratory Study . 15
Bibliography. 21

European foreword
This document (prEN 18206:2025) 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 standardization request addressed to CEN by the European
Commission.
Introduction
This document has been prepared by the experts of CEN/TC 454 'Algae and algae products'.
The European Committee for Standardization (CEN) was requested by the European Commission (EC) 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 interest in algae and algae-based products or intermediates has increased significantly in Europe as
a valuable source including but not limited to, carbohydrates, 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 textile, cosmetics, biopolymers, biofuel and fertilizer/biostimulants. Standardization was
identified as having an important role in order to promote the use of algae and algae products.
The work of CEN/TC 454 should improve 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 will promote and support
commercialisation of the European algae industry.
Given the importance of carotenoids in algae and algae products, it is important to have a good
standardized method to determine the amount of the carotenoids. Since there are a lot of carotenoids,
only the determination of beta carotene, fucoxanthin and lutein is incorporated in this standard.

Text in grey is fixed (based on CEN/TC 454 Decision 5-2019 taken by CEN/TC 454 on 2019-01-29 /
Subject: CEN/TC 454 – Approval of the General Introduction Text).
Available at http://ec.europa.eu/growth/tools-databases/mandates/index.cfm?fuseaction=refSearch.search#
1 Scope
This document specifies a laboratory method for the determination of the carotenoid content in
microalgae. The method is based on the development of the chlorophyll a standard, which resulted in
EN 18034. This method has been validated for the microalgae species Nannochloropsis and
Phaodactlylum. This standard is only validated for beta carotene, fucoxanthin and lutein content, but
could be used for other carotenoids as well. Given small adaptations to be able to measure smaller
concentrations, this method could also be used for macro algae.
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
3 Terms and definitions
For the purpose of this document, the terms and definitions given in EN 17399 and the following apply.
3.1
carotenoid
accessory pigment of the class of tetraterpenoids including carotenes and xanthophylls
3.2
carotene
unoxygenated (oxygen free) carotenoid
EXAMPLE α-carotene, β-carotene, and lycopene
3.3
carotenoid standard
solution with defined concentration of the carotenoid of interest that is used to determine the analyte of
interest with the help of standard curves
Note 1 to entry: This is explained in further detail in Annex A.
3.4
pigment
any colouring matter in the cells and tissues of plants
3.5
xanthophyll
carotenoid molecule containing oxygen
EXAMPLE lutein, zeaxanthin.
4 Principle
The carotenoids are extracted from the samples with methanol and separated using HPLC. The separated
components are analysed at 440 nm using a photodiode array detector. This method specifically aims at
determining beta carotene, fucoxanthin and lutein content.
5 Apparatus
Usual laboratory apparatus and, in particular, the following:
5.1 Analytical balance, with an accuracy of preferably 0,01 mg.
5.2 High Performance Liquid Chromatograph (HPLC) with a Photodiode Array detector (PDA).
5.3 Centrifuge.
5.4 Vortex.
5.5 Spectrophotometer.
5.6 Fume hood.
6 Reagents and materials
6.1 HPLC reagents
All reagents should be HPLC grade. For the triple gradient (see 8.5.2):
6.1.1 Mixture of methanol with a volume fraction of 80 % and 0,5M ammonium acetate buffer in
purified water, e.g. Milli-Q, with a volume fraction of 20 %.
6.1.2 Mixture of acetonitrile with a volume fraction of 90 %, and purified water, e.g. Milli-Q with a
volume fraction of 10 %.
6.1.3 Ethyl acetate
For the double gradient (see 8.5.3):
6.1.4 Mixture of methanol with a volume fraction of 70 %, and 0,028 M TBAA
(tetrabutylammonium acetate) in purified water, e.g. Milli-Q.
6.1.5 Methanol
6.1.6 Glass beads, ∅ 0,75 mm to 1 mm
6.2 HPLC column
For the triple gradient (see 8.5.2:):
6.2.1 C18 stationary phase, e.g. Nova-pak 4 μm (3,9 × 150 mm)
For the double gradient (see 8.5.3):
6.2.2 C8 stationary phase e.g. Zorbax Eclipse XDB-C8 3,5 μm (4,6 × 150 mm)
6.3 Pyrex screw capped tubes, 10 mL (1 / test portion) and 20 mL (1 / test portion)
6.4 Micropipettes
6.5 Pasteur pipettes
6.6 HPLC vials and caps
6.7 Syringe with volume of 1 mL and filters (0.20 μm, ∅ 15 mm)
6.8 Methanol with a volume fraction of ⩾ 99.0 %
6.9 Quartz cuvettes
6.10 Carotenoid standards: for the identification and quantification of carotenoids (in particular: beta
carotene, fucoxanthin and lutein content), that shall comply to the following quality parameters:
— a concentration range from 0,5 to 1,5 μg/ml;
— dissolved in solvent;
— transported on dry ice by the supplier;
— stored after arrival at least at −20 °C and preferably at −80 °C.
Recommendations for carotenoid standards are given in Annex A.
6.11 Aluminium foil
7 Sampling and preparation of the sample
7.1 Sampling
The sample received by the laboratory shall not be damaged or changed during transport or storage
Sampling is not part of the method specified in this document.
7.2 Sample treatment
Sample treatment is not parts of the method specified in this document. A recommended sample handling
procedure is given in 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.
The samples shall be protected from light at any time before and during the analysis, by using e.g.
aluminium foil.
Samples shall not be stored for over one month, because storage can degrade carotenoids, even when the
samples are frozen.
8 Procedure
8.1 Extraction
The whole extraction procedure, except for the weighing, shall be performed under a fume hood.
The extraction procedure shall be conducted through the following steps:
1) Weigh a test portion of approximately 25 mg of the test sample in a 10 mL pyrex screw capped tube
(i.e. weight test portion);
2) Record the weight to the nearest 0,01 mg;
3) Add approximately 850 mg glass beads;
4) Add 5 mL methanol;
5) V
...