FprEN 18196

SLOVENSKI STANDARD
01-julij-2025
Alge in izdelki iz alg - Določanje anorganskega arzena v algah in izdelkih iz alg z
anionsko izmenjavo (HPLC-ICP-MS)
Algae and algae products - Determination of inorganic arsenic in algae and algae
products by anion-exchange (HPLC-ICP-MS)
Algen und Algenprodukte - Bestimmung von anorganischem Arsen in Algen und
Algenprodukten durch Anionenaustausch (HPLC-ICP-MS)
Algues et produits d’algues - Détermination de l’arsenic inorganique dans les algues et
les produits d’algues par échange d’anions et spectrométrie de masse à plasma à
couplage inductif (CLHP/ICP-SM)
Ta slovenski standard je istoveten z: prEN 18196
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 18196
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2025
ICS
English Version
Algae and algae products -Determination of inorganic
arsenic in algae and algae products by anion-exchange
(HPLC-ICP-MS)
Algues et produits à base d'algues - Détermination de Algen und Algenprodukte - Bestimmung von
la teneur en arsenic inorganique des algues et produits anorganischem Arsen in Algen und Algenprodukten
à base d'algues par échange d'anions (HPLC-ICP-MS) durch Anionenaustausch (HPLC-ICP-MS)
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 18196:2025 E
worldwide for CEN national Members.

prEN 18196:2025 (E)
Contents Page
European foreword . 3
1 Scope . 4
2 Normative references . 4
3 Terms and definitions . 4
4 Abbreviated terms . 4
5 Principle . 4
6 Reagents . 5
6.1 General. 5
7 Apparatus and equipment . 6
7.1 General. 6
8 Procedure . 7
8.1 Sampling . 7
8.2 Sample preparation . 7
8.3 Water bath extraction . 7
8.4 Determination of inorganic arsenic by HPLC-ICP-MS . 8
8.4.1 General. 8
8.4.2 Preparation of apparatus . 8
8.4.3 Calibration . 8
8.4.4 Determination of samples and blank solution . 8
8.4.5 HPLC sequence . 8
8.4.6 Typical HPLC-ICP-MS settings . 9
8.4.7 Resolution criterium . 10
9 Calculation . 10
9.1 Integration of peaks . 10
9.2 Inorganic arsenic in test solutions . 10
9.3 Calculation of inorganic arsenic in the samples . 10
10 Precision . 11
10.1 General. 11
10.2 Repeatability . 11
10.3 Reproducibility . 11
11 Reporting . 11
Annex A (informative) Results of Interlaboratory study for inorganic arsenic . 12
Annex B (informative) Supplementary information about chromatographic conditions . 14
Bibliography . 16
prEN 18196:2025 (E)
European foreword
This document (prEN 18196:2025) 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.
prEN 18196:2025 (E)
1 Scope
This document describes a method for the determination of inorganic arsenic in algae and algae products
by anion-exchange HPLC-ICP-MS following water bath extraction. The method is specifically designed for
seaweeds containing inorganic arsenic and arsenosugar 408 (i.e. sulfate-arsinoylriboside). The peaks of
inorganic arsenic and arsenosugar 408 are separated by gradient elution.
The method was initially tested and evaluated on the algae species Ascophyllum nodosum, Fucus
vesiculosus and Saccharina latissima. Given the limited number of participating laboratories in the
interlaboratory studies, this document is only validated for Ascophyllum nodosum and Saccharina
latissima, but it can also be used for other algae species.
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
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 17399 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/
4 Abbreviated terms
As(III) Arsenite
As(V) Arsenate
AsSug_408 Arsenosugar 408
HPLC High performance liquid chromatography
ICP-MS Inductively coupled plasma mass spectrometry
5 Principle
Inorganic arsenic consists of arsenite (As(III)) and arsenate (As(V)). A representative test portion of the
sample is treated with a diluted nitric acid and hydrogen peroxide solution in a heated water bath. Hereby
the arsenic species are extracted into solution and As(III) is oxidized to As(V).
This standard was developed with respect to standards EN 16802, EN 17374 and CEN/TS 17706. It
shares the same extraction procedure with these standards. The detection procedure was modified by
changing the isocratic elution condition into a gradient elution.
The inorganic arsenic is selectively separated from other arsenic compounds using anion exchange HPLC
coupled online to the element-specific detector ICP-MS for the determination of the mass fraction of
inorganic arsenic. External calibration with solvent matrix-matched standards is used for quantification
of the amount of inorganic arsenic and the presence of arsenosugar 408 (AsSug_408).
prEN 18196:2025 (E)
6 Reagents
6.1 General
The concentration of arsenic species in the reagents and water used shall be low enough to not affect the
results of the determination. Reagents should be of minimum p.a. quality where possible. Special facilities
should be used in order to avoid contamination during the steps of preparation and measurement (e.g.
uses of laminar flow benches or comparable clean room facilities).
NOTE 1 When using a method of high sensitivity like ICP-MS, the control of the blank levels of water, acid and
other reagents is very important. Generally ultra-pure water complying with ISO 3696 grade 1 (i.e. electrical
conductivity below 0,1 μS/cm at 25 °C) and acid of high purity, e.g. cleaned by sub- boiling distillation, are used.
NOTE 2 Estimate the total volume of the prepared reagents needed for the and produce only this amount.
6.2 Nitric acid (HNO ), concentrated, ≥ 65 % (mass fraction), mass concentration of approximately ρ
(HNO3) 1,4 g/ml
Use only nitric acid available with high purity or perform a clean-up by a sub-boiling distillation in order
to avoid potential contamination.
6.3 Hydrogen peroxide, H O not less than 30 % (mass fraction)
2 2
High purity is essential to avoid potential contamination. Commercially available hydrogen peroxide for
analysis should be tested for contamination of arsenic prior to use.
6.4 Ammonium carbonate, (NH ) CO , mass fraction w ≥ 99,999 %, for production of mobile phase
4 2 3
solution
6.5 Standard solution As(V), with an arsenic mass concentration of 1 000 mg/l
The use of commercial standards of arsenic, As(III) and/or As(V), with a mass concentration of
1,000 mg/l is recommended.
NOTE By preparing the standard in the extraction solution (6.10) all arsenite will be completely oxidized to
arsenate.
6.6 Monomethylarsenous acid (MMA), optional
6.7 Dimethylarsinic acid (DMA), optional
6.8 Arsenobetaine (AB), optional
6.9 Sulfate-arsinoylriboside (AsSug_408), optional
NOTE Arsenic standards are used for checking chromatographic separation
6.10 Extraction solution, 0,1 mol/l HNO in 3 % (V/V) H O
3 2 2
Pour 800 ml of H O and then 6,5 ml of HNO (6.2) and thereafter 100 ml of H O (6.3) into a 1 000 ml
2 3 2 2
volumetric flask. Fill it up to the mark with H O. This solution should be prepared on the same day of use.
prEN 18196:2025 (E)
6.11 Ammonium carbonate mobile phase
EXAMPLE 200 mmol/l ammonium carbonate mobile phase.
For a volumetric flask of 1000 ml, dissolve 19,22 g of ammonium carbonate (6.4) in water. If necessary,
filter the mobile phase solution through a 0,45 µm filter prior to use.
6.12 Calibration solutions
Prepare a range of standards including a blank calibration solution that covers the linear range (e.g. 2-
50 µg/l) of the analyte to be determined by diluting the analyte stock solution with extraction solution
(6.10).
Appropriate matrix matching of the calibration solutions shall be performed by using the extraction
solution (6.10) for the final dilution step, which furthermore will prevent reduction of arsenate to
arsenite. Transfer an aliquot of the calibration solutions to HPLC vials prior to analysis.
The quantitative oxidation of arsenite to arsenate in the standard solutions should be verified (e.g. visual
inspection of chromatogram by looking for an additional peak or a reduced intensity of the arsenate
peak).
6.13 Solution for checking chromatographic separation, either a solution containing the different
arsenic compounds (e.g. 10 μg/l), monomethylarsenous acid (MMA), dimethylarsinic acid (DMA),
arsenobetaine (AB), Sulfate-arsinoylriboside (AsSug_408) and arsenate (As(V)), and chloride (e.g.
100 mg/l) or an extract of a reference material certified for at least inorganic As or AsSug_408 could be
used to check sufficient separation.
7 Apparatus and equipment
7.1 General
To minimize the contamination, all apparatus and equipment that come into direct contact with the
sample and the solutions shall be carefully pre-treated. It is recommended to avoid the use of glassware,
since this may cause contamination with arsenate.
WARNING — Some auto sampler systems use syringes made of glass. In this case, you can only check for
contamination and minimize it.
7.2 Laboratory grinder, capable of grinding to a particle size of 90 % less than 0,5 mm
7.3 Analytical balance, accuracy of 1 mg
7.4 Filtering device, for filtration of mobile phase, pore size 0,45 µm, optional
7.5 Water bath, capable of programming of the temperature at 90 °C
7.6 Centrifuge, for minimum 4 000 min-1 (2 010 g)
7.7 Single use syringe filters (0,45 µm) or HPLC vials with filters, compatible with acidic solutions
for filtering of test solutions prior to analysis
7.8 Plastic volumetric flasks, for preparation of mobile phase and calibration solutions
7.9 Plastic centrifugation tubes with screw
...