FprEN 18197

SLOVENSKI STANDARD
01-julij-2025
Alge in izdelki iz alg - Določanje aminokislinskega profila mikro- in makroalg
Algae and algae products - Determination of the amino acid profile of micro- and
macroalgae
Algen und Algenprodukte - Bestimmung des Aminosäuren Profils von Mikro- und
Makroalgen
Algues et produits à base d'algues - Détermination du profil en acides aminés des micro
et macroalgues
Ta slovenski standard je istoveten z: prEN 18197
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 18197
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2025
ICS
English Version
Algae and algae products - Determination of the amino
acid profile of micro- and macroalgae
Algues et produits à base d'algues - Détermination du Algen und Algenprodukte - Bestimmung des
profil en acides aminés des micro et macroalgues Aminosäuren Profils von Mikro- und Makroalgen
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 18197:2025 E
worldwide for CEN national Members.

prEN 18197:2025 (E)
Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Principle . 6
5 Reagents . 7
6 Reagents and standard preparation . 8
6.1 Reagents common to UV and MS detection . 8
6.2 Reagents specific for UV detection . 10
6.3 Reagents specific for MS detection . 11
7 Apparatus . 12
8 Procedure. 13
8.1 Procedure with UV detection . 13
8.1.1 General . 13
8.1.2 Sample preparation . 13
8.1.3 Cystine Calibration standards preparation . 13
8.1.4 Hydrolysis (of samples and cystine standards) . 14
8.1.5 Neutralization and dilution (of samples and converted cystine standards) . 14
8.1.6 Amino acids calibration standards preparation (not needed to be acid hydrolysed) . 14
8.1.7 Derivatization (of samples, cystine standards and amino acids standards) . 14
8.1.8 UHPLC separation and UV detection . 15
8.1.9 Peak identification and integration . 16
8.2 Procedure with MS detection . 16
8.2.1 General . 16
8.2.2 Sample preparation . 16
8.2.3 Cystine Calibration standards preparation . 17
8.2.4 Hydrolysis (of samples and cystine standards) . 17
8.2.5 Neutralization and dilution (of samples and converted cystine standards) . 17
8.2.6 Amino acids calibration standards preparation (not needed to be acid hydrolysed) . 18
8.2.7 Derivatization (of samples, cystine standards and amino acids standards) . 18
8.2.8 UHPLC separation and MS detection . 18
8.2.9 Peak identification and integration . 21
9 Calculation and expression of results . 21
9.1 Calibration curve . 21
9.2 Amino acid calculation . 22
10 Precision . 23
10.1 General . 23
10.2 Repeatability . 23
10.3 Reproducibility . 23
11 Test report . 25
prEN 18197:2025 (E)
Annex A (informative) Precision data . 26
Annex B (informative) Chromatogram examples . 33
Annex C (informative) Equivalence between UV and MS methods . 35
Annex D (informative) Calculation of nitrogen-to-protein conversion factor. 38
Bibliography . 40

prEN 18197:2025 (E)
European foreword
This document (prEN 18197:2025) 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 18197:2025 (E)
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/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 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.
Determining amino acids is important for the industry to measure specific amino acids and establish the
whole amino acid profile of algae and algae products. Moreover, total amino acids can be used to calculate
the protein content of a sample with accuracy.
The goal of this document is to give algae producers and algae products industries a recommendation for
an analysis method for amino acids which can be utilized for algae. The document relies strongly on
standards ISO 4214 | IDF 254 Milk and milk products - Determination of amino acids in infant formula and
other dairy products [1], AOAC 2018.06, Total Amino Acids in Infant Formulas and Adult Nutritionals [2],
and AACC 07-50.01, Total Amino Acids by UHPLC-UV [3], and adds the possibility to use mass
spectrometry for the determination of amino acids.
prEN 18197:2025 (E)
1 Scope
This document describes a method for determining the amino acid profile of micro- and macroalgae.
It specifies a method for the quantitative determination, in one single analysis, of the following amino
acids: alanine, arginine, aspartic acid (combined with asparagine), cystine (dimer of cysteine, combined
with cysteine), glutamic acid (combined with glutamine), glycine, histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, proline, serine, threonine, tyrosine and valine.
This method does not apply to the determination of tryptophan.
2 Normative references
The following document is 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 17399:2024, 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 purposes of this document, the terms and definitions in EN 17399 apply.
ISO and IEC maintain terminological 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 Principle
Proteins are hydrolysed in 6 mol/l hydrochloric acid (HCl) for 24 h at 110 °C in the presence of phenol
and 3-3′-dithiodipropionic acid (DDP). Phenol is added to prevent halogenation of tyrosine. DDP is added
to convert cystine and cysteine to S-2-carboxyethylthiocysteine (XCys) as described in Reference [4].
After neutralization, amino acids and XCys are derivatized with 6-aminoquinolyl-N-hydroxy-
succinimidyl carbamate (AQC). Derivatized amino acids are separated using reversed phase UHPLC and
detected by UV at 260 nm or by mass spectrometry. For UV detection, norvaline is added as an internal
standard, while heavy labelled amino acids are used for MS detection.
NOTE Fluorescence detection can be used provided equivalence has been demonstrated.
During acid hydrolysis, glutamine (Gln) and asparagine (Asn) are converted to glutamic acid (Glu) and
aspartic acid (Asp), respectively. Thus, Glu values represent the combined values of Glu and Gln, and Asp
values represent the combined values of Asp and Asn. Cys2 values represent the combined values of
cysteine and cystine since both are converted to XCys by DDP.
prEN 18197:2025 (E)
5 Reagents
Use only reagents of recognized analytical grade.
Commercial references are only a guideline. Equivalent chemicals or materials can be used provided their
equivalence has been demonstrated.
Before using chemicals, refer to the safety data sheets and ensure that the safety precautions are applied.
Phenol is toxic if swallowed, in contact with skin or if inhaled. It can cause severe skin burns and eye
damage, is suspected of causing genetic defects, may cause damage to organs through prolonged or
repeated exposure, and is toxic to aquatic life with long lasting effects.
5.1 AccQ·Tag™ Ultra Derivatization Kit (Waters 186003836 ). ®
As an alternative derivatizing buffer, di-sodium tetraborate decahydrate (CAS Registry Number 1303-
96-4) can be used.
As an alternative tagging reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (CAS 148757-94-
2) can be used.
5.2 AccQ·Tag™ Ultra Eluent A concentrate (Waters 1860038381).
As alternative reagents, acetonitrile (CAS 75-05-8), ammonium formate (CAS 540-69-2) and formic acid
(CAS 64-18-6) can be used.
NOTE This reagent is only required for UV detection.
5.3 AccQ·Tag™ Ultra Eluent B (Waters 1860038391).
As alternative reagents, acetonitrile (CAS 75-05-8) and formic acid (CAS 64-18-6) can be used.
NOTE This reagent is only required for UV detection.
5.4 Phenol (CAS 108-95-2).
5.5 3,3′-Dithiodipropionic acid (CAS 1119-62-6).
5.6 Amino acid standard solution
An amino acid standard solution that contains the following 17 amino acids at 2,5 mmol/l each (except
L-cystine at 1,25 mmol/l) in 0,1 mol/l HCl: L-alanine, L-arginine, L-aspartic acid, L-cystine, L-glutamic acid,
L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,
L-threonine, L-tyrosine and L-valine.
5.7 Heavy-labelled amino acid standard solution
A heavy-labelled amino acid standard solution that contains the following 17 heavy-labelled amino acids
13 15 13
at 2,5 mmol/l each (except L-cystine at 1,25 mmol/l) in 0,1 mol/l HCl: l-alanine ( C , N), L-arginine ( C ,
3 6
15 13 15 13 15 13 15 13 15
N ), L-aspartic acid ( C , N), L-cystine ( C , N ), L-glutamic acid ( C , N), L-glycine ( C , N), L-
4 4 6 2 5 2
This is an example of a suitable product available commercially. This information is given for the convenience of
users of this document and does not constitute an endorsement by CEN of the product named. The alternative
reagents listed in this document have been shown to lead to the same results. ®
CAS Registry Number is a trademark of CAS corporation. This information is given for the convenience of users
of this document and does not constitute an endorsement by CEN of the product named. Equivalent products may
be used if they can be shown to lead to the same results.
prEN 18197:2025 (E)
13 15 13 15 13 15 13 15 13
histidine ( C , N ), L-isoleucine ( C , N), L-leucine ( C , N), L-lysine ( C , N ), L-methionine ( C ,
6 3 6 6 6 2 5
15 13 15 13 15 13 15 13 15
N), L-phenylalanine ( C , N), L-proline ( C , N), L-serine ( C , N), L-threonine ( C , N), L-tyrosine
9 5 3 4
13 15 13 15 1)
( C , N) and L-valine ( C , N). This standard solution is provided by Euristop (MSK-A2-1.2) . If
9 5
another supplier is selected, verify that the labelling is similar to that described in this section. If this is
not the case, the masses of the internal standards shall be adapted accordingly in Table 8.
NOTE This reagent is only required for MS detection.
5.8 l-cystine (CAS 56-89-3).
5.9 Heavy-labelled l-cystine (13C6, 15N2) (CAS 1252803-65-8).
NOTE This reagent is only required for MS detection.
5.10 Norvaline (CAS 6600-40-4).
NOTE This reagent is only required for UV detection.
5.11 Sodium hydroxide pellets, reagent grade (CAS 1310-73-2).
5.12 Sodium hydroxide solution (CAS 1310-73-2), substance concentration c = 1 mol/l.
5.13 Sodium hydroxide solution (optional) (CAS 1310-73-2), c = 6 mol/l.
5.14 Hydrochloric acid fuming 37 % (CAS 7647-01-0), c = 12 mol/l, GR grade for analysis.
5.15 Hydrochloric acid solution (CAS 7647-01-0), c = 1 mol/l.
5.16 Hydrochloric acid solution (CAS 7647-01-0), c = 0,1 mol/l.
5.17 Laboratory water, with a resistivity of 18,2 MΩ-cm (ultra-pure water).
NOTE Use gradient grade for liquid chromatography (LC) for UV detection or LC-MS grade for MS detection.
5.18 Acetonitrile (CAS 75-05-8).
NOTE Use gradient grade for liquid chromatography (LC) for UV detection or LC-MS grade for MS detection.
5.19 Formic acid (CAS 64-18-6).
5.20 Ammonium formate (CAS 540-69-2).
NOTE This reagent is only required for UV detection.
6 Reagents and standard preparation
6.1 Reagents common to UV and MS detection
6.1.1 Sodium hydroxide (NaOH) solutions, c = 6 mol/l, c = 0,2 mol/l and c = 0,05 mol/l.
For the c = 6 mol/l solution, weigh out 24 g of sodium hydroxide (5.11) into a 100 ml volumetric flask.
Dissolve in about 80 ml of water. Allow to cool down and dilute to the mark with water. Optional: use a
commercially available equivalent (5.13).
For the c = 0,2 mol/l solution, pipet 20 ml of 1 mol/l NaOH (5.12) into a 100 ml volumetric flask and make
up to the mark with water.
prEN 18197:2025 (E)
For the c = 0,05 mol/l solution, pipet 5 ml of 1 mol/l NaOH (5.12) into a 100 ml volumetric flask and make
up to the mark with water.
6.1.2 Hydrochloric acid (HCl) solution, c = 0,2 mol/l.
Pipet 20 ml of 1 mol/l HCl (5.12) into a 100 ml volumetric flask and make up to the mark with water.
6.1.3 DDP solution, mass concentration ρ = 20 g/l (in NaOH, c = 0,2 mol/l).
Into a 50 ml volumetric flask, weigh out 1000 mg of DDP and make up to the mark with 0,2 mol/l NaOH
(6.1.1).
6.1.4 Phenol/HCl solution, ρ = 1 g/l (in HCl, c = 12 mol/l).
Into a 100 ml volumetric flask, weigh out 100 mg of phenol and make up to the mark with 12 mol/l HCl
(5.14)
NOTE In some matrices, the addition of phenol was shown to have negligible impact on amino acids, including
tyrosine. Phenol can be omitted from this solution provided equivalence between the two methods (with or without
phenol) is demonstrated in the matrix of interest.
1)
6.1.5 AccQ·Tag™ Ultra Derivatization kit.
Prepare the reagents included in the kit following the manufacturer’s instructions.
1)
6.1.5.1 AccQ·Tag™ Ultra Borate buffer (reagent 1)
Ready-to-use solution. An alternative reagent is sodium tetraborate in water solution (ρ = 50 g/l). Into a
100 ml volumetric flask, weigh 5 g of sodium tetraborate decahydrate, dissolve and make up to the mark
with water.
1)
6.1.5.2 AccQ·Tag™ Ultra reagent (vial 2A and 2B).
Reconstitute AccQ·Tag™ Ultra reagent (vial 2A) according to the manufacturer’s instructions as follows:
a) Preheat a heating block to 55 °C.
TM
b) Tap vial 2A lightly before opening to ensure all AccQ·Tag Ultra reagent powder is at the bottom of
the vial.
TM
c) Rinse a clean micropipette by drawing and discarding 1 ml of AccQ·Tag Ultra reagent diluent from
vial 2B (ready-to-use solution). Repeat twice.
TM
d) Draw 1,0 ml from vial 2B and transfer it to the AccQ·Tag Ultra reagent powder in vial 2A. Cap the
vial tightly.
e) Vortex mix for approximately 10 s.
TM
f) Heat vial 2A on top of the preheated heating block until the AccQ·Tag Ultra reagent powder is
dissolved. Do not heat the reagent for longer than 10 min.
TM
Once reconstituted, the AccQ·Tag Ultra reagent concentration is approximately 10 mmol/l. Store the
TM
reconstituted AccQ·Tag Ultra reagent in a desiccator at room temperature for up to one week.
TM
CAUTION — AccQ·Tag Ultra reagent reacts with atmospheric moisture. Seal the container tightly when
not in use. Do not refrigerate. Do not use discoloured reagent, especially if it is yellow or green.
prEN 18197:2025 (E)
The following alternative reagent can be used. Into a 4 ml vial, weigh out approximately 3,0 mg to 4,0 mg
of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Continue with step c) above using LC-grade
acetonitrile instead of the AccQ·Tag™ Ultra reagent diluent.
6.1.6 Cystine calibration standards
NOTE Check purity of cystine by MS (full scan) or UV prior to using it as a standard.
6.1.6.1 Cystine stock solution, c = 10 mmol/l.
Weigh out 240 mg cystine into a 100 ml volumetric flask and make up to the mark with 0,05 mol/l NaOH
(6.1.1). Store this solution at −20 °C for up to one month as 1 ml aliquots.
6.1.6.2 Cystine solution, c = 1 mmol/l.
Add 900 µl of 0,05 mol/l NaOH (6.1.1) to 100 µl of cystine stock solution (6.1.6.1). Prepare this solution
freshly for each analysis.
6.1.7 Amino acid (AA) calibration standards (with exception of cystine)
6.1.7.1 AA stock solution, c = 2,5 mmol/l.
Amino acid standard solution is ready-to-use and contains 2,5 mmol/l of each amino acid (although
present in this solution, cystine is not used for quantification and is prepared separately, see 6.1.6).
Store this calibration standard stock solution at −20 °C for up to six months as 60 µl aliquots.
6.1.7.2 AA solution 1, c = 0,5 mmol/l.
Add 200 μl of 0,1 mol/l HCl (5.16) to 50 μl of AA stock solution (6.1.7.1). Prepare this solution freshly for
each analysis.
6.1.7.3 AA solution 2, c = 0,05 mmol/l.
Add 180 μl of 0,1 mol/l HCl (5.16) to 20 μl of AA solution 1 (6.1.7.1). Prepare this solution freshly for each
analysis.
6.1.8 Wash solvents.
Use gradient grade for liquid chromatography (LC) reagents.
a) The weak needle wash solvent is 50 ml/l acetonitrile in water.
b) The strong needle wash solvent is 950 ml/l acetonitrile in water.
c) The seal wash solvent is 500 ml/l acetonitrile in water.
6.2 Reagents specific for UV detection
6.2.1 Norvaline (Nva) internal standards
Norvaline internal standards are only used for UV detection.
6.2.1.1 Nva stock solution, c = 10 mmol/l.
Weigh out 117,16 mg Nva into a 100 ml volumetric flask and make up to the mark with 0,1 mol/l HCl
(5.16).
prEN 18197:2025 (E)
6.2.1.2 Nva solution, c = 2,5 mmol/l.
Pipet 2,5 ml of Nva stock solution (6.2.1.1) into a 10 ml volumetric flask and make up to the mark with
0,1 mol/l HCl (5.16).
Store both Nva solutions at −20 °C for up to six months as 2 ml aliquots.
6.2.2 Eluent A (Solvent A), for UV detection.
Use gradient grade for liquid chromatography (LC) reagents.
Prepare Eluent A from AccQ·Tag™ Ultra Eluent A concentrate as follows:
a) Measure 850 ml of water into a 1 l graduated cylinder.
b) In a separate graduated cylinder, measure 150 ml of AccQ·Tag™ Ultra Eluent A concentrate.
c) Add the concentrate to the water and mix thoroughly.
Eluent A concentrate, once opened, shall be stored tightly capped at around 4 °C. Dilute Eluent A is stable
for one week at room temperature.
As an alternative for Eluent A concentrate, the following solution can be used. Mix 840 ml of 200 mmol/l
ammonium formate solution (12,61 g ammonium formate in 1 l of water), 110 ml of acetonitrile and
50 ml of formic acid. Prepare Eluent A from the concentrate as described above.
6.2.3 Eluent B (Solvent B), for UV detection.
AccQ·Tag™ Eluent B is supplied as a working solution. No additional preparation is required.
As an alternative for Eluent B, the following solution can be used. Add 13,2 ml formic acid to 1 l
acetonitrile (gradient grade for liquid chromatography).
6.3 Reagents specific for MS detection
6.3.1 Heavy-labelled cystine internal standard (IS-Cys2), c = 0,5 mmol/l.
Heavy-labelled cystine internal standard is only required for MS detection.
Weigh out 12,2 mg of labelled cystine (5.9) into a 100 ml volumetric flask and make up to the mark with
0.05 mol/l NaOH (6.1.1). Store this solution at −20 °C for up to three months as 4,5 ml aliquots.
6.3.2 Heavy-labelled amino acid internal standards (IS-AA)
Heavy-labelled amino acid internal standards are only required for MS detection. Although present in this
solution, heavy-labelled cystine is not used for quantification and is prepared separately (6.3.1).
6.3.2.1 IS-AA stock solution, c = 2,5 mmol/l.
Heavy-labelled amino acid internal standard solution is ready-to-use and contains 2,5 mmol/l of each
heavy-labelled amino acid.
6.3.2.2 IS-AA solution, c = 0,1 mmol/l.
Add 1 ml of 2,5 mmol/l IS-AA stock solution (6.3.2.1) into a 25 ml volumetric flask and make up to the
mark with 0,1 mol/l HCl (5.16).
Store 4,5 ml of this internal standard solution at -20 °C for up to 6 months as 375 µl aliquots.
prEN 18197:2025 (E)
6.3.2.3 IS-AA solution, c= 0,02 mmol/l.
Add 20 ml of 0,1 mmol/l IS-AA solution (6.3.2.2) into a 100 ml volumetric flask and make up to the mark
with 0.1 mol/l HCl (5.16).
Store this internal standard solution at -20 °C for up to 3 months as 4,5-ml aliquots.
6.3.3 Mobile phase A, for MS detection.
Add 1 ml formic acid to 1 l MS grade water.
6.3.4 Mobile phase B, for MS detection.
Add 1 ml formic acid to 1 l MS grade acetonitrile.
7 Apparatus
7.1 UHPLC system coupled with a UV or MS detector .
7.2 Chromatography columns
For UV detection
1)
ACQUITY UPLC™ BEH C18 Column, 130 Å, 1,7 μm, 2,1 mm × 150 mm (Waters 186002353 ) or
equivalent, provided baseline separation of the amino acids is achieved.
For MS detection
A shorter column such as ACQUITY UPLC™ BEH C18 Column, 130Å, 1.7 µm, 2.1 mm X 100 mm (Waters
1) 1)
186002352 ) or AccQ.Tag Ultra C18, 1.7 µm, 2.1 mm X 100 mm (Waters 186003837 ) can be used.
7.3 Adjustable micropipettes, of volume 10 µl, 20 µl, 200 µl and 1 000 µl and tips.
7.4 Vortex mixer.
7.5 Analytical balance, with a precision of 0,1 mg.
7.6 Heating block, able to maintain a temperature of 55 °C ± 2 °C.
7.7 Laboratory oven, able to maintain a temperature of 110 °C ± 2 °C.
7.8 Syringe filter, 0,45 µm Polyvinylidene fluoride (PVDF) Millex®-HV (e.g. Millipore SLHV013NL1)
or equivalent).
7.9 Syringes, of 2 ml volume.
7.10 Borosilicate glass tubes (e.g. Pyrex), of 10 ml volume with screw cap.
7.11 Microtubes, of volume 1,5 ml, 2 ml and 5 ml.
7.12 Vial with screw cap, of volume 2 ml.
7.12.1 Glass screw neck total recovery vial, 12 mm × 32 mm (Waters 186000384C1) or equivalent).

Single- or triple-quadrupole detectors have been successfully used for MS detection.
prEN 18197:2025 (E)
8 Procedure
8.1 Procedure with UV detection
8.1.1 General
Samples (clause 8.1.2) and cystine calibration standards (clause 8.1.3) are prepared on the first day and
hydrolyzed at 110 °C overnight (clause 8.1.4). The amino acid calibration standards (clause 8.1.6) are
prepared on the second day. All solutions are then derivatized (8.1.7) and analyzed by UHPLC-UV (8.1.8).
This section follows this order of operations.
8.1.2 Sample preparation
The test sample shall be dry i.e. with a moisture content < 15 %, and preferably below 10 %,
homogeneous, and finely ground according to clauses 3.7, 3.9, and 3.13 of EN 17605:2022.
Weigh out 30-40 mg ± 5 mg algae powder samples into a 10 ml glass tube with screw cap. Report the
sample mass to 0,1 mg.
To each tube, add water, DDP, HCl, internal standard and phenol/HCl according to Table 1.
Add the phenol/HCl solution under the hood. Bubble nitrogen through the sample solution in the glass
tube for about 30 s. Close tubes with screw caps and vortex. Make sure the caps are perfectly clean (i.e.
devoid of any particle) to ensure tightness and avoid evaporation during hydrolysis.
Table 1 — Preparation of the sample tubes
Solution Amount
Sample, mg 30 ± 5
Water, μl 770
DDP solution (6.1.3), μl 600
0,2 mol/l HCl (6.1.2), μl 600
Nva stock solution (6.2.1.1), μl 500
Phenol/HCl solution (6.1.4), μl 2 500
8.1.3 Cystine Calibration standards preparation
Table 2 describes how to prepare calibration standards for converted cystine at 0 pmol/µl to 10 pmol/µl
with Nva at 10 pmol/µl (all are final concentrations after derivatization).
Add the phenol/HCl solution under the hood. Bubble nitrogen through the solution in the glass tube for
about 30 s. Close tubes with screw caps and vortex. Make sure the caps are perfectly clean (i.e. devoid of
any particle) to ensure tightness and avoid evaporation during hydrolysis.
Table 2 — Final cystine concentration after derivatization
Solution 5 pmol/ 2,5 pmol/ 1 pmol/ 0,5 pmol/ 0,1 pmol/ 0 pmol/
µl
µl µl µl µl µl
a a b b b
Cystine solution, μl 250 125 500 250 50 0
Water, μl 550 675 300 550 750 800
DDP solution (6.1.3), μl 600 600 600 600 600 600
0,2 mol/l HCl (6.1.2), μl 600 600 600 600 600 600
prEN 18197:2025 (E)
Solution 5 pmol/ 2,5 pmol/ 1 pmol/ 0,5 pmol/ 0,1 pmol/ 0 pmol/
µl
µl µl µl µl µl
Nva stock solution (6.2.1.1), μl 500 500 500 500 500 500
Phenol/HCl solution (6.1.4), μl 2 500 2 500 2 500 2 500 2500 2 500
a
10 mmol/l cystine stock solution (6.1.6.1).
b
1 mmol/l cystine solution (6.1.6.2).
8.1.4 Hydrolysis (of samples and cystine standards)
Place tubes in an oven at 110 °C ± 2 °C for 24 h ± 0,5 h.
8.1.5 Neutralization and dilution (of samples and converted cystine standards)
Take the tubes out of the oven. Allow hydrolysates to cool down and particles to settle down prior to
taking an aliquot. When transferring aliquots, pipet about 1 cm below the top of the liquid. Perform
neutralization under the hood.
Transfer 0,1 ml of the hydrolysate sample solution into a 1,5 ml microtube, add 0,1 ml of 6 mol/l NaOH
(6.1.1) and then 0,8 ml of 0,1 mol/l HCl (5.16). Mix well and filter through 0,45 µm membrane filter into
another 1,5 ml microtube.
8.1.6 Amino acids calibration standards preparation (not needed to be acid hydrolysed)
Table 3 shows how to prepare 0,5 ml calibration standards at 0 pmol/µl to 25 pmol/µl with norvaline at
10 pmol/µl (all are final concentration after derivatization).
The amino acid solutions are stable for one week when stored at 4 °C ± 2 °C.
Table 3 — Final amino acid concentrations after derivatization (UV detection)
Solution 25 pmol/ 10 pmol/ 5 pmol/ 1 pmol/ 0,5 pmol/ 0,1 pmol/ 0 pmol/
µl µl µl µl µl µl µl
a b b c c c
Amino acid solution, μl 50 100 50 100 50 10 0
Nva solution (6.2.1.2), μl 20 20 20 20 20 20 20
0,1 mol/l HCl (5.16), μl 430 380 430 380 430 470 480
a
2,5 mmol/l AA stock solution (6.1.7.1).
b
0,5 mmol/l AA solution 1 (6.1.7.2).
c
0,05 mmol/l AA solution 2 (6.1.7.3).
8.1.7 Derivatization (of samples, cystine standards and amino acids standards)
Derivatization converts free amino acids into highly stable derivatives. Standards and samples are
derivatized following the manufacturer’s instructions, as follows:
— Preheat a heating block to 55 °C.
— With a micropipette, add 70 μl of AccQ·Tag™ Ultra Borate buffer (reagent 1, see 6.1.5.1) to a clean
12 mm × 32 mm glass screw neck total recovery vial.
— Add 10 μl of calibration standard (8.1.5), neutralized sample solution (8.1.4), or neutralized
converted cystine standard (8.1.4) to the vial.
— Vortex mix briefly.
prEN 18197:2025 (E)
— Add 20 μl of reconstituted AccQ·Tag™ Ultra reagent (6.1.5.2) to the sample vial.
— Mix the solution immediately by pipetting up and down several times. Mix by vortex immediately for
several seconds, cap the vial and tap it to ensure no bubble is trapped.
— Let stand for 1 min at room temperature.
— Heat the vial in a heating block for 10 min at 55 °C ± 1 °C.
— Vortex mix briefly to remove the condensation.
8.1.8 UHPLC separation and UV detection
Perform UHPLC separation as follows:
— Prime solvent lines for 5 min.
— Prime wash/sample syringes for four cycles.
— Allow the chromatographic system to stabilize before injecting standards and samples. Make sure
the system pressure and initial conditions are stable before performing injections.
— Before starting a series of analyses, inject two blanks (water) to condition the column.
— Inject 1 µl of each derivatized calibration standards, one blank injection (water), and then 1 µl of each
derivatized sample solution. After the sample series, inject 1 blank followed by 1 μl of each
derivatized calibration standards. Blank injections can be added throughout the injection series to
verify that there is no carryover. Perform single injections.
— Perform UHPLC separation and UV detection under the following conditions:
— column length: 150 mm (see section 7.2);
— column temperature: 50 °C;
— UV detector: 260 nm;
— injection volume: 1 μl;
— flow rate: 0,4 ml/min;
— mobile phase A: Eluent A (6.2.2);
— mobile phase B: Eluent B (6.2.3);
— eluent gradient: See Table 4.
NOTE Fluorescence detection can be used provided equivalence has been demonstrated.
Operating conditions can vary depending on the apparatus. Follow the supplier’s instructions. Examples
1)
of wash solvents used with Waters UPLC systems are given in section 6.1.8.
prEN 18197:2025 (E)
Table 4 — Eluent gradient for UV (150 mm column)
Time % A % B Curve
0,00 99,9 0,1
5,50 99,9 0,1 2
15,22 90,9 9,1 7
20,47 78,8 21,2 6
21,26 40,4 59,6 6
21,29 10 90 6
22,84 10 90 6
26,00 99,9 0,1 6
32,00 99,9 0,1 6
8.1.9 Peak identification and integration
Identify the amino acids peaks in the sample solution by comparison with the retention times of the
corresponding peaks obtained in the calibration standards (see Annex B for example). Check that peaks
are separated with a good resolution (baseline separation). If this is not the case, adapt the
chromatographic conditions (gradient, temperature, tubing length, etc.) accordingly.
If a peak has not been integrated correctly, call the recorded data and reintegrate.
To verify system stability, inject a mid-level standard a minimum of three times (5 × for United States
Pharmacopoeia (USP) requirements) and ensure response and retention times have a coefficient of
variation, Cv < 2.
To check that the derivatization reagent was present in sufficient amount (excess), verify that the 6-
aminoquinoline (AMQ) peak is present in the chromatogram. The response of the excess reagent is
present in the chromatography as this large AMQ peak first to elute. The response should be equal to that
of the 25 pmol/μl standard or the reaction and sample should be flagged and discarded. The
derivatization peak (seen prior to lysine at approximately 17 min) can be ignored.
8.2 Procedure with MS detection
8.2.1 General
Samples (clause 8.2.2) and cystine calibration standards (clause 8.2.3) are prepared on the first day and
hydrolyzed at 110 °C overnight (clause 8.2.4). The amino acid calibration standards (clause 8.2.6) are
prepared on the second day. All solutions are then derivatized (8.2.7) and analyzed by UHPLC-MS (8.2.8).
This section follows this order of operations
8.2.2 Sample preparation
Weigh out 30 ± 5 mg algae powder samples into a 10 ml glass tube with screw cap. Report the sample
mass to 0,1 mg.
To each tube, add water, DDP, HCl, internal standard and phenol/HCl according to Table 5.
Add the phenol/HCl solution under the hood. Bubble nitrogen through the sample solution in the glass
tube for about 30 s. Close tubes with screw caps and vortex. Make sure the caps are perfectly clean (i.e.
devoid of any particle) to ensure tightness and avoid evaporation during hydrolysis.
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Table 5 — Preparation of the sample tubes
Solution Amount
Sample, mg 30 ± 5
Water, μl 770
DDP solution (6.1.3), μl 600
0,2 mol/l HCl (6.1.2), μl 600
IS-Cys2 stock solution (6.3.1), µl 500
Phenol/HCl solution (6.1.4), μl 2500
8.2.3 Cystine Calibration standards preparation
Table 6 describes how to prepare calibration standards for converted cystine at 0 pmol/µl to 5 pmol/µl
with heavy labelled cystine at 0,5 pmol/µl (all are final concentrations after derivatization).
Add the phenol/HCl solution under the hood. Bubble nitrogen through the solution in the glass tube for
about 30 s. Close tubes with screw caps and vortex. Make sure the caps are perfectly clean (i.e. devoid of
any particle) to ensure tightness and avoid evaporation during hydrolysis.
Table 6 — Final cystine concentration after derivatization
Solution 5 pmol/ 2,5 pmol/ 1 pmol/ 0,5 pmol/ 0,1 pmol/ 0 pmol/
µl
µl µl µl µl µl
a a b b b
Cystine solution, μl 250 125 500 250 50 0
Water, μl 550 675 300 550 750 800
DDP solution (6.1.3), μl 600 600 600 600 600 600
0,2 mol/l HCl (6.1.2), μl 600 600 600 600 600 600
IS-Cys2 stock solution (6.3.1),
500 500 500 500 500 500
µl
Phenol/HCl solution (6.1.4), μl 2 500 2 500 2 500 2 500 2500 2 500
a
10 mmol/l cystine stock solution (6.1.6.1).
b
1 mmol/l cystine solution (6.1.6.2).
8.2.4 Hydrolysis (of samples and cystine standards)
Place tubes in an oven at 110 °C ± 2 °C for 24 h ± 0,5 h.
8.2.5 Neutralization and dilution (of samples and converted cystine standards)
Take the tubes out of the oven. Allow hydrolysates to cool down and particles to settle down prior to
taking an aliquot. When transferring aliquots, pipet about 1 cm below the top of the liquid. Perform
neutralization under the hood.
For converted cystine standards, transfer 0,1 ml of the hydrolysate sample solution into a 1,5 ml
microtube, add 0,1 ml of 6 mol/l NaOH (6.1.1) and then 0,8 ml of 0,1 mol/l HCl (5.16).
For algae powder samples, transfer 0,1 ml of the hydrolysate sample solution into a 1,5 ml microtube,
add 0,1 ml of 6 mol/l NaOH (6.1.1), 0,3 ml of 0,1 mol/l HCl (5.16), and 0,5 ml of 0,02 mol/l IS-AA (6.3.2.3).
Mix well and filter through 0,45 µm membrane filter into another 1,5 ml microtube.
prEN 18197:2025 (E)
8.2.6 Amino acids calibration standards preparation (not needed to be acid hydrolysed)
Table 7 shows how to prepare 0,5 ml calibration standards at 0 pmol/µl to 25 pmol/µl with with heavy
labelled amino acids at 1 pmol/µl (all are final concentration after derivatization).
The amino acid solutions are stable for one week when stored at 4 °C ± 2 °C.
Table 7 — Final amino acid concentrations after derivatization (MS detection)
Solution 25 pmol/ 10 pmol/ 5 pmol/ 1 pmol/ 0,5 pmol/ 0,1 pmol/ 0 pmol/
µl µl µl µl µl µl µl
a b b c c c
Amino acid solution, μl 50 100 50 100 50 10 0
IS-AA solution (6.3.2.2), μl 50 50 50 50 50 50 50
0,1 mol/l HCl (5.16), μl 400 350 400 350 400 440 450
a
2,5 mmol/l AA stock solution (6.1.7.1).
b
0,5 mmol/l AA solution 1 (6.1.7.2).
c
0,05 mmol/l AA solution 2 (6.1.7.3).
8.2.7 Derivatization (of samples, cystine standards and amino acids standards)
Derivatization converts free amino acids into highly stable derivatives. Standards and samples are
derivatized following the manufacturer’s instructions, as follows:
— Preheat a heating block to 55 °C.
— With a micropipette, add 70 μl of AccQ·Tag™ Ultra Borate buffer (reagent 1, see 6.1.5.1) to a clean
12 mm × 32 mm glass screw neck total recovery vial.
— Add 10 μl of calibration standard (8.2.5), neutralized sample solution (8.2.4), or neutralized
converted cystine standard (8.2.4) to the vial.
— Vortex mix briefly.
— Add 20 μl of reconstituted AccQ·Tag™ Ultra reagent (6.1.5.2) to the sample vial.
— Mix the solution immediately by pipetting up and down several times. Mix by vortex immediately for
several seconds, cap the vial and tap it to ensure no bubble is trapped.
— Let stand for 1 min at room temperature.
— Heat the vial in a heating block for 10 min at 55 °C ± 1 °C.
— Vortex mix briefly to remove condensation.
8.2.8 UHPLC separation and MS detection
Perform UHPLC separation as follows:
— Prime solvent lines for 5 min.
— Prime wash/sample syringes for four cycles.
— Allow the chromatographic system to stabilize before injecting standards and samples. Make sure
the system pressure and initial conditions are stable before performing injections.
prEN 18197:2025 (E)
— Before starting a series of analyses, inject two blanks (water) to condition the column.
— Inject 1 µl of each derivatized calibration standards, one blank injection (water), and then 1 µl of each
derivatized sample solution. After the sample series, inject one blank followed by 1 μl of each
derivatized calibration s
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