EN ISO 13903:2005

Animal feeding stuffs - Determination of amino acids content (ISO 13903:2005)

ISO 13903:2005 describes the determination of free (synthetic and natural) and total (peptide-bound and free) amino acids in feeding stuffs, using an amino acid analyser or HPLC equipment. It is applicable to the following amino acids: sum of cystine and cysteine; methionine; lysine; threonine; alanine; arginine; aspartic acid; glutamic acid; glycine; histidine; isoleucine; leucine; phenylalanine; proline; serine; tyrosine; valine.
The method does not distinguish between the salts of amino acids, nor does it differentiate between D and L forms of amino acids. It is not valid for the determination of tryptophan or hydroxy analogues of amino acids.
Limits of quantification depend on the chromatographic equipment, but levels as low as: 0,3 g/kg total lysine; 0,25 g/kg total methionine; 0,35 g/kg total cystine plus cysteine; 0,2 g/kg total threonine; 0,035g/kg free lysine; 0,035g/kg free methionine; and 0,03g/kg free threonine can typically be analysed.

Futtermittel - Bestimmung des Aminosäuregehalts (ISO 13903:2005)

Diese Internationale Norm beschreibt die Bestimmung der freien (synthetischen und natürlichen) Aminosäuren und der Gesamtaminosäuren (peptidgebundene und freie) in Futtermitteln unter Verwendung eines Aminosäurenanalysators oder einer HPLC Anlage. Sie gilt für folgende Aminosäuren:
-   Summe von Cystin und Cystein;
-   Methionin;
-   Lysin;
-   Threonin;
-   Alanin;
-   Arginin;
-   Asparaginsäure;
-   Glutaminsäure;
-   Glycin;
-   Histidin;
-   Isoleucin;
-   Leucin;
-   Phenylalanin;
-   Prolin;
-   Serin;
-   Tyrosin;
-   Valin.
Das Verfahren unterscheidet nicht zwischen Aminosäuresalzen und auch nicht zwischen D- und L-Formen von Aminosäuren. Es gilt nicht für die Bestimmung von Tryptophan oder hydroxylhaltigen Analogen von Aminosäuren.
Die Grenzen der quantitativen Bestimmung hängen von der chromatographischen Einrichtung ab, jedoch so geringe Gehalte wie 0,3 g/kg Gesamtlysin, 0,25 g/kg Gesamtmethionin, 0,35 g/kg Gesamtcystin plus  cystein, 0,2 g/kg Gesamtthreonin, 0,035 g/kg freies Lysin, 0,035 g/kg freies Methionin und 0,03 g/kg freies Threonin können gewöhnlich analysiert werden.
ANMERKUNG   Es könnte eine noch niedrigere Bestimmungs- oder Nachweisgrenze erreichbar sein, aber das ist von den Anwendern zu bestätigen.

Aliments des animaux - Détermination de la teneur en acides aminés (ISO 13903:2005)

L'ISO 13903:2005 décrit le dosage des acides aminés libres (de synthèse et naturels) et totaux (dans des peptides et libres) dans les aliments des animaux, au moyen d'un analyseur d'acides aminés ou d'un équipement de chromatographie liquide à haute performance (CLHP). Elle s'applique aux acides aminés suivants: la somme de la cystine et de la cystéine, la méthionine, la lysine, la thréonine, l'alanine, l'arginine, l'acide aspartique, l'acide glutamique, la glycine, l'histidine, l'isoleucine, la leucine, la phénylalanine, la proline, la sérine, la tyrosine et la valine.
Cette méthode ne distingue pas les sels d'acides aminés, pas plus qu'elle ne fait la différence entre les formes D et L des acides aminés. Elle n'est pas valable pour le dosage du tryptophane ou des hydroxy-analogues d'acides aminés.
Les limites de quantification dépendent de l'équipement chromatographique, mais on peut généralement analyser des niveaux aussi bas que 0,3 g/kg pour la lysine totale, 0,25 g/kg pour la méthionine totale, 0,35 g/kg pour la cystine plus la cystéine totales, 0,2 g/kg pour la thréonine totale, 0,035 g/kg pour la lysine libre, 0,035 g/kg pour la méthionine libre et 0,03 g/kg pour la thréonine libre.

Krma - Določevanje amino kislin (ISO 13903:2005)

General Information

• Status: Published
• Publication Date: 14-May-2005
• Withdrawal Date: 29-Nov-2005
• ICS: 65.120 - Animal feeding stuffs
• Technical Committee: CEN/TC 327 - Animal feeding stuffs - Methods of sampling and analysis
• Drafting Committee: CEN/TC 327 - Animal feeding stuffs - Methods of sampling and analysis
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 15-May-2005
• Completion Date: 15-May-2005
• Ref Project: SIST EN ISO 13903:2005 - Animal feeding stuffs - Determination of amino acids content (ISO 13903:2005)

Standards Content (Sample)

SLOVENSKI STANDARD
01-september-2005
.UPD'RORþHYDQMHDPLQRNLVOLQ,62
Animal feeding stuffs - Determination of amino acids content (ISO 13903:2005)
Futtermittel - Bestimmung des Aminosäuregehalts (ISO 13903:2005)
Aliments des animaux - Détermination de la teneur en acides aminés (ISO 13903:2005)
Ta slovenski standard je istoveten z: EN ISO 13903:2005
ICS:
65.120 Krmila Animal feeding stuffs
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 13903
NORME EUROPÉENNE
EUROPÄISCHE NORM
May 2005
ICS 65.120
English version
Animal feeding stuffs - Determination of amino acids content
(ISO 13903:2005)
Aliments des animaux - Détermination de la teneur en Futtermittel - Bestimmung des Aminosäuregehalts (ISO
acides aminés (ISO 13903:2005) 13903:2005)
This European Standard was approved by CEN on 19 April 2005.
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 Central Secretariat 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 Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,
Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2005 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 13903:2005: E
worldwide for CEN national Members.

Foreword
This document (EN ISO 13903:2005) has been prepared by Technical Committee ISO/TC 34
"Agricultural food products" in collaboration with Technical Committee CEN/TC 327 "Animal
feeding stuffs - Methods of sampling and analysis", 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 2005, and conflicting national
standards shall be withdrawn at the latest by November 2005.

According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary,
Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

Endorsement notice
The text of ISO 13903:2005 has been approved by CEN as EN ISO 13903:2005 without any
modifications.
INTERNATIONAL ISO
STANDARD 13903
First edition
2005-05-15
Animal feeding stuffs — Determination
of amino acids content
Aliments des animaux — Détermination de la teneur en acides aminés

Reference number
ISO 13903:2005(E)
©
ISO 2005
ISO 13903:2005(E)
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ii © ISO 2005 – All rights reserved

ISO 13903:2005(E)
Contents Page
Foreword. iv
1 Scope. 1
2 Principle . 1
2.1 Free amino acids. 1
2.2 Total amino acids. 2
3 Reagents and materials. 2
4 Apparatus. 4
5 Procedure. 4
5.1 Preparation of test sample. 4
5.2 Determination of free amino acids in feeding stuffs and premixtures. 4
5.3 Determination of total amino acids . 5
5.4 Chromatography . 6
6 Calculation of results. 7
7 Precision . 8
7.1 Interlaboratory tests . 8
7.2 Repeatability. 8
7.3 Reproducibility . 8
8 Use of reference materials . 8
9 Observations on the method . 8
Annex A (informative) Results of interlaboratory tests . 10
Annex B (informative) Examples of chromatograms. 15
Bibliography . 17

ISO 13903:2005(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 13903 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 10, Animal
feeding stuffs.
[1]
ISO 13903 is based on Commission Directive 98/64/EC of September 1998 .

iv © ISO 2005 – All rights reserved

INTERNATIONAL STANDARD ISO 13903:2005(E)

Animal feeding stuffs — Determination of amino acids content
1 Scope
This International Standard describes the determination of free (synthetic and natural) and total
(peptide-bound and free) amino acids in feeding stuffs, using an amino acid analyser or HPLC equipment. It is
applicable to the following amino acids:
 sum of cystine and cysteine;
 methionine;
 lysine;
 threonine;
 alanine;
 arginine;
 aspartic acid;
 glutamic acid;
 glycine;
 histidine;
 isoleucine;
 leucine;
 phenylalanine;
 proline;
 serine;
 tyrosine;
 valine.
The method does not distinguish between the salts of amino acids, nor does it differentiate between D and L
forms of amino acids. It is not valid for the determination of tryptophan or hydroxy analogues of amino acids.
Limits of quantification depend on the chromatographic equipment, but levels as low as: 0,3 g/kg total lysine;
0,25 g/kg total methionine; 0,35 g/kg total cystine plus cysteine; 0,2 g/kg total threonine; 0,035 g/kg free
lysine; 0,035 g/kg free methionine; and 0,03 g/kg free threonine can typically be analysed.
NOTE A lower limit of quantification or detection might be achievable but this is to be validated by the users.
2 Principle
2.1 Free amino acids
The free amino acids are extracted with dilute hydrochloric acid. Co-extracted nitrogenous macromolecules
are precipitated with sulfosalicylic acid and removed by filtration. The filtered solution is adjusted to pH 2,20.
The amino acids are separated by ion exchange chromatography and determined by reaction with ninhydrin
with photometric detection at 570 nm.
ISO 13903:2005(E)
2.2 Total amino acids
The procedure chosen depends on the amino acids under investigation. Cyst(e)ine and methionine shall be
oxidized to cysteic acid and methionine sulphone, respectively, prior to hydrolysis. Tyrosine shall be
determined in hydrolysates of unoxidized samples. All the other amino acids listed in Clause 1 may be
determined in either the oxidized or unoxidized sample.
Oxidation is performed at 0 °C with a performic acid/phenol mixture. Excess oxidation reagent is decomposed
with sodium disulfite. The oxidized or unoxidized sample is hydrolysed with hydrochloric acid (c = 6 mol/l) for
23 h. The hydrolysate is adjusted to pH 2,20. The amino acids are separated by ion exchange
chromatography and determined by reaction with ninhydrin, using photometric detection at 570 nm (440 nm
for proline).
3 Reagents and materials
Use only reagents of recognized analytical grade, unless otherwise specified.
3.1 Water, double distilled water or water of equivalent quality shall be used (conductivity < 10 µS).
3.2 Hydrogen peroxide, w = 30 %.
3.3 Formic acid, w = 98 % to 100 %.
3.4 Hydrochloric acid, density approximately 1,19 g/ml.
3.5 2,2'-Thiodiethanol (thiodiglycol)
3.6 Light petroleum, boiling rate 40 °C to 60 °C
3.7 Norleucine, or any other compound suitable for use as internal standard.
3.8 Nitrogen gas (< 10 parts per million oxygen).
3.9 Amino acids.
3.9.1 Standard substances listed under Clause 1.
Use pure compounds containing no water of crystallization. Dry under vacuum over P O or H SO for
2 5 2 4
1 week prior to use.
3.9.2 Cysteic acid.
3.9.3 Methionine sulfone.
3.10 Sodium hydroxide solution I, c = 7,5 mol/l.
Dissolve 300 g of NaOH (3.6) in water and make up to 1 l.
3.11 Sodium hydroxide solution II, c = 1 mol/l.
Dissolve 40 g of NaOH in water (3.1) and make up to 1 l.
3.12 Formic acid-phenol solution.
Mix 889 g of formic acid (3.3) with 111 g of water (3.1) and add 4,73 g of phenol.
2 © ISO 2005 – All rights reserved

ISO 13903:2005(E)
3.13 Hydrolysis mixture, c = 6 mol/l HCl containing 1 g of phenol per litre.
Add 1 g of phenol to 492 ml of HCl (3.4) and make up to 1 l with water (3.1).
3.14 Extraction mixture, c = 0,1 mol/l HCl containing 2 % thiodiglycol.
Take 8,2 ml of HCl (3.4), dilute with approximately 900 ml of water (3.1). Add 20 ml of thiodiglycol (3.5) and
make up to 1 l with water. Do not mix 3.
...