EN ISO 13904:2005

Animal feeding stuffs - Determination of tryptophan content (ISO 13904:2005)

ISO 13904:2005 describes the determination of the total and free tryptophan in feeding stuffs (e.g. complete and complementary feeds, supplementary feeds, raw materials, ingredients, premixtures and concentrates. It does not distinguish between D- and L-forms.

Futtermittel - Bestimmung des Tryptophangehalts (ISO 13904:2005)

Diese Internationale Norm beschreibt die Bestimmung des Gehalts an Gesamttryptophan und freiem Tryptophan in Futtermitteln. Bei der Analyse wird nicht zwischen D  und L Formen unterschieden.

Aliments des animaux - Détermination de la teneur en tryptophane (ISO 13904:2005)

L'ISO 13904:2005 décrit le dosage du tryptophane total et libre des aliments pour animaux. Elle ne fait pas la distinction entre les formes D et L.

Krma - Določevanje triptofana (ISO 13904:2005)

General Information

• Status: Withdrawn
• Publication Date: 30-Apr-2005
• Withdrawal Date: 08-Mar-2016
• 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: 9960 - Withdrawal effective - Withdrawal
• Completion Date: 09-Mar-2016
• Ref Project: SIST EN ISO 13904:2005 - Animal feeding stuffs - Determination of tryptophan content (ISO 13904:2005)

Relations

• Replaced By: EN ISO 13904:2016 - Animal feeding stuffs - Determination of tryptophan content (ISO 13904:2016)
• Effective Date: 16-Mar-2016

Standards Content (Sample)

SLOVENSKI STANDARD
01-september-2005
.UPD'RORþHYDQMHWULSWRIDQD,62
Animal feeding stuffs - Determination of tryptophan content (ISO 13904:2005)
Futtermittel - Bestimmung des Tryptophangehalts (ISO 13904:2005)
Aliments des animaux - Détermination de la teneur en tryptophane (ISO 13904:2005)
Ta slovenski standard je istoveten z: EN ISO 13904:2005
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 13904
NORME EUROPÉENNE
EUROPÄISCHE NORM
May 2005
ICS 65.120
English version
Animal feeding stuffs - Determination of tryptophan content (ISO
13904:2005)
Aliments des animaux - Détermination de la teneur en Futtermittel - Bestimmung des Tryptophangehalts (ISO
tryptophane (ISO 13904:2005) 13904:2005)
This European Standard was approved by CEN on 4 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 13904:2005: E
worldwide for CEN national Members.

Foreword
This document (EN ISO 13904:2005) 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 13904:2005 has been approved by CEN as EN ISO 13904:2005 without any
modifications.
INTERNATIONAL ISO
STANDARD 13904
First edition
2005-05-01
Animal feeding stuffs — Determination
of tryptophan content
Aliments des animaux — Détermination de la teneur en tryptophane

Reference number
ISO 13904:2005(E)
©
ISO 2005
ISO 13904:2005(E)
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In
the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.

©  ISO 2005
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail [e-mail]
Web www.iso.org
Published in Switzerland
ii © ISO 2005 – All rights reserved

ISO 13904:2005(E)
Contents Page
Foreword. iv
1 Scope. 1
2 Principle . 1
3 Reagents and materials. 1
4 Apparatus. 2
5 Procedure. 3
5.1 Preparation of samples . 3
5.2 Determination of free tryptophan (extract). 3
5.3 Determination of total tryptophan (hydrolysate) . 3
5.4 HPLC determination. 4
6 Calculation of results. 4
7 Precision . 5
7.1 Interlaboratory test . 5
7.2 Repeatability. 5
7.3 Reproducibility . 5
8 Test report. 5
Annex A (informative) Results of an interlaboratory test. 6
Annex B (informative) Observations on the method. 8
Bibliography . 9

ISO 13904:2005(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 13904 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 10, Animal
feeding stuffs.
iv © ISO 2005 – All rights reserved

INTERNATIONAL STANDARD ISO 13904:2005(E)

Animal feeding stuffs — Determination of tryptophan content
1 Scope
This International Standard describes determination of the total and free tryptophan content in feeding stuffs
(e.g. complete and complementary feeds, supplementary feeds, raw materials, ingredients, premixtures and
concentrates). It does not distinguish between D- and L-forms.
2 Principle
For the determination of the total tryptophan, the sample is hydrolysed under alkaline conditions with
saturated barium hydroxide solution and heated to 110 °C for 20 h. After hydrolysis, an internal standard is
added.
For the determination of free tryptophan, the sample is extracted under mild acidic conditions in the presence
of an internal standard.
The tryptophan and the internal standard in the hydrolysate or in the extract are determined by reversed
phase C HPLC with fluorescence detection.
3 Reagents and materials
Use only reagents of recognized analytical grade, unless otherwise specified.
3.1 Double-distilled water, or water of equivalent purity (conductivity < 10 µS/cm).
3.2 Standard substance: tryptophan (purity/content W 99 %) dried under vacuum over phosphorus
pentoxide.
3.3 Internal standard substance: αα-methyltryptophan (purity/content W 99 %), dried under vacuum over
αα
phosphorus pentoxide.
3.4 Barium hydroxide octahydrate.
Care should be taken not to expose the Ba(OH)⋅8H O excessively to air in order to avoid formation of BaCO ,
2 2 3
which could disturb the determination (see observation in B.3).
3.5 Sodium hydroxide.
3.6 Orthophosphoric acid, w = 85 %.
3.7 Concentrated hydrochloric acid, ρ = 1,19 g/ml.
3.8 Methanol, HPLC grade.
3.9 Light petroleum, boiling range 40 °C to 60 °C.
3.10 Sodium hydroxide solution, c = 1 mol/l.
Dissolve 40,0 g of NaOH (3.5) in water (3.1) and make up to 1 l with water (3.1).
ISO 13904:2005(E)
3.11 Hydrochloric acid, c = 6 mol/l.
Take 492 ml of HCl (3.7) and make up to 1 l with water (3.1).
3.12 Hydrochloric acid, c = 1 mol/l.
Take 82 ml of HCl (3.7) and make up to 1 l with water (3.1).
3.13 Hydrochloric acid, c = 0,1 mol/l.
Take 8,2 ml of HCl (3.7) and make up to 1 l with water (3.1).
3.14 Orthophosphoric acid, c = 0,5 mol/l.
Take 34 ml of orthophosphoric acid (3.6) and make up to 1 l with water (3.1).
3.15 Concentrated tryptophan solution (3.2), c = 0,000 5000 g/ml.
In a 500 ml volumetric flask, dissolve 0,25 g of tryptophan (3.2) (weighed to the nearest 0,1 mg) in
hydrochloric acid (3.13) and make up to the mark with hydrochloric acid (3.13). Store at –18 °C for a maximum
of four weeks.
3.16 Concentrated internal standard solution, c = 0,000 54 g/ml.
In a 500 ml volumetric flask, dissolve 0,27 g of α-methyltryptophan (3.3) (weighed to the nearest 0,1 mg) in
hydrochloric acid (3.13) and make up to the mark with hydrochloric acid (3.13). Store at –18 °C for a maximum
of four weeks.
3.17 Calibration standard solution of tryptophan and internal standard.
Take 2,00 ml of the concentrated tryptophan solution (3.15) and 2,00 ml of concentrated internal standard
solution (α-methyltryptophan) (3.16). Dilute with water (3.1) and methanol (3.8) to approximately the same
volume and to approximately the same concentration of methanol (10 % to 30 %) as the finished hydrolysate.
This solution shall be prepared freshly before use.
Protect from direct sunlight during preparation.
3.18 Ethanolamine > 98 %.
3.19 1,1,1-Trichloro-2-methyl-2-propanol solution.
Add 1 g of 1,1,1-trichloro-2-methyl-2-propanol to 100 ml of methanol (3.8).
3.20 Mobile phase for HPLC.
Dissolve 3,00 g of acetic acid in 900 ml of water (3.1) and add 50,0 ml of 1,1,1-trichloro-2-methyl-2-propanol
solution (3.19). Adjust the pH to 5,00 using ethanolamine (3.18). Make up to 1 000 ml with water (3.1).
4 Apparatus
Usual laboratory apparatus and, in particular, the following.
4.1 HPLC equipment with a spectrofluorimetric detector.
4.2 Liquid chromatographic column, 125 mm × 4 mm, with C , 3 µm packing, or equivalent.
4.3 pH-meter.
2 © ISO 2005 – All rights reserved

ISO 13904:2005(E)
4.4 Polypropylene flask, of capacity 125 ml, with wide neck and screw cap.
4.5 Membrane filter, 0,45 µm.
4.6 Autoclave, capable of being maintained at (110 ± 2) °C, [(140 ± 10) kPa (1,4 ± 0,1) bar].
A pressure-tight covered dish that may be put into a drying oven adjustable to (110 ± 2) °C may be used.
4.7 Mechanical shaker or magnetic stirrer.
4.8 Vortex mixer.
5 Procedure
5.1 Preparation of samples
Grind the sample to pass through a 0,5 mm sieve. Samples hig
...