SIST EN ISO 13904:2016
(Main)Animal feeding stuffs - Determination of tryptophan content (ISO 13904:2016)
Animal feeding stuffs - Determination of tryptophan content (ISO 13904:2016)
This International Standard specifies a method for determination of the total and free tryptophan (Trp)
content in feeding stuffs (e.g. complete and complementary feeds, supplementary feeds, raw materials,
ingredients, and concentrates) and determination of free tryptophan in commercial pure substances
and premixtures containing more than 2 % of tryptophan.
It does not distinguish between D- and L-forms.
Futtermittel - Bestimmung des Tryptophangehalts (ISO 13904:2016)
Diese Internationale Norm legt ein Verfahren zur Bestimmung des Gehaltes an Gesamttryptophan und an freiem Tryptophan (Trp) in Futtermitteln (z. B. Allein- und Ergänzungsfuttermittel, Milchaustausch-Futtermittel, Rohstoffe, Futterbestandteile und Konzentrate) und zur Bestimmung von freiem Tryptophan in handelsüblichen Reinsubstanzen und Vormischungen, die mehr als 2 % Tryptophan enthalten, fest.
Bei dem Verfahren wird nicht zwischen D und L Formen unterschieden.
Aliments des animaux - Détermination de la teneur en tryptophane (ISO 13904:2016)
ISO 13904:2016 décrit une méthode de dosage du tryptophane total et libre (Trp) dans les aliments pour animaux (par exemple aliments complets et complémentaires, compléments alimentaires, matières premières, ingrédients et concentrés) et une méthode de dosage du tryptophane libre dans les substances pures du commerce et les prémélanges contenant plus de 2 % de tryptophane.
Elle ne fait pas la distinction entre les formes D et L.
Krma - Določevanje triptofana (ISO 13904:2016)
Ta mednarodni standard določa metodo za določevanje celotnega in prostega triptofana (Trp) v krmi (npr. polnovredna in dopolnilna krma, dodatna krma, surovine, sestavine in koncentrati) ter določevanje prostega triptofana v komercialnih čistih snoveh in premiksih, ki vsebujejo več kot 2 % triptofana.
Standard ne razlikuje med oblikama D in L.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 13904:2016
01-maj-2016
1DGRPHãþD
SIST EN ISO 13904:2005
.UPD'RORþHYDQMHWULSWRIDQD ,62
Animal feeding stuffs - Determination of tryptophan content (ISO 13904:2016)
Futtermittel - Bestimmung des Tryptophangehalts (ISO 13904:2016)
Aliments des animaux - Détermination de la teneur en tryptophane (ISO 13904:2016)
Ta slovenski standard je istoveten z: EN ISO 13904:2016
ICS:
65.120 Krmila Animal feeding stuffs
SIST EN ISO 13904:2016 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 13904:2016
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SIST EN ISO 13904:2016
EN ISO 13904
EUROPEAN STANDARD
NORME EUROPÉENNE
March 2016
EUROPÄISCHE NORM
ICS 65.120 Supersedes EN ISO 13904:2005
English Version
Animal feeding stuffs - Determination of tryptophan
content (ISO 13904:2016)
Aliments des animaux - Dosage du tryptophane (ISO Futtermittel - Bestimmung des Tryptophangehalts (ISO
13904:2016) 13904:2016)
This European Standard was approved by CEN on 30 January 2016.
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 CEN-CENELEC Management Centre 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 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2016 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 13904:2016 E
worldwide for CEN national Members.
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SIST EN ISO 13904:2016
EN ISO 13904:2016 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 13904:2016
EN ISO 13904:2016 (E)
European foreword
This document (EN ISO 13904:2016) has been prepared by Technical Committee ISO/TC 34 “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 September 2016, and conflicting national standards
shall be withdrawn at the latest by September 2016.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent
rights.
This document supersedes EN ISO 13904:2005.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 13904:2016 has been approved by CEN as EN ISO 13904:2016 without any modification.
3
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SIST EN ISO 13904:2016
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SIST EN ISO 13904:2016
INTERNATIONAL ISO
STANDARD 13904
Second edition
2016-02-15
Animal feeding stuffs —
Determination of tryptophan content
Aliments des animaux — Dosage du tryptophane
Reference number
ISO 13904:2016(E)
©
ISO 2016
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SIST EN ISO 13904:2016
ISO 13904:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
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Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved
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SIST EN ISO 13904:2016
ISO 13904:2016(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Principle . 1
3 Reagents and materials . 1
4 Apparatus . 3
5 Procedure. 4
5.1 Preparation of samples . 4
5.1.1 Feeding stuffs . 4
5.1.2 Commercial pure substances and premixtures containing more than 2 %
of tryptophan . 4
5.2 Determination of free tryptophan (extract) . 4
5.2.1 Feeding stuffs . 4
5.2.2 Commercial pure substances and premixtures containing more than 2 %
of tryptophan . 4
5.3 Determination of total tryptophan (hydrolysates) . 5
5.4 HPLC determination . 5
6 Calculation of results . 6
6.1 Feeding stuffs . 6
6.2 Commercial pure products and premixtures containing more than 2 % of tryptophan . 6
6.2.1 Control of the calibration. 6
6.2.2 Calculation . 6
7 Precision . 7
7.1 Interlaboratory test. 7
7.1.1 Feeding stuffs . 7
7.1.2 Commercial pure substances and premixtures containing more than 2 %
of tryptophan . 7
7.2 Repeatability . 7
7.2.1 Feeding stuffs . 7
7.2.2 Commercial pure substances and premixtures containing more than 2 %
of tryptophan . 7
7.3 Reproducibility . 7
7.3.1 Feeding stuffs . 7
7.3.2 Pure products and premixtures containing more than 2 % of tryptophan . 8
8 Test report . 8
Annex A (informative) Results of an interlaboratory test . 9
Annex B (informative) Observations on the method .11
Annex C (informative) Sample preparation: Example for analyses in pure products
and premixtures .12
Annex D (informative) Results of an interlaboratory test for tryptophan in pure products
and premixtures containing more than 2 % of tryptophan .13
Bibliography .14
© ISO 2016 – All rights reserved iii
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SIST EN ISO 13904:2016
ISO 13904:2016(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 10, Animal
feeding stuffs.
This second edition cancels and replaces the first edition (ISO 13904:2005), which has been
technically revised.
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SIST EN ISO 13904:2016
INTERNATIONAL STANDARD ISO 13904:2016(E)
Animal feeding stuffs — Determination of tryptophan
content
1 Scope
This International Standard specifies a method for determination of the total and free tryptophan (Trp)
content in feeding stuffs (e.g. complete and complementary feeds, supplementary feeds, raw materials,
ingredients, and concentrates) and determination of free tryptophan in commercial pure substances
and premixtures containing more than 2 % of tryptophan.
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. For commercial pure substances and premixtures containing more
than 2 % of tryptophan, it is possible to add the internal standard after the extraction.
The tryptophan and the internal standard in the hydrolysate or in the extract are determined by
reversed phase C HPLC with fluorescence detection.
18
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 and control substance: tryptophan (purity ≥99 %) dried under vacuum
over phosphorus pentoxide.
The two products are considered as 100 % pure. Control substance shall come from another
manufacturer than the standard substance (see 3.17.2).
NOTE The control of the purity of the standard substance can be performed by measuring the absorbance
-3
of a solution of tryptophan at 280 nm. Prepare a solution of about 5 mg/l in HCl 10 N from a stock solution and
-3
measure the Optical Density (OD) at 280 nm versus HCl 10 N. Then, the concentration of tryptophan is:
+06
C = OD/5 630 * 10
where
5 630 is the molar extinction coefficient of tryptophan in water at 280 nm;
C is expressed in μmole/l.
The standard substance purity is then (C/C )*100 where C is the theoretical concentration of the diluted
0 0
solution, expressed in μmole/l (about 25 μmole/l).
The control of the purity is performed every 6 months of use; it shall be ≥99 %.
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SIST EN ISO 13904:2016
ISO 13904:2016(E)
3.3 Internal standard substance: α-methyltryptophan (purity ≥99 %), dried under vacuum over
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
2 2
BaCO , which could disturb the determination (see observation in B.3).
3
3.5 Sodium hydroxide.
3.6 Orthophosphoric acid, w = 85 %.
3.7 Concentrated hydrochloric acid, ρ = 1,19 g/ml.
20
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).
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 standard solution and control solution (3.2), c = 0,50 g/l.
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 approximately
-18 °C for a maximum of four weeks.
3.16 Concentrated internal standard solution, c = 0,54 g/l.
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
approximately -18 °C for a maximum of four weeks.
3.17 Calibration standard solutions of tryptophan and internal standard.
3.17.1 Calibration standard solution for the analysis of tryptophan in feeding stuffs, (c = 0,010 g/l).
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)
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SIST EN ISO 13904:2016
ISO 13904:2016(E)
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.
3.17.2 Calibration standard solution of tryptophan for the analysis of tryptophan in commercial
pure substances and premixtures containing more than 2 % of tryptophan, (c = 0,010 g/l).
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 it with hydrochloric acid 0,1 mol/l (3.13) in a 100 ml volumetric flask. Fill up to the mark.
This solution shall be prepared freshly before use.
Protect from direct sunlight during preparation.
For verification of the calibration standard solution, it is possible to use a control solution of tryptophan.
This control solution is prepared and analyzed as it is described for the calibration standard solution,
but shall come from another manufacturer than the standard substance (3.2). The recovery of
tryptophan in the control solution sample shall be between 99 % and 101 %.
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).
NOTE 1,1,1-trichloro-2-methyl-2-propanol solution (3.19) could be considered critical for environmental
reasons and may require special disposal.
3.20 Mobile phase for HPLC.
Dissolve 3,00
...
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