SIST EN ISO 13903:2005

SLOVENSKI STANDARD
SIST EN ISO 13903:2005
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
SIST EN ISO 13903:2005 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 13903:2005

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SIST EN ISO 13903:2005
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.

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SIST EN ISO 13903:2005

EN ISO 13903:2005 (E)





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.

2

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SIST EN ISO 13903:2005

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

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SIST EN ISO 13903:2005
ISO 13903:2005(E)
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©  ISO 2005
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ii © ISO 2005 – All rights reserved

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SIST EN ISO 13903:2005
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

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SIST EN ISO 13903:2005
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 .

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SIST EN ISO 13903:2005
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.
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SIST EN ISO 13903:2005
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.
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SIST EN ISO 13903:2005
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 3.4 and 3.5 directly.
3.15 5-Sulfosalicylic acid, β = 6 %.
Dissolve 60 g of 5-sulfosalicylic acid dihydrate in water (3.1) and make up to 1 l with water.
3.16 Oxidation mixture (performic acid-phenol).
Mix 0,5 ml of hydrogen peroxide (3.2) with 4,5 ml of formic acid-phenol solution (3.12) in a small beaker.
Incubate at between 20 °C and 30 °C for 1 h in order to form performic acid, then cool in an ice-water bath
(15 min) before adding to the sample.
Avoid contact with skin and wear protective clothing.
+
3.17 Citrate buffer, c = 0,2 mol/l Na , pH 2,20.
Dissolve 19,61 g of sodium citrate dihydrate, 5 ml of thiodiglycol (3.5), 1 g of phenol and 16,50 ml of HCl (3.4)
in approximately 800 ml of water (3.1). Adjust the pH to 2,20. Make up to 1 l with water.
3.18 Elution buffers, prepared according to conditions for the analyser used (4.9).
3.19 Ninhydrin reagent, prepared according to conditions for the analyser used (4.9).
3.20 Standard solutions of amino acids.
These solutions shall be stored below 5 °C.
3.20.1 Stock standard solution of amino acids (3.9.1), c = 2,5 µmol/ml of each in hydrochloric acid.
These may be obtained commercially.
3.20.2 Stock standard solution of cysteic acid and methionine sulfone, c = 1,25 µmol/ml.
Dissolve 0,211 5 g of cysteic acid (3.9.2) and 0,226 5 g of methionine sulphone (3.9.3) in citrate buffer (3.17)
in a 1 l graduated flask and make up to mark with citrate buffer. Store below 5 °C for not more than 12 months.
This solution shall not be used if the stock standard solution (3.20.1) contains cysteic acid and methionine
sulfone.
3.20.3 Stock standard solution of the internal standard e.g. norleucine, c = 20 µmol/ml.
Dissolve 0,656 0 g of norleucine (3.7) in citrate buffer (3.17) in a graduated flask and make up to 250 ml with
citrate buffer. Store below 5 °C for no more than 6 months.
3.20.4 Calibration solution of standard amino acids, for use with hydrolysates, c = 0,1 µmol/ml of cysteic
acid and methionine sulfone and c = 0,2 µmol/ml of the other amino acids.
Dissolve 2,2 g of sodium chloride in 100 ml beaker with 30 ml of citrate buffer (3.17). Add 4,00 ml of stock
standard solution of amino acids (3.20.1), 4,00 ml of stock standard solution of cysteic acid and methionine
sulfone (3.20.2), and 0,50 ml of stock standard solution of internal standard (3.20.3) if used. Adjust the pH to
2,20 with sodium hydroxide (3.11). Transfer quantitatively to a 50 ml graduated flask and make up to the mark
with citrate buffer (3.17) and mix. Store below 5 °C for not more than 3 months. See also 9.1.
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SIST EN ISO 13903:2005
ISO 13903:2005(E)
3.20.5 Calibration solution of standard amino acids, for use with hydrolysates prepared according to
5.3.3.2 and for use with extracts (5.2).
Prepare the calibration solution according to 3.20.4 but omitting sodium chloride. Store below 5 °C for not
more than 3 months.
4 Apparatus
Usual laboratory apparatus and, in particular, the following.
4.1 Round bottomed flask, of capacity 100 ml or 250 ml, fitted with a reflux condenser.
4.2 Borosilicate glass bottle, of capacity 100 ml, with screw cap with rubber/teflon liner (e.g. Duran,
Schott) for use in the oven.
4.3 Oven, with forced ventilation and a temperature regulator, with an accuracy better than ± 2 °C.
4.4 pH-meter, reading to three decimal places.
4.5 Membrane filter, 0,2 µm.
4.6 Centrifuge.
4.7 Rotary vacuum evaporator.
4.8 Mechanical shaker or magnetic stirrer.
4.9 Amino acid analyser or HPLC equipment with ion exchange column, device for ninhydrin,
post-column derivatization and photometric detector
The column is filled with sulfonated polystyrene resins capable of separating the amino acids from each other
and from other ninhydrin-positive materials. The flow in the buffer and ninhydrin lines is provided by pumps
having a flow stability of ± 0,5 % in the period covering both the standard calibration run and the analysis of
the sample.
With some amino acid analysers, hydrolysis procedures may be used in which the hydrolysate has a sodium
concentration of c = 0,8 mol/l and contains all the residual formic acid from the oxidation step. Others do not
give a satisfactory separation of certain amino acids if the hydrolysate contains excess formic acid and/or high
sodium ion concentrations. In this case, reduce the volume of acid by evaporation to approx. 5 ml after the
hydrolysis and prior to pH adjustment. The evaporation shall be performed under vacuum at 40 °C maximum.
5 Procedure
5.1 Preparation of test sample
Grind the sample until it passes through a 0,5 mm sieve. Samples high in moisture shall either be air-dried at
a temperature not exceeding 50 °C or freeze-dried prior to grinding. Samples with a high fat content shall be
extracted with light petroleum (3.6) prior to grinding.
5.2 Determination of free amino acids in feeding stuffs and premixtures
Weigh, to the nearest 0,2 mg, an appropriate amount (1 g to 5 g) of the prepared test sample (5.1) into a
conical flask and add 100,0 ml of extraction mixture (3.14). Shake the mixture for 60 min using a mechanical
shaker or a magnetic stirrer (4.8). Allow the sediment to settle and pipette 10,0 ml of the supernatant solution
into a 100 ml beaker.
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SIST EN ISO 13903:2005
ISO 13903:2005(E)
Add 5,0 ml of sulfosalicylic acid solution (3.15), with stirring, and continue to stir with the aid of magnetic stirrer
for 5 min. Filter or centrifuge the supernatant in order to remove any precipitate. Place 10,0 ml of the resulting
solution into a 100 ml beaker and adjust the pH to 2,20 using sodium hydroxide solution (3.11). Transfer to a
volumetric flask of appropriate volume using citrate buffer (3.17), and make up to the mark with the buffer
solution.
If an internal standard is being used, add 1,00 ml of internal standard (3.20.3) for each 100 ml of final solution
and make up to the mark with the buffer solution (3.17).
Proceed to the chromatography step according to 5.4.
If the extracts are not
...