UGANDA STANDARD  
      US ISO 13903
First Edition
   2009 -09-04
 
Reference numbe r 
   US ISO 13903:2005
 © UNBS 200 9 
  
Animal feeding stuffs — Determination of amino acids content 
 US ISO 13903:2005  
 
© UNBS  2009 – All rights reserved Compliance with this standard does not, of itself confer immunity from legal obligations 
A Uganda Standard does not purport to include all necessary provisions of a contract. Users are 
responsible for its correct application  
© UNBS 2009  
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilised in any 
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permission from UNBS. 
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    US ISO 13903: 2005  
© UNBS  2009 - All rights reserved National foreword 
Uganda National Bureau of Standards (UNBS) is a parastatal under the Ministry  of Tourism, Trade and 
Industry established by the Act of Parliament of 198 3, of the Laws of Uganda.  UNBS is mandated to co-
ordinate the elaboration of standards and is  
(a) a member of International Organisation for Standardisation (ISO) and  
(b) a contact point for the WHO/FAO Codex A limentarius Commission on Food Standards, and  
(c) the National Enquiry Point on TBT/SPS Agr eements of the World Trade Organisation (WTO). 
The work of preparing Uganda Standards is carried out through Technical Committees. A Technical 
Committee is established to deliberate on standards in a gi ven field or area and consists of representatives of 
consumers, traders, academician s, manufacturers, governme nt and other stakeholders. 
Draft Uganda Standards adopted by the Technical Committ ee are widely circulated to stakeholders and the 
general public for comments. The committee reviews the comments before recommending the draft standards 
for approval and declaration as Uganda Standards by the National Standards Council. 
This Uganda Standard, US ISO 13903:2005,  Animal feeding stuffs — Determination of amino acids content,
is identical with and  has been reproduced from an International Standard ISO 13903:2005,  Animal feeding
stuffs — Determination of amino acids content, and has been adopted as a Uganda Standard. 
Wherever the words, "International Standard" a ppear, they should be replaced by "Uganda Standard."   
 
  
 Reference numbe r
ISO 13903:2005(E)
© ISO 2005
  
 
 INTERNATIONAL 
STANDARD  ISO
13903
First edition
2005-05-15
Animal feeding stuffs — Determination 
of amino acids content 
Aliments des animaux — Détermination de la teneur en acides aminés 
 ISO 13903:2005(E) 
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ii © ISO 2005 – All rights reserved
  ISO 13903:2005(E) 
© ISO 2005 – All rights reserved  iii
 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) 
iv © ISO 2005 – All rights reserved
 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 . 
ISO 13903 is based on Commission Directive 98/64/EC of September 1998
 [1]. 
 INTERNATIONAL STANDARD ISO 13903:2005(E)
 
© ISO 2005 – All rights reserved  1
 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 © ISO 2005 – All rights reserved
 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 P2O5 or H2SO4 for 
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. ISO 13903:2005(E) 
© ISO 2005 – All rights reserved  3
 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. ISO 13903:2005(E) 
4 © ISO 2005 – All rights reserved
 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. ISO 13903:2005(E) 
© ISO 2005 – All rights reserved  5
 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 being examined the same day, they shall be stored below 5 °C. 
5.3 Determination of total amino acids 
5.3.1 Oxidation 
Weigh, to the nearest 0,2 mg, from 0,1 g to 1 g of the prepared test sample (5.1) into 
 a 100 ml round-bottomed flask (4.1) for open hydrolysis (5.3.2.3), or 
 a 250 ml round-bottomed flask (4.1) if a low sodium concentration is required (5.3.3.2), or 
 a 100 ml bottle, fitted with a screw cap (4.2), for closed hydrolysis (5.3.2.4). 
The weighed test portion shall have a nitrogen content of about 10 mg and a moisture content not exceeding 
100 mg. 
Place the flask/bottle in an ice-water bath and cool to 0 °C. Add 5 ml of oxidation mixture (3.16) and mix using 
a glass spatula with a bent tip. Seal the flask/bottle containing the spatula with an airtight film, place the 
ice-water bath containing the sealed container in a refrigerator at 0 °C and leave for 16 h. After 16 h, remove 
from the refrigerator and decompose the excess oxidation reagent by the addition of 0,84 g of sodium disulfite. 
Proceed to 5.3.2.1. 
5.3.2 Hydrolysis 
5.3.2.1 Hydrolysis of oxidized samples 
To the oxidized sample prepared according to 5.3.1, add 25 ml of hydrolysis mixture (3.13), taking care to 
wash down any sample residue adhering to the sides of the vessel and the spatula. Depending on the 
hydrolysis procedure being used, proceed according to 5.3.2.3 or 5.3.2.4. 
5.3.2.2 Hydrolysis of unoxidized samples 
Weigh, to the nearest 0,2 mg, into a 100 ml or 250 ml round-bottom flask (4.1) or into a 100 ml bottle fitted 
with a screw cap (4.2) from 0,1 g to 1 g of the prepared sample (5.1). The weighed sample portion shall have a nitrogen content of about 10 mg. Carefully add 25 ml of hydrolysis mixture (3.13) and mix with the sample. 
Proceed according to either 5.3.2.3 or 5.3.2.4. 
5.3.2.3 Open hydrolysis 
Add three glass beads to the mixture in the flask (prepared in accordance with 5.3.2.1 or 5.3.2.2) and boil with 
continuous bubbling under reflux for 23 h. On completion of hydrolysis, wash the condenser down with 5 ml of 
citrate buffer (3.17). Disconnect the flask and cool it in an ice bath. Proceed according to 5.3.3. ISO 13903:2005(E) 
6 © ISO 2005 – All rights reserved
 5.3.2.4 Closed hydrolysis 
Place the bottle containing the mixture prepared in accordance with 5.3.2.1 or 5.3.2.2 in an oven (4.3) set at 
110 °C. During the first hour, in order to prevent a build up of pressure (due to the evolution of gaseous substances) and to avoid explosion, place the screw cap over the top of the vessel. Do not close the vessel with the cap. After 1 h, close the vessel with the cap and leave in the oven (4.3) for 23 h. On completion of hydrolysis, remove the bottle from the oven, carefully open the cap of the bottle and place the bottle in an 
ice-water bath. Leave to cool. 
Depending on the procedure for pH adjustment (5.3.3), quantitatively transfer the contents of the bottle to a 
250 ml beaker or a 250 ml round-bottom flask, using citrate buffer (3.17). 
Proceed according to 5.3.3. 
5.3.3 Adjustment of pH 
5.3.3.1 Depending on the sodium tolerance of the amino acid analyser (4.9), proceed according to 
5.3.3.2 or 5.3.3.3 for the pH adjustment. 
5.3.3.2 When amino acid analysers requiring a low sodium concentration are used (when the acid 
volume has to be reduced) and for chromatographic systems (4.9) requiring a low sodium concentration, it is 
advisable to use an internal stock standard solution (3.20.3). 
In this case, add 2,00 ml of the internal stock standard solution (3.20.3) to the hydrolysate before the 
evaporation. 
Add two drops of 1-octanol to the hydrolysate obtained in accordance with 5.3.2.3 or 5.3.2.4. 
Using a rotary evaporator (4.7), reduce the volume to 5 ml to 10 ml under vacuum at 40 °C. If the volume is 
accidentally reduced to less than 5 ml, the hydrolysate shall be discarded and the analysis recommenced. 
Adjust the pH to 2,20 with sodium hydroxide solution II (3.11) and proceed to 5.3.4. 
5.3.3.3 For chromatographic systems (4.9) not requiring a low sodium concentration, take the 
hydrolysates obtained in accordance with 5.3.2.3 or 5.3.2.4 and partly neutralize them by carefully adding, 
with stirring, 17 ml of sodium hydroxide solution I (3.10), ensuring that the temperature is kept below 40 °C. 
Adjust the pH to 2,20 at room temperature using sodium hydroxide solution I (3.10) and finally sodium 
hydroxide solution II (3.11). Proceed to 5.3.4. 
5.3.4 Sample solution for chromatography 
Quantitatively transfer the pH-adjusted hydrolysate (5.3.3.2 or 5.3.3.3) with citrate buffer (3.17) to a 200 ml 
graduated flask, and make up to the mark with the buffer. 
If an internal standard has not already been used, add 2,00 ml of internal standard (3.20.3) and make up to 
the mark with citrate buffer (3.17). Mix thoroughly. 
Proceed to the chromatography step (5.4). If the sample solutions are not to be examined the same day, they shall be stored below 5 °C. 
5.4 Chromatography 
Before conducting the chromatography, bring the extract (5.2) or hydrolysate (5.3.4) to room temperature. 
Shake the mixture and filter a suitable amount through a 0,2 µm membrane filter (4.5). Subject the resulting clear solution to ion exchange chromatography, using an amino acid analyser or HPLC equipment (4.9). ISO 13903:2005(E) 
© ISO 2005 – All rights reserved  7
 The injection may be performed manually or automatically. It is important that the same quantity of ± 0,5 % 
solution is added to the column for the analysis of standards and samples except when an internal standard is 
used, and that the sodium:amino acid ratios in the standard and sample solutions are as similar as is practicable. 
In general, the frequency of the calibration runs depends on the stability of the ninhydrin reagent and the 
analytical system. Dilute the standard or sample with citrate buffer (3.17) to give a peak area of the standard of 30 % to 200 % of the sample amino acid peak area. 
The chromatogram of amino acids will vary slightly according to the type of analyser employed and resin used. 
The chosen system shall be capable of separating the amino acids from each other and from the 
ninhydrin-positive materials. In the range of operation the chromatographic system shall give a linear response to changes in the amounts of amino acids added to the column. 
During the chromatography step, the valley:peak height ratios mentioned below apply, when an equimolar 
solution (of the amino acids being determined) is analysed. This equimolar solution shall contain at least 30 % 
of the maximum load of each amino acid that can be accurately measured with the amino acid analyser 
system (4.9). 
For separation of threonine-serine, the valley:peak height ratio of the lower of the two overlapping amino acids 
on the chromatogram shall not exceed 2:10 (if only cyst(e)ine, methionine, threonine and lysine are 
determined, insufficient separation from adjoining peaks will adversely influence the determination). For all other amino acids, the separation shall be better than 1:10. 
The system shall ensure that lysine is separated from 'lysine artifacts' and ornithine. 
6 Calculation of results 
The area of the sample and standard peaks is measured for each individual amino acid and the amount, w, in 
grams of amino acid per kilogram of sample, is calculated: 
ee
c 1000A cMVwAm×× ×=×× 
If an internal standard is used, multiply by ic
ieA
A 
where 
Ae is the peak area of the hydrolysate or extract; 
Ac is the peak area of the calibration standard solution; 
Aie is the peak area of the internal standard in the hydrolysate or extract; 
Aic is the peak area, of the internal standard in the calibration standard solution; 
M is the molecular mass of the amino acid being determined; 
c is the concentration of standard, in µmol/ml; 
m is the sample mass, in grams (corrected to original mass if dried or defatted); 
Ve is the volume of total hydrolysate (5.3.4), in millilitres, the calculated total dilution volume of extract 
(6.1), in millilitres. ISO 13903:2005(E) 
8 © ISO 2005 – All rights reserved
 Cystine and cysteine are both determined as cysteic acid in hydrolysates of the oxidized sample, but 
calculated as the sum of cystine and cysteine by using M = 120,15 ( = 0,5 × 240,30) (C6H12N2O4S2, 
M = 240,30). 
Methionine is determined as methionine sulfone in hydrolysates of the oxidized sample, but calculated as 
methionine by using M of methionine = 149,21. 
Added free methionine is determined after extraction as methionine; for the calculation, the same value of M is 
used. 
The total dilution volume of extracts ( Ve) for determination of free amino acids (5.2) is calculated as follows: 
()ef
e10 510010 10VV+=× ×  
where Vef is the volume of final extract, in millilitres. 
The results may be expressed as a percentage: result in percent = 0,1 × result in grams per kilogram. 
7 Precision 
7.1 Interlaboratory tests 
Details of interlaboratory tests on the precision of the method are summarized in Annex A. The values derived 
from these interlaboratory tests may not be applicable to concentration ranges and matrices other than those 
given. 
7.2 Repeatability 
The absolute difference between two independent single test results, obtained using the same method on identical test material in the same laboratory by the same operator using the same equipment within a short interval of time, will in not more than 5 % of cases be greater than the repeatability limit r (= 2,83 s
r) given in 
Table A.2 for the first test, and in Tables A.6 to A.10 for the second test 
7.3 Reproducibility 
The absolute difference between two single test results, obtained using the same method on identical test material in different laboratories with different operators using different equipment, will in not more than 5 % of 
cases be greater than the reproducibility limit R (= 2,83 s
R) given in Table A.4 for the first test, and in 
Tables A.6 to A.10 for the second test 
8 Use of reference materials 
The correct application of the method shall be verified by making replicate measurements of certified 
reference materials when available. Calibration with certified amino acid calibration solution is recommended. 
9 Observations on the method 
9.1 Because of differences between amino acid analysers, the final concentrations of the calibration 
solutions of standard amino acids (see 3.20.4 and 3.20.5) and of the hydrolysate (see 5.3.4) shall be taken as 
a guideline. 
The range of linear response of the apparatus shall be checked for all amino acids. ISO 13903:2005(E) 
© ISO 2005 – All rights reserved  9
 The standard solution is diluted with citrate buffer to give peak areas in the middle of the range. 
9.2 Where high-performance liquid chromatographic equipment is used to analyse the hydrolysates, the 
experimental conditions shall be optimized in accordance with the manufacturer's recommendations. 9.3 By applying the method to feeding stuffs containing more than 1 % chloride (concentrate, mineral feeds, 
supplementary feeds), underestimation of methionine could occur and special treatment shall be carried out in 
the following way (the Slump method). After addition of 5 ml of the oxidation mixture in 5.3.1, add 12,5 ml of 
water and stir the sample mixture on a stirrer for 15 min. Afterwards follow the normal procedure. This 
procedure is not quantitative for determination of the sum of cysteine and cystine. 
 ISO 13903:2005(E) 
10 © ISO 2005 – All rights reserved
 Annex A  
(informative) 
 
Results of interlaboratory tests 
A.1 First test 
The method has been tested in a collaborative test made at the international level in 1990 using four different 
feeding stuffs (mixed pig feed, broiler compound, protein concentrate and premixture). The results, after 
elimination of outliers, are given in Table A.1. The repeatability expressed as within-laboratory standard 
deviation of this test is given in Tables A.2 and A.3. The results for the between-laboratory standard deviation 
for this test are given in Tables A.4 and A.5. 
Table A.1 — Amino acids content 
Amino acid 
threonine sum of cystine and 
cysteine methionine lysine Reference material 
g/kg g/kg g/kg g/kg 
Mixed pig feed 6,94 
n = 15 3,01 
n = 17 3,27 
n = 17 9,55 
n = 13 
Broiler compound 9,31 
n = 16 3,92 
n = 18 5,08 
n = 18 13,93 
n = 16 
Protein concentrate 22,32 
n = 16 5,06 
n = 17 12,01 
n = 17 47,74 
n = 15 
Premixture 58,42 
n = 16 — 90,21 
n = 16 98,03 
n = 16 
n is the number of participating laboratories. 
 
Table A.2 — Within-laboratory standard deviation (sr) 
Amino acid 
threonine sum of cystine and 
cysteine methionine lysine Reference material 
g/kg g/kg g/kg g/kg 
Mixed pig feed 0,13 
n = 15 0,10 
n = 17 0,11 
n = 17 0,26 
n = 13 
Broiler compound 0,20 
n = 16 0,11 
n = 18 0,16 
n = 18 0,28 
n = 16 
Protein concentrate 0,48 
n = 16 0,13 
n = 17 0,27 
n = 17 0,99 
n = 15 
Premixture 1,30 
n = 16 — 2,19 
n = 16 2,06 
n = 16 
n is the number of participating laboratories. ISO 13903:2005(E) 
© ISO 2005 – All rights reserved  11
 Table A.3 — Coefficient of variation  (%) for within-laboratory standard deviation (sr) 
Amino acid 
threonine sum of cystine and 
cysteine methionine lysine Reference material 
g/kg g/kg g/kg g/kg 
Mixed pig feed 1,9 
n = 15 3,3 
n = 17 3,4 
n = 17 2,8 
n = 13 
Broiler compound 2,1 
n = 16 2,8 
n = 18 3,1 
n = 18 2,1 
n = 16 
Protein concentrate 2,7 
n = 16 2,6 
n = 17 2,2 
n = 17 2,4 
n = 15 
Premixture 2,2 
 n = 16 — 2,4 
n = 16 2,1 
n = 16 
n is the number of participating laboratories. 
 
Table A.4 — Between-laboratory standard deviation (sR) 
Amino acid 
threonine sum of cystine and 
cysteine methionine lysine Reference material 
g/kg g/kg g/kg g/kg 
Mixed pig feed 0,28 
n = 15 0,30 
n = 17 0,23 
n = 17 0,30 
n = 13 
Broiler compound 0,48 
n = 16 0,34 
n = 18 0,55 
n = 18 0,75 
n = 16 
Protein concentrate 0,85 
n = 16 0,62 
n = 17 1,57 
n = 17 1,24 
n = 15 
Premixture 2,49 
n = 16 — 6,20 
n = 16 6,62 
n = 16 
n is the number of participating laboratories. 
 
Table A.5 — Coefficient of variation  (%) for between-laboratory standard deviation  (sR) 
Amino acid 
threonine sum of cystine and 
cysteine methionine lysine Reference material 
[g/kg] [g/kg] [g/kg] [g/kg] 
Mixed pig feed 4,1 
n = 15 9,9 
n = 17 7,0 
n = 17 3,2 
n = 13 
Broiler compound 5,2 
n = 16 8,8 
n = 18 10,9 
n = 18 5,4 
n = 16 
Protein concentrate 3,8 
n = 16 12,3 
n = 17 13,0 
n = 17 3,0 
n = 15 
Premixture 4,3 
n = 16 — 6,9 
n = 16 6,7 
n = 16 
n is the number of participating laboratories. ISO 13903:2005(E) 
12 © ISO 2005 – All rights reserved
 A.2 Second test 
Blind duplicate pairs of broiler finisher feed, broiler starter feed, corn, fishmeal and poultry meal samples were 
submitted to twenty-three laboratories. The results are given in Tables A.6 to A.10. 
Table A.6 — Method performance for analysis of amino acids in poultry meal 
Amino acid No. of single 
determinations Mean sr Standard deviation 
of repeatability sR Standard 
deviation of 
reproducibility 
    %  % 
Alanine 42 4,26 0,087 2,04 0,210 4,93 
Arginine 46 4,35 0,144 3,31 0,420 9,66 
Aspartic acid 44 4,92 0,132 2,68 0,376 7,64 
Cystine plus cysteine 42 0,81 0,037 4,57 0,143 17,65 
Glutamic acid 46 7,97 0,216 2,71 0,728 9,13 
Glycine 38 6,90 0,085 1,23 0,286 4,14 
Histidine 38 1,31 0,036 2,75 0,242 18,47 
Isoleucine 46 2,24 0,060 2,68 0,261 11,65 
Leucine 46 4,09 0,101 2,47 0,310 7,58 
Lysine 46 3,63 0,112 3,09 0,360 9,92 
Methionine 40 1,17 0,025 2,14 0,140 11,97 
Phenylalanine 44 2,33 0,082 3,52 0,215 9,23 
Proline 34 4,53 0,102 2,25 0,283 6,25 
Serine 44 2,76 0,116 4,20 0,347 12,57 
Threonine 44 2,32 0,073 3,15 0,212 9,14 
Valine 44 2,82 0,090 3,19 0,361 12,80 
 
Table A.7 — Method performance for analysis of amino acids in broiler finisher feed 
Amino acid No. of single 
determinations Mean sr Standard deviation 
of repeatability sR Standard 
deviation of 
reproducibility 
    %  % 
Alanine 44 1,17 0,032 2,74 0,099 8,46 
Arginine 44 1,28 0,030 2,34 0,110 8,59 
Aspartic acid 46 1,68 0,047 2,80 0,121 7,20 
Cystine plus cysteine 38 0,32 0,010 3,13 0,036 11,25 
Glutamic acid 44 3,25 0,052 1,60 0,226 6,95 
Glycine 46 1,27 0,029 2,28 0,085 6,69 
Histidine 34 0,50 0,020 4,00 0,099 19,80 
Isoleucine 42 0,76 0,024 3,16 0,052 6,84 
Leucine 46 1,66 0,044 2,65 0,105 6,33 
Lysine 44 1,07 0,037 3,46 0,096 8,97 
Methionine 38 0,53 0,006 1,13 0,040 7,55 
Phenylalanine 44 0,87 0,038 4,37 0,127 14,60 
Proline 36 1,39 0,044 3,17 0,124 8,92 
Serine 44 0,94 0,044 4,68 0,127 13,51 
Threonine 40 0,73 0,020 2,74 0,060 8,22 
Valine 44 0,92 0,035 3,80 0,117 12,72 ISO 13903:2005(E) 
© ISO 2005 – All rights reserved  13
 Table A.8 — Method performance for analysis of amino acids in broiler starter feed 
Amino acid No. of single 
determinations Mean sr Standard deviation 
of repeatability sR Standard 
deviation of 
reproducibility 
    %  % 
Alanine 46 1,28 0,027 2,11 0,100 7,81 
Arginine 46 1,57 0,042 2,68 0,129 8,22 
Aspartic acid 40 2,29 0,035 1,53 0,137 5,98 
Cystine plus cysteine 38 0,35 0,006 1,71 0,056 16,00 
Glutamic acid 46 4,04 0,072 1,78 0, 339 8,39 
Glycine 46 1,27 0,034 2,68 0,090 7,09 
Histidine 40 0,65 0,018 2,77 0,100 15,38 
Isoleucine 46 0,95 0,019 2,00 0,098 10,32 
Leucine 46 1,97 0,033 1,68 0,124 6,29 
Lysine 46 1,35 0,032 2,37 0,122 9,04 
Methionine 42 0,62 0,013 2,10 0,063 10,16 
Phenylalanine 42 1,12 0,025 2,23 0,101 9,02 
Proline 36 1,47 0,060 4,08 0,128 8,71 
Serine 44 1,12 0,028 2,50 0,153 13,66 
Threonine 44 1,12 0,024 2,73 0,087 9,89 
Valine 40 1,11 0,019 1,71 0,098 8,83 
 
Table A.9 — Method performance for analysis of amino acids in corn 
Amino acid No. of single 
determinations Mean sr Standard deviation 
of repeatability sR Standard 
deviation of 
reproducibility 
    %  % 
Alanine 44 0,61 0,009 1,48 0,049 8,03 
Arginine 44 0,40 0,013 3,25 0,038 9,50 
Aspartic acid 44 0,54 0,015 2,78 0,045 8,33 
Cystine plus cysteine 38 0,18 0,007 3,89 0,025 13,89 
Glutamic acid 40 1,51 0,036 2,38 0,094 6,23 
Glycine 46 0,33 0,010 3,03 0,030 9,09 
Histidine 42 0,27 0,019 7,04 0,063 23,33 
Isoleucine 44 0,28 0,015 5,38 0,041 14,62 
Leucine 44 0,99 0,019 1,92 0,069 6,97 
Lysine 44 0,26 0,008 3,08 0,034 13,08 
Methionine 42 0,18 0,010 5,56 0,021 11,67 
Phenylalanine 40 0,38 0,009 2,37 0,073 19,21 
Proline 34 0,73 0,019 2,60 0,044 6,03 
Serine 40 0,39 0,007 1,79 0,040 10,26 
Threonine 44 0,29 0,012 4,14 0,034 11,72 
Valine 46 0,38 0,009 2,37 0,061 16,05 ISO 13903:2005(E) 
14 © ISO 2005 – All rights reserved
 Table A.10 — Method performance for analysis of amino acids in fishmeal 
Amino acid No. of single 
determinations Mean sr Standard deviation 
of repeatability sR Standard 
deviation of 
reproducibility 
    %  % 
Alanine 42 3,50 0,073 2,09 0223 6,37 
Arginine 46 3,40 0,117 3,00 0,280 7,18 
Aspartic acid 46 5,22 0,108 2,07 0,327 6,26 
Cystine plus cysteine 38 0,48 0,019 3,96 0,091 18,96 
Glutamic acid 46 7,37 0,063 0,85 0,347 4,71 
Glycine 46 3,84 0,059 1,54 0,215 5,60 
Histidine 38 1,37 0,033 2,41 0,176 12,85 
Isoleucine 46 2,32 0,049 2,11 0,238 10,26 
Leucine 46 4,07 0,079 1,94 0,276 6,78 
Lysine 44 4,22 0,117 2,77 0,335 7,94 
Methionine 42 1,61 0,030 1,86 0, 156 9,69 
Phenylalanine 40 2,29 0, 037 1,62 0, 176 7,69 
Proline 36 2,62 0,079 3,02 0,326 12,44 
Serine 42 2,21 0,048 2,17 0,248 11,22 
Threonine 44 2,28 0, 081 3,55 0,244 10,70 
Valine 44 2,78 0,063 2,27 0,311 11,19 
 ISO 13903:2005(E) 
© ISO 2005 – All rights reserved  15
 Annex B  
(informative) 
 
Examples of chromatograms 
 
Key 
X retention time, min 
Y voltage, volts 
Figure B.1 — Amino acid standard  (570 nm)  ISO 13903:2005(E) 
16 © ISO 2005 – All rights reserved
 
 
Key 
X retention time, min 
Y voltage, volts 
Figure B.2 — Oxidized sample of pig feed (570 nm)  
 ISO 13903:2005(E) 
© ISO 2005 – All rights reserved  17
 Bibliography 
[1] Commission Directive 98/64/EC of 3 September 1998, establishing Community methods of analysis for 
the determination of amino acids, crude oils and fats, and olaquindox in feeding stuffs 
[2] L LAMES  and F ONTAINE , Journal of AOAC International , 77, No. 6, 1994, pp. 1362-1402 
 ISO 13903:2005(E) 
ICS  65.120 
Price based on 17 pages 
© ISO 2005 – All rights reserved