UGANDA STANDARD  
      US ISO 13904
First Edition
  2009 -09-04
 
Reference numbe r 
   US ISO 13904:2005
 © UNBS 200 9 
  
Animal feeding stuffs — Determination of tryptophan content 
 US ISO 13904:2005  
 
© 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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Tel: 256 41 505 995 
Fax: 256 41 286 123 
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    US ISO 13904: 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 13904:2005,  Animal feeding stuffs — Determination of tryptophan content,
is identical with and  has been reproduced from an International Standard ISO 13904:2005, Animal feeding
stuffs — Determination of tryptophan 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 13904:2005(E)
© ISO 2005
  
 
 INTERNATIONAL 
STANDARD  ISO
13904
First edition
2005-05-01
Animal feeding stuffs — Determination 
of tryptophan content 
Aliments des animaux — Détermination de la teneur en tryptophane 
 ISO 13904:2005(E) 
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ii © ISO 2005 – All rights reserved
  ISO 13904:2005(E) 
© ISO 2005 – All rights reserved  iii
 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) 
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 13904 was prepared by Technical Committee ISO/TC 34, Food products , Subcommittee SC 10, Animal 
feeding stuffs . 
 INTERNATIONAL STANDARD ISO 13904:2005(E)
 
© ISO 2005 – All rights reserved  1
 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 C18 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: αααα-methyltryptophan  (purity/content W 99 %), dried under vacuum over 
phosphorus pentoxide. 
3.4 Barium hydroxide octahydrate . 
Care should be taken not to expose the Ba(OH)2⋅8H2O excessively to air in order to avoid formation of BaCO3, 
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 , ρ20 = 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) 
2 © ISO 2005 – All rights reserved
 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 C18, 3 µm packing, or equivalent. 
4.3 pH-meter . ISO 13904:2005(E) 
© ISO 2005 – All rights reserved  3
 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 high in moisture shall be either air-dried at a 
temperature not exceeding 50 °C or freeze-dried prior to grinding. Samples with high fat content shall be 
extracted with light petroleum (3.9) prior to grinding. 
5.2 Determination of free tryptophan (extract)  
Weigh, to the nearest 1 mg, an appropriate amount (1 g to 5 g) of the prepared sample (5.1) into a conical 
flask. Add 100,0 ml of hydrochloric acid, (3.13) and 5,00 ml of concentrated internal standard solution (3.16). Shake or mix for 60 min using a mechanical shaker or a magnetic stirrer (4.7). Allow the sediment to settle and pipette 10,0 ml of the supernatant solution into a beaker. Add 5 ml of orthophosphoric acid (3.14). Adjust the pH to 3,0 using sodium hydroxide (3.10). Add sufficient methanol (3.8) to give a concentration of between 10 % and 30 % of methanol in the final volume. Transfer to a volumetric flask of appropriate volume and dilute 
with water (3.1) to a volume necessary for the chromatography [approximately the same volume as the 
calibration standard solution (3.17)]. 
Filter a few millilitres of the solution through a 0,45 µm membrane filter (4.5) before injection on the HPLC 
column. Proceed to the chromatography step according to 5.4. 
Protect the standard solution and extracts against direct sunlight. If it is not possible to analyse the extracts 
the same day, the extracts may be stored at 5 °C for a maximum of three days. 
5.3 Determination of total tryptophan (hydrolysate)  
Weigh, to the nearest 0,2 mg, from 0,1 g to 1 g of the prepared sample (5.1) into the polypropylene flask (4.4). 
The weighed test portion should have a nitrogen content of about 10 mg. Add 8,4 g of barium hydroxide octahydrate (3.4) and 10 ml of water (3.1). Mix on a vortex mixer (4.8) or magnetic stirrer (4.7). Leave the 
Teflon-coated magnet in the mixture. Wash down the walls of the vessel with 4 ml of water (3.1). Put on the 
screw cap and close the flask loosely. Transfer to an autoclave (4.6) which contains boiling water, and steam 
for 30 min to 60 min. Close the autoclave and autoclave at (110 ± 2) °C for 20 h. 
Before opening the autoclave, reduce the temperature to just under 100 °C. In order to avoid crystallization of 
Ba(OH)
2·8H2O, add to the warm mixture 30 ml of water (3.1) which is at room temperature. Shake or stir 
gently. Add 2,00 ml of concentrated internal standard solution ( α-methyltryptophan) (3.16). Cool the vessel in 
a water/ice bath for 15 min. 
Then, add 5 ml of orthophosphoric acid (3.14). Keep the vessel in the cooling bath and neutralize with 6 mol/l 
HCl (3.11) whilst stirring and adjust the pH to 3,0 using 1 mol/l HCl (3.12). Add sufficient methanol to give a 
concentration of between 10 % and 30 % of methanol in the final volume. Transfer to a volumetric flask of appropriate volume and dilute with water (3.1) to the defined volume necessary for the chromatography (for example 100 ml). The addition of methanol should not cause precipitation. ISO 13904:2005(E) 
4 © ISO 2005 – All rights reserved
 Filter a few millilitres of the solution through a 0,45 µm membrane filter (4.5) before injection on the HPLC 
column. Proceed to the chromatography step according to 5.4. 
Protect the standard solution and hydrolysates against direct sunlight. If it is not possible to analyse the 
hydrolysates the same day, they may be stored at 5 °C for a maximum of three days. 
5.4 HPLC determination 
The following conditions for isocratic elution are offered for guidance; other conditions may be used, provided 
they yield equivalent results (see also observations B.1 and B.2): 
Liquid chromatographic column (4.2): 125 mm × 4 mm, with C18, 3 µm packing or equivalent 
Column temperature: Room temperature 
Mobile phase (3.20): 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. 
Flow rate: 1 ml/min 
Total run time: approximately 34 min 
Detection wavelength: excitation: 280 nm; emission: 356 nm 
Injection volume: 20 µl 
6 Calculation of results 
The content of tryptophan, w, in grams per 100 g of sample, is calculated by 
is,cal try,sam try try is,sam
is,sam try,cal is,cal100 AA V c V
wAA V m×× × × ×
=×× × 
where 
Ais,cal is the peak area of the internal standard in the calibration standard solution (3.17); 
Atry,sam is the peak area of tryptophan in the extract (5.2) or hydrolysate (5.3); 
Vtry is the volume, in millilitres (2 ml), of concentrated tryptophan solution (3.15) added to the 
calibration solution (3.17); 
ctry is the concentration, in grams per millilitre (= 2,50), of concentrated tryptophan solution (3.15) 
added to the calibration solution (3.17); 
Vis,sam is the volume, in millilitres, of concentrated internal standard solution (3.16) added at the 
extraction (5.2) (= 5,00 ml) or to the hydrolysate (5.3) (= 2,00 ml); 
Ais,sam is the peak area of the internal standard in the extract (5.2) or hydrolysate (5.3); 
Atry,cal is the peak area of the tryptophan calibration standard solution (3.17); 
Vis,cal is the volume, in millilitres (= 2,00 ml), of the concentrated internal standard solution (3.16) 
added to the calibration standard solution (3.17); 
m is the sample mass, in grams (corrected to the original mass if dried and/or defatted). ISO 13904:2005(E) 
© ISO 2005 – All rights reserved  5
 7 Precision 
7.1 Interlaboratory test 
Details of an interlaboratory test on the precision of the method are summarized in Annex A. The values 
derived from this interlaboratory test 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 not in more than 5 % of cases be greater than the repeatability limit r given in Tables A.1 
to A.3. 
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 not in more than 5 % of cases be greater than the reproducibility limit R given in Tables A.1 to A.3. 
8 Test report 
The test report shall specify: 
a) all information necessary for the complete identification of the sample; b) the sampling method used, if known; c) the test method used, with reference to this International Standard; d) all operating details not specified in this International Standard, or regarded as optional, together with 
details of any incidents which may have influenced the test result(s); 
e) the test result(s) obtained, or, if the repeatability has been checked, the final quoted result obtained. ISO 13904:2005(E) 
6 © ISO 2005 – All rights reserved
 Annex A  
(informative) 
 
Results of an interlaboratory test 
An interlaboratory test was arranged within the European Union in which three samples were analysed by up 
to 12 laboratories to certify the method for hydrolysis. Five replicate analyses were performed on each sample. 
The results are given in Table A.1. 
Table A.1 
Sample 1 Sample 2 Sample 3  
Pig feed Pig feed supplemented 
with L-tryptophan Feed concentrate for 
pigs 
Number of laboratories submitting results 12 12 12 
Number of test results from remaining laboratories 50 55 50 
Mean value x, g/kg 2,42 3,40 4,22 
Repeatability standard deviation, sr, g/kg 0,05 0,05 0,08 
Repeatability relative standard deviation, % 1,9 1,6 1,9 
Repeatability limit, r (=2,8 sr), g/kg 0,14 0,14 0,22 
Reproducibility standard deviation, sR, g/kg 0,15 0,20 0,09 
Reproducibility relative standard deviation, % 6,3 6,0 2,2 
Reproducibility limit, R (=2,8 sR), g/kg 0,42 0,56 0,25 
Another collaborative study was arranged in which two samples were analysed by up to 13 laboratories to 
certify the method for extraction of free tryptophan . Five replicate analyses were performed on each sample. 
The results are given in Table A.2. 
Table A.2 
Sample 4 Sample 5  
Wheat and soya mixture Wheat and soya mixture 
(= sample 4) with added 
tryptophan (0,457 g/kg) 
Number of laboratories submitting results 12 12 
Number of test results from remaining laboratories 55 60 
Mean value x, g/kg 0,391 0,931 
Repeatability standard deviation, sr, g/kg 0,005 0,012 
Repeatability relative standard deviation, % 1,34 1,34 
Repeatability limit, r (=2,8 sr), g/kg 0,014 0,034 
Reproducibility standard deviation, sR, g/kg 0,018 0,048 
Reproducibility relative standard deviation, % 4,71 5,11 
Reproducibility limit, R (=2,8 sR), g/kg 0,05 0,134 ISO 13904:2005(E) 
© ISO 2005 – All rights reserved  7
 Another interlaboratory test was arranged in which four samples were analysed by up to seven laboratories 
with the aim of a tryptophan certification for hydrolysis. The results are given in Table A.3. Five replicate analyses were performed on each sample. 
Table A.3 
Sample 1 Sample 2 Sample 3 Sample 4  
Mixed pig feed
(CRM 117)) Low-fat fish 
meal 
(CRM 118) Soybean 
meal 
(CRM 119) Skimmed milk 
powder  
(CRM 120) 
Number of laboratories submitting results 7 7 7 7 
Number of test results from remaining laboratories 25 30 30 30 
Mean value x, g/kg 2,064 8,801 6,882 5,236 
Repeatability standard deviation, sr, g/kg 0,021 0,101 0,089 0,040 
Repeatability relative standard deviation, % 1,04 1,15 1,30 0,76 
Repeatability limit, r (=2,8 sr), g/kg 0,059 0,283 0,249 0,112 
Reproducibility standard deviation, sR, g/kg 0,031 0,413 0,283 0,221 
Reproducibility relative standard deviation, % 1,48 4,69 4,11 4,22 
Reproducibility limit, R (=2,8 sR), g/kg 0,087 1,156 0,792 0,619 ISO 13904:2005(E) 
8 © ISO 2005 – All rights reserved
 Annex B  
(informative) 
 
Observations on the method 
B.1 The following special chromatographic conditions may give better separation between tryptophan and 
α-methyltryptophan. 
Isocratic elution followed by gradient column cleaning: 
Liquid chromatographic column: 125 mm × 4 mm, with C18, 5 µm packing or equivalent 
Column temperature: 32 °C 
Mobile phase: A: 0,01 mol/l KH2PO4/methanol, 95 + 5 (volume fraction) 
 B: Methanol 
Gradient programme: 0 min 100 % A 0 % B 
 15 min 100 % A 0 % B 
 17 min 60 % A 40 % B  19 min 60 % A 40 % B  21 min 100 % A 0 % B  33 min 100 % A 0 % B 
Flow rate: 1,2 ml/min 
Total run time: approximately 33 min 
B.2 The chromatography will vary according to the type of HPLC and column packing material used. The 
system chosen should be capable of giving baseline separation between the tryptophan and the internal 
standard. Moreover it is important that degradation products are well separated from the tryptophan and the internal standard. Hydrolysates without the internal standard should be run in order to check the baseline under the internal standard for impurities. It is important that the run time is sufficiently long for the elution of 
all the degradation products, otherwise late eluting peaks may interfere with subsequent chromatographic 
runs. 
The chromatographic system should give linear response over the range of operation. The linear response 
should be measured with a constant (normal) concentration of the internal standard and varying concentrations of tryptophan. It is important that the size of both the tryptophan and internal standard peaks are within the linear range of the HPLC/fluorescence system. If either the tryptophan and/or the internal 
standard peak(s) is (are) too small or too high, the analysis should be repeated with another sample size 
and/or a changed final volume. 
B.3 With age, barium hydroxide becomes more difficult to dissolve. This results in an unclear solution for 
the HPLC determination, which may produce low results for tryptophan. ISO 13904:2005(E) 
© ISO 2005 – All rights reserved  9
 Bibliography 
[1] Commission Directive 2000/45/EC of 6 July 2000, establishing Community methods of analysis for the 
determination of vitamin A, vitamin E and tryptophan in feedingstuffs ISO 13904:2005(E) 
ICS  65.120 
Price based on 9 pages 
© ISO 2005 – All rights reserved