UGANDA STANDARD  
      US ISO 6866
First Edition
  2009 -09-04
 
Reference numbe r 
  US ISO 6866:1985
 © UNBS 200 9 
  
Animal feeding stuffs — Determination of free and total 
gossypol 
 US ISO 6866:1985  
 
© 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 
form or by any means, electronic or mechanical, in cluding photocopying and microfilm, without prior written 
permission from UNBS. 
Requests for permission to reproduce this document should be addressed to 
The Executive Director 
Uganda National Bureau of Standards 
P.O. Box 6329 
Kampala  
Uganda 
Tel: 256 41 505 995 
Fax: 256 41 286 123 
E-mail: unbs@infocom.co.ug
     US ISO 6866:1985  
© 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 6866:1985,  Animal feeding stuffs — Determination of free and total 
gossypol, is identical with and  has been reproduced from an International Standard ISO 6866:1985, Animal
feeding stuffs — Determination of free and total gossypol, and has been adopted as a Uganda Standard. 
Wherever the words, "International Standard" a ppear, they should be replaced by "Uganda Standard." z - International Standard 
INTERNATIONAL ORGANIZATION FOR STANDARDIZATION.MEX~YHAPO~HAR OPTAHM3AUMR I-IO CTAHCIAPTM3AI_IMI4@0RGANISATION INTERNATIONALE DE NORMALISATION 
Animal feeding stuffs - Determination of free and total 
gossypol 
Aliments des animaux - Dosage du goss ypol libre et du goss ypol total 
First edition - 1985-11-15 
UDC 636.085.19 : 543.8 Ref. No. ISO 68664985 (E) 
Descriptors : agricultural products, animal feeding products, Chemical analysis, determination of content, gossypol. 
CD 
8 
0 
m - Price based on 3 pages 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. Esch 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, govern- 
mental and non-governmental, in liaison with ISO, also take part in the work. 
Draft International Standards adopted by the technical committees are circulated to 
the member bodies for approval before their acceptance as International Standards by 
the ISO Council. They are approved in accordance with ISO procedures requiring at 
least 75 % approval by the member bodies voting. 
International Standard ISO 6866 was prepared by Technical Committee ISO/TC 34, 
Agricultural food products. 
Users should note that all International Standards undergo revision from time to time 
and that any reference made herein to any other International Standard implies its 
latest edition, unless otherwise stated. 
0 International Organkation for Standardkation, 1985 Q 
Printed in Switzerland INTERNATIONAL STANDARD ISO 6866-1985 (E) 
Animal feeding stuffs - Determination of free and total 
gossypol 
1 Scope 
This International Standard specifies a method for the deter- 
mination of the content of free and total gossypol and 
chemically related substances in animal feeding stuffs. 
2 Field of application 
The method is applicable to cottonseed and cottonseed meals 
and cakes, and to compound feeding stuffs containing these 
substances. 
The detection limit for free gossypol is 20 mg/kg and that for 
total gossypol is 50 mg/kg. 
3 References 
ISO 6497, Animal feeding stuffs - Sampling. 1) 
I S 0 6498, Animal feeding s tuffs - Prepara tion of test samples. 
4 Principle 
Extraction of the gossypol in the presence of 3-aminopropan-l- 
01, either with a mixture of 2-propanol and hexane for the deter- 
mination of free gossypol or with dimethylformamide for the 
determination of total gossypol. Conversion of the gossypol in- 
to gossypol-dianiline using aniline. Measurement of the absorb- 
ante at the wavelength of maximum absorbance (between 435 
and 445 nm). 
5 Reagents 
All reagents shall be of recognized analytical grade. The water 
used shall be distilled water or water of at least equivalent purity. 
5.1 2-Propanollrz-hexane, 60 + 40 mixture by volume. 
5.2 Solvent A. 
Place in a 1 000 ml volumetric flask approximately 500 ml of the 
2-propanol/hexane mixture (5.11, 2 ml of 3-aminopropan-1-01, 
8 ml of glacial acetic acid and 50 ml of water. Make up to 
volume with the 2-propanollhexane mixture. 
This reagent is stable for one week only. 5.3 Solvent 5. 
Place in a 100 ml volumetric flask 2 ml of 3-aminopropan-1-01 
and 10 ml of glacial acetic acid. Cool to room temperature and 
make up to volume with NJV-dimethylformamide. 
This reagent is stable for one week only. 
5.4 Aniline. 
If the absorbance determined in the blank test exceeds 0,022, 
distil the aniline over zinc dust, discarding the first and last 
10 % fractions of distillate. Store in a stoppered brown glass 
bottle in a refrigerator (0 to 4 “CL 
The reagent is stable for several months. 
6 Apparatus 
Usual laboratory apparatus and in particular 
6.1 Tumbler mixer, having a rotational frequency of 
approximately 35 min -1, or other shaker. 
6.2 Spectrometer, suitable for making measurements at 
wavelengths between 435 and 445 nm, with cells of Optical path 
length 10 mm. 
6.3 Volumetric flasks, of capacities 25 and 50 mI. 
6.4 Conical flasks, of capacities 50, 100 and 250 ml. 
7 Sarnpling 
Take the laboratory Sample in accordance with ISO 6497. 
1) At present at the Stage of draft. ISO 68664985 (EI 
8 Procedure 8.3.1.8 Measure the absorbance of the blank test Solution 
(flask F; 8.3.1.7) against the reference Solution (flask E; 
8.1 Preparation of the test Sample 
Prepare the test Sample in accordance with ISO 6498. 8.3.1.5) and the absorbance of the test Solution (flask D; 
8.3.1.7) against the reference Solution (flask C; 8.3.1.3) using 
the spectrometer (6.2) at the wavelength of maximum absorb- 
ante (between 435 and 445 nm). 
8.2 Test Portion 
The mass of the test Portion depends on the expected gossypol 
content. For the determination of free gossypol in cottonseed 
and cottonseed meals and cakes the mass of the test Portion 
should not exceed 1 g; for compound feeding stuffs, it may be 
as much as 5 g. For the determination of total gossypol, the 
mass of the test Portion should be between 0,5 and 5 g. 
NOTE - The mass of the test portion should be large enough to give 
the quantity of free gossypol indicated in 8.3.1.2 or the quantity of total 
gossypol indicated in 8.3.2.4 in each aliquot Portion of the filtrate. 
8.3 Determination 
8.3.1 Determination of free gossypol 
8.3.1.1 Transfer the test Portion (8.2) to a 250 ml conical 
flask, the bottom of which has been covered with glass beads. 
Add, by means of a pipette, 50 ml of solvent A (5.2), stopper 
the flask and mix, using the tumbler mixer (6.1), for 1 h. Filter 
through a dry filter Paper, collecting the filtrate in a 100 ml 
conical flask. During filtration, cover the funnel with a 
watch-glass. 
8.3.1.2 Transfer, by means of a pipette, identical aliquot por- 
tions sf the filtrate, containing 50 to 100 pg of gossypol, to 
each of two 25 ml volumetric flasks (6.3) (C and D). If 
necessary, make up to 10 ml with solvent A (5.2). 
8. 3.1.3 Make up the contents of flask C to volume 
2- ,propanol/ hexane mixture (5.1 ) and mix. 
This Solution will be used as the reference 
which the test Solution will be measured. 8.3.1.9 Subtract the absorbance of the blank test Solution 
from that of the test Solution to obtain the corrected ab- 
sorbance. 
8.3.2 Determination of total gossypol 
8.3.2.1 Transfer the test Portion (8.2) into a 50 ml volumetric 
flask and add 10 ml of solvent B (5.3). 
8.3.2.2 Transfer 10 ml of solvent B (5.3) to a second 50 ml 
volumetric flask (6.3). 
8.3.2.3 Heat the two flasks (8.3.2.1 and 8.3.2.2) for 30 min on 
a boiling water-bath. 
Cool to room temperature and make up to volume with the 
2-propanol/hexane mixture (5.1). Mix and leave to settle for 10 
to 15 min, then filter and collect the filtrates in 50 ml conical 
flasks (6.4). 
8.3.2.4 Transfer, by means of a pipette, 2 ml of the filtrate ob- 
tained from the test portion, containing 40 to 200 pg of 
gossypol, into each of two 25 ml volumetric flasks and 2 ml of 
the filtrate obtained from the second flask into each of two 
other 25 ml volumetric flasks. 
Make up the contents of one of the flasks containing the test 
Solution and of one flask from the blank test to volume with the 
2-propanol/hexane mixture (5.1). 
Solution against 
These solutions will be used as reference solutions. 
8.3.1.4 Transfer, by means of a pipette, 10 ml of solvent A 
(5.2) into each of two 25 ml volumetric flasks (E and F). 
8.3.1.5 Make up the contents of flask E to volume with the 
2-propanoll hexane mixture (5.1) and mix. 8.3.2.5 Add 2,0 ml of the aniline (5.4) to the remaining test 
and blank test solutions. Heat for 30 min on a boiling water- 
bath to develop the colour. Cool to room temperature, make up 
to volume with the 2-propanol/hexane mixture (5.1), mix and 
leave for 1 h. 
This Solution will be used as the reference Solution against 
which the blank test Solution will be measured. 
8.3.1.6 Add 2,0 ml of the aniline (5.4) to flasks D and F. Heat 
for 30 min on a boiling water-bath to develop the colour. 8.3.2.6 Measure the absorbances of the solutions as specified 
in 8.3.1.8 and calculate the corrected absorbance as specified 
in 8.3.1.9. 
84 . Number of determinations 
8.3.1.7 Cool to room temperature, make up to volume with 
the 2-propanol/hexane mixture (5.1), mix and leave for 1 h. Carry out two determinations on the same test Sample. 
2 ISO 6866-1985 (El 
9 Expression of results 9.2 Repeatability 
9.1 Method of calculation and formula The differente between the results of two determinations, 
carried out simultaneously or in rapid succession on the same 
The free or total gossypol content, expressed in milligrams per Sample, shall not exceed 
kilogram of the product as received, is equal to 
A x 1250 x 1000 A x 1,25 ZZ x 106 
axmxV amV 15 % (relative) of the mean for gossypol contents less than 
500 mglkg; 
75 mg/kg (absolute) for gossypol contents not less than 
500 mg/kg and not more than 750 mg/kg; 
where 10 % (relative) of the mean for gossypol contents more 
than 750 mglkg. 
A is the corrected absorbance (8.3.1.9 or 8.3.2.6); 
m is the mass, in grams, of the test Portion (8.2); 10 Test report 
V is the volume, in millilitres, of the aliquot Portion of 
filtrate taken in 8.3.1.2 or 8.3.2.4; The test report shall show the method used and the result 
obtained. lt shall also mention any operating details not 
a is the specif ic mass absorbance coeff icient specified in this International Standard, or regarded as 
(62,5 cm-‘mg-1-I in the case of free gossypol; optional, together with details of any incidents likely to have 
60,O cm - 1. g - 1. I in the case of total gossypol). influenced the result. 
Take as the result the arithmetic mean of the two determina- 
tions (see 8.4) if the condition for repeatability (sec 9.2) is met. The test report shall include all the information necessary for 
the complete identification of the Sample. This page intentionally left blank This page intentionally left blank This page intentionally leff blank