UGANDA STANDARD  
      US ISO 14565
First Edition
   2008-09-04
 
Reference numbe r
  US ISO 14565:2000
 © UNBS 2008
  
Animal feeding stuffs — Determination of vitamin A content — 
Method using high-performance liquid chromatography 
 US ISO 14565:2000  
 
© UNBS  2008 – 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 2008  
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 14565: 2000  
© UNBS  2008 - 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 14565:2000,  Animal feeding stuffs —  Determination of vitamin A content 
— Method using high-performance liquid chromatography, is identical with and  has been reproduced from
an International Standard ISO 14565:2000,  Animal feeding stuffs — Determination of vitamin A content — 
Method using high-performance liquid chromatography, andhas been adopted as a Uganda Standard.
Wherever the words, "International Standard" a ppear, they should be replaced by "Uganda Standard." Reference number
ISO14565:2000(E)
©ISO2000INTERNATIONAL
STANDARDISO
14565
Firstedition
2000-12-01
Animal feedingstuffs— Determinationof
vitamin Acontent— Methodusinghigh-
performanceliquidchromatograph y
Aliments des animaux — Déterminationde la teneur envitamine A—
Méthode par chromatographieliquideà hauteperformanceISO 14565:2000(E)
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but shall not
be edited unless thetypefaces which are embedded arelicensedto andinstalled onthecomputer performing the editing. In downloading thisfile, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat accepts no liability in thi s
area.
Adobe is atrademark of AdobeSystems Incorporated.
Details of thesoftware products usedtocreate this PDF file can be found in the General Info relative to the file; the PDF-creation parameters
were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the unlikely eventthat aproblem relatingto it is found, pleaseinform theCentralSecretariat at the address given below.
© ISO 2000
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic
or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO's member bodyin thecountry of the requester.
ISOcopyright office
Case postale 56 /Gb7CH-1211 Geneva 20
Tel. + 4122 749 0111
Fax + 41 22 749 09 47E-mail copyright@iso.chWeb www.iso.ch
Printed inSwitzerland
ii © ISO2000 –All rights reservedISO 14565:2000(E)
© ISO2000 –All rights reserved iiiForeword
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 hasthe right to be represented on that committee. International organizations, governmental and non-governmental, inliaison with ISO, also take part in the work. ISO collaborates closely with the International ElectrotechnicalCommission(IEC) onall matters of electrotechnicalstandardization.
InternationalStandards are drafted in accordance with the rules givenintheISO/IEC Directives, Part3.
Draft International Standards adopted by the technical committees are circulated to the member bodies for voting.
Publicationas anInternationalStandardrequires approval byat least 75% of themember bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this International Standard may be the subject of
patent rights. ISO shall not beheldresponsiblefor identifyinganyor allsuch patent rights.
International Standard ISO14565 was prepared by Technical Committee ISO/TC 34, Food products ,
SubcommitteeSC 10, Animalfeedingstuffs .
Annex A of this International Standardis for informationonly.INTERNATIONAL STANDARD ISO 14565:2000(E)
© ISO2000 –All rights reserved 1Animalfeedingstuffs —DeterminationofvitaminAcontent —
Methodusinghigh-performance liquidchromatography
1 Scope
This International Standardspecifies amethodfor the determination of thetotal vitaminA (retinol) content of animal
feeding stuffs and pet foods using high-performance liquid chromatography. The vitamin A content is the content ofall-trans-retinyl alcohol and cis-isomers determined by the method described in this International Standard, and is
expressedinInternational Units per kilogram (IU/kg).
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of
this International Standard. For dated references, subsequent amendments to, or revisions of, any of thesepublications do not apply. However, parties to agreements based on this International Standard are encouraged to
investigate the possibility of applying the most recent editions of the normative documents indicated below. For
undated references, the latest edition of the normative documents referred to applies. Members of ISO and IECmaintain registers of currentlyvalid InternationalStandards.
ISO3696:1987, Water for analytical laboratory use —Specification and test methods .
ISO 6498, Animalfeedingstuffs —Preparation of test samples .
3 Term and definition
For the purposes of this International Standard, thefollowing term anddefinition apply.
3.1
vitamin Acontentcontent of all- trans-retinyl alcohol and cis-isomers determinedin accordance withthis InternationalStandard
NOTE The vitamin Acontent is expressed in International Units per kilogram (IU/kg); 1 IUof vitamin A is equal to 0,300 /G6dg
of all-trans-retinol.
4P r i n c i p l e
The sample is saponified with ethanolic potassium hydroxide solution and the vitaminA is extracted into light
petroleum. Thelight petroleum is removed byevaporationandtheresidue is dissolved in 2-propanol. The vitaminAconcentration in the 2-propanol extract is determined byreverse-phase liquid chromatography using conditions thatgive a single peak for allretinol isomers.ISO 14565:2000(E)
2 © ISO2000 –All rights reserved5 Reagents
Useonlyreagents of recognized analyticalgrade.
5.1 Water , complying with at least grade3 in accordance with ISO3 696.
5.2 Potassium hydroxidesolution (KOH).
Dissolve500g of potassium hydroxidein water (4.1) and diluteto 1litre.
5.3 Ethanol ,w(C2H5OH) =95 % (byvolume), or equivalent industrialmethylated spirit.
5.4 2-Propanol (C3H7OH).
5.5 Light petroleum , boiling range 40 /Gb0Cto60 /Gb0C.
Theresidue onevaporationshall be less than20mg/l.
5.6 Vitamin Astandardsubstances
5.6.1 All -trans-retinylacetate , vitaminA acetate(C 22H32O2), 328.5g/mol, with apurityof at least 90%.
5.6.2 All -trans-retinol, vitamin A alcohol(C 20H30O), 286,5g/mol, with apurityof at least 90%
5.7 Methanol ,HP L Cg ra de .
5.8 Mobilephaseforliquid chromatography .
Mix together methanol(5.7) and water (4.1) intheproportions 770+30 (byvolume).If necessary, filter througha membranefilter (6.6).
5.9 Sodium sulfate (Na
2SO4), anhydrous.
5.10 Sodium ascorbate solution ,/G72=100g/l.
5.11 Inert gas ,e .g .ni tro ge n .
6 Apparatus
Usual laboratoryapparatus and, in particular, thefollowing.
6.1 High-performanceliquid chromatograph , consisting of the following.
6.1.1 Pump , set todeliver aconstant eluent volumeflow rate of 1ml/min.
6.1.2 HPLC injection device.
6.1.3 Column , length 250mm, internal diameter 4,6mm, packed with a stationary phase consisting of octadecyl
(C18) groups bondedtosilica.
A column with at least 4 000 theoretical plates and a k/Ga2value of 0,6, both with respect to all -trans-retinol, has been
found to be satisfactory. The particle size shall be not smaller than 5 /G6dm and not greater than 10 /G6dm. Other
systemsmaybe usedprovidedthat a satisfactoryseparation of vitaminAfrom other co-extractives is achieved.ISO 14565:2000(E)
© ISO2000 –All rights reserved 36.1.4 Detector , allowing the measurement of ultraviolet radiation at 325nm, and equipped with integrator/data-
handlingsystem.
6.2 UV (or UV-Visible) spectrometer , capable of measuring absorbance at the wavelengths defined in 9.6,
equipped with quartzcells of 10mm pathlength.
6.3 Boiling waterbath .
6.4 Rotaryvacuum evaporator , with water bathat 40 /Gb0C.
6.5 Extraction apparatus (seeFigure1) consistingof thefollowing:
/Gbeacylinder of 1litrecapacityfitted with aground glass neck andstopper;
/Gbeaground glass joint, fitting thecylinder andequipped with an adjustable tubepassingthroughthecentre; and
/Gbeaside-arm.
The adjustable tube should have a U-shaped lower end and a jet at the opposite end so that the upper liquid layer
inthecylinder maybetransferredto aseparatingfunnel of 1litrecapacity.
Other extraction equipment such as conical flasks and separating funnels may be used in place of the apparatus
shown inFigure1, providedthat satisfactoryrecoveries of vitaminAare achieved.
6.6 Membranefilter , 0,45µm poresize, for filtration of mobilephase(5.8) andsampletest solutions.
7 Sampling
It is important that the laboratory receive a sample which is truly representative and has not been damaged or
changedduringtransport or storage.
Sampling is not part of the method specified in this International Standard. A recommended sampling method is
given in ISO 6497 [4].
Store thesample insuch awaythat deteriorationandchange in its compositionare prevented.
8 Preparationoftestsample
Prepare the test sampleinaccordance withISO6498.Just prior to starting the analysis, grind a portion of the well-mixed laboratory sample so that it passes through a
sieve with1mm apertures.Mix thoroughly.
Homogenizecanned pet foods. Mincesemi-moist pet foods to pass through aplate with4mm apertures.ISO 14565:2000(E)
4 © ISO2000 –All rights reservedKey
1 Cylinder, of capacity1litre, with ground-glass neck 5 Bottle,ofcapacity1litre, with ground-glassjoint
2 Lightpetroleum layer 6 Side-arm
3 Aqueous layer+ saponified feed 7 Adjustable tub
4J e t
Figure1—Exampleof extraction apparatusISO 14565:2000(E)
© ISO2000 –All rights reserved 59 Procedure
9.1 General
Because of the sensitivity of vitamin A to UV radiation and air, perform all operations away from natural and strong
fluorescent light and as rapidly as is consistent with accurate working. Use amber glassware where possible.
Complete each assay within one working day. Carry out the saponification and extraction of the all -trans-retinyl
acetate standardandthefeedingstuff samples at the sametime.
9.2 Saponification
Weigh, to the nearest 0,1g, approximately 50g of the prepared test sample (see clause8) into a 1litre conicalflask.
Add 200mlof ethanol(5.3). Swirltheflask contents to dispersethesample.Add 2mlof sodium ascorbate(5.10) and 50ml of potassium hydroxide solution(5.2) and mix byswirling.Fit areflux condenser totheflask andimmersetheflask intheboiling water bath (6.3).Allowthe contents of the flask to reflux for 60min, swirling occasionally.
Removeandcooltheflask toroom temperatureas rapidlyas possible under astream of cold water.
9.3 Extraction of vitamin A(retinol)
Transfer thecontents of theflask tothe extractioncylinder (see6.5).
Rinse the flask with two 25ml portions of ethanol or industrial methylated spirit (5.3) and transfer the rinsings to the
cylinder.
Repeat the rinsing of the flask with two 125ml portions of light petroleum (5.5) and one 250ml portion of water
(4.1), each timetransferringtherinsings tothecylinder.
Stopper thecylinder and shakewellfor 1min, takingcaretorelease anypressurefrom timetotime.
Cool the cylinder under a stream of cold water while waiting for the two liquid phases to separate, before removing
thestopper.
When the layers have separated, remove the stopper, wash the sides of the stopper with a few millilitres of light
petroleum (5.5) and insert the adjustable tube (see 6.5), positioning the lower open end so that it is just above thelevel of the interface.
By application of a slight pressure of inert gas (5.11) to the side-arm tube, transfer the upper, light petroleum layer
toa 1litreseparatingfunnel(see6.5).
Add 125mlof light petroleum (5.5) tothecylinder, stopper andshake wellfor 1min.Allow the layers to separate and transfer the upper layer to the separating funnel using the adjustable tube
(see6.5) as before.
Again, add125mlof light petroleum (5.5) tothecylinder, stopper andshakewell for 1min.Again, allow the layers to separate and transfer the upper layer to the separating funnel using the adjustable tube
as before.
Wash the combined light petroleum extracts with four 100ml portions of water using at first only gentle inversion
then onlygentle shaking inorder tokeep emulsionformationto aminimum.ISO 14565:2000(E)
6 © ISO2000 –All rights reservedTransfer the washed extract through a medium/fast filter paper containing 60g of anhydrous sodium sulfate (5.9),
into aflask suitablefor vacuum evaporation.
Rinse the separating funnel with two 20ml portions of light petroleum (5.5) and add the rinsings, through the filter,
totheevaporationflask.
Wash the filter further with two 25ml portions of light petroleum (5.5) and collect the washings in the evaporation
flask.
Evaporatethe light petroleum extract todryness under vacuum at atemperaturenot exceeding40 °C.
Restoreatmospheric pressurebyadmittinginert gas (5.11).
9.4 High-performanceliquidchromatography
9.4.1Dissolve the residue in the minimum volume of 2-propanol (5.4) and transfer quantitatively to a 20ml
volumetric flask.
Rinse the evaporation flask with three small portions of 2-propanol (5.4), transferring the rinsings to the volumetric
flask. Dilute to volume with 2-propanol (5.4) and mix. For material containing more than 100000IU/kg, further
dilutionmaybenecessary.
NOTE IU= International Unit; 1 IUof vitamin Aisequal to 0,300 /G6dgofa ll-trans-retinol.
If necessary, filter thesampleextract througha membranefilter (6.6).
9.4.2If delayis unavoidable, store the extract under inert gas (5.11) inarefrigerator at atemperatureof 4 °Ca n d
then allow it toreturntoroom temperatureinthedark.
9.4.3Inject 10 /G6dl of the sample extract onto the column of the liquid chromatograph (6.1) and measure the area
of theretinolpeak.
9.4.4Calculate the mean peak area from replicate injections of the sample extract and determine the retinol
concentration of the extract byreference to the mean peak area found from replicate injections of a retinol standardof similar concentration. Theretention timeof retinolis about 5min. Make alternate injections of sample extract andstandardsolution.
9.5 Hydrolysis of all -trans-retinyl acetatefor calibration
Prepare a solution of all- trans-retinyl acetate (5.6) in ethanol (5.3) so that 1ml contains approximately 15000 IU of
vitamin A.
NOTE 1 IUof vitaminAis equalto 0,344 /G6dgofa ll-trans-retinylacetate.
Using a5ml burette with0,02mlsubdivisions, transfer 2,5ml /Gb10,02ml of this solutiontoa 150mlflask.
Add 20ml of ethanol(5.3), 1mlof potassium hydroxidesolution(5.2) and5ml of sodium ascorbatesolution(5.10).
Fit acondenser totheflask. Immersetheflask intheboiling water bathand allow toreflux for 60min.
Cool the flask to room temperature under a stream of cold water and transfer the contents to a separating funnel
(see6.5).
Rinse the flask with 50ml of water (4.1), followed by 25ml of ethanol (5.3), adding the rinsings to the separating
funnel.
Extract the aqueous phase with one 80ml portion of light petroleum (5.5) and then with two 50ml portions of light
petroleum (5.5).ISO 14565:2000(E)
© ISO2000 –All rights reserved 7Combine the light petroleum extracts, then wash with two 50ml portions of water. Add 2g of anhydrous sodium
sulfate(5.9).
Transfer the light petroleum extract quantitatively to a 250ml volumetric flask and dilute to volume. The retinol
concentration of this solution(solution 1) is approximately150IU/ml.
9.6 Standardizationofretinol solutionforcalibration
Pipette 5ml /Gb10,03ml of solution 1 (9.5) into a 50ml volumetric flask and remove the solvent, at ambient
temperature, witha stream of inertgas (5.11).
Dissolvetheresidue in 2-propanol(5.4), andthendilutetovolumewith2-propanol.
Measure the absorbance ( A) of the solution, using 2-propanol as reference, at wavelengths of 310nm, 325nm and
334nm. The absorbance values will be approximately 0,7 to 0,8. If necessary, an intermediate dilution may be
used.
Usingthefollowingequation, calculatethecorrected absorbanceat 325nm:
A325,corr=6,815 /Gb4A325–2,555 /Gb4A310–4,26/Gb4A334
IfA325,corr/A325is less than0,97, use the valueof A325,corrfor thestandardization; otherwiseuse A325.
The retinolconcentration of solution1 is given by:
concentration (IU/ml) = A325/Gb4183IU/ml,or
concentration (IU/ml) = A325,corr /Gb4183IU/ml.
9.7 Preparation of aretinol standardforchromatography
Prepare aretinolsolutionin2-propanol (5.4) theconcentration of which is approximatelythe same as that expected
in the sample extract (9.4.1). For each 1000IU of vitamin A per kilogram of sample, a retinol concentration of
2,5IU/mlis expected intheextract.
Evaporate an aliquot volume of solution 1 (9.5) to dryness at ambient temperature with a stream of inert gas (5.11).
Dissolve the residue in the appropriate volume of 2-propanol to provide the required retinol concentration and mixit.
If necessary, filter thestandardsolutionthroughamembranefilter (6.6).
Alternatively for calibration purposes, a standard solution of vitamin A (retinol) in 2-propanol (5.4) prepared by
diluting a stock standard solution of all -trans-retinol made by dissolving an appropriate quantity of all -trans-retinol
standardsubstance (5.6.2) directlyin2-propanol(5.4) maybe used.
In this case, check the vitamin A standard by measuring the absorbance of the standard solution in quartz cells
(6.2) at wavelengths of 300nm, 325nm, 350nm and 370nm against 2-propanol as reference. Determine theA/A
325ratio at each of the wavelengths for all -trans-retinol. If the ratio does not exceed 0,602, 0,432 and 0,093 at
300 nm,350nm and370nm respectively,thestandardsubstance is suitablefor use(see references [5], [6]).
NOTE The use of a multiple point calibration may be used to facilitate analysis in cases where, for example, the expected
concentration ofvitamin Ain the sample extractisunknown.ISO 14565:2000(E)
8 © ISO2000 –All rights reserved10 Expressionofresults
Calculate the numerical value of the vitaminAcontent of thetest samplebythe equation:
A20 000cwm/G3d/Gb4
where
wAis the numerical value of the vitamin A content of the test sample, in International Units per kilogram
(IU/kg);
cis the numericalvalue of theretinolconcentration of the extract, in International Units per millilitre(IU/ml);
mis the numericalvalue of themass of thetest sample, in grams.
11 Precision
11.1 Interlaboratorytests
Details of interlaboratory tests on the precision of the method are given in annex A. The values derived from these
tests maynot be applicabletoconcentrationranges andmatrices other thanthosegiven.
Table1—Repeatabilitylimit (r)and reproducibilitylimit (R)
Sample Vitamin Acontent rR
IU/kg IU/kg IU/kg
Pelleted poultryfeed 9888 860 2068
Cattleration 10812 2099 3549
Fishfood 15879 2800 4672
Semi-moist pet food 33250 1536 2446
Pig concentrateration 40661 4049 7941
11.2 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,
willinnot morethan 5% of cases exceedtherepeatabilitylimit ( r) mentioned in or derivedfrom Table1.
11.3 Reproducibility
The absolute difference between two single test results obtained using the same method on identical test material
in different laboratories by different operators using different equipment, will in not more than 5% of cases exceed
the reproducibilitylimit ( R) mentioned in or derivedfrom Table1.ISO 14565:2000(E)
© ISO2000 –All rights reserved 912 Testreport
Thetest report shallspecify:
/Gbeall information necessaryfor the complete identification of the sample;
/Gbethesamplingmethodused, if known;
/Gbethetest methodused, withreferencetothis International Standard;
/Gbeall operating details not specified in this International Standard, or regarded as optional, together with details of
anyincidents whichmayhave influenced the test result(s);
/Gbethetest result obtained, or thetwo test results obtained if therepeatabilityhas beenchecked.ISO 14565:2000(E)
10 © ISO2000 –All rights reservedAnnex A
(informative)
Results ofinterlaboratorytests
The precision of the method was established by interlaboratory tests carried out in accordance with ISO5725[1]1).
The results of these tests have been published (see reference [7]). In the tests 12 to 14 laboratories participated
and samples were investigated of semi-moist pet food, pelleted poultry feed, cattle ration, pig concentrate ration
andfishfood.
TableA.1 —Statisticalresultsof interlaboratorytests
Parameter Samplea
12345
Number of laboratories 14 12 13 12 13
Number of individualresults 56 24 26 24 26
Number of acceptedresults 56 24 26 24 26
Mean vitaminAcontent,IU/kg 33250 9888 10812 40661 15879
Repeatabilitystandard deviation( sr), IU/kg 668 304 742 1431 989
Repeatabilitycoefficient of variation, % 2,0 3,1 6,9 3,5 6,2
Repeatabilitylimit ( r)[r=2 , 8 /Gb4sr), IU/kg 1536 860 2099 4049 2800
Reproducibilitystandard deviation ( sR),IU/kg 864 731 1254 2806 1651
Reproducibilitycoefficient of variation, % 2,6 7,4 11,6 6,9 10,4
Reproducibilitylimit ( R)[R=2,8 /Gb4sR), IU/kg 2446 2068 3549 7941 4672
a1:semi-moistpetfood;
2: pelleted poultryfeed;
3: cattleration;
4: pig concentrate ration;
5: fish food.
1)ISO5725:1986(nowwithdrawn) was used toobtaintheprecisiondata.ISO 14565:2000(E)
© ISO2000 –All rights reserved 11Bibliography
[1] ISO 5725:1986, Precision of test methods —Determination of repeatability and reproducibility for a
standard testmethodby inter-laboratory tests .
[2] ISO 5725-1:1994, Accuracy (trueness and precision) of measurement methods and results —Part 1:
General principles and definitions .
[3] ISO 5725-2:1994, Accuracy (trueness and precision) of measurement methods and results —Part 2: Basic
methodfor the determination of repeatability andreproducibility of a standardmeasurement method .
[4] ISO 6497, Animalfeedingstuffs —Sampling .
[5] Takashima, Y. et al. Stability of retinol analogs. Chem. Pharm. Bull. ,27, 1979, pp. 1553.
[6] Bolliger, M.R. et al. The monograph of vitamin A in the European Pharmacopoeia. Pharm. Acta. Helv. ,52
(8),1977,pp.161-174.
[7] AnalyticalMethods Committee, Analyst,110,1985, pp.1019-1026.ISO 14565:2000(E)
ICS 65.120
Pricebased on11 pages
© ISO2000 –All rights reserved