EN ISO 14939:2001
(Main)Animal feeding stuffs - Determination of carbadox content - Method using high-performance liquid chromatography (ISO 14939:2001)
General Information
- Abstract
This International Standard specifies a high-performance liquid chromatographic (HPLC) method for the
determination of the carbadox content in premixtures and animal feeding stuffs.
The method is applicable to animal feeding stuffs with a mass fraction of carbadox of 0,5 mg/kg (limit of
quantification) to 100 mg/kg, and to premixtures with a mass fraction of carbadox up to 10 %.
The lower limit of detection is 0,1 mg/kg.
NOTE 1 For animal feeding stuffs the mass fraction of carbadox is expressed in milligrams per kilogram, and for premixtures
as a percentage by mass.
NOTE 2 Carbadox is a chemotherapeuticum belonging to the quinoxaline group. Carbadox is used as a growth-promoting
feed additive for piglets.
- Status
- Published
- Publication Date
- 14-Aug-2001
- Technical Committee
- CEN/TC 327 - Animal feeding stuffs - Methods of sampling and analysis
- Drafting Committee
- CEN/TC 327 - Animal feeding stuffs - Methods of sampling and analysis
- Current Stage
- 9092 - Decision on results of review/2YR ENQ - revise - Review Enquiry
- Start Date
- 18-Sep-2007
- Completion Date
- 18-Sep-2007
Overview
EN ISO 14939:2001 - "Animal feeding stuffs - Determination of carbadox content - Method using high-performance liquid chromatography (HPLC)" is a CEN-adopted International Standard that specifies a validated HPLC procedure to quantify carbadox in premixtures and animal feeding stuffs. The method covers feeds with carbadox mass fractions from 0.5 mg/kg (limit of quantification) to 100 mg/kg, premixtures up to 10%, and has a lower limit of detection of 0.1 mg/kg. Carbadox is a quinoxaline chemotherapeuticum used as a growth-promoting feed additive for piglets.
Key topics and technical requirements
- Analytical technique: Reverse-phase HPLC with UV detection at 365 nm; post-column derivatization with NaOH and detection at 420 nm is an alternative for confirmation.
- Sample types & ranges: Applicable to premixtures (up to 10% carbadox) and animal feeding stuffs (0.5–100 mg/kg; LOD 0.1 mg/kg).
- Extraction and cleanup: Carbadox is extracted using an acetonitrile:methanol (1:1) solvent. Feeding stuffs are prewetted with water and extracts are purified on a short aluminium oxide column; premixtures are directly diluted.
- Mobile phase & reagents: Mobile phase based on sodium acetate solution and acetonitrile; specific reagent preparations and standard solutions (stock and working) are specified.
- Apparatus requirements: HPLC pump (0.5–1.5 ml/min), injection loops (20–100 µl), UV detector (365 nm) or diode-array detector for confirmation, specified guard and analytical C18 columns.
- Interferences & confirmation: Potential interferences from dimetridazole, nitrofurazone, and sulfadimidine are noted. Confirmation can be performed by co‑chromatography, diode-array spectra or post-column derivatization.
- Quality & safety: Method includes requirements for standard materials, light-sensitive handling (carbadox and some standards), and preparation/storage of stock solutions. The standard contains annexes with a flowchart and interlaboratory precision data.
Applications and users
- Who uses it: Analytical laboratories, feed manufacturers, regulatory agencies, and quality-control teams involved in feed safety and compliance testing.
- Practical uses: Routine quantification of carbadox residues in premixtures and finished feeds, regulatory monitoring, supplier quality control, and investigation of suspected contamination or non‑compliance.
- Value: Provides a standardized, comparable HPLC method with defined limits of detection/quantification and documented precision for feed safety programs.
Related standards
- ISO 6498:1998 - referenced for preparation of test samples (sampling and sample prep guidance).
- Adopted by CEN (EN ISO 14939:2001) for harmonized use across European national standards bodies.
Keywords: EN ISO 14939:2001, carbadox, animal feeding stuffs, HPLC, high-performance liquid chromatography, feed analysis, premixtures, limit of detection, feed additive.
Relations
- Effective Date
- 25-Aug-2026
- Consolidated By
ISO 14939:2001 - Animal feeding stuffs — Determination of carbadox content — Method using high-performance liquid chromatography - Effective Date
- 12-Feb-2026
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Frequently Asked Questions
EN ISO 14939:2001 is a standard published by the European Committee for Standardization (CEN). Its full title is "Animal feeding stuffs - Determination of carbadox content - Method using high-performance liquid chromatography (ISO 14939:2001)". This standard covers: This International Standard specifies a high-performance liquid chromatographic (HPLC) method for the determination of the carbadox content in premixtures and animal feeding stuffs. The method is applicable to animal feeding stuffs with a mass fraction of carbadox of 0,5 mg/kg (limit of quantification) to 100 mg/kg, and to premixtures with a mass fraction of carbadox up to 10 %. The lower limit of detection is 0,1 mg/kg. NOTE 1 For animal feeding stuffs the mass fraction of carbadox is expressed in milligrams per kilogram, and for premixtures as a percentage by mass. NOTE 2 Carbadox is a chemotherapeuticum belonging to the quinoxaline group. Carbadox is used as a growth-promoting feed additive for piglets.
This International Standard specifies a high-performance liquid chromatographic (HPLC) method for the determination of the carbadox content in premixtures and animal feeding stuffs. The method is applicable to animal feeding stuffs with a mass fraction of carbadox of 0,5 mg/kg (limit of quantification) to 100 mg/kg, and to premixtures with a mass fraction of carbadox up to 10 %. The lower limit of detection is 0,1 mg/kg. NOTE 1 For animal feeding stuffs the mass fraction of carbadox is expressed in milligrams per kilogram, and for premixtures as a percentage by mass. NOTE 2 Carbadox is a chemotherapeuticum belonging to the quinoxaline group. Carbadox is used as a growth-promoting feed additive for piglets.
EN ISO 14939:2001 is classified under the following ICS (International Classification for Standards) categories: 65.120 - Animal feeding stuffs. The ICS classification helps identify the subject area and facilitates finding related standards.
EN ISO 14939:2001 has the following relationships with other standards: It is inter standard links to ISO 6498:1998, ISO 14939:2001. Understanding these relationships helps ensure you are using the most current and applicable version of the standard.
EN ISO 14939:2001 is available in PDF format for immediate download after purchase. The document can be added to your cart and obtained through the secure checkout process. Digital delivery ensures instant access to the complete standard document.
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Futtermittel - Bestimmung des Carbadoxgehaltes - Hochleistungs-flüssigchromatographisches Verfahren (ISO 14939:2001)Aliments des animaux - Détermination de la teneur en carbadox - Méthode par chromatographie liquide a haute performance (ISO 14939:2001)Animal feeding stuffs - Determination of carbadox content - Method using high-performance liquid chromatography (ISO 14939:2001)65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN ISO 14939:2001SIST EN ISO 14939:2001en01-december-2001SIST EN ISO 14939:2001SLOVENSKI
STANDARD
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN ISO 14939August 2001ICS 65.120English versionAnimal feeding stuffs - Determination of carbadox content -Method using high-performance liquid chromatography (ISO14939:2001)Aliments des animaux - Détermination de la teneur encarbadox - Méthode par chromatographie liquide à hauteperformance (ISO 14939:2001)Futtermittel - Bestimmung des Carbadoxgehaltes -Hochleistungs-flüssigchromatographisches Verfahren (ISO14939:2001)This European Standard was approved by CEN on 15 August 2001.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the Management Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2001 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 14939:2001 ESIST EN ISO 14939:2001
2EN ISO 14939:2001 (E)ForewordThe text of the International Standard ISO 14939:2001 has been prepared by TechnicalCommittee ISO/TC 34 "Agricultural food products" in collaboration with Technical CommitteeCEN/TC 327 "Animal feeding stuffs - Methods of sampling and analysis", the secretariat ofwhich is held by NEN.This European Standard shall be given the status of a national standard, either by publicationof an identical text or by endorsement, at the latest by February 2002, and conflicting nationalstandards shall be withdrawn at the latest by February 2002.According to the CEN/CENELEC Internal Regulations, the national standards organizations ofthe following countries are bound to implement this European Standard: Austria, Belgium,Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy,Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the UnitedKingdom.Endorsement noticeThe text of the International Standard ISO 14939:2001 was approved by CEN as a EuropeanStandard without any modification.SIST EN ISO 14939:2001
ReferencenumberISO14939:2001(E)©ISO2001INTERNATIONALSTANDARDISO14939Firstedition2001-08-15Animalfeedingstuffs—Determinationofcarbadoxcontent—Methodusinghigh-performanceliquidchromatographyAlimentsdesanimaux—Déterminationdelateneurencarbadox—MéthodeparchromatographieliquideàhauteperformanceSIST EN ISO 14939:2001
ISO14939:2001(E)PDFdisclaimerThisPDFfilemaycontainembeddedtypefaces.InaccordancewithAdobe'slicensingpolicy,thisfilemaybeprintedorviewedbutshallnotbeeditedunlessthetypefaceswhichareembeddedarelicensedtoandinstalledonthecomputerperformingtheediting.Indownloadingthisfile,partiesacceptthereintheresponsibilityofnotinfringingAdobe'slicensingpolicy.TheISOCentralSecretariatacceptsnoliabilityinthisarea.AdobeisatrademarkofAdobeSystemsIncorporated.DetailsofthesoftwareproductsusedtocreatethisPDFfilecanbefoundintheGeneralInforelativetothefile;thePDF-creationparameterswereoptimizedforprinting.EverycarehasbeentakentoensurethatthefileissuitableforusebyISOmemberbodies.Intheunlikelyeventthataproblemrelatingtoitisfound,pleaseinformtheCentralSecretariatattheaddressgivenbelow.©ISO2001Allrightsreserved.Unlessotherwisespecified,nopartofthispublicationmaybereproducedorutilizedinanyformorbyanymeans,electronicormechanical,includingphotocopyingandmicrofilm,withoutpermissioninwritingfromeitherISOattheaddressbeloworISO'smemberbodyinthecountryoftherequester.ISOcopyrightofficeCasepostale56CH-1211Geneva20Tel.+41227490111Fax+41227490947E-mailcopyright@iso.chWebwww.iso.chPrintedinSwitzerlandii©ISO2001–AllrightsreservedSIST EN ISO 14939:2001
ISO14939:2001(E)©ISO2001–AllrightsreservediiiContentsPageForeword.iv1Scope.12Normativereference.13Principle.14Reagents.25Apparatus.46Sampling.57Preparationoftestsample.58Procedure.58.1General.58.2Preparationofspikedsample.58.3Extraction.68.4Columnchromatography.78.5HPLCanalysis.79Confirmation.89.1General.89.2Co-chromatography.89.3Diodearraydetector.99.4Post-columnderivatization.1010Calculationofresults.1010.1General.1010.2Feedingstuffscontaining0,1mg/kgto10mg/kgofcarbadox.1010.3Feedingstuffscontaining10mg/kgto100mg/kgofcarbadox.1110.4Premixturescontainingupto10%ofcarbadox.1111Precision.1111.1Interlaboratorytest.1111.2Repeatability.1111.3Reproducibility.1212Testreport.12AnnexA(informative)Flowchart.13AnnexB(informative)Resultsofinterlaboratorytest.14Bibliography.16SIST EN ISO 14939:2001
ISO14939:2001(E)iv©ISO2001–AllrightsreservedForewordISO(theInternationalOrganizationforStandardization)isaworldwidefederationofnationalstandardsbodies(ISOmemberbodies).TheworkofpreparingInternationalStandardsisnormallycarriedoutthroughISOtechnicalcommittees.Eachmemberbodyinterestedinasubjectforwhichatechnicalcommitteehasbeenestablishedhastherighttoberepresentedonthatcommittee.Internationalorganizations,governmentalandnon-governmental,inliaisonwithISO,alsotakepartinthework.ISOcollaboratescloselywiththeInternationalElectrotechnicalCommission(IEC)onallmattersofelectrotechnicalstandardization.InternationalStandardsaredraftedinaccordancewiththerulesgivenintheISO/IECDirectives,Part3.DraftInternationalStandardsadoptedbythetechnicalcommitteesarecirculatedtothememberbodiesforvoting.PublicationasanInternationalStandardrequiresapprovalbyatleast75%ofthememberbodiescastingavote.AttentionisdrawntothepossibilitythatsomeoftheelementsofthisInternationalStandardmaybethesubjectofpatentrights.ISOshallnotbeheldresponsibleforidentifyinganyorallsuchpatentrights.InternationalStandardISO14939waspreparedbyTechnicalCommitteeISO/TC34,Foodproducts,SubcommitteeSC10,Animalfeedingstuffs.AnnexesAandBofthisInternationalStandardareforinformationonly.SIST EN ISO 14939:2001
INTERNATIONALSTANDARDISO14939:2001(E)©ISO2001–Allrightsreserved1Animalfeedingstuffs—Determinationofcarbadoxcontent—Methodusinghigh-performanceliquidchromatography1ScopeThisInternationalStandardspecifiesahigh-performanceliquidchromatographic(HPLC)methodforthedeterminationofthecarbadoxcontentinpremixturesandanimalfeedingstuffs.Themethodisapplicabletoanimalfeedingstuffswithamassfractionofcarbadoxof0,5mg/kg(limitofquantification)to100mg/kg,andtopremixtureswithamassfractionofcarbadoxupto10%.Thelowerlimitofdetectionis0,1mg/kg.NOTE1Foranimalfeedingstuffsthemassfractionofcarbadoxisexpressedinmilligramsperkilogram,andforpremixturesasapercentagebymass.NOTE2Carbadoxisachemotherapeuticumbelongingtothequinoxalinegroup.Carbadoxisusedasagrowth-promotingfeedadditiveforpiglets.2NormativereferenceThefollowingnormativedocumentcontainsprovisionswhich,throughreferenceinthistext,constituteprovisionsofthisInternationalStandard.Fordatedreferences,subsequentamendmentsto,orrevisionsof,anyofthesepublicationsdonotapply.However,partiestoagreementsbasedonthisInternationalStandardareencouragedtoinvestigatethepossibilityofapplyingthemostrecenteditionsofthenormativedocumentsindicatedbelow.Forundatedreferences,thelatesteditionofthenormativedocumentreferredtoapplies.MembersofIECandISOmaintainregistersofcurrentlyvalidInternationalStandards.ISO6498:1998,Animalfeedingstuffs—Preparationoftestsamples.3PrincipleCarbadoxisextractedfromthesamplewithamixtureofacetonitrileandmethanol.Animalfeedsareprewettedwithwater.Theextractofanimalfeedsispurifiedthroughashortaluminiumoxidecolumn.Theextractofpremixturesisdirectlydilutedwithamixtureofwater,acetonitrileandmethanol.Thefinalextractisanalysedbyreverse-phaseHPLCwithUVdetectionatawavelengthof365nm(seereferences[1]to[3]).Thepresenceofdimetridazole,nitrofurazoneorsulfadimidinesodiumcaninterferewiththedeterminationofcarbadox.Alternatively,carbadoxmaybedeterminedafterpost-columnderivatizationwithsodiumhydroxidewithdetectionatawavelengthof420nm.SIST EN ISO 14939:2001
ISO14939:2001(E)2©ISO2001–Allrightsreserved4ReagentsUseonlyreagentsofrecognizedanalyticalgrade.4.1Water,demineralizedordeionized,withresistivityofatleast10Mcm,orwaterofatleastequivalentpurity.4.2Extractionsolvent:mixtureofacetonitrileandmethanol(1:1byvolume).Combineequalvolumesofacetonitrileandmethanol.Mixwellandallowtoadjusttoroomtemperaturebeforeuse.4.3Dilutionsolvent:mixtureofextractionsolvent(4.2)andwater(4.1)(70:30byvolume).Mix70mlofextractionsolvent(4.2)with30mlofwater(4.1).4.4Aceticacid,volumefraction,w(CH3CO2H)=10%.Dilute10mlofglacialaceticacidto100mlwithwater.4.5Sodiumacetatesolution,c(C2H3NaO2)=0,01mol/l,pH=6,0.Weigh0,82gofwater-freesodiumacetateintoa1000mlone-markvolumetricflask.Dissolvein700mlofwater.AdjustthepHtopH=6,0withaceticacid(4.4).Dilutetothemarkwithwaterandmix.4.6MobilephaseforHPLC.Combine825mlofsodiumacetatesolution(4.5)and175mlofacetonitrileandmix.Filtertheeluentthrougha0,22µmfilterusingasolventfiltrationsystem(5.2),anddegasfor10mininanultrasonicbath(5.3)beforeuse.4.7Carbadoxstandardmaterial,3-(2-quinoxalinylmethylene)carbazicacidmethyesterN,N-dioxide(CASnumber6804-07-5).WARNING—Becauseofthesensitivityofcarbadoxtolight,conductalloperationsintheabsenceofdaylightorartificialwhitelight.Avoidinhalationofandexposuretothetoxiccarbadoxstandardmaterialandsolutionsthereof.Workinafumecupboardwhenhandlingthesolventsandsolutions.Wearsafetyglassesandprotectiveclothing.4.8Carbadoxstocksolution(approximately100µg/ml).Weigh10mg1mgofcarbadox(4.7),tothenearest0,1mg,intoa100mlone-markvolumetricflask.Dissolveinextractionsolvent(4.2),dilutetothemarkandmix.Calculatetheconcentrationtakingintoaccountthepurityofthestandardmaterial.Preparefresheverymonth.Storeinthedarkat0°Cto8°C.4.9Carbadoxworkingsolutions(approximately2µg/mland10µg/ml).Pipette1,0mland5,0mlofthecarbadoxstocksolution(4.8)intoseparate50mlone-markvolumetricflasks.Dilutetothemarkwithdilutionsolvent(4.3)andmix.Preparefreshforeachseriesofsamples.4.10Carbadoxworkingsolutions(approximately0,4µg/mland2µg/ml).Pipette1,0mlofthecarbadoxstocksolution(4.8)intoa50mlone-markvolumetricflask,dilutetothemarkwithmobilephase(4.6)andmix.Pipette10mlofthissolution(2µg/ml)intoa50mlone-markvolumetricflask,dilutetothemarkwithmobilephase(4.6)andmix.Preparefreshforeachseriesofsamples.4.11Dimetridazolestandardmaterial,1,2-dimethyl-5-nitro-1H-imidazole(CASnumber551-92-8).WARNING—Becauseofthesensitivityofdimetridazoletolight,conductalloperationsintheabsenceofdaylightorartificialwhitelight.AvoidinhalationofandexposuretothetoxicdimetridazolestandardSIST EN ISO 14939:2001
ISO14939:2001(E)©ISO2001–Allrightsreserved3materialandsolutionsthereof.Workinafumecupboardwhenhandlingthesolventsandsolutions.Wearsafetyglassesandprotectiveclothing.4.12Dimetridazolestocksolution(approximately100µg/ml).Weigh10mg1mgofdimetridazole(4.11),tothenearest0,1mg,intoa100mlone-markvolumetricflask.Dilutetothemarkwithmethanolandmix.Calculatetheconcentrationtakingintoaccountthepurityofthestandardmaterial.Preparefresheverymonth.Storeinthedarkat0°Cto8°C.4.13Dimetridazoleworkingsolution(approximately20µg/ml).Pipette2,0mlofthedimetridazolestocksolution(4.12)intoa10mlone-markvolumetricflask.Dilutetothemarkwithwaterandmix.Preparefreshforeachseriesofsamples.4.14Sulfadimidinestandardmaterial,sodiumsaltof4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzenesulfonamide(CASnumber1981-58-4).WARNING—Avoidinhalationofandexposuretothetoxicsulfadimidinestandardmaterialandsolutionsthereof.Workinafumecupboardwhenhandlingthesolventsandsolutions.Wearsafetyglassesandprotectiveclothing.4.15Sulfadimidinestocksolution(approximately200µg/ml).Weigh10mg1mgofsulfadimidinestandardmaterial(4.14),tothenearest0,1mg,intoa50mlone-markvolumetricflask.Dilutetothemarkwithmethanolandmix.Calculatetheconcentrationtakingintoaccountthepurityofthestandardmaterial.Preparefresheverymonth.Storeinthedarkat0°Cto8°C.4.16Sulfadimidineworkingsolution(approximately20µg/ml).Pipette1,0mlofsulfadimidinestocksolution(4.15)intoa10mlone-markvolumetricflask.Dilutetothemarkwithwaterandmix.Preparefreshforeachseriesofsamples.4.17Nitrofurazonestandardmaterial,5-nitro-2-furaldehydesemicarbazone(CASnumber59-87-0).WARNING—Becauseofthesensitivityofnitrofurazonetolight,conductalloperationsintheabsenceofdaylightorartificialwhitelight.Avoidinhalationofandexposuretothetoxicnitrofurazonestandardmaterialandsolutionsthereof.Workinafumecupboardwhenhandlingthesolventsandsolutions.Wearsafetyglassesandprotectiveclothing.4.18Nitrofurazonestocksolution(approximately100µg/ml).Weigh10mg1mgofnitrofurazone(4.17),tothenearest0,1mg,intoa100mlone-markvolumetricflask.Dilutetothemarkwithmethanolandmix.Calculatetheconcentrationtakingintoaccountthepurityofthestandardmaterial.Preparefresheverymonth.Storeinthedarkat0°Cto8°C.4.19Nitrofurazoneworkingsolution(approximately20µg/ml).Pipette2,0mlofnitrofurazonestocksolution(4.18)intoa10mlone-markvolumetricflask.Dilutetothemarkwithwaterandmix.Preparefreshforeachseriesofsamples.4.20Neutralaluminiumoxide,activity1.Fortotalde-activation0%to1%ofwaterisnecessary.4.21Sodiumhydroxidesolution,c(NaOH)=0,5mol/l.Weigh20gofsodiumhydroxideintoa1litreone-markvolumetricflaskanddissolvein10mlofwater.Dilutetothemarkwithwaterandmix.SIST EN ISO 14939:2001
ISO14939:2001(E)4©ISO2001–Allrightsreserved5ApparatusUsuallaboratoryapparatusand,inparticular,thefollowing.5.1pH-meter.5.2Solventfiltrationsystem,allglassapparatussuitablefor0,22µmfilters.5.3Ultrasonicbath.5.4Rotaryshaker,horizontalrotation,rotationfrequency250min1to300min1.5.5Glassmicrofibrefilter,diameter15cm.5.6Glasswool.5.7Glasscolumnforchromatography,length30cm,internaldiameter10mm,restrictedattheendandfittedwithawadofglasswool(5.6),oranequivalentcolumnwithaninternaldiameterof10mm.5.8Filtrationsystem,equippedwithpolyvinylidenedifluoride(PVDF)filtersorpolytetrafluorethylene(PTFE)filtersofporesize0,45µm.5.9Wat
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