commiss direct ec juli establish commun method analysi determin vitamin vitamin tryptophan feedingstuff text eea relev commiss european commun regard treati establish european commun regard council direct eec juli introduct commun method sampl analysi offici control feedingstuff amend act access austria finland sweden articl thereof direct eec stipul offici control feedingstuff purpos check complianc requir aris law regul administr provis govern qualiti composit carri commun method sampl analysi council direct eec novemb addit feedingstuff amend commiss regul ec novemb stipul vitamin vitamin content label substanc ad premixtur feedingstuff council direct eec april market compound feedingstuff amend commiss direct ec council direct eec septemb feedingstuff intend nutrit purpos amend direct ec stipul amino acid set feedingstuff label commun method analysi establish check substanc measur provid direct accord opinion stand committe feedingstuff adopt direct articl member state provid analys conduct view offici control vitamin vitamin tryptophan content feedingstuff premixtur carri method set annex hereto articl member state bring forc august law regul administr provis compli provis direct immedi inform commiss thereof appli measur septemb member state adopt measur refer direct accompani refer time offici public procedur refer adopt member state articl direct enter forc dai public offici journal european commun articl direct address member state brussel juli commiss david byrn member commiss oj oj oj oj oj oj oj oj annex part determin vitamin purpos scope method determin vitamin retinol feedingstuff premixtur vitamin includ tran retinyl alcohol ci isom determin method content vitamin express intern unit iu kg iu correspond activ μg tran vitamin alcohol μg tran vitamin acet μg tran vitamin palmit limit determin iu vitamin kg principl sampl hydrolys ethanol potassium hydroxid solut vitamin extract light petroleum solvent remov evapor residu dissolv methanol dilut requir concentr content vitamin determin revers phase high perform liquid chromatographi rp hplc uv fluoresc detector chromatograph paramet chosen separ tran vitamin alcohol ci isom reagent ethanol light petroleum boil rang methanol potassium hydroxid solut ml sodium ascorb solut ml observ sodium sulfid na sodium sulfid solut mol glycerol observ phenolphthalein solut ml ethanol propanol mobil phase hplc mixtur methanol water exact ratio determin characterist column emploi nitrogen oxygen free tran vitamin acet extra pure certifi activ iu stock solut tran vitamin acet weigh nearest mg mg vitamin acet ml graduat flask dissolv propanol make mark solvent nomin concentr solut iu vitamin ml exact content determin tran vitamin palmit extra pure certifi activ iu stock solut tran vitamin palmit weigh nearest mg mg vitamin palmit ml graduat flask dissolv propanol make mark solvent nomin concentr solut iu vitamin ml exact content determin di tert butyl methylphenol bht observ apparatu vacuum rotari evapor amber glasswar flat bottom conic flask ml ground glass socket graduat flask ground glass stopper narrow neck ml separ funnel conic ml ground glass stopper pear shape flask ml ground glass socket allihn condens jacket length mm ground glass joint adapt ga feed pipe pleat filter paper phase separ diamet mm schleicher amp schuell hy hplc equip inject system liquid chromatograph column mm mm μm pack equival perform criterion singl peak retinol isom hplc condit uv fluoresc detector variabl wavelength adjust spectrophotomet mm quartz cell water bath magnet stirrer extract apparatu figur consist glass cylind capac fit ground glass neck stopper ground glass insert equip side arm adjust tube pass centr adjust tube shape lower end jet opposit end upper liquid layer cylind transfer separ funnel procedur note vitamin sensit uv light oxid oper carri absenc light amber glasswar glasswar protect aluminium foil oxygen flush nitrogen extract air liquid replac nitrogen avoid excess pressur loosen stopper time time prepar sampl grind sampl pass mm mesh siev take care avoid gener heat grind carri immedi weigh saponif loss vitamin saponif depend vitamin content weigh nearest sampl ml flat bottom conic flask add success swirl ml ethanol approxim mg bht ml sodium ascorb solut ml sodium sulfid solut fit condens flask immers flask water bath magnet stirrer heat boil reflux minut add ml potassium hydroxid solut condens reflux min stir slow stream nitrogen rins condens approxim ml water cool content flask room temperatur extract transfer decant saponif solut quantit rins total volum ml water ml separ funnel extract apparatu rins saponif flask success ml ethanol ml light petroleum transfer rins separ funnel extract apparatu proport water ethanol combin solut shake vigor minut settl minut extract separ funnel layer separ observ transfer light petroleum layer separ funnel repeat extract ml light petroleum ml light petroleum wash combin extract separ funnel gentli swirl avoid format emuls ml portion water repeat shake ml portion water water remain colourless addit phenolphthalein solut wash time suffici filter wash extract dry pleat filter phase separ remov suspend water ml graduat flask rins separ funnel filter ml light petroleum make mark light petroleum mix extract extract apparatu layer separ observ replac stopper glass cylind ground glass insert posit shape lower end adjust tube level interfac applic pressur nitrogen line side arm transfer upper light petroleum layer ml separ funnel add ml light petroleum glass cylind stopper shake layer separ transfer upper layer separ funnel repeat extract procedur ml light petroleum ml portion light petroleum add light petroleum layer separ funnel wash combin light petroleum extract proce prepar sampl solut hplc pipett aliquot portion light petroleum solut ml pear shape flask evapor solvent dryness rotari evapor reduc pressur bath temperatur exceed restor atmospher pressur admit nitrogen remov flask rotari evapor remov remain solvent stream nitrogen dissolv residu immedi volum ml methanol concentr vitamin rang iu ml iu ml determin hplc vitamin separ revers phase column concentr measur mean uv detector nm fluoresc detector excit nm emiss nm inject aliquot portion μl methanol solut obtain elut mobil phase calcul peak height area inject sampl solut peak height area inject calibr solut hplc condit condit offer guidanc condit provid give equival result gt tabl gt calibr prepar work standard solut pipett ml vitamin acet stock solut ml vitamin palmit stock solut ml flat bottom conic flask hydrolys addit bht subsequ extract light petroleum make ml light petroleum evapor ml extract rotari evapor dryness remov remain solvent stream nitrogen redissolv residu ml methanol nomin concentr solut iu vitamin ml exact content determin work standard solut freshli prepar pipett ml work standard solut ml graduat flask make mark methanol mix nomin concentr dilut work standard solut iu vitamin ml prepar calibr solut calibr graph transfer ml dilut work standard solut seri ml graduat flask make mark methanol mix nomin concentr solut iu vitamin ml inject μl calibr solut time determin peak height area peak height area plot calibr graph result uv control uv standardis standard solut vitamin acet stock solut pipett ml vitamin acet stock solut ml graduat flask make mark propanol nomin concentr solut iu vitamin ml pipett ml dilut vitamin acet solut ml graduat flask make mark propanol nomin concentr solut iu vitamin ml measur uv spectrum solut propanol spectrophotomet nm nm extinct maximum nm nm calcul vitamin content gt pic file quot en ep quot gt vitamin palmit stock solut pipett ml vitamin palmit stock solut ml graduat flask make mark propanol nomin concentr solut iu vitamin ml pipett ml dilut vitamin palmit solut ml graduat flask make mark propanol nomin concentr solut iu vitamin ml measur uv spectrum solut propanol spectrophotomet nm nm extinct maximum nm nm calcul vitamin content gt pic file quot en ep quot gt vitamin work standard solut pipett ml undilut vitamin work standard solut prepar ml graduat flask make mark propanol pipett ml solut ml graduat flask make mark propanol nomin concentr solut iu vitamin ml measur uv spectrum solut propanol spectrophotomet nm nm extinct maximum nm nm calcul vitamin content gt pic file quot en ep quot gt calcul result height area vitamin peak sampl solut determin concentr sampl solut iu ml refer calibr graph vitamin content iu kg sampl formula gt pic file quot en ep quot gt vitamin concentr sampl solut iu ml volum sampl solut ml volum aliquot ml mass test portion observ sampl low vitamin concentr combin light petroleum extract saponif charg amount weigh sampl solut hplc determin weight sampl analysi fat phase separ occur add approxim ml ethanol break emuls cod liver oil pure fat saponif time extend minut hydroquinon bht normal phase column separ retinol isom approxim mg ascorb acid sodium ascorb solut approxim mg edta sodium sulfid solut repeat differ result parallel determin carri sampl exce rel higher result result collabor studi gt tabl gt figur extract apparatu gt pic file quot en ep quot gt part determin vitamin purpos scope method determin vitamin feedingstuff premixtur content vitamin express mg dl tocopherol acet kg mg dl tocopherol acet correspond mg dl tocopherol vitamin limit determin mg vitamin kg principl sampl hydrolys ethanol potassium hydroxid solut vitamin extract light petroleum solvent remov evapor residu dissolv methanol dilut requir concentr content vitamin determin revers phase high perform liquid chromatographi rp hplc fluoresc uv detector reagent ethanol light petroleum boil rang methanol potassium hydroxid solut ml sodium ascorb solut ml observ sodium sulfid na sodium sulfid solut mol glycerol observ phenolphthalein solut ml ethanol mobil phase hplc mixtur methanol water exact ratio determin characterist column emploi nitrogen oxygen free dl tocopherol acet extra pure certifi activ stock solut dl tocopherol acet weigh nearest mg mg dl tocopherol acet ml graduat flask dissolv ethanol make mark solvent ml solut mg dl tocopherol acet uv control stabilis observ dl tocopherol extra pure certifi activ weigh nearest mg mg dl tocopherol ml graduat flask dissolv ethanol make mark solvent ml solut mg dl tocopherol uv control stabilis observ di tert butyl methylphenol bht observ apparatu vacuum rotari evapor amber glasswar flat bottom conic flask ml ground glass socket graduat flask ground glass stopper narrow neck ml separ funnel conic ml ground glass stopper pear shape flask ml ground glass socket allihn condens jacket length mm ground glass joint adapt ga feed pipe pleat filter paper phase separ diamet mm schleicher amp schuell hy hplc equip inject system liquid chromatograph column mm mm μm pack equival fluoresc uv detector variabl wavelength adjust spectrophotomet mm quartz cell water bath magnet stirrer extract apparatu figur consist glass cylind capac fit ground glass neck stopper ground glass insert equip side arm adjust tube pass centr adjust tube shape lower end jet opposit end upper liquid layer cylind transfer separ funnel procedur note vitamin sensit uv light oxid oper carri absenc light amber glasswar glasswar protect aluminium foil oxygen flush nitrogen extract air liquid replac nitrogen avoid excess pressur loosen stopper time time prepar sampl grind sampl pass mm mesh siev take care avoid gener heat grind carri immedi weigh saponif loss vitamin saponif depend vitamin content weigh nearest sampl ml flat bottom conic flask add success swirl ml ethanol approxim mg bht ml sodium ascorb solut ml sodium sulfid solut fit condens flask immers flask water bath magnet stirrer heat boil reflux minut add ml potassium hydroxid solut condens reflux min stir slow stream nitrogen rins condens approxim ml water cool content flask room temperatur extract transfer decant saponif solut quantit rins total volum ml water ml separ funnel extract apparatu rins saponif flask success ml ethanol ml light petroleum transfer rins separ funnel extract apparatu proport water ethanol combin solut shake vigor minut settl minut extract separ funnel layer separ observ transfer light petroleum layer separ funnel repeat extract ml light petroleum ml light petroleum wash combin extract separ funnel gentli swirl avoid format emuls ml portion water repeat shake ml portion water water remain colourless addit phenolphthalein solut wash time suffici filter wash extract dry pleat filter phase separ remov suspend water ml graduat flask rins separ funnel filter ml light petroleum make mark light petroleum mix extract extract apparatu layer separ observ replac stopper glass cylind ground glass insert posit shape lower end adjust tube level interfac applic pressur nitrogen line side arm transfer upper light petroleum layer ml separ funnel add ml light petroleum glass cylind stopper shake layer separ transfer upper layer separ funnel repeat extract procedur ml light petroleum ml portion light petroleum add light petroleum layer separ funnel wash combin light petroleum extract proce prepar sampl solut hplc pipett aliquot portion light petroleum solut ml pear shape flask evapor solvent dryness rotari evapor reduc pressur bath temperatur exceed restor atmospher pressur admit nitrogen remov flask rotari evapor remov remain solvent stream nitrogen dissolv residu immedi volum ml methanol concentr dl tocopherol rang μg ml μg ml determin hplc vitamin separ revers phase column concentr measur fluoresc detector excit nm emiss nm uv detector nm inject aliquot portion μl methanol solut obtain elut mobil phase calcul peak height area inject sampl solut peak height area inject calibr solut hplc condit condit offer guidanc condit provid give equival result gt tabl gt calibr dl tocopherol acet dl tocopherol dl tocopherol acet standard prepar work standard solut transfer pipett ml dl tocopherol acet stock solut ml flat bottom conic flask hydrolys subsequ extract light petroleum make ml light petroleum evapor ml extract rotari evapor dryness remov remain solvent stream nitrogen redissolv residu ml methanol nomin concentr solut μg dl tocopherol ml equival μg dl tocopherol acet ml work standard solut freshli prepar prepar calibr solut calibr graph transfer ml work standard solut seri ml graduat flask make mark methanol mix nomin concentr solut μg ml dl tocopherol acet μg ml μg ml dl tocopherol inject μl calibr solut time determin peak height area peak height area plot calibr graph uv standardis dl tocopherol acet stock solut dilut ml dl tocopherol acet stock solut ml ethanol measur uv spectrum solut ethanol spectrophotomet nm nm absorpt maximum nm gt pic file quot en ep quot gt dilut extinct obtain dl tocopherol standard prepar work standard solut transfer pipett ml dl tocopherol stock solut ml graduat flask dissolv methanol make mark methanol nomin concentr solut μg dl tocopherol ml equival μg dl tocopherol acet ml work standard solut freshli prepar prepar calibr solut calibr graph transfer ml work standard solut seri ml graduat flask make mark methanol mix nomin concentr solut μg ml dl tocopherol μg ml μg ml dl tocopherol acet inject μl calibr solut time determin peak height area peak height area plot calibr graph uv standardis dl tocopherol stock solut dilut ml dl tocopherol stock solut ml ethanol measur uv spectrum solut ethanol spectrophotomet nm nm absorpt maximum nm gt pic file quot en ep quot gt dilut extinct obtain calcul result height area vitamin peak sampl solut determin concentr sampl solut μg ml calcul tocopherol acet refer calibr graph vitamin content mg kg sampl formula gt pic file quot en ep quot gt vitamin concentr sampl solut μg ml volum sampl solut ml volum aliquot ml mass test portion observ sampl low vitamin concentr combin light petroleum extract saponif charg amount weigh sampl solut hplc determin weight sampl analysi fat phase separ occur add approxim ml ethanol break emuls spectrophotometr measur dl tocopherol acet dl tocopherol solut add approxim mg bht solut solut refriger storag life maximum week hydroquinon bht normal phase column separ tocopherol approxim mg ascorb acid sodium ascorb solut approxim mg edta sodium sulfid solut repeat differ result parallel determin carri sampl exce rel higher result result collabor studi gt tabl gt figur extract apparatu gt pic file quot en ep quot gt part determin tryptophan purpos scope method determin total free tryptophan feedingstuff distinguish form principl determin total tryptophan sampl hydrolyz alkalin condit satur barium hydroxid solut heat oc hour hydrolysi intern standard ad determin free tryptophan sampl extract mild acid condit presenc intern standard tryptophan intern standard hydrolys extract determin hplc fluoresc detect reagent doubl distil water water equival qualiti conduct amp lt ìs cm standard substanc tryptophan puriti content amp gt dri vacuum phosphor pentoxid intern standard substanc methyl tryptophan puriti content amp gt dri vacuum phosphor pentoxid barium hydroxid octa hydrat care expos ba excess air order avoid format baco disturb determin observ sodium hydroxid ortho phosphor acid hydrochlor acid ml methanol hplc grade light petroleum boil rang sodium hydroxid solut mol dissolv naoh water make water hydrochlor acid mol ml hcl make water hydrochlor acid mol ml hcl make water hydrochlor acid mol ml hcl make water ortho phosphor acid mol ml ortho phosphor acid make water concentr solut tryptophan μmol ml ml volumetr flask dissolv tryptophan hydrochlor acid make mark hydrochlor acid store oc maximum week concentr intern standard solut μmol ml ml volumetr flask dissolv methyl tryptophan hydrochlor acid make mark hydrochlor acid store oc maximum week calibr standard solut tryptophan intern standard ml concentr solut tryptophan ml concentr intern standard methyl tryptophan solut dilut water methanol approxim volum approxim concentr methanol finish hydrolys solut prepar freshli protect direct sunlight prepar acet acid trichloro methyl propanol ethanolamin amp gt solut trichloro methyl propanol ml methanol mobil phase hplc acet acid ml water ml solut trichloro methyl propanol methanol ml adjust ph ethanolamin make ml water apparatu hplc equip spectrofluorimetr detector liquid chromatograph column mm mm μm pack equival ph meter polypropylen flask capac ml wide neck screw cap membran filter μm autoclav oc bar mechan shaker magnet stirrer vortex mixer procedur prepar sampl sampl ground pass mm siev sampl high moistur air dri temperatur exceed oc freez dri prior grind sampl high fat content extract light petroleum prior grind determin free tryptophan extract weigh nearest mg amount prepar sampl conic flask add ml hydrochlor acid mol ml concentr intern standard solut shake mix min mechan shaker magnet stirrer sediment settl pipett ml supernat solut beaker add ml ortho phosphor acid mol adjust ph sodium hydroxid mol add suffici methanol give concentr methanol final volum transfer volumetr flask volum dilut water volum chromatographi approxim volum calibr standard solut filter ml solut μm membran filter inject hplc column proce chromatographi step paragraph protect standard solut extract direct sunlight analys extract dai extract store oc maximum dai determin total tryptophan hydrolys weigh nearest mg prepar sampl polypropylen flask weigh sampl portion nitrogen content mg add barium hydroxid octa hydrat ml water mix vortex mixer magnet stirrer leav teflon coat magnet mixtur wash wall vessel ml water put screw cap close flask loos transfer autoclav boil water steam minut close autoclav autoclav oc hour open autoclav reduc temperatur oc order avoid crystallis ba add warm mixtur ml water room temperatur shake stir gentli add ml concentr intern standard methyl tryptophan solut cool vessel water ic bath minut add ml ortho phosphor acid mol vessel cool bath neutralis hcl mol whilst stir adjust ph hcl mol add suffici methanol give concentr methanol final volum transfer volumetr flask volum dilut water defin volum chromatographi ml addit methanol precipit filter ml solut μm membran filter inject hplc column proce chromatographi step paragraph protect standard solut hydrolys direct sunlight analys hydrolys dai store oc maximum dai hplc determin condit isocrat elut offer guidanc condit provid yield equival result observ gt tabl gt calcul result gt pic file quot en ep quot gt peak area intern standard calibr standard solut peak area tryptophan extract hydrolys volum ml ml concentr tryptophan solut ad calibr solut concentr μmol ml concentr tryptophan solut ad calibr solut volum ml concentr intern standard solut ad extract ml hydrolys ml peak area intern standard extract hydrolys peak area tryptophan calibr standard solut volum ml ml concentr intern standard solut ad calibr standard solut sampl weight correct origin weight dri defat mw molecular weight tryptophan repeat differ result parallel determin carri sampl exce rel highest result result collabor studi commun collabor studi fourth intercomparison arrang sampl analys laboratori certifi method hydrolysi replic analys perform sampl result tabl gt tabl gt commun collabor studi intercomparison arrang sampl analys laboratori certifi method extract free tryptophan replic analys perform sampl result tabl gt tabl gt commun intercomparison studi arrang sampl analys laboratori aim tryptophan certif hydrolysi result replic analys perform sampl gt tabl gt observ special chromatograph condit give separ tryptophan methyl tryptophan gt tabl gt chromatographi vari type hplc column pack materi chosen system capabl give baselin separ tryptophan intern standard import degrad product separ tryptophan intern standard hydrolys intern standard run order check base line intern standard impur import run time suffici long elut degrad product late elut peak interfer subsequ chromatograph run rang oper chromatograph system give linear respons linear respons measur constant normal concentr intern standard vari concentr tryptophan import size tryptophan intern standard peak linear rang hplc fluoresc system tryptophan intern standard peak small high analysi repeat sampl size chang final volum barium hydroxid ag barium hydroxid difficult dissolv result unclear solut hplc determin produc low result tryptophan conduct feedingstuff work group verband deutscher landwirtschaftlich untersuchung und forschungsanstalten vdlufa conduct feedingstuff work group verband deutscher landwirtschaftlich untersuchung und forschungsanstalten vdlufa