commiss direct ec septemb establish commun method analysi determin amino acid crude oil fat olaquindox feedingstuff amend direct eec 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 april market compound feedingstuff amend commiss direct ec council direct eec septemb feedingstuff intend nutrit purpos amend direct ec stipul amino acid crude oil fat set feedingstuff label council direct eec novemb addit feedingstuff amend commiss direct ec stipul olaquindox content label substanc ad compound feedingstuff commiss direct eec novemb establish commun method analysi offici control feedingstuff amend commiss direct eec set method analysi inter alia determin crude oil fat modifi method 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 amino acid crude oil fat olaquindox content feedingstuff carri method set annex hereto articl annex commiss direct eec point determin crude oil fat replac part annex direct articl member state bring forc law regul administr provis compli direct decemb immedi inform commiss thereof appli measur januari member state adopt measur refer direct accompani refer time offici public procedur refer adopt member state member state commun commiss text main provis domest law adopt field govern direct articl direct enter forc dai public offici journal european commun articl direct address member state brussel septemb commiss franz fischler member commiss oj oj oj oj oj oj oj oj oj annex part determin amino acid purpos scope method determin free synthet natur total peptid bound free amino acid feedingstuff amino acid analyz applic amino acid cyst in methionin lysin threonin alanin arginin aspart acid glutam acid glycin histidin isoleucin leucin phenylalanin prolin serin tyrosin valin method distinguish salt amino acid differenti form amino acid valid determin tryptophan hydroxi analogu amino acid principl free amino acid free amino acid extract dilut hydrochlor acid coextract nitrogen macromolecul precipit sulfosalicyl acid remov filtrat filter solut adjust ph amino acid separ ion exchang chromatographi determin reaction ninhydrin photometr detect nm total amino acid procedur chosen depend amino acid investig cyst in methionin oxidis cysteic acid methionin sulphon prior hydrolysi tyrosin determin hydrolys unoxid sampl amino acid list paragraph determin oxidis unoxidis sampl oxid perform perform acid phenol mixtur excess oxid reagent decompos sodium disulphit oxidis unoxidis sampl hydrolys hydrochlor acid mol hour hydrolys adjust ph amino acid separ ion exchang chromatographi determin reaction ninhydrin photometr detect nm nm prolin reagent doubl distil water water equival qualiti conduct num gt mw gt den gt amino acid kg sampl intern standard multipli gt num gt gt den gt gt tabl gt cystin cystein determin cysteic acid hydrolys oxid sampl calcul cystin mw mw methionin determin methionin sulphon hydrolys oxid sampl calcul methionin mw methionin ad free methionin determin extract methionin calcul mw total dilut volum extract determin free amino acid calcul ml gt num gt ml ml gt den gt ml gt num gt vml gt den gt ml volum final extract evalu method method test intercomparison made intern level feedingstuff mix pig feed broiler compound protein concentr premixtur result elimin outlier standard deviat tabl gt tabl gt repeat repeat express laboratori standard deviat mention intercomparison tabl gt tabl gt gt tabl gt reproduc result laboratori standard deviat mention intercomparison tabl gt tabl gt gt tabl gt refer materi correct applic method verifi make replic measur certifi refer materi calibr certifi amino acid calibr solut recommend observ differ amino acid analys final concentr calibr solut standard amino acid hydrolys guidelin rang linear respons apparatu check amino acid standard solut dilut citrat buffer give peak area middl rang high perform liquid chromatograph equip analys hydrolys experiment condit optimis accord manufactur recommend appli method feedingstuff chlorid concentr miner feed supplementari feed underestim methionin occur special treatment part determin crude oil fat purpos scope method determin crude oil fat feedingstuff cover analysi oil seed oleagin fruit defin council regul eec septemb procedur depend natur composit feedingstuff reason carri analysi procedur directli extract crude oil fat method applic feed materi plant origin includ scope procedur procedur total crude oil fat method applic feed materi anim origin compound feed materi oil fat complet extract prior hydrolysi gluten yeast potato protein product subject process extrus flake heat interpret result case higher result obtain procedur procedur result obtain procedur accept true principl procedur sampl extract light petroleum solvent distil residu dri weigh procedur sampl treat heat hydrochlor acid mixtur cool filter residu wash dri submit determin procedur reagent light petroleum boil rang bromin residu evapor mg ml sodium sulfat anhydr hydrochlor acid mol hcl filtrat aid kieselguhr hyflo supercel apparatu extract apparatu fit siphon soxhlet apparatu reflux rate produc cycl hour siphon type reflux rate ml minut extract thimbl free matter solubl light petroleum poros consist requir point dry oven vacuum oven set air oven set procedur procedur point weigh sampl nearest mg transfer extract thimbl cover fat free wad cotton wool place thimbl extractor extract hour light petroleum collect light petroleum extract dry weigh flask fragment pumic stone distil solvent dry residu maintain flask half hour dry oven leav cool desicc weigh dry minut ensur weight oil fat remain constant loss weight success weigh mg procedur weigh sampl nearest mg point place ml beaker ml conic flask add ml hydrochlor acid fragment pumic stone cover beaker watch glass fit conic flask reflux condens bring mixtur gentl boil low flame hot plate hour product stick side contain cool add quantiti filtrat aid suffici prevent loss oil fat filtrat filter moisten fat free doubl filter paper wash residu cold water neutral filtrat obtain check filtrat oil fat presenc sampl extract light petroleum procedur hydrolysi place doubl filter paper residu watch glass dry half hour air oven place doubl filter paper dry residu extract thimbl cover fat free wad cotton wool place thimbl extractor proce paragraph point express result express weight residu percentag sampl repeat differ result parallel determin carri sampl analyst exce absolut content crude oil fat lower rel highest result content absolut content observ product high content oil fat difficult crush unsuit draw homogen reduc test sampl proce weigh sampl nearest mg mix anhydr sodium sulfat extract light petroleum point make extract obtain ml light petroleum mix ml solut place small dry weigh flask fragment pumic stone distil solvent dry proce paragraph point elimin solvent extract residu left thimbl crush residu fine mm return extract thimbl add sodium sulfat proce paragraph point calcul content oil fat percentag sampl formula mass gram residu extract aliquot part extract mass gram residu extract product low oil fat test sampl increas pet food high content water mix anhydr sodium sulfat prior hydrolysi extract procedur paragraph effect hot water place cold water wash residu filtrat dry time extend feedingstuff excess dry avoid lead low result microwav oven pre extract procedur prior hydrolysi extract procedur recommend crude oil fat content greater extent depend natur feedingstuff natur oil fat feedingstuff part determin olaquindox hydroxyethyl carbamoyl methylquinoxalin dioxyd purpos scope method determin olaquindox feedingstuff lower limit determin mg kg principl sampl extract water methanol mixtur content olaquindox determin revers phase high perform liquid chromatographi hplc uv detector reagent methanol methanol hplc grade water hplc grade mobil phase hplc water methanol mixtur standard substanc pure olaquindox hydroxyethyl carbamoyl methylquinoxalin dioxyd olaquindox stock standard solut ìg ml weigh nearest mg mg olaquindox ml graduat flask add ca ml water place flask min ultrason bath ultrason treatment bring solut room temperatur make mark water mix wrap flask aluminum foil store refriger solut prepar fresh month olaquindox intermedi standard solut ìg ml transfer ml stock standard solut ml graduat flask make mark mobil phase mix wrap flask aluminium foil store refriger solut prepar fresh dai calibr solut seri ml graduat flask transfer ml intermedi standard solut make mark mobil phase mix wrap flask aluminium foil solut correspond ìg olaquindox ml solut prepar fresh dai apparatu ultrason bath mechan shaker hplc equip variabl wavelength ultraviolet detector diod arrai detector liquid chromatograph column mm mm ìm pack equival membran filter ìm procedur note olaquindox light sensit carri procedur subdu light amber glasswar gener blank feed analys check olquindox interf substanc present recoveri test carri analys blank feed fortifi addit quantiti olaquindox similar present sampl fortifi level mg kg transfer ml stock standard solut ml conic flask evapor solut ca ml add blank feed mix leav min mix time proceed extract step note purpos method blank feed similar type sampl olaquindox detect extract weigh nearest approxim sampl transfer ml conic flask add ml methanol place flask minut ultrason bath add ml water leav ultrason bath minut remov flask ultrason bath shake minut shaker filter fold filter transfer ml filtrat ml graduat flask make mark water mix aliquot filter membran filter observ proce hplc determin hplc determin paramet condit offer guidanc condit provid give equival result gt tabl gt check stabil chromatograph system inject time calibr solut ìg ml constant peak height retent time achiev calibr graph inject calibr solut time determin peak height area concentr plot calibr graph peak height area calibr solut ordin concentr ìg ml abscissa sampl solut inject sampl extract time volum calibr solut determin peak height area olaquindox peak calcul result height area olaquindox peak sampl solut determin concentr sampl solut ìg ml refer calibr graph olaquindox content mg kg sampl formula gt num gt gt den gt olaquindox concentr sampl extract ìg ml mass test portion valid result ident ident analyt confirm chromatographi diod arrai detector spectra sampl extract calibr solut ìg ml compar chromatographi sampl extract fortifi addit amount calibr solut amount ad olaquindox similar amount olaquindox found sampl extract height olaquindox peak enhanc take account amount ad dilut extract peak width half height origin width olaquindox peak unfortifi sampl extract diod arrai detect result evalu criteria wavelength maximum absorpt sampl standard spectra record peak apex chromatogram margin determin resolv power detect system diod arrai detect typic nm nm sampl standard spectra record peak apex chromatogram part spectrum rang rel absorb criterion met maxima present observ point deviat spectra exce absorb standard analyt nm spectra upslop apex downslop peak produc sampl extract part spectrum rang rel absorb criterion met maxima present observ point deviat spectra exce absorb spectrum peak apex criteria met presenc analyt confirm repeat differ result parallel determin carri sampl exce rel higher result olaquindox content mg kg recoveri fortifi blank sampl recoveri result collabor studi ec collabor studi arrang piglet feed sampl includ blank feed analys laboratori result gt tabl gt observ method valid feed mg kg olaquindox obtain satisfactori result take smaller sampl weight dilut extract reach concentr rang calibr graph oil fat undergo subsequ qualiti test replac fragment pumic stone glass bead