twelfth commiss direct ec decemb establish commun analysi method offici control feedingstuff commiss european commun regard treati establish european commun regard council direct eec juli introduct commun method sampl analysi offici control feedingstuff amend regul eec articl thereof direct eec requir offici control feedingstuff purpos check complianc requir aris qualiti composit provis laid law regul administr action carri commun sampl analysi method commun analysi method addit robenidin methyl benzoqu establish check complianc condit anim feedingstuff measur provid direct accord opinion stand committe feedingstuff adopt direct articl member state requir analys conduct offici check feedingstuff identifi robenidin methyl benzoqu content made method annex hereto articl member state bring forc law regul administr provis requir complianc direct novemb latest forthwith inform commiss thereof member state adopt provis refer direct accompani refer time offici public procedur refer adopt member state articl direct enter forc dai public offici journal european commun brussel decemb commiss rené steichen member commiss oj oj annex determinaton robenidin bi chlorobenzyliden amino guanidin hydrochlorid purpos scope method determin robenidin feedingstuff lower limit determin mg kg principl sampl extract acidifi methanol extract dri aliquot portion subject clean aluminium oxid column robenidin elut column methanol concentr made suitabl volum mobil phase content robenidin determin revers phase high perform liquid chromatographi hplc uv detector reagent methanol acidifi methanol transfer ml hydrochlor acid ml ml graduat flask make mark methanol mix solut freshli prepar acetonitril hplc grade molecular siev type mesh bead mm bead crystallin alumino silic diamet pore mm aluminium oxid acid activ grade column chromatographi transfer aluminium oxid suitabl contain add ml water stopper shake approxim minut store stopper contain potassium dihydrogen phosphat solut mol dissolv potassium dihydrogen phosphat water hplc grade ml graduat flask make mark mix di sodium hydrogen phosphat solut mol dissolv anhydr dihydr dodecahydr di sodium hydrogen phosphat water hplc grade ml graduat flask make mark mix hplc mobil phase mix reagent ml acetonitril ml water hplc grade ml potassium di hydrogen phosphat solut ml di sodium hydrogen phosphat solut filter mm filter dega solut ultrasonif minut standard substanc pure robenidin bi chlorobenzyliden amino guanidin hydrochlorid robenidin stock standard solut mg ml weigh nearest mg mg robenidin standard substanc dissolv acidifi methanol ml graduat flask make mark solvent mix wrap flask aluminium foil store dark place robenidin intermedi standard solut mg ml transfer ml stock standard solut ml graduat flask make mark mobil phase mix wrap flask aluminium foil store dark place calibr solut seri ml calibr flask transfer ml intermedi standard solut make mark mobil phase mix solut correspond mg ml robenidin solut freshli prepar apparatu glass column construct amber glass fit stopcock reservoir approxim ml capac intern diamet mm length mm laboratori wrist action shaker rotari film evapor hplc equip variabl wavelength ultraviolet detector diod arrai detector oper rang mm liquid chromatograph column mm mm mm pack equival glass fibr filter paper whatman gf equival membran filter mm membran filter mm procedur note robenidin light sensit amber glasswar oper gener blank feed analys check robenidin interf substanc present recoveri test carri analys blank feed fortifi addit quantiti robenidin similar present sampl fortifi level mg kg transfer ml stock standard solut ml conic flask evapor solut ml stream nitrogen add blank feed mix wait minut proceed extract step note purpos method blank feed similar type sampl analysi robenidin detect extract weigh nearest approxim prepar sampl transfer ml conic flask add ml acidifi methanol stopper shake hour shaker filter solut glass fibr filter paper collect filtrat ml conic flask add molecular siev stopper shake minut filter immedi glass fibr filter paper retain solut purif step purif prepar aluminium oxid column insert small glass wool plug lower end glass column tamp glass rod weigh prepar aluminium oxid transfer column care minim exposur atmospher stage gentli tap load column lower end settl aluminium oxid sampl purif transfer column pipett ml sampl extract prepar rest pipett tip close column wall solut absorb aluminium oxid elut robenidin column ml methanol flow rate ml minut collect eluat ml round bottom flask evapor methanol solut dryness reduc pressur mean rotari film evapor dissolv residu ml mobil phase transfer quantit ml graduat flask rins flask ml portion mobil phase transfer rins graduat flask make mark solvent mix aliquot filter mm reserv solut hplc determin hplc determin paramet condit offer guidanc condit provid yield equival result liquid chromatograph column hplc mobil phase flow rate ml minut detector wavelength nm inject volum ml check stabil chromatograph system inject calibr solut mg ml time constant peak height retent time achiev calibr graph inject calibr solut time measur peak height area concentr plot calibr curv peak height area calibr solut ordin concentr mg ml abscissa sampl solut inject sampl extract time volum calibr solut determin peak height area robenidin peak calcul result height area robenidin peak sampl solut determin concentr sampl solut mg ml refer calibr graph content robenidin mg kg sampl formula robenidin concentr sampl solut mg ml mass test posit gram valid result ident ident analyt confirm chromatographi diod arrai detector spectra sampl extract calibr solut mg ml compar chromatographi sampl extract fortifi addit amount calibr solut amount ad robenidin similar estim amount robenidin found sampl extract height robenidin peak enhanc take account amount ad dilut extract peak width half maximum height approxim origin width 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 approxim 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 apex criteria met presenc analyt confirm repeat differ result parallel determin carri sampl exce higher result robenidin content higher mg kg recoveri fortifi blank sampl recoveri result collabor studi collabor studi arrang commun sampl poultri rabbit feed meal pellet form analys laboratori duplic analys perform sampl result tabl quot quot id quot quot gt mg kg gt id quot quot gt gt id quot quot gt gt id quot quot gt gt id quot quot gt quot gt id quot quot gt sr mg kg gt id quot quot gt gt id quot quot gt gt id quot quot gt gt id quot quot gt quot gt id quot quot gt cvr gt id quot quot gt gt id quot quot gt gt id quot quot gt gt id quot quot gt quot gt id quot quot gt sr mg kg gt id quot quot gt gt id quot quot gt gt id quot quot gt gt id quot quot gt quot gt id quot quot gt cvr gt id quot quot gt gt id quot quot gt gt id quot quot gt gt id quot quot gt quot gt id quot quot gt recoveri gt id quot quot gt gt id quot quot gt gt id quot quot gt gt id quot quot gt quot quot sr standard deviat repeat cvr coeffici variat repeat sr standard deviat reproduc cvr coeffici variat reproduc gt determin methyl benzoqu benzyloxi butyl methoxycarbonyl quinolon purpos scope method determin methyl benzoqu feedingstuff lower limit determin mg kg principl methyl benzoqu extract sampl methanol methanesulfon acid solut extract purifi dichloromethan ion exchang chromatographi dichloromethan methyl benzoqu content determin revers phase high perform liquid chromatographi hplc uv detector reagent dichloromethan methanol hplc grade hplc mobil phase mixtur methanol water hplc grade filter mm filter dega solut ultrasonif minut methanesulfon acid solut dilut ml methanesulfon acid ml methanol hydrochlor acid solut dilut ml hydrochlor acid ml ml water cation exchang resin amberlit cg na mesh resin pretreat slurri resin ml hydrochlor acid solut heat hot plate boil stir continu cool decant acid filter filter paper vacuum wash resin ml portion water ml methanol rins resin ml portion methanol dry pass air filter cake store dri resin stopper bottl standard substanc pure methyl benzoqu benzyloxi butyl methoxycarbonyl quinolon methyl benzoqu stock standard solut mg ml weigh nearest mg mg standard substanc dissolv methanesulfon acid solut ml graduat flask make mark mix methyl benzoqu intermedi standard solut mg ml transfer ml methyl benzoqu stock standard solut ml graduat flask make mark methanol mix calibr solut transfer ml methyl benzoqu intermedi standard solut seri ml graduat flask make mark mobil phase mix solut concentr mg ml methyl benzoqu solut freshli prepar apparatu laboratori shaker rotari film evapor glass column mm mm fit stopcock reservoir approxim ml capac hplc equip variabl wavelength ultraviolet detector diod arrai detector liquid chromatograph column mm mm mm pack equival membran filter mm membran filter mm procedur gener blank feed analys check methyl benzoqu interf substanc present recoveri test carri analys blank feed fortifi addit quantiti methyl benzoqu similar present sampl fortifi level mg kg add ml stock standard solut blank feed mix wait minut proceed extract step note purpos method blank feed similar type sampl analysi methyl benzoqu detect extract weigh nearest approxim prepar sampl transfer ml conic flask add ml methanesulfon acid solut shake mechan minut filter solut filter paper retain filtrat liquid liquid partit step liquid liquid partit transfer ml separ funnel ml hydrochlor acid solut ml filtrat obtain add ml dichloromethan funnel shake minut layer separ run lower dichloromethan layer ml round bottom flask repeat extract aqueou phase ml portion dichloromethan combin extract round bottom flask evapor dichloromethan extract dryness rotari evapor oper reduc pressur dissolv residu ml methanol stopper flask retain extract ion exchang chromatographi ion exchang chromatographi prepar cation exchang column insert plug glass wool lower end glass column prepar slurri treat cation exchang resin ml hydrochlor acid pour glass column settl run excess acid resin surfac wash column water effluent neutral litmu transfer ml methanol column drain resin surfac column chromatographi mean pipett carefulli transfer extract obtain column rins round bottom flask portion ml methanol transfer wash column run extract resin surfac wash column ml methanol ensur flow rate exce ml minut discard effluent elut methyl benzoqu column ml methanesulfon acid solut collect column eluat ml conic flask liquid liquid partit transfer eluat obtain litr separ funnel rins conic flask ml methanol combin wash content separ funnel add ml hydrochlor acid solut ml dichloromethan shake minut phase separ run lower dichloromethan layer ml round bottom flask repeat extract aqueou phase ml portion dichloromethan combin extract round bottom flask evapor dichloromethan extract dyr rotari evapor oper reduc pressur dissolv residu flask approxim ml methanol transfer solut quantit ml graduat flask rins round bottom flask portion ml methanol transfer graduat flask make mark methanol mix aliquot portion filter membran filter reserv solut hplc determin hplc determin paramet condit offer guidanc condit provid give equival result liquid chromatograh column hplc mobil phase methanol water mixtur flow rate ml minut detect wavelength nm inject volum ml check stabil chromatograph system inject calibr solut mg ml time contant peak height area retent time achiev calibr graph inject calibr solut time measur peak height area concentr plot calibr graph peak height area calibr solut ordin concentr mg ml abscissa sampl solut inject sampl extract time volum calibr solut determin peak height area methyl benzoqu peak calcul result determin concentr sampl solut mg ml height area methyl benzoqu peak sampl solut refer calibr graph content methyl benzoqu mg kg sampl formula methyl benzoqu concentr sampl solut mg ml mass test portion gram valid result ident ident analyt confirm chromatographi diod arrai detector spectra sampl extract calibr solut mg ml compar chromatographi sampl extract fortifi addit amount intermedi standard solut amount ad methyl benzoqu similar estim amount methyl benzoqu sampl extract height methyl benzoqu peak enhanc take account amount ad dilut extract peak width half maximum height approxim origin width 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 approxim 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 apex criteria met presenc analyt confirm repeat differ result parallel determin carri sampl exce rel higher result methyl benzoqu content mg kg recoveri fortifi blank sampl recoveri result collabor studi sampl analys laboratori duplic analys perform sampl result quot quot id quot quot gt mg kg gt id quot quot gt gt id quot quot gt gt id quot quot gt gt id quot quot gt gt id quot quot gt quot gt id quot quot gt sr mg kg gt id quot quot gt gt id quot quot gt gt id quot quot gt gt id quot quot gt gt id quot quot gt quot gt id quot quot gt cvr gt id quot quot gt gt id quot quot gt gt id quot quot gt gt id quot quot gt gt id quot quot gt quot gt id quot quot gt sr mg kg gt id quot quot gt gt id quot quot gt gt id quot quot gt gt id quot quot gt gt id quot quot gt quot gt id quot quot gt cvr gt id quot quot gt gt id quot quot gt gt id quot quot gt gt id quot quot gt gt id quot quot gt quot gt id quot quot gt recoveri gt id quot quot gt gt id quot quot gt gt id quot quot gt gt id quot quot gt gt id quot quot gt quot quot detect sr standard deviat repeat cvr coeffici variat repeat sr standard deviat reproduc cvr coeffici variat reproduc gt