eleventh commiss direct eec juli establish commun analysi method offici control feedingstuff commiss european commun regard treati establish european econom 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 halofuginon establish check complianc condit anim nutrit measur provid direct accord opinion stand committe feedingstuff adopt direct articl member state requir analys halofuginon content purpos offici check feedingstuff made method annex hereto articl member state bring forc law regul administr provis compli direct june immedi inform commiss thereof member state adopt provis refer direct accompani refer time offici public procedur refer adopt member state articl direct address member state brussel juli commiss rené steichen member commiss oj oj annex determin halofuginon dl tran bromo chloro hydroxi piperidyl acetonyl quinazolin hydrobromid purpos scope method determin halofuginon feedingstuff lower limit determin mg kg principl treatment hot water halofuginon extract free base ethyl acet subsequ partit hydrochlorid aqueou acid solut extract purifi ion exchang chromatographi content halofuginon determin reserv phase high perform liquid chromatographi hplc uv detector reagent acetonitril hplc grade amberlit xad resin ammonium acet ethyl acet acet acid glacial halofuginon standard substanc dl tran brome chloro hydroxi piperidyl acetonyl quinazolin hydrobromid halofuginon stock standard solut mg ml weight nearest mg mg halofuginon ml graduat flask dissolv ammonium acet buffer solut make mark buffer solut mix solut stabl week store dark calibr solut seri ml graduat flask transfer ml stock standard solut make mark mobil phase mix solut concentr mg ml halofuginon solut freshli prepar hydrochlor acid approxim ml methanol silver nitrat sodium ascorb sodium carbon sodium chlorid edta ehtylenediaminetetraacet acid disodium salt water hplc grade sodium carbon solut ml sodium chlorid satur sodium carbon solut ml dissolv sodium carbon water dilut add sodium chlorid solut satur hydrochlor acid approxim mol dilut ml hci water ammonium acet buffer solut approxim dissolv ammonium acet ml acet acid water dilut amberlit xad resin prepar wash quantiti amberlit water chlorid ion remov silver nitrat test perform discard aqueou phase wash resin ml methanol discard methanol store resin fresh methanol silver nitrat solut approxim mol dissolv silver nitrat ml water hplc mobil phase mix ml acetonitril ml ammonium acet buffer solut ml water adjust ph acet acid filter mm filter dega solut ultrasonif minut solut stabl month store dark close contain apparatu ultrason bath rotari film evapor centrifug hplc equip variabl wavelength ultraviolet detector diod arrai detector liquid chromatograph column mm mm mm packag equival column glass column mm mm fit sinter glass filter stopcock glass fibr filter diamet mm membran filter mm membran filter mm procedur note halofuginon free base unstabl alkalin ethyl acet solut remain ethyl acet minut gener blank feed analys check halofuginon interf substanc present recoveri test carri analys blank feed fortifi addit quantiti halofuginon 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 halofuginon detect extract weigh nearest prepar sampl ml centrifug tube add sodium ascorb edta ml water mix place tube minut water bath cool room temperatur add ml sodium carbon solut mix add immedi ml ethyl acet shake vigor hand second place tube minut ultrason bath loosen stopper centrifug minut decant ethyl acet phase glass fibr filter ml separ funnel repeat extract te sampl portion ml ethyl acet wash combin extract minut ml sodium chlorid satur sodium carbon solut discard aqueou layer extract organ layer min ml hydrochlor acid run lower acid layer ml separ funnel extract organ layer minut ml hydrochlorid acid combin extract wash combin acid extract swirl approxim second ml ethyl acet quantit transfer aqueou layer ml round bottom flask discard organ phase evapor remain ethyl acet acid solut rotari film evapor temperatur water bath exce vacuum approxim mbar residu ethyl acet remov minut clean prepar amberlit column xad column prepar sampl extract transfer prepar amberlit glass column methanol add small plug glass wool top resin bed drain methanol column wash resin ml water stop flow liquid reach top resin bed column equilibr minut column run dry sampl clean transfer extract quantit top prepar amberlit column elut discard eluat rate elut exce ml min rins round bottom flask ml hydrochlorid acid wash resin column blow remain acid solut stream air discard wash add ml methanol column ml elut collect eluat ml round bottom flask leav remain methanol minut equilibr resin continu elut rate exceed ml min collect eluat round bottom flask evapor methanol rotari film evapor temperatur waterbath exce transfer residu quantit ml calibr flask mobil phase make mark mobil phase mix aliquot filter membran filter reserv solut hplc determin hplc determin paramet condit offer guidanc condit provid yield equival result liquid chromatograph column hplc mobil phase flow rate ml min detect wavelength nm inject volum ml check stabil chromatograph system inject calibr solut mg ml time constant 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 halofuginon peak calcul result determin concentr sampl solut mg ml height area halofuginon peak sampl solut refer calibr graph content halofuginon mg kg sampl formula halofuginon 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 calibr solut amount ad halofuginon similar estim amount halofuginon found sampl extract height halofuginon peak enhanc take account amount ad dilut extract peak width half maximum height origin width diod arrai detect result evalu criteria wavelength maximum asbsorpt 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 apex criteria met presenc analyt confirm repeat differ result parallel determin carri sampl exce mg kg halofuginon content mg kg recoveri fortifi blank sampl recoveri result collabor studi collabor studi arrang sampl analys laboratori result tabl oj analyst pp