commiss direct eec novemb amend annex seventh direct eec establish commun method analysi 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 act access spain portug articl thereof seventh commiss direct eec march establish commun method analysi offici control feedingstuff amend direct eec prescrib method determin aflatoxin ground adapt method light advanc scientif technic knowledg advis method control low limit aflatoxin fix council direct eec decemb fix maximum permit level undesir substanc product anim nutrit amend direct eec advis method determin aflatoxin presenc interf substanc citru pulp measur provid direct accord opinion stand committe feedingstuff adopt direct articl annex direct eec amend accord annex direct articl member state bring forc octob law regul administr provis compli provis direct fortwith 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 novemb commiss rai mac sharri member commiss oj oj oj oj oj annex part dimension thin layer chromatograph method text point purpos scope replac text purpos scope method make determin level aflatoxin raw materi straight feedingstuff method appli presenc citru pulp lower limit determin mg kg ppb presenc interf substanc repeat analysi method high perform liquid chromatographi ii part dimension thin layer chromatograph method text replac determin aflatoxin high perform liquid chromatograph method purpos scope method determin aflatoxin anim feedingstuff includ citru pulp lower limit determin mg kg ppb principl sampl extract chloroform extract filter aliquot portion purifi florisil cartridg cartridg final separ determin achiev high perform liquid chromatographi hplc revers phase column post column derivat iodin water fluoresc detect note mycotoxin extrem toxic sustanc manipul perform design fume cupboard special precaut toxin dry form electrostat natur result tendenc dispers work area reagent chloroform stabil ethanol mass observ methanol hplc grade preatat aceton acetonitril hplc grade elut solvent prepar dai remov air solvent ultrason mixtur aceton water mixtur water methanol mixtur water aceton mobil phase hplc mixtur water methanol acetonitril nb composit mobil phase solvent adjust depend characterist hplc column satur iodin solut add iodin ml water mix min filter membran filter protect satur solut light prevent photodegrad acid wash celit equival florisil cartridg water sep pak equival cartridg water sep pak equival inert ga nitrogen aflatoxin standard solut chloroform concentr mg ml check concentr solut determin absorpt spectrum solut nm mean spectrophotomet measur absorb maximum nm calcul concentr aflatoxin microgram millilitr solut formula concentr mg ml aflatoxin stock standard solut chloroform transfer quantit ml aflatoxin standard solut ml volumetr flask adjust mark chloroform store solut cool place dark seal wrap aluminium foil aflatoxin calibralt solut hplc nb alcid wash glasswar prepar solut apparatu calibr solut ng ml volumetr flask stock standard solut warm room temperatur aluminium foil hour transfer ml stock standard solut ng aflatoxin ml volumetr flask evapor solut dryness current inet ga dissolv residu obtain approxim ml water aceton mixtur make mark water aceton mixtur mix calibr solut ng ml transfer quantit ml calibr solut ml volumetr flask make mark water aceton mixtur mix calibr solut ng ml transfer quantit ml calibr solut ml volumetr flask make mark water aceton mixtur mix solut refer refer standard repetit inject hplc calibr solut ng ml transfer quantit ml calibr solut ml volumetr flask make mark water aceton mixtur mix ampoul mixtur aflatoxin concentr approxim mg ml ml chloroform chromatograph test solut transfer content ampoul glass stopper test tube screw cap vial transfer ml solut glass stopper test tube acid rins evapor chloroform stream inert ga redissolv ml water aceton mixtur reagent confirmatori test sodium chlorid satur solut sodium sulphat anhydr granular apparatu caution acid wash glasswar aqueou aflatoxin solut loss care glasswar dispos glasswar autosampl vial pasteur pipett laboratori glasswar come contact aqueou solut aflatoxin soak dilut acid sulfur acid mol hour rins distil water remov trace acid time check ph paper practic treatment round bottom flask volumetr flask measur cylind vial tube calibr solut final extract vial autosampl pasteur pipett transfer calibr solut extract grinder mixer siev apertur size mm iso mechan shaker rotari vacuum evapor equip ml round bottom flask high perform liquid chromatograph injector loop suitabl inject ml manufactur instruct partial complet loop fill hplc analyt column mm mm pack puls free pump deliveri iodin post column reagent valco dead volum tee stainless steel quot mm spiral reaction coil teflon stainless steel dimens mm mm found combin mm mm hplc column thermostat control water bath adjust capabl temperatur regul fluoresc detector excit nm emiss nm wavelenth filter instrument emiss wavelength gt nm detect ng aflatoxin back pressur advis restrictor teflon stainless steel coil connect outlet detector suppress air bubbl flow cell strip chart record electron integr option flute filter paper diamet cm macherei nagel equival membran filter pore size mm millipor hawp equival ml glass stopper conic flask glass column intern diamet approxim cm length approxim cm equip luer tip luer chloroform resist stopcock bio rad analytichem baker equival chemic resist syring ml luer connector syring suitabl hplc inject ml ml microsyring prepar calibr solut check accuraci weigh ml glass stopper calibr tube spectrophotomet suitabl make measur uv region spectrum equip confirmatori test acid rins ml separ funnel teflon stopcock heat block procedur prepar sampl grind sampl pass siev test portion weigh prepar test sampl conic flask extract add celit ml chloroform ml water test portion stopper flask shake minut mechan shaker filter flute filter paper collect ml filtrat aliquot filtrat dilut ml chloroform concentr aflatoxin greater ng ml clean procedur carri signific interrupt caution protect laboratori analys adequ daylight achiev effect egmond van heisterkamp paulsch food addit contamin iso oj uv absorb foil window combin subdu light direct sunlight ii curtain blind combin artifici light fluoresc tube accept aflatoxin solut protect light dark aluminium foil florisil sep pak purif prepar column cartridg assembl attach stopcock shorter stem florisil cartridg figur wash cartridg remov aid take ml chloroform pass ml stopcock rapidli cartridg syring attach longer stem cartridg glass column pass reamin ml chloroform cartridg column close stopcock remov syring purif add filtrat collect column cartridg assembl drain graviti rins ml chloroform ml methanol discard eluat oper ensur column cartridg assembl run dry elut aflatoxin ml aceton water mixtur collect eluat round bottom flask rotari evapor concentr eluat rotari evapor aceton distil nb approxim ml liquid remain flask point experi shown evapor harm ml liquid remain signific amount aceton residu aceton lead loss aflatoxin cartridg add ml methanol swirl flask dissolv aflatoxin side flask add ml water mix disconnect discard cartridg rins glass column water retain purif step sep pak purif prepar column cartridg assembl attach stopcock shorter stem cartridg figur prime cartridg remov air pass ml methanol stopcock rapidli cartridg syring air bubbl cartridg visibl light spot greyish background ml water pass ml cartridg avoid introduct air cartridg switch methanol water attach longer stem cartridg glass column pass remain ml water cartridg column close stopcock remov syring purif transfer extract collect quantit glass column rins flask ml water methanol mixtur drain graviti oper ensur column cartridg assembl run dry air bubbl develop constrict cartridg stop flow tap top glass column remov air bubbl continu elut ml water methanol mixtur discard eluat elut aflatoxin ml water aceton mixtur collect eluat ml volumetr flask make mark water mix result test solut chromatographi caution filtrat final extract prior hplc consid cellulos filter lead loss aflatoxin teflon filter accept high perform liquid chromatographi figur set equip suffici time condit stabil instrument note flow rate mobil phase post column reagent indic adjust depend characterist hplc column note detector respons aflatoxin depend temperatur compens made drift figur inject fix amoung aflatoxin refer standard regular interv inject aflatoxin peak valu refer standard correct respons provid differ respons consecut refer standard small lt inject made interrupt interrupt inject interrupt inject interrupt refer standard calibr curv linear pass origin amount aflatoxin sampl extract determin directli refer adjac standard hplc pump set set hplc pump give flow ml min mm mm hplc column mobil phase post column pump set set pump give flow ml min iodin satur water solut rough guid flow approxim ml min advis combin flow ml min mobil phase fluoresc detector set fluoresc detector exc nm em nm filter instrument gt nm adjust detector attenu obtain approxim full scale deflect record pen ng aflatoxin injector solut inject ml amount instruct manufactur injector check chromatograph separ inject chromatograph test solut vallei sum peak height adjac peak check stabil system seri analys inject refer standard stabl peak area achiev nb peak respons aflatoxin consecut inject differ proce delai check linear check linear inject aflatoxin calibr solut inject refer standard correct drift respons nb peak respons refer standard differ minut correct drift formula calibr graph linear pass origin standard error estim valu found differ nomin valu requir fulfil continu delai identifi correct sourc problem continu inject sampl extract inject purifi sampl extract sampl extract repeat inject refer standard sequenc refer standard extract extract refer standard extract extract refer standard confirmatori test treatment extract add ml sodium chlorid solut final extract obtain extract time ml chloroform minut separ funnel pour combin chloroform extract approxim sodium sulphat ml test tube small funnel diamet cm piec cottonwool constrict cover approxim sodium sulphat wash sodium sulphat layer ml chloroform collect wash test tube evapor chloroform extract dryness test tube heat block redissolv ml chloroform prepar deriv thin layer chromatographi annex council direct eec method point calcul result calcul aflatoxin content mm kg present sampl formula aflatoxin content mg kg ve vm vf vc amount aflatoxin ng repres peak sampl calcul sampl st st st sampl peak area aflatoxin sampl st peak area aflatoxin result preceed inject refer standard st peak area aflatoxin result inject refer standard st inject amount aflatoxin refer standard ng vm volum inject sampl extract ml ve final volum sampl extract ml allow dilut made mass sampl vf volum filtrat transfer florisil cartridg ml vc volum chloroform extract sampl ml procedur protocol formula reduc aflatoxin content mg kg calcul result peak height measur repeat reproduc observ precis collabor studi carri intern level mix feed stuff gave result repeat reproduc tabl term repeat defin largest ratio signific probabl level comparison read sampl laboratori similar condit term reproduc similarli defin compar laboratori accord iso commiss decis eec tabl term coeffici variat tabl repeat reproduc express ratio coeffici variat laboratori level cvr cvr mg kg amp cv coeffici variat stabil chloroform adsorpt characterist florisil cartridg chang stabil ethanol verifi accord chloroform accuraci correct applic method verifi make replic measur certifi refer materi perform method verifi recoveri experi made fortifi blank sampl deviat actual express percentag actual lie limit figur column cartridg assembl mobil phase pump inject valv guard column hplc analyt column satur iodin solut reagent pump joint thermostat control bath spiral reaction coil fluoresc detector restrictor wast strip chart record integr figur flow diagram lc system iodin post column derivat respons adjac refer standard time refer standard extract calibr extract calibr refer standard extract calibr figur compens drift aflatoxin respons inject refer standard regular interv