commiss regul ec novemb amend regul ec lai commun refer method analysi spirit drink commiss european commun regard treati establish european commun regard council regul eec lai gener rule definit descript present spirit drink amend act access austria finland sweden articl thereof commiss regul ec decemb lai commun refer method analysi spirit drink describ method annex method analysi determin tran anethol anise flavour spirit drink glycyrrhiz acid chalcon pasti egg yolk egg liqueur liqueur egg valid internation recognis procedur part research project support commiss method recognis commun refer method ad annex regul ec measur provid regul accord opinion implement committe spirit drink adopt regul articl annex regul ec amend summari annex term quot quot point vi vii ix delet chapter vi vii ix annex regul ad chapter iii articl regul enter forc seventh dai public offici journal european commun regul bind entireti directli applic member state brussel novemb commiss franz fischler member commiss oj oj annex anethol ga chromatograph determin tran anethol spirit drink scope method suitabl determin tran anethol anise flavour spirit drink capillari ga chromatographi norm refer iso water analyt laboratori specif test method principl tran anethol concentr spirit determin ga chromatographi gc quantiti intern standard allylanisol estragol estragol natur present sampl ad test sampl tran anethol refer solut concentr dilut ethanol solut inject directli gc system extract sampl prepar analysi liqueur larg amount sugar reagent materi analysi reagent puriti water grade defin iso refer chemic store cold oc light aluminium contain tint amber glass reagent bottl stopper prefer fit aluminium seal tran anethol quot thaw quot crystallin state case temperatur exce oc ethanol vol ca methoxi propenyl benzen tran anethol ca allylanisol estragol ca suggest intern standard ethanol vol add distil water ethanol vol prepar standard solut standard solut store room temperatur oc light aluminium contain tint amber glass reagent bottl stopper prefer fit aluminium seal tran anethol allylanisol practic insolubl water dissolv tran anethol allylanisol ethanol addit ethanol stock solut freshli prepar week standard solut stock solut tran anethol concentr weigh mg tran anethol ml volumetr flask mg ml add ethanol make volum vol ethanol mix intern standard solut stock solut intern standard estragol concentr weigh mg estragol ml volumetr flask mg ml add vol ethanol make volum vol ethanol mix solut check linear respons flame ionis detector fid linear respons fid check analysi take account rang concentr tran anethol spirit procedur analysi unknown sampl spirit analys dilut time condit analysi method stock solut concentr tran anethol sampl analys prepar ml stock solut pipett separ ml volumetr flask pipett flask ml intern standard solut make volum vol ethanol mix blank solut solut standard solut ml standard solut pipett ml volumetr flask add ml intern standard solut make volum vol ethanol mix apparatu equip capillari ga chromatograph fit flame ionis detector fid integr data handl system capabl measur peak height area automat sampler equip manual sampl inject split splitless injector capillari column length intern diamet mm film thick μm stationari phase ffap modifi tpa polyethylen glycol cross link porou polym common laboratori equip grade volumetr glasswar analyt balanc precis mg chromatographi condit column type dimens gc condit anethol intern standard separ interf substanc typic condit column carrier ga analyt helium flow rate ml min injector temperatur oc detector temperatur oc oven temperatur condit isotherm oc run time minut inject volum μl split sampl sampl store room temperatur light cold procedur sampl screen estragol ensur estragol natur present sampl blank analysi carri addit intern standard estragol natur present intern standard chosen instanc menthol pipett ml sampl ml volumetr flask make volum vol ethanol mix prepar unknown sampl pipett ml sampl ml volumetr flask add ml intern standard solut make volum vol ethanol mix blank pipett ml intern standard solut ml volumetr flask make volum vol ethanol mix linear test prior commenc analysi linear respons fid check success analys triplic linear standard solut integr peak area peak height inject plot graph mother solut concentr versu ratio tran anethol peak height area divid estragol peak height area linear plot obtain determin inject blank solut standard solut linear standard act qualiti control sampl chosen refer probabl concentr tran anethol unknown unknown insert linear qualiti control sampl unknown sampl ensur analyt stabil calcul respons factor measur peak area integr data system peak height manual integr tran anethol intern standard peak respons factor rfi calcul respons factor calcul rfi ci area heighti area heighti ci ci concentr tran anethol standard solut ci concentr intern standard standard solut areai area height tran anethol peak areai area height intern standard peak rfi calcul sampl solut analysi linear respons test solut inject linear respons test solut analysi sampl inject unknown sampl solut calcul result formula calcul concentr tran anethol ci ci area heighti area heighti rfi ci unknown tran anethol concentr ci concentr intern standard unknown area heighti area height tran anethol peak area heighti area height intern standard peak rfi respons coeffici calcul tran anethol concentr express gram litr decim place qualiti assur control chromatogram anethol intern standard separ interf substanc rfi calcul result inject solut coeffici variat cv standard deviat minu rfi averag accept calcul calcul concentr tran anethol sampl select qualiti control linear control solut calcul result analysi linear solut select intern qualiti control sampl iqc minu theoret result unknown sampl accept treatment spirit sampl larg amount sugar liqueur sampl prior gc analysi extract alcohol spirit drink larg amount sugar order determin tran anethol concentr capillari ga chromatographi principl aliquot liqueur sampl ad intern standard concentr similar analyt tran anethol liqueur ad sodium phosphat dodecahydr anhydr ammonium sulphat result mixtur shaken chill layer develop upper alcohol layer remov aliquot alcohol layer dilut ethanol solut note intern standard ad stage ad result solut analys ga chromatographi reagent materi extract reagent puriti greater ammonium sulphat anhydr ca sodium phosphat dibas dodecahydr ca apparatu equip conic flask separ flask refriger procedur sampl screen estragol ensur estragol natur present sampl blank extract analysi carri addit intern standard estragol natur present intern standard chosen extract pipett ml ethanol conic flask weigh flask mg intern standard add ml sampl add ammonium sulphat anhydr dibas sodium phosphat dodecahydr stopper conic flask shake flask minut mechan shake devic teflon coat magnet stir bar teflon absorb analyt note ad salt dissolv complet place stopper flask refriger amp lt hour time distinct liquid layer solid residu alcohol layer clear replac refriger clear separ achiev alcohol layer clear carefulli aliquot ml disturb aqueou layer place amber vial close secur prepar extract sampl analys extract reach room temperatur ml alcohol layer attemper extract sampl pipett ml volumetr flask make volum ethanol mix determin follow procedur outlin calcul result formula calcul result gt refer graphic gt mi weight intern standard milligram volum unknown sampl ml rfi respons factor areai area tran anethol peak areai area intern standard peak result express gram litr decim place qualiti control assur follow procedur outlin method perform characterist precis statist result interlaboratori test tabl give valu anethol data obtain intern method perform studi carri internation agre procedur gt tabl gt gt tabl gt sampl type pasti blind duplic pasti blind duplic pasti blind duplic pasti blind duplic pasti singl duplic pasti singl duplic gt tabl gt sampl type ouzo split level ani blind duplic anise flavour liqueur duplic anise flavour liqueur duplic vi glycyrrhiz acid determin glycyrrhiz acid high perform liquid chromatographi scope method suitabl determin glycyrrhiz acid anise flavour spirit drink high perform liquid chromatographi hplc regul eec specifi anise flavour spirit call quot pasti quot glycyrrhiz acid litr norm refer iso water analyt laboratori specif test method principl glycyrrhiz acid concentr determin high perform liquid chromatographi hplc uv detect standard solut test sampl filter separ inject directli hplc system reagent materi analysi reagent hplc grade absolut ethanol water grade defin iso ethanol vol ca ammonium glycyrrhizin nh glycyrrhiz acid ammonium salt mol wt ca puriti mol wt glycyrrhiz acid glacial acet acid ch cooh ca methanol ch ca ethanol vol ml oc vol ethanol ml water ml prepar hplc elut solut elut solvent part volum water part volum acet acid dega elut solvent minut note water microfilt advis filter prepar elut solvent filter organ solvent pore size equal μm elut solvent methanol prepar standard solut standard solut freshli prepar month refer solut weigh nearest mg mg ammonium glycyrrhizin ml volumetr flask add vol ethanol dissolv ammonium glycyrrhizin dissolv make mark vol ethanol filter filter organ solvent standard solut check linear respons instrument stock solut prepar weigh nearest mg mg ammonium glycyrrhizin ml volumetr flask add vol ethanol dissolv ammonium glycyrrhizin dissolv make mark vol ethanol solut ammonium glycyrrhizin prepar pipet ml ml ml ml stock solut separ ml volumetr flask make mark vol ethanol mix filter solut filter organ solvent apparatu equip separ system high perform liquid chromatograph pump system enabl achiev maintain constant program rate flow great precis uv spectrophotometr detect system set nm solvent degass system comput integr record perform compat rest system column materi stainless steel glass intern diamet mm length mm stationari phase cross link silica prefer spheric octadecyl function group maximum particl size μm laboratori equip analyt balanc precis mg grade volumetr glasswar micromembran filtrat arrang small volum chromatographi condit elut characterist flow rate ml minut solvent solvent detect uv nm procedur prepar spirit sampl filter filter organ solvent pore diamet μm determin chromatographi condit stabilis inject μl refer solut inject μl sampl solut compar chromatogram identifi glycyrrhiz acid peak retent time measur area height calcul concentr decim figur equat gt refer graphic gt concentr gram litr glycyrrhiz acid spirit analys concentr gram litr ammonium glycyrrhizin refer solut area height glycyrrhiz acid peak spirit analys area height glycyrrhiz acid peak refer solut puriti refer ammonium glycyrrhizin mass mole glycyrrhiz acid mass mole ammonium glycyrrhizin method perform characterist precis statist result interlaboratori test tabl give valu glycyrrhiz acid data obtain intern method perform studi carri internation agre procedur gt tabl gt gt tabl gt sampl type pasti blind duplic pasti split level pasti blind duplic pasti blind duplic pasti blind duplic vii chalcon high perform liquid chromatographi method verifi presenc chalcon pasti scope method suitabl determin chalcon present anise flavour drink chalcon natur color flavonoid famili present liquoric root glycyrrhiza glabra anise flavour spirit call quot pasti quot chalcon regul eec norm refer iso water analyt laboratori specif test method principl refer liquoric extract solut prepar presenc absenc chalcon determin high perform liquid chromatographi hplc uv detect reagent materi analysi reagent hplc grade ethanol vol water grade defin iso ethanol vol ca acetonitril ch cn ca refer substanc glycyrrhiza glabra liquoric quot sweet root quot coars ground liquoric root glycyrrhiza glabra averag dimens rodlik particl length mm thick mm sodium acet ch coona ca glacial acet acid ch cooh ca prepar solut ethanol volum ml vol ethanol ml water ml solvent acetonitril acetonitril hplc analyt puriti dega solvent sodium acet buffer solut ph weigh sodium acet add glacial acet acid make ml water volumetr flask prepar refer extract glycyrrhiza glabra weigh ground liquoric root glycyrrhiza glabra place round bottom distil flask add ml vol ethanol boil reflux hour filter set filtrat recov liquoric extract filter place round bottom distil flask add ml vol ethanol boil reflux hour filter set filtrat liquoric root extract perform time success combin filtrat evapor solvent phase rotari evapor residu extract ml vol ethanol apparatu equip separ system high perform liquid chromatograph pump system capabl achiev maintain constant program rate flow high pressur uv visibl spectrophotometr detect system set nm solvent degass system column oven set temperatur comput integr record perform compat rest separ system column materi stainless steel glass intern diamet mm stationari phase cross link silica octadecyl deriv function group particl size μm cross link phase common laboratori equip includ analyt balanc precis mg distil apparatu reflux condens compris ml round bottom flask standardis ground glass joint cm long reflux condens heat sourc pyrogen reaction involv extract matter avoid arrang rotari evapor apparatu filtrat set buchner funnel chromatographi condit elut characterist solvent shift gradient minut shift gradient minut equal strength minut stabilis column inject equal strength minut flow rate ml minut uv detector set detector set nm detect presenc chalcon nm detect glycyrrhiz acid note chang wavelength nm nm carri second begin peak elut glycyrrhiz acid procedur prepar spirit sampl filter filter organ solvent pore diamet μm prepar residu liquoric extract make ten dilut vol ethanol analysi determin inject μl prepar liquoric extract perform analysi chromatographi condit inject μl sampl anise flavour spirit sampl perform analysi chromatographi condit compar chromatogram great similar chromatogram chalcon exit zone detect nm analysi condit figur characterist chromatogram pasti figur chromatogram obtain method show presenc chalcon quot pasti quot peak chalcon peak glycyrrhiz acid gt pic file quot en tif quot gt method perform characterist precis result interlaboratori test tabl perform recognit presenc absenc chalcon pasti anise flavour spirit data obtain intern method perform studi carri internation agre procedur gt tabl gt gt tabl gt gt tabl gt sampl type pasti blind duplic pasti singl sampl pasti singl sampl quot pasti quot chalcon blind duplic quot pasti quot chalcon blind duplic anise flavour liqueur chalcon blind duplic anise flavour liqueur chalcon blind duplic ouzo chalcon singl sampl ouzo chalcon singl sampl ani chalcon blind duplic quot pasti quot chalcon blind duplic ix egg yolk determin egg yolk concentr spirit drink photometr method scope method suitabl determin egg yolk concentr rang egg liqueur liqueur egg norm refer iso water analyt laboratori specif test method principl ethanol solubl phosphoru compound found egg yolk extract assai photometr phosphoru molybd complex reagent materi doubl distil water diatomac earth ethanol vol ca magnesium acet ca solut sulphur acid ca sulphur acid potassium dihydrogen phosphat ca kh po solut reagent phosphat determin dissolv ammonium molybd ca nh mo ml water oc dissolv vessel ammonium vanad ca nh vo ml hot water cool add ml concentr nitric acid ca combin cool solut ml volumetr flask make ml mark apparatu equip ml conic flask ultrason bath magnet stirrer ml volumetr flask oc water bath filter whatman equival porcelain platinum crucibl boil water bath hot plate muffl furnac ml volumetr flask ml volumetr flask spectrophotomet set nm cm cuvett sampl sampl store room temperatur prior analysi procedur sampl prepar weigh sampl ml conic flask add gradual ml ethanol small portion swirl addit place ultrason bath minut stir mixtur magnet stirrer minut room temperatur transfer content flask ml volumetr flask wash ethanol adjust calibr mark ethanol place flask oc water bath adjust calibr mark oc add small amount diatomac earth filter discard ml transfer ml filtrat porcelain platinum crucibl filtrat concentr gentl evapor boil water bath addit ml magnesium acet solut place crucibl hot plate heat dry ash residu heat incandesc oc muffl furnac ash white minimum half hour left overnight ash ml sulphur acid transfer wash distil water ml volumetr flask fill mark room temperatur distil water ml aliquot ash solut prepar sampl solut photometr phosphat assai photometr phosphat assai compar solut place ml sulphur acid ml volumetr flask fill mark distil water add ml aliquot solut contain ml volumetr flask ml sulphur acid ml phosphat reagent make ml distil water stopper loos insert stopper shake heat boil water bath minut cool oc water bath minut fill cm cuvett compar solut sampl solut add ml aliquot ash solut contain ml volumetr flask ml sulphur acid ml phosphat reagent make ml distil water stopper loos insert stopper shake heat boil water bath minut cool oc water bath minut yellow solut develop immedi analys spectrophotometr cm cuvett nm compar solut calibr curv construct calibr curv add ml aliquot phosphat reagent ml volumetr flask ml sulphur acid ml potassium dihydrogen phosphat solut make ml mark distil water stopper loos insert stopper shake heat boil water bath minut cool oc water bath minut analys spectrophotometr cm cuvett nm compar solut construct calibr curv gt tabl gt express result egg yolk content calcul formula gt refer graphic gt convers factor total egg yolk mg establish calibr curv densiti mass unit volum ml egg base liqueur oc weight egg base liqueur dilut factor ml aliquot ash solut method perform characterist precis statist result interlaboratori test tabl valu egg yolk data obtain intern method perform studi carri internation agre procedur gt tabl gt gt tabl gt sampl type advocaat blind duplic advocaat blind duplic advocaat blind duplic advocaat dilut split level advocaat blind duplic