commiss regul ec april amend regul eec determin commun method analysi wine commiss european commun regard treati establish european commun regard council regul eec march common organis market wine amend regul ec articl thereof annex commiss regul eec amend regul ec describ method analysi method analysi malic acid chapter prove imprecis accur method develop method develop analysi cyanid deriv sensit easier appli method determin ethyl carbam wine develop intern level method valid accord internation recognis criteria method ensur control wine qualiti authent prevent disput due applic outdat unreli method analysi descript method approv intern vine wine offic incorpor regul measur provid regul accord opinion manag committe wine adopt regul articl annex regul eec amend chapter malic acid replac annex regul chapter cyanid deriv replac annex ii regul annex iii regul ad chapter articl regul enter forc seventh dai public offici journal european commun regul bind entireti directli applic member state brussel april commiss franz fischler member commiss oj oj oj oj annex quot malic acid enzymat method principl presenc malat dehydrogenas mdh malic acid malat oxidis nicotinamid adenin dinucleotid nad oxaloacet oxaloacet form split pyruv carbon dioxid gt refer graphic gt quantiti nadh form proport concentr malic acid measur wavelength nm reagent test combin approxim determin bottl ml solut consist hepe buffer hydroxyethyl piperazin ethanesulfon acid ph stabilis bottl mg nad lyophilis bottl mdh lyophilis prepar solut content bottl undilut bring solut dissolv content bottl ml doubl distil water dissolv content bottl ml doubl distil water bring solut stabil solut content bottl stabl year store solut stabl week store month store solut stabl dai store apparatu spectrophotomet permit measur made nm wavelength nadh absorpt maximum fail spectrophotomet discontinu spectrum sourc permit measur made nm absolut absorb measur involv set calibr solut refer extinct coeffici nadh wavelength scale spectral absorb apparatu check glass cuvett optic path length cm prefer dispos cuvett micropipett pipet volum rang ml prepar sampl malat analysi carri directli wine preliminari decoloris amount malat cuvett μg wine dilut yield malat concentr depend apparatu dilut tabl gt tabl gt procedur spectrophotomet adjust wavelength nm determin absorb cm cuvett air set absorb cuvett optic path water pipett cuvett gt tabl gt mix minut measur absorb refer test solut add gt tabl gt mix wait reaction complet minut measur absorb refer test solut calcul absorb differ refer δαt test δαe solut final calcul differ differ gt refer graphic gt note time need complet enzym activ vari batch time guidanc recommend determin batch malic acid react rapidli enzym transform tartar acid slowli explain slight side reaction correct mean extrapol appendix express result gener formula calcul concentr mg gt refer graphic gt volum test solut ml ml volum sampl ml ml pm molecular mass substanc determin malic acid pm optic path cuvett cm cm absorpt coeffici nadh nm mmol cm nm mmol cm nm mmol cm sampl dilut prepar multipli result dilut factor malic acid concentr milligram litr mg decim place accuraci detail interlaboratori trial accuraci method summaris appendix valu deriv interlaboratori trial applic rang analyt concentr matric appendix repeat absolut differ individu result obtain ident matter submit trial oper apparatu shortest time interv exce repeat case mg reproduc absolut differ individu result obtain ident matter submit trial laboratori exce reproduc case mg comment view degre accuraci method valu malic acid mg confirm analyt method measur principl przyborski al mitteilungen klosterneuburg wine sampl cuvett exce ml avoid inhibit enzymat activ polyphenol appendix deal side reaction side reaction gener due secondari reaction enzym presenc enzym sampl matrix interact element matrix factor enzymat reaction normal reaction absorb reach constant time gener minut depend speed specif enzymat reaction secondari reaction occur absorb reach constant increas regularli time type process commonli call quot side reaction quot side reaction occur absorb solut measur regular interv minut requir time standard solut reach final absorb elaps absorb increas regularli measur made extrapol back mean graph calcul determin absorb observ final enzym ad substrat concentr calcul basi differ absorb extrapol time af ai figur side reaction gt pic file quot en ep quot gt appendix statist result interlaboratori trial gt tabl gt quot annex ii quot cyanid deriv caution compli safeti measur handl chemic chloramin pyridin potassium cyanid hydrochlor acid phosphor acid dispos product proper complianc environment rule forc caution hydrocyan acid releas distil acidifi wine principl total free hydrocyan acid wine releas acid hydrolysi separ distil react chloramin pyridin glutacon dialdehyd form determin colorimetri basi blue color dimethyl barbitur acid apparatu distil apparatu distil apparatu determin alcohol content wine ml round bottom flask standardis ground joint water bath thermostat control spectrophotomet allow absorb measur wavelength nm glass cell singl cell optic path mm reagent phosphor acid po chloramin solut clnna solut dimethyl barbitur acid dissolv dimethyl barbitur acid ml pyridin ml hydrochlor acid ml add ml distil water potassium cyanid kcn potassium iodid ki solut silver nitrat solut agno procedur distil put ml wine ml distil water ml phosphor acid glass bead ml flask place flask immedi distil apparatu taper tube conduct distil ml calibr flask ml water immers calibr flask ic water collect ml distil ml liquid total calibr flask flush taper tube condens millilitr distil water bring distil fill calibr line distil water measur put ml distil ml conic flask ground glass stopper add ml chloramin solut seal stopper second add ml dimethyl barbitur acid solut seal stopper leav minut measur absorb control ml distil water ml distil wavelength nm cell optic path mm determin calibr curv argentometr titrat potassium cyanid dissolv kcn carefulli measur ml distil water ml calibr flask add ml potassium iodid solut titrat silver nitrat solut stabl yellowish colour obtain take ml silver nitrat solut mg kcn calcul concentr kcn sampl standard curv prepar standard solut establish concentr kcn procedur set prepar standard solut ml hydrocyan acid ml hcn cong ml kcn dilut solut introduc ml ml ml ml ml dilut sampl solut ml calibr flask fill calibr line distil water solut prepar μg μg μg μg μg hydrocyan acid litr titrat ml sampl solut obtain continu valu obtain absorb regard standard solut function hydrocyan acid content produc straight line pass origin express result hydrocyan acid express microgram litr μg decim place method calcul read hydrocyan acid content calibr curv sampl dilut multipli result dilut factor repeat reproduc white wine μg approxim xi μg approxim xi red wine μg approxim xi μg approxim xi xi averag concentr hcn wine xi μg white wine xi μg red wine quot annex iii determin ethyl carbam wine select detect method ga chromatographi mass spectrometri applic determin ethyl carbam concentr μg caution compli safeti measur handl chemic ethanol aceton carcinogen product ethyl carbam dichloromethan dispos solvent proper complianc environment rule forc principl propyl carbam ad sampl intern standard solut dilut water ml solid phase extract column ethyl carbam propyl carbam elut dichloromethan eluat concentr vacuum rotari evapor concentr analys ga chromatographi gc detect mass spectrometri fragmentometri sim select ion monitor mode apparatu chromatograph condit ga chromatogram mass spectromet gc ms sampl filter data treatment system equival capillari column fuse silica mm intern diamet μm carbowax oper injector helium ga vector ml minut inject splitless method temperatur programm minut rise minut minut rise maintain temperatur minut specif retent time ethyl carbam minut propyl carbam minut ga chromatogram spectromet gc ms interfac transfer line mass spectromet paramet manual tune perfluorotributylamin optimis lower mass sensit sim acquisit mode solvent delai acquisit start time minut dwell time ion ms vacuum rotari evapor concentr system similar kuderna danish nb rate recoveri ethyl carbam test sampl process flask pear shape ml singl ground neck concentr tube ml graduat teflon coat joint cork reagent aceton qualiti lc nb check batch gc ms absenc respons ion dichloromethan nb analys batch gc ms fold concentr check absenc respons ion ethanol anhydr ethyl carbam ec standard solut stock solut mg ml weigh mg ec puriti amp gt ml volumetr flask dilut aceton standard work solut μg ml transfer ml stock ec solut ml volumetr flask dilut aceton mark propyl carbam pc standard solut stock solut mg ml weigh mg pc reagent grade ml volumetr flask dilut aceton mark standard work solut μg ml transfer ml stock pc solut ml volumetr flask dilut aceton mark intern standard solut pc ng ml transfer ml standard pc work solut ml volumetr flask dilut water mark standard calibr solut ec pc dilut ec standard work solut pc standard work solut dichloromethan obtain ng ec ng pc ml ng ec ng pc ml ng ec ng pc ml ng ec ng pc ml ng ec ng pc ml test sampl ng ec ml ethanol transfer ml ec standard work solut ml volumetr flask dilut ethanol mark solid phase extract column dispos materi pre pack diatomac earth capac ml nb analysi check batch extract column recoveri ec pc absenc respons ion prepar ng ec ml test sampl analys ml test sampl recoveri ng ec ml satisfactori absorb particl diamet irregular lead slow flow affect recoveri ec pc test sampl obtain trial chang column correct calibr recoveri curv quantifi ec obtain correct calibr curv prepar standard solut ethanol dichloromethan analys ml standard calibr solut establish calibr curv ec pc ratio extract standard prepar test sampl place volum test materi separ ml beaker wine vol alcohol ml wine maximum vol alcohol ml beaker add ml intern standard pc solut water obtain total volum ml extract extract carri extractor hood adequ ventil transfer sampl prepar head extract column rins beaker ml water transfer rins water column leav liquid absorb minut elut ml dichloromethan collect eluat ml conic flask evapor eluat ml water bath rotari evapor nb boil dry transfer concentr residu ml graduat tube pasteur pipett rins flask ml dichloromethan transfer rins liquid tube concentr sampl ml weak nitrogen stream transfer concentr auto sampler flask gc ms analysi gc ms analysi calibr curv inject μl standard calibr solut gc ms plot graph ec pc area ratio ion respons vertic axi quantiti ec ng ml horizont axi ng ml ec quantif inject μl concentr extract prepar gc ms system calcul ec pc area ratio ion establish concentr ec ng ml extract intern standard calibr curv calcul ec concentr test sampl ng ml divid quantiti ec ng extract test sampl volum ml confirm ec ident determin respons ion period ec retent respons featur main fragment ch molecular ion presenc ec confirm rel ratio ion ratio ec standard extract concentr order obtain suffici respons ion collabor analysi tabl show individu result practic entrain sampl type wine cochran test led elimin pair result wine alcohol strength vol wine alcohol strength vol laboratori rel reproduc rsdr decreas concentr ethyl carbam increas perform method determin ethyl carbam ec alcohol beverag gc ms gt tabl gt rich wine capillari column desir rich wine temperatur programm minut desir