ࡱ>  oqdefghijklmnbjbjVV 4<< >!>!..qOqOqOOOO8OQO"ET3U3U3U3U~W~W~W$nmqO`>W@~W``..3U3U drrr`.3UqO3Ur`rrIF( #3UPٯBO{ kNz0j##*qOMp~W2Zr \]*~W~W~WX~W~W~W````~W~W~W~W~W~W~W~W~W>! G-:Nitriti i nitrati kao prekursori N-nitrozamina u paatetama u konzervi Nitrites, nitrates as N-nitrosamines precursors in canned liver patties Pavlini, I.1, Bevardi, M.2, Maruai, N.3, Vida ek, S.3, Kolari, S.4, Medi, H.3 Nitriti i nitrati se koriste u proizvodnji mesnih preraevina i imaju va~nu ulogu ne samo kao konzervansi ve i u formiranju karakteristi ne crvene boje i specifi nog okusa proizvoda od mesa te inhibiraju rast i razvoj patogene bakterije Clostridium botulinum. Nitrozamini su kancerogeni i toksi ni spojevi koji mogu nastati u mesu i mesnim proizvodima kao produkt reakcije nitrita s proteinima mesa nakon ato se mesu ili mesnim proizvodima dodaju nitriti, odnosno nitrati kao konzervansi. `tetnost nitrozamina dokazana je u mnogim objavljenim znanstvenim radovima tijekom zadnjih 30.-ak godina te je stoga potrebna stalna sustavna kontrola na prisutnost N-nitrozamina, jer meso i mesni proizvodi kao glavni izvor proteina ine veliki udio u prehrani kako odrasle populacije tako i djece. U Republici Hrvatskoj se takva kontrola ne provodi, a isto tako u dosadaanjoj legislativi nisu propisane najviae dopuatene koli ine za te spojeve (NDK). Identifikacija i kvantifikacija natri-nitrita i natrij-nitrata raena je kromatografskom tehnikom visokou inkovite tekuinske kromatografije (engl. High Performance Liquid Chromatography-HPLC) s DAD detektorom serija 1200 (Agilent), dok su N-nitrozamini odreeni praenjem karakteristi nih iona u vezanom sustavu plinske kromatografije i spektrometrije masa (GC-MS-SIM). Dobiveni rezultati kretali su se u rasponu od 0.0001 mg/kg - 0.0099 mg/kg. Klju ne rije i: nitrit, nitrat, N-nitorzamini, paateta u konzervi Nitrites and nitrates are used as curing agents in meat products and they are very important not only as preservatives, but also for color and flavor formation. They are growth inhibitors of Clostridium botulinum. N-nitrosamines are carcinogenic and toxic compounds that can occur in meat and meat products as the reaction product of nitrite with proteins of meat after the meat or meat products are added nitrites, and nitrates as preservatives. N-nitrosamines harm has been proved in many scientific papers published over the last 30.-odd years, and therefore requires constant systematic control of the presence of N-nitrosamines as meat and meat products as the main source of protein form a large share of food to the adult population and children. In the Republic of Croatia, such control is not implemented, as well as in the previous legislation is not prescribed maximal allowed concentration (MAC). Identification and quantification of sodium nitrites and nitrates were done by High Performance Liquid Chromatography-HPLC with DAD detector series 1200 (Agilent). The presence of N-nitrosamines was determined by gas chromatography and mass spectrometry system (GC-MS-SIM). The obtained results were ranging from 0.0001 mg/kg - 0.0099 mg/kg. Key words: nitrite, nitrate, N-nitrosamines, canned liver patty U prehrani suvremenog ovjeka dominiraju ne samo meso ve i kobasi arski proizvodi, proizvodi od suaenog i dimljenog mesa, paateta, konzerve, te razni mesni specijaliteti. Prilikom procesiranja mesa ili mesnih preraevina dolazi do reakcije izmeu dodanih nitrita s proteinima mesa, te kao produkt reakcije nitrozacije mogu nastati atetni i kancerogeni spojevi N-nitrozamini (Hui, 1992; Pearson i Dutson, 1990; Boanir i sur.,2003) Toksi nost i kancerogenost nitrozamina dokazana je i opisana u mnogim do sad objavljenim znanstvenim radovima tijekom zadnjih 30.-ak godina (Jakszyn i sur., 2006). Reakcija nitrozacije, prilikom koje nastaju nitrozamini, direktno je ovisna o koli ini amina, koli ini dodanih nitrita, pH, temperaturi, redoks potencijalu, te prisutnosti drugih kemijskih spojeva ili agensa. Osim kao konzervansi, nitriti mogu nastati i enzimskom redukcijom iz nitrata (prisutnih uglavnom u namirnicama biljnog podrijetla) tijekom procesiranja ili dodatkom nitrata namirnicama, dr. sc. Iva Pavlini,Hrvatski centar za poljoprivredu, hranu i selo, Hondlova 2/11, 10 000 Zagreb 2 M. Bevardi, Zavod za javno zdravstvo Andrija `tampar, Mirogojska 16, 10 000 Zagreb 3 dr. sc. Helga Medi, dr.sc. Sanja Vida ek, Nives Maruai, dipl. ing., Prehrambeno-biotehnoloaki fakultet, Pierottijeva 6, 10 000 Zagreb 4 Sanja Kolari, dipl. ing., Dr~avni inspektorat, Svetoaimunska cesta 25, 10 000 Zagreb ato predstavlja indirektnu opasnost od nastanka atetnih spojeva nitrozamina. Nitrati, za razliku od nitrita, imaju manju reaktivnost i nemaju direktan utjecaj na meso i mesne preraevine kad se dodaju kao konzervans ve predstavljaju zalihu nitrita, a samim tim i opasnost od nastanka nitrozamina (Hui, 1992; Pearson i Dutson, 1990). Zbog toga je sve vei pritisak na proizvoa e hrane, odnosno mesnu industriju da se reducira koli ina dodanih nitrita i nitrata kako bi se smanjila izlo~enost ljudi N-nitrozo spojevima ato je bio zaklju ak Ameri ke nacionalne akademije za znanost joa 1981. godine. Meso nije samo va~an izvor energije za naa organizam, ve je i prava riznica proteina, vitamina, posebno A i B grupe, minerala, fosfora, magnezija i kalija, te elemenata u tragovima, kao ato su ~eljezo, cink i selen. Proteini iz mesa sudjeluju u izgradnji naaih stanica i tkiva (~ivci, miaii i krv) (Mandi, 2007). Meso sadr~i punovrijedne proteine i masti koje su naaem organizmu neophodne za normalno funkcioniranje. Masnoe koje sadr~i meso va~an su izvor energije za ekstremne napore i nosa i su vitamina topivih u mastima: A, D, E i K. Prema tvrdnjama veine nutricionista, va~no je da uravnote~ena prehrana sadr~i umjerenu koli inu mesa, zajedno sa slo~enim ugljikohidratima (uklju ujui cjelovito zrnje), voem, povrem i umjerenim koli inama mlijeka i mlije nih proizvoda. U inci mesa na zdravlje ovise i o na inu konzumiranja mesa, veli ini porcija i o samoj vrsti mesa. Na stav potroaa a prema mesu utje u brojni imbenici poput cijene i dostupnosti, no glavne razlike u koli ini i tipu mesa koje se konzumira u razli itim zemljama pripisuju se razlikama u kulturi i tradicionalnom na inu prehrane. Meso i mesni proizvodi svrstani su prema postojeem Pravilniku u sedam skupina unutar kojih postoje detaljnije podjele, odnosno podskupine i to: Proizvodi od svje~eg mesa; Kobasice; Suhomesnati proizvodi; Polugotova i gotova jela od mesa; Konzerve; Slanina; Ostali proizvodi od mesa. Uporaba aditiva u tehnologiji proizvodnje mesnih proizvoda postaje sve eaa, a poznata je dugi niz godina. Fosfati i polifosfati su tako naj eae koriateni funkcionalni aditivi u mesnoj industriji. Kao takvi zaslu~ni su za vezanje vode, poboljaanje teksture proizvoda i stabilizaciju proizvoda (Branen, Devidson i Salminen, 2002). Isto tako, koriste se sol (NaCl), aeer, nitrati/nitriti, askorbinska kiselina i dr. Postavlja se pitanje mogunosti kontrole s obzirom na koli inu dodanih aditiva u gotovim proizvodima. Da je kontrola dodanih aditiva neophodna govori nam injenica, da se meu dozvoljenim aditivima nalaze i supstance, koje u poveanoj koli ini mogu biti toksi ne ili ak kancerogene. Kao primjer se navodi dodatak natrijeva i kalijeva nitrita, ija je primjena naaim Pravilnikom dozvoljena u salamurenim mesnim proizvodima. O toksi nosti nitrita ima u literaturi mnogo podataka. Ipak je najva~nije upozoriti na opasnost koja nastaje neminovnim i stalno prisutnim procesom redukcije nitrita u kancerogene nitrozamine. Proces redukcije nitrita u nitrozamine odvija se ve u samoj sirovini, ali i u gotovim proizvodima, te kasnije u probavnom traktu konzumenta uz sudjelovanje crijevnih bakterija. S druge strane jedini su u inkoviti konzervansi koji sprije e rast bakterije Clostridium botulinum, bakterije iju su toksini paralizirajui za ~iv ani sustav (Issenberg, 1976). Konzervansi se dodaju radi stabiliziranja proizvoda, produljenja njegove odr~ivosti i sprje avanja kvarenja, odnosno mikrobioloake kontaminacije. Na taj na in se produ~uje trajnost namirnice i omoguuje transport lako pokvarljive robe na vee udaljenosti (Walker, 1990). Sam proces prerade, odnosno proizvodnje mesnih proizvoda uklju uje stvaranje karakteristi ne crvene boje, te specifi an okus i teksturu preraenih mesnih proizvoda koji se upravo po tome razlikuju od svje~eg mesa. Kemija prerade mesa uklju uje mnoge kemijske reakcije (Reinik, 2007). Istra~ivanjima je potvreno da su nitriti zaslu~ni za crvenu boju mesa i njenu toplinsku stabilnost. Hoagland je joa 1910 godine predlo~io slijedei niz kemijskih reakcija tijekom prerade (dorade) mesa: KNO3 (saltpetre nitrate) ! reakcija s mikroorganizmima ! KNO2 (nitrit) KNO2 + H+ ! HNO2 + K+ 2HNO2 ! N2O3 + H2O N2O3 ! NO + NO2 NO + mioglobin ! NO-mioglobin Sredinom 20. stoljea postao je sasvim jasan mehanizam nastanka karakteristi ne crvene boje preraenog mesa. Upotreba nitrita prilikom prerade mesa u koli ini od 156 ppm daje ~eljenu boju, a dodadak natrij-nitrita u toj koli ini je slu~beno dopuatena u Sjedinjenim Ameri kim Dr~avama joa po etkom 20. stoljea, dok se kasnije postepeno po inje koristiti u proizvodnji i preradi mesa i mesnih proizvoda i u mnogim drugim zemljama (Girard, 1996). Godine 1940. prvi put je provedeno istra~ivanje koje je dokazalo utjecaj nitrita na nastajanje specifi nog okusa preraenih (suhomesnatih) mesnih proizvoda. Nakon toga, iste godine, otkrivene su tri glavne uloge nitrita: utjecaj na boju, okus i njegova antibakterijska svojstva, ali mehanizam djelovanja joa uvijek nije bio poznat. Ubrzo nakon toga, otkriveno je, da je kod mesa i mesnih proizvoda najvea koli ina nitrita tijekom samog procesa proizvodnje. Ta je spoznaja potakla istra~ivanja o sudbini nitrita u mesu i mesnim proizvodima, njihovom toksi nom djelovanju i uvjetima nastanka toksi nih produkata (Watson, 2002; Cammack i sur., 2002). Prilikom procesiranja mesa i mesnih proizvoda koli ina nitrita u rasponu od 1-5% izgubi se prilikom stvaranja duaikovog oksida (NO) ili se ve~e na lipide mesa. Od 1-10% se oksidira do nitrata, 5-10% ostaju kao slobodni nitriti dok 5-15% reagira sa sulfhidridnim spojevima ili mioglobinom, a 20-30% ostaje vezano na proteine (Hui, 1992). Tijekom procesiranja i skladiatenja mesa dolazi do gubitka nitrita, ato ga ini nesigurnim aditivom. Napredak u definiranju uloge nitrita u preraenim mesnim proizvodima uklju ivao je potvrdu o meudjelovanju mnogih faktora i razjaanjenju mehanizma inhibitornog u inka nitrita, a tijekom 1970.-ih i 1980.-ih godina slu ajno otkrie toksi nih i kancerogenih spojeva N-nitrozamina postaje od velikog zna aja (Jakszyn i sur., 2006). Osim za postizanje i o uvanje karakteristi ne boje, okusa i teksture, nitriti/nitrati se dodaju prilikom prerade i proizvodnje mesa i mesnih proizvoda i u kombinaciji s drugim faktorima kako bi smanjili ili sprije iti rast i razvoj patogenih bakterija kao npr. Clostridium botulinum (Anonimno, 2003). Spore Clostridium botulinum se nalaze svugdje u tlu i na taj na in predstavljaju potencijalnu opasnost za ~ivotinje i prehrambeni lanac. Otporne su na mnoge tretmane zagrijavanja koji se koriste prilikom proizvodnje hrane, te se zato mogu nai u mnogim vrstama hrane. Konzumacijom takve hrane u kojoj se razmno~ava C. botulinum, te proizvodi neurotoksine (proteini koji nastaju prilikom razmno~avanja i tvorbe vegetativnih stanica), toksin se absorbira u probavnom traktu i nepovratno ve~e na periferni ~ivac, te inhibira otpuatanje neurotransmitora. Simptomi botulinske infekcije uklju uju mu ninu, povraanje, umor, nesvjesticu, paralizu miaia i poteakoe disanja, a razvijaju se unutar 12-72 sata nakon konzumacije kontaminirane hrane. Ukoliko ne nastupi smrt, oporavak traje nekoliko mjeseci, a stupanj smrtnosti danas iznosi 10%. U mnogim zemljama upravo je konzumacija mesnih proizvoda glavni uzrok pojave botulizma jer je upravo meso idealan medij za rast i razvoj, te proizvodnju toksina Clostridium botulinum (Cassens i sur.,1978; Perez Rodriguez, 1998; Pegg i Shahidi, 2004). Spoznaja da je dodatak odreene koli ine nitrita potreban za sprije avanje rasta i razvoja Clostridium botulinum kod kratkotrajnih, konzerviranih i preraenih mesnih proizvoda dokazana je 70.-ih godina proalog stoljea. Ta je koli ina razli ita i potrebno ju je odrediti za svaki pojedini proizvod. Takoer se pretpostavlja da je rast C. botulinum ovisan o tzv.  rezidualnoj koli ini dodanih nitrita, a va~nom se smatra i razina ~eljeza prisutna u mesu, iako se to ne mo~e sa sigurnoau tvrditi, dok je sasvim jasno da sudbina nitrita dodanih mesu ovisi o nekoliko faktora: pH, vrsti mesnih proizvoda, temperaturi skladiatenja, eventualnom tretiranju zagrijavanjem, te prisutnosti drugih tvari (npr. limunske kis.). Smatra se i da duaikov-oksid inaktivira ~eljezo-sulfat koji je prisutan u mesu i to je vjerojatni mehanizam inhibicije rasta i razvoja Clostridium botulinum dodatkom nitrita suhomesnatim i preraenim mesnim proizvodima (Roberts, 1995). Mnogi su pokusi do sada raeni kako bi se utvrdila minimalna koli ina nitrita koja osigurava inhibitorni u inak na klijanje i rast spora C. botulinum prilikom proizvodnje i konzerviranja mesnih proizvoda zbog opasnosti od nastanka kancerogenih nitrozamina. Znanstveni odbor za hranu (Scientific Committee on Food) utvrdio je 1990. godine da je 50-100 mg nitrita kao NaNO2/kg mesnih proizvoda dovoljno za veinu proizvoda, dok je za proizvode s duljim rokom trajanja i malom koli inom soli potrebno dodati 50-150 mg nitrita/kg kako bi se inhibirao rast Clostridium botulinum (Anonimno, 2006). Nitriti i nitrati dozvoljeni su za upotrebu u zemljama Europske unije kao kalij-nitrit i natrij-nitrit (E 249 i E 250) i kalij-nitrat i natrij-nitrat (E 251 i E 252). Njihova uporaba propisana je direktivom Europske unije i sve viae se te~i smanjivanju koli ina nitrata i nitrita potrebnih za o uvanje mikrobioloake sigurnosti hrane (Anonimno, 2005). Nitrati su, osim kao dodaci hrani (prehrambeni aditivi), u prirodi prisutni kao dio duai nog ciklusa. Imaju va~nu ulogu u prehrani, te rastu i razvoju bilja. Povre tako sadr~i znatan udio nitrata, a zna ajan udio sadr~e i uljarice i gomoljasto bilje. Lisnato povre kao npr. salata ili apinat sadr~e najvee koncentracije nitrata. Unos nitrata kod ljudi uglavnom se odvija egzogeno konzumacijom povra i ostalih proizvoda koji sadr~e nitrate, te putem pitke vode dok nitriti uglavnom nastaju konverzijom iz nitrata, dok se neki nitriti konzumiraju kao dodaci hrani. Voe i povre uz meso i mesne preraevine predstavlja zna ajan segment u prehrani ljudi, a njihovom konzumacijom unosi se u organizam od 11-41% dnevnog unosa nitrata (Anonimno, 2008b). Znanstveni odbor za hranu (Scientific Committee on Food-SCF) je 2002. godine utvrdio prihvatljiv dnevni unos (ADI) za nitrate, a iznosi 0-3.7 mg/kg, tj. te~./dan, ato je ekvivalentno unosu od 222 mg nitrata/dan za odraslu osobu od 60 kg, dok ADI za nitrite iznosi 0-0.07 mg/kg, tj. te~./dan. Odrasla osoba prosje no konzumira dnevno 400g razli itog povra pa je tako prosje an unos nitrata 157 mg/kg. Uzevai u obzir i konzumaciju voa koje sadr~i manje nitrata ukupan unos nitrata kree se od 81-106 mg/dan za veinu EU populacije ato je unutar ADI vrijednosti. Problem predstavlja manji dio populacije koji konzumira samo lisnato povre u velikim koli inama i tada dolazi do prekora enja ADI vrijednosti (Anonimno, 2006). Nitrati se kod ljudi nakon unosa brzo apsorbiraju iz gastrointestinalnog trakta u krvnu plazmu uz pomo crijevne mikroflore. Oko 25% nitrata iz plazme prolazi kroz ~lijezde slinovnice koncentrira se i izlu uje kroz slinu. U ustima, bakterijskom redukcijom nastaje oko 20% nitrita iz nitrata. U kiselom mediju ~eluca iz nitrita nastaje duaikov oksid i drugi metaboliti. Veina apsorbiranih nitrata se odmah izlu uje urinom. Toksikoloakim istra~ivanjima na eksperimentalnim ~ivotinjama utvreno je da je natrij-nitrit oko 10 puta toksi niji od natrij-nitrata (Gray i Randall, 1979). Kao ato je ve ranije spomenuto, istra~ivanja o pojavi kancerogenih spojeva N-nitrozamina i njihovoj etiologiji u proteklih 50.-ak godina zapo ela su otkriem dviju va~nih spoznaja. Prva se~e u 1956. godinu kada su britanski znanstvenici izvijestili o pronalasku kancerogenog N-nitrosodimethylamine (NDMA) kod eksperimentalnih ~ivotinja. Nadalje, u Norveakoj je ranih 1960.-ih potvreno da je NDMA bio glavni uzro nik smrti stoke koja je hranjena ribljim obrocima kojima su dodani nitriti. Nitriti su reagirali sa aminima ribljeg obroka i proizveli letalnu dozu NDMA (Preussmann i Stewart, 1984). N-nitrozamini se mogu podijeliti u 2 grupe na osnovi njihovih fizikalnih svojstava: Hlapivi N-nitrozamini (nitrozirani dialkil amini i cikli ki spojevi manje molekulske mase); Ne hlapivi N-nitrozamini (hidroksilirani amini ili polifunkcionalne tvari) (Walker, 1990) N-nitrozamini nastaju kemijskom reakcijom nitrozacijskog agensa i sekundarnih ili tercijarnih amina. Oksidi duaika (NOx) u oksidacijskom stanju +3 ili +4 slu~e kao nitrozacijski agensi, a najviae istra~ivan nitrozacijski agens koji sudjeluje u stvaranju nitrozamina koji nastaju u hrani je nitratni anhidrid (N2O3). Nitratni anhidrid brzo nastaje u kiselo vodenoj otopini i to u slijedeoj reakciji: 2NO2- + 2H+ ! N2O3 + H2O Dalje se N2O3 spaja sa slobodnim parom elektrona neprotoniranih sekundarnih amina reakcijom nukleofilne supstitucije i nastaju N-nitrozamini prema slijedeoj reakciji: R2NH + N2O3 ! R2N-N=O + HNO2 Stupanj nitrozacije je ovisan o pH i uvjetovan je koncentracijama nitrita i i amina: Rate = k [amine] x [nitrite]2 S obzirom da nitratni anhidrid reagira s neprotoniranim aminima, brzina (stupanj) nitrozacije sekundarnih amina je obrnuto proporcionalna lu~natosti amina. Optimalan pH za nitrozaciju veinu sekundarnih amina je izmeu 2.5-3.5. Radi se o dovoljno kiselom mediju kao ato je i u ljudskom ~elucu, a koji pogoduje stvaranju nitrozamina iz prisutnih amina i nitratnog anhidrida (Hui, 1992; Walker, 1990; Sen, Seaman i McPherson, 1980). N-nitrozamini esto su prisutni u suhomesnatim i preraenim mesnim proizvodima, jer im se prilikom procesiranja, kao ato je ve ranije naglaaeno, dodaju konzervansi u obliku nitrita ili nitrata. Nitrati se pomou enzima nitrat-reduktaze (prisutnog kod mnogih bakterija) reduciraju u nitrite i tako stupaju u daljnje kemijske reakcije sa aminima tijekom procesa proizvodnje, skladiatenja ili kuhanja, te tako nastaju nitrozamini (Girard, 1996; Walker, 1990). Najva~niji inhibitori reakcija nitrozacije u hrani su tvari s redoks potencijalom kao to su: askorbati (askorbinska kiselina-vitamin C), vitamin E, selen i dr. Askorbinska kiselina inhibira stvaranje nitrozamina mehanizmom kompeticije nitrostabilnih prekursora (amini) sa nitrozacijskim agensom. Kompletna inhibicija jedina je u inkovita i to u prisustvu vee koli ine askorbinske kiseline i/ili u anaerobnim uvjetima. Produkt koji nastaje reakcijom izmeu nitrita i askorbinske kiseline je vjerojatno odgovoran ne samo za nitrozacijske reakcije ve i za gubitak nitrita za vrijeme suaenja (pr~enja) mesa (Chow i Hong, 2002; Lu i sur.,1986). Vitamin E je zaslu~an za smanjenu produkciju reakcijskog produkta NO+, dok selen smanjuje produkciju reaktivnog ONOO- produkta. U zadnje vrijeme zahtjeva se smanjenje upotrebe nitrata i nitrita prilikom konzerviranja i procesiranja mesnih proizvoda tj. upotreba minimalnih koli ina koje e osigurati inhibiciju bakterijskog rasta. U svrhu prevencije stvaranja N-nitrozo spojeva u Sjedinjenim Ameri kim Dr~avama i nekim drugim zemljama predla~e se i dodatak od 500 mg/kg natrij-askorbata (Cassens, 1990; Schuddeboom, 1993). Materijali i metode U ovom radu ispitivani su uzorci konzervnih mesnih proizvoda-jetrene paatete od 3 razli ita proizvoa a. Uzorci jetrene paatete kupljeni su u nekoliko hrvatskih trgovina i jednom austrijskom supermarketu tijekom 2008. godine, te su dopremljeni u prostorije Laboratorija za zdravstvenu ispravnost i kvalitetu hrane, Odjela za analize hrane i predmeta ope uporabe, Slu~be za zdravstvenu ekologiju, Zavoda za javno zdravstvo  Dr. Andrija `tampar u Zagrebu, za daljnju analizu. Uzorci jetrene paatete odabrani su metodom slu ajnog odabira i za potrebe analize podijeljeni su u nekoliko grupa. Deklarirani sastav jetrenih paateta koriatenih za analizu: P r o i z v o  a  1 (Hrvatska): svinjsko mesno tkivo, voda, svinjska jetra 15%, svinjsko meso, bjelan evine mlijeka, za ini, kuhinjska sol, poja iva  okusa (E621), konzervans (250), sadr~i mlijeko; P r o i z v o  a  2 (Hrvatska): svinjsko mesno tkivo, voda, svinjska jetra 15%, svinjsko meso, emulgator (bjelan evine soje, lecitin, karagenan, ksantan guma), kuhinjska sol, za ini, konzervans (E250). Mo~e sadr~avati ~itarice, jaja, mlijeko, celer i goruaicu u tragovima; P r o i z v o  a  3 (Austrija): svinjsko meso 36%, slanina, svinjska jetrica 25%, voda, kuhinjska sol, za ini, dekstroza, saharoza, emulgator E471, E472c, poja iva  okusa: E261, konzervans: E250, kuhano. Uzorci jetrene paatete su pripremljeni i analizirani neposredno nakon dopremanja u Laboratorij. Odreivanje nitrita i nitrata Nakon vaganja i obrade uzoraka paatete, slijedila je izolacija pomou ekstrakcije toplom vodom, te su zatim uzorci paatete podvrgnuti postupku filtracije kako bi se pro istili prije kvantifikacije, a sve prema akreditiranoj metodi. Identifikacija i kvantifikacija natri-nitrita i natrij-nitrata raena je kromatografskom tehnikom visokou inkovite tekuinske kromatografije (engl. High Performance Liquid Chromatography-HPLC) s DAD detektorom serija 1200 (Agilent) Kolona: C18 Mobilna faza: 10% 0.01 M oktalamin 90% 5 mM tetrabutil amonij hidrogensulfat pH=6,5 Valna duljina: 214 nm Temperatura penice: 30C Protok: 1 mL/min (84). Parametri validacije metode Tablica 1. Parametri validacije primijenjene metode ParametarDobiveni kriterij prihvatljivostiRasipanje rezultata3.71%Iskoriatenje99.2 5%Ponovljivost mjerenja0.11%Ponovljivost pripreme uzorka2.71%LinearnostKe"0.999Intermedijarna preciznost3.33%Granica kvantifikacije1 mg/kg Priprema uzoraka Nakon vaganja to no odreene koli ine uzorka jetrene paatete (25 0,01 grama), uzorak je usitnjen i preba en u tikvicu za destilaciju uz dodatak natrij-klorida (NaCl). Nakon toga uzorak se destilirao postupkom klasi ne destilacije vodenom parom i sakupljala se odreena koli ina destilata. Nakon zavraene destilacije prikupljenom destilatu se dodala 6N sulfatna kiselina (H2SO4) i bezvodni natrij-sulfat (Na2SO4), te se tijekom postupka redestilacije sakupljala odreena koli ina redestilata kojem se na kraju postupka dodao kalij-hidroksid (KOH) i natrij-klorid (NaCl). Tako pripremljeni redestilat se dalje ekstrahirao organskim otapalom diklormetanom u lijevku za odjeljivanje, a prikupljena koli ina ekstrahiranog odjeljenog uzorka se uparila do suhog na rotavaporu, nakon ega je uslijedilo pro iaavanje, identifikacija i kvantifikacija N-nitrozamina metodom vezanog sustava plinske kromatografije s masenom spektrometrijom (Crosby i sur., 1972). Odreivanje N-nitrozamina Nakon obrade uzoraka, identifikacija i kvantifikacija N-nitrozamina je provedena vezanim sustavom plinske kromatografije i spektrometrije masa (engl. Gas Chromatography-Mass Spectrometry, GC-MS). Identifikacija i kvantifikacija N-nitrozamina Nakon izolacije N-nitrozamina ekstrakcijom pogodnim organskim otapalom-diklormetanom, pro iaavanja uzoraka pomou SPE kolona, kvantifikacija pojedinih spojeva N-nitrozamina napravljena je analizom pripremljenog uzorka pomou vezanog sustava plinskog kromatografa i masenog detektora (GC-MS, Shimadzu). Primijenjena je tzv. SIM tehnika analize zbog identifikacije odabranih i poznatih analita. 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N4n8\$&^d( $$Ifa$gdfsgduFU$a$gduFUmperatura injektora: 45C Temperatura detektora: 225C Detektor: detektor za masenu spektrometriju (MSD) MSD Mode: SIM Rang: 10C-280C Plin nosioc: helij Inlet Mode: Split Split flow: 1.5 ml/min Injektiranje uzorka: ru no Volumen injektiranja: 1l Vrijeme analize: 20 min Parametri validacije metode Za analizu uzoraka koriatena je metoda za odreivanje hlapivih N-nitrozamina, ali je navedena metoda unaprijeena i poboljaana u tome ato je za identifikaciju tra~enih spojeva N-nitrozamina koriaten sofisticiraniji ureaj (vezani sustav plinskog kromatografa i masenog detektora) zbog sigurne identifikacije spojeva i selektivnosti, osjetljivosti i preciznosti analize, a uzorci su prethodno pro iaeni pomou tzv. SPE kolona (Crosby i sur., 1972; Groenen i sur., 1976; Grebel, Young i Suffet HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TG8-4JRVB84-7&_user=10&_coverDate=06%2F02%2F2006&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_searchStrId=1482078122&_rerunOrigin=google&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=c2b4855fd91358d6643d26a7ee1d5469&searchtype=a" \l "aff1" , 2006). Stoga su provedeni osnovni parametri validacije koji uklju uju: linearnost, preciznost, to nost, selektivnost, granica detekcije i granica kvantifikacije (Anonimno, 1996). Tablica 2. Parametri validacije primijenjene metode ParametarDobiveni kriterij prihvatljivostiSelektivnostPrema informacijiPreciznostRSDd"5.3%Iskoriatenje92 5%Ponovljivost mjerenja0.19%LinearnostKe"0.0997Granica detekcije0,1 /kgGranica kvantifikacije0,3 /kg REZULTATI I RASPRAVA Mnoge do sada objavljene studije pokazale su da se i mali udio nitrita dodanih mesu kao konzervans, javlja kasnije u obliku hlapivih N-nitrozamina, pa su stoga uzorci paatete sva tri navedena proizvoa a analizirani na udio prisutnih natrij-nitrita (NaNO2) i natrij-nitrata (NaNO3), kako bi se dokazala veza s nastankom hlapivih N-nitrozamina. Poznato je da nitrit nakon dodatka odreenom supstratu vrlo brzo stupa u kemijske reakcije jer mo~e djelovati kao oksidacijski, redukcijski ili nitrozacijski agens, pa se tako pretvara u razli ite oblike kao ato su nitrat, nitratna kiselina, duaikov oksid, duaik(II)-oksid, a kao dokaz te reaktivnosti su promjene u boji, okusu i trajnosti mesnih proizvoda, dok odreen udio dodanog nitrita nestaje ovisno o formulaciji proizvoda, pH, temperaturi, te duljini trajanja tijekom prerade i skladiatenja. Tako npr. nakon proizvodnje nekog suhomesnatog proizvoda manje od 50% dodanog nitrita mo~e se dokazati kemijskom analizom. Zbunjujua je injenica da dodatkom razli ite koli ine nitrita u dva uzorka, nakon prerade, nalazimo iste koli ine zaostalog (rezidualnog) nitrita u oba uzorka. Znanstvene studije su pokazale da nitrit mo~e stupiti u reakciju s proteinima, lipidima, pigmentima i drugim sastojcima mesa, a udio koji ostaje a da nije stupio u kemijsku reakciju ostaje u slobodnom obliku (kao NO2- i HNO2) i naziva se slobodni ili rezidualni nitrit, dok se nitrit koji stupa u kemijsku reakciju sa sastojcima mesa naziva vezani nitrit. Analiti kim metodama se mo~e detektirati samo slobodni (rezidualni) nitrit. Koli ina slobodnog (rezidualnog) nitrita ovisi raznim faktorima kao ato su: pH, temperatura procesiranja i skladiatenja. Gubitak nitrita je pak funkcija vremena i temperature. Tijekom skladiatenja dolazi do smanjenja koncentracije nitrita, pa tako mesni proizvodi na tr~iatu sadr~e u prosjeku 5  30 mg/kg slobodnih nitrita (Sharat, D. i Gangolli, 1995; Pegg i Shahidi, 2004). U Tablici 3 prikazane su koli ine slobodnih natrij-nitrita (NaNO2) i nastalih natrij-nitrata (NaNO3) koji su nakon obrade uzoraka detektirani metodom visoko tla ne tekuinske kromatografije (HPLC). Naime, na deklaraciji kako hrvatskih tako i austrijskih paateta navedena je prisutnost konzervansa oznake E-250 ato potvruje prisutnost natrij-nitrita. Rezultati pokazuju razlike u koli inama prisutnih Na-nitrita kao i nastalih Na-nitrata ovisno o vrstama analiziranih proizvoda, odnosno proizvoa u. Razlike u koli inama su direktno povezane sa koli inama koriatenog NaNO2 tijekom procesa proizvodnje, mada postoje i neka odstupanja, a ato je najvjerojatnije razlika u samom procesu proizvodnje, kvalitete sirovine ili kvalitete samog aditiva. Poznato je da se nitrati mogu nai samo u onim mesnim proizvodima kojima su prethodno dodani nitriti; otprilike 20% dodanih nitrita reakcijom oksidacije prelazi u nitrate dva sata nakon procesiranja, a nastavlja se tijekom skladiatenja. Klju nu ulogu u konverziji nitrita u nitrate kod suhomesnatih mesnih proizvoda ima dodatak askorbata ili njegovog izomera eritorbata dodatkom natrij-askorbata kao konzervansa, no nitrati nastaju i bez njegovog dodatka ali u manjoj koli ini kod ostalih mesnih proizvoda (Pegg i Shahidi, 2004; Schuddeboom, 1993). Kasnih 60.-ih godina proalog stoljea upotreba nitrita u mesnim proizvodima doala je u pitanje, ak je Ameri ka vlada razmatrala mogunost zabrane tog konzervansa zbog znanstveno dokazane povezanosti s nastankom N-nitrozamina usprkos povoljnom djelovanju zbog ega se i koristi. Kao ato je ve spomenuto, nitriti reagiraju u odreenim uvjetima s aminima ili aminokiselinama mesa prilikom toplinske obrade. Pri tome naj eae nastaju spojevi N-dimetilaminnitrozoamin (NDMA) i N-pirolidinnitrozoamin (NPYR) koji su pokusima na eksperimentalnim ~ivotinjama dokazano kancerogeni, mutageni i teratogeni. Viae od 90% spojeva od poznatih 300-tinjak spojeva N-nitrozamina je do sada testirano na ~ivotinjama, te je dokazano da uzrokuju razli ite vrste tumora, meutim, ne postoji neki vrsti dokaz izmeu pojave tumora kod ljudi i izlo~enosti N-nitrozo spojevima. Zbog mnogih indirektnih dokaza o posebnoj povezanosti s pojavom tumora kod ljudi, mo~e se nedvojbeno ustvrditi da se radi o moguem etioloakom uzro niku uzevai u obzir injenicu da je 35% svih tumora u ljudi uzrokovano konzumacijom odreene hrane. U Sjedinjenim Ameri kim Dr~avama nitrit je 1998. godine proglaaen kao razvojni i reproduktivan toksikant (Honikel, 2008; Nabrzyski i Gajewska,1994). Utjecaj razli itih uvjeta skladiatenja na djelovanje nitrita i nitrata, te njihov utjecaj na nastajanje N-nitrozamina u 3 razli ita mesna proizvoda od iznutrica koji se u Poljskoj redovito konzumiraju, analizirali su Domanska-Blicharz i sur. (2004). `to se pak vrijednosti N-nitrozamina ti e, svje~i uzorci sporadi no sadr~avali su male koli ine N-nitrozodimetilamina (NDMA), dok su u svim uzorcima uvanim 72 sata na +4 do +8C bili prisutni spojevi NDMA, a prosje ne vrijednosti iznosile su 2.6 g/kg, 2.8 g/kg i 3.0 g/kg. Prosje ne vrijednosti N-nitrozopiperidina (NPIP) bile su 4.1 g/kg, 1.9 g/kg i 6.6 g/kg (ovisno o vrsti proizvoda). U nekim proizvodima pronaeni su i spojevi N-nitrozodibutilamin (NDBA) i N-nitrozomorfolin (NMOR). Rezultati poljskih znanstvenika potvrdili su da na promjene koli ina nitrita i nitrata kao i na nastanak N-nitrozamina u razli itim mesnim proizvodima uvelike utje u uvjeti skladiatenja. Naime, viae temperature pogoduju razvoju bakterija tako da dolazi do naglog smanjenja koli ina nitrita pri sobnoj i viaim temperaturama i to zbog bakterijske aktivnosti, dok se vrijednosti nitrata djelovanjem bakterija smanjuju ali ne u zna ajnoj mjeri. Koli ine nitrita smanjuju se i zbog reakcija nitrita sa sirovinama mesnih proizvoda iz kojih su proizvedeni, a to uklju uje i na in proizvodnje, duljinu skladiatenja, dodatne sastojke koji se koriste, a va~na je injenica da li se nitriti dodaju odjednom ili postepeno. Iz rezultata oba istra~ivanja vidljiva je korelacija izmeu razine nitrita i nitrata i sadr~aja N-nitrozamina. Stvaranje N-nitrozamina je jedan opse~an i joa uvijek nedovoljno razjaanjen proces koji rezultira razli itim interakcijama izmeu nitrozacijskih aktivatora i/ili inhibitora, a postoji i niz drugih faktora koji utje u na pojavu N-nitrozamina. Rezultati ovih dvaju istra~ivanja nedvojbeno dokazuju da su za nastanak N-nitrozamina  odgovorni nitriti, nitrati, primarni ili sekundarni amini ili amidi, proteini, peptidi ili aminokiseline koji se nalaze ili nastaju u navedenim mesnim proizvodima i koji pod utjecajem mikrobioloake aktivnosti postaju prekursori iz kojih dalje kao rezultat kemijskih reakcija nastaju N-nitrozamini (Pegg i Shahidi, 2004; Mirvish, 1995; Tannenbaum, 1985; Epley, Addis i Warthesen, 2009). U dostupnoj znanstvenoj literaturi postoje joa mnoga istra~ivanja koja ukazuju na opasnost od poveane izlo~enosti atetnim N-nitrozaminima putem konzumacije razli itih prehrambenih proizvoda, a osobito putem povra, te mesa i mesnih proizvoda. Naime, u veini Europskih zemalja postoje bolje ili loaije studije vezane uz na in prehrane pu anstava, a provodi se i sustavna kontrola hrane (monitoring), kako na prisutnost N-nitrozamina, tako i na prisutnost ostalih kontaminanata u svrhu procjene mogueg atetnog utjecaja na osobe koje ih konzumiraju, a s ciljem zaatite zdravlja pu anstva. Znanstvenici u Finskoj pratili su utjecaj izmeu unosa nitrita, nitrata i N-dimetilaminnitrozoamina (NDMA), te rizika od pojave tumora gastrointestinalnog trakta. Dobiveni rezultati pokazali su da nema zna ajne veze izmeu unosa nitrita i nitrata i pojave tumora gastrointestinalnog trakta, te unosa NDMA i pojave tumora ~eluca. Zaklju ak te studije bio je da N-nitrozo spojevi mogu inducirati pojavu tumora probavnog trakta. Isto tako joa je od 70.-tih godina proalog stoljea poznato da se N-nitrozo spojevi u organizmu sintetiziraju in vivo tijekom probave u kiselom mediju ~eluca ili bakterijskom aktivnoau tijekom prolaska kroz probavni sustav (Penttil, Rsnen i Kimppa, 1990). U jednom od novijih istra~ivanja iz 2007. godine Sabranek i Bacus pokuaali su ukazati na mogunost proizvodnje tzv. prirodnih odnosno organskih mesnih proizvoda bez direktnog dodatka nitrita ili nitrata koristei kao nadomjestak prirodne dodatke kao ato su: vinski ocat, limunov sok, prah od vianje, prah od celera, ru~marin i dr. Rok trajanja preraenih mesnih proizvoda s dodanim nitritima dokazano je du~i nego kod onih kojima su dodani prirodni izvori nitrata, ali je isto tako dokazano da dodatkom praha celera sa ili bez 0.28% vianjinog praha dolazi do 50%-tnog smanjenja koli ine rezidualnih nitrita. Sigurnost mesnih proizvoda kojima se dodaju samo prirodni dodaci su vrlo upitna iz dva razloga, s jedne strane nitrit je vrlo u inkovit aditiv u suzbijanju C. botulinum, dok je koncentracija rezidualnih (slobodnih) nitrita je dobro poznati rizi an faktor pri nastanku N-nitrozamina. U oba slu aja, ulazna i rezidualna koli ina nitrita, mora se strogo kontrolirati kako bi se osigurala sigurnost proizvoda ato je u slu aju proizvoda s prirodnim izvorom nitrata vrlo teako. Razlog tome je ato u nekim proizvodima nastaje vea, a u nekima manja koli ina rezidualnih nitrita tijekom skladiatenja, a va~an parametar predstavlja, odnosno veza izmeu vremena uvanja proizvoda i temperature iz razloga ato utje e na redukciju nitrata u nitrite uz pomo starter kultura zbog ega je koncentracija nitrita promjenjiva. ZAKLJU AK Iz svega navedenog mo~e se zaklju iti da je bez dodatka nitrita vrlo teako osigurati mikrobioloaki ispravne mesne proizvode, meutim, kao posljedica dodavanja nitrita nastaje potencijalna opasnost od nastanka toksi nih N-nitrozamina zbog reakcija slobodnih-rezidualnih nitrita koji ostaju slobodni tijekom prerade mesa. Stoga je, va~no nai onu  idealnu koli inu koja e inhibirati rast i razvoj C. botulinum, te osigurati mikrobioloaku sigurnost i prihvatljive senzorske karakteristike proizvoda, a istovremeno nee ostati previae slobodnih (rezidualnih) nitrita koji dalje ovisno o pH, dodatku tzv. reduktanata kao ato su askorbati ili eritrobati, dodatku soli, fosfata, termalnim uvjetima procesiranja, stupaju u razne ne~eljene reakcije. Joa uvijek nije sasvim jasno u kojoj mjeri N-nitrozamini utje u na nastanak razli itih vrsta tumora kod ljudi, iako je njihova toksi nost i kancerogenost nedvojbeno dokazana pokusima na eksperimentalnim ~ivotinjama. Stoga je va~no provoditi stalnu i sustavnu kontrolu mesa i mesnih proizvoda na prisutnost slobodnih nitrita i nitrata, te na eventualnu prisutnost svakako atetnih N-nitrozamina. Isto tako, potrebno je zakonski propisati maksimalno dozvoljene koli ine istih kao ato je to slu aj u EU zakonodavstvu. Kako bi se pak mogla utvrditi kolika je stvarna izlo~enost i opasnost od navedenih spojeva potrebno je imati podatke o konzumaciji i prehrambenim navikama pu anstva, ato u Republici Hrvatskoj joa uvijek ne postoji. Potrebno je analizirati i ostale vrste mesnih proizvoda, poglavito one u konzervama, jer su to proizvodi koji se dulje skladiate, pa stoga postoji opravdana opasnost od nastanka N-nitrozamina Svakako se mora istaknuti da je primjena visokospecifi ne tehnike za identifikaciju i kvantifikaciju N-nitrozamina, te specifi ni na in ekstrakcije i pro iaavanja uzoraka uporabom posebnih kolona klju ne u ovom radu i predstavljaju proizvod novijeg doba i zna ajan napredak u analiti kim procesima. LITERATURA Anonimno (1996): Validation of analytical procedures. ICH harmonised tripartite guideline. Anonimno (2003): The effects of Nitrites/Nitrates on the Microbiological Safety of Meat Products. The EFSA Journal, Parma. Anonimno (2005): EC Directive 95/2, Annex III, dio C, EC Directive 2005/69. Anonimno (2006): Guidance of the Scientific Committee on a request from EFSA. EFSA Journal, Parma. Anonimno (2007): Pravilnik o proizvodima od mesa (2007), Narodne novine broj 01, (N.N. 01/07). 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(1995): Role of N-nitroso compounds (NOC) and N-nitrosation in etiology of gastric, esophageal, nasopharyngeal and bladder cancer and contribution to cancer of known exposures to NOC. Canc. Lett. 93, 17-48. Nabrzyski, M., R. Gajewska (1994): The content of nitrates and nitrites in fruits, vegetables and other foodstuffs. Rocz. PZH 45, 167-180. Pearson, M. A., R. T. Dutson (1990): Meat and Health, Advances in meat research, Elsevier Applied Science, London/New York. Pavlini Prokurica, I. (2010): Praenje nastanka N-nitrozamia u hrani ~ivotinjskog podrijetla. Doktorska rasprava. PBF, Zagreb. Pegg, R. B., F. Shahidi (2004): Nitrite Curing of Meat: The N-Nitrosamine Problem and Nitrite Alternatives. Food & Nutrition Press, USA. Penttil, P. L., L. Rsnen, S. Kimppa (1990) Nitrate, nitrite and N-nitroso compounds in Finnish foods and the estimation of the dietary intakes. Z. Lebensm. Unters. Forsch. 190, 336-340. Perez Rodriguez, M. L. (1998): Effect of adding smoke flavouring to frankfurters on nitrite and nitrate levels. Food Research International 31, 581-585. Preussmann, R., B. W. Stewart (1984): N-nitroso carcinogens, Chemical Carcinogens. ACS Monograph 2, Washington. Reinik, M. (2007): Nitrates, nitrites, N-nitrosamines and polycyclic aromatic hydrocarbons in food: analytical methods, occurrence and dietary intake. Dissertation, University of Tartu. Roberts, T. A. (1995): The microbiological role of nitrite and nitrata. J. Sci. Food Agric. 26, 1755-1760. Sabranek, G. J., N. J. Bacus (2007): Cured meat products without direct addition of nitrate or nitrite, what are the issues?. Meat Sci. 77, 136-147. Schuddeboom, L. J. (1993): Nitrates and nitrites in foodstuffs. Council of Europe Press, Publishing and Documentation Service, ISBN 92-871-2424-8. Sen, N. P., S. Seaman, M. McPherson (1980): Further studies on the occurrence of volatile and non-volatile nitrosamines in foods. IARC Sci. Publ. 31, 457-465. Sharat, D., Gangolli, I. (1995) Nitrate, nitrite and N-nitroso compounds. Developments in Food Science 37, 685-704. Tannenbaum, S. R. (1985): Diet and exposure to N-nitroso compound. Princess Takamatsu Symp. 16, 67-75. Walker, R. (1990): Nitrates, nitrites and N-nitroso compounds: a review of the occurrence in food and diet and toxicological implications. Food Addit. Contam. 7, 717-768. Watson, D. H. (2002): Food chemical safety: Additives, CRC Press, Florida. Tablica 3. Koli ine nitrita i nitrata u uzorcima jetrene paatete izra~ene u mg/kg Table 3. Nitrite and nitrate concentrations in canned liver patties Podrijetlo proizvodaUzorakXrasponSDProizvoa  1Grupa 1 n=15NaNO22,952,73-3,220,25NaNO361,2058,7-65,13,42Grupa 2 n=15NaNO23,123,00-3,190,11NaNO369,363,9-72,14,68Proizvoa  2Grupa 1 n=15NaNO24,594,21-4,790,33NaNO325,6324,4-27,51,64Grupa 2 n=15NaNO25,254,90-5,450,30NaNO325,9323,8-27,61,94Proizvoa  3Grupa 1 n=15NaNO236,936,5-37,20,36NaNO352,8752,2-54,11,07Grupa 2 n=15NaNO237,4336,7-38,40,87NaNO353,0752,7-53,40,35 Tablica 4. Koli ine N-nitrozamina u uzorcima jetrene paatete (g/kg) Table 4. N-nitrosamines concentrations in canned liver patties (g/kg) PODRIJETLO PROIZVODAProizvoa  1 (n=30)0 DANAXrasponSDEtMet3,70,5-5,81,1diEtND--morfolin1,00,4-2,80,6diProp2,20,8-4,91,5piperidin1,40,6-8,21,6diButND--UKUPNO8,3ND-21,71,2Proizvoa  2 (n=30)EtMet1,10,5-2,00,4diEt0,60,3-0,80,2morfolin0,70,4-1,40,2diProp0,60,3-0,80,1piperidin0,60,3-1,30,2diButND--UKUPNO3,6ND-6,30,2Proizvoa  3 (n=30)EtMet1,60,8-3,91,0diEtND--morfolin0,20,1-0,90,2diPropND--piperidin0,20,1-0,30,1diBut3,00,8-9,92,8UKUPNO5,0ND-151,0EtMet  N-etilmetilnitrozoamin; diEt  N-dietilnitrozoamin; morfolin  N-nitrozomorfolin; diProp  N-dipropilnitrozoamin; piperidin  N-nitrozopiperidin; diBut  N-dibutilnitrozoamin  Slika 1. Kromatogram uzorka  odreivanje nitrata i nitrita  Slika 2. 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