ࡱ> TWQRSY "bjbj fC\\= 2<<$!$!$!8!8!8!8p!L"|8! f":####777$p&L$!7^777##׼7R #$!#7 h#ѻ[0 r+r8r$!t7777777777 7777r777777777<> z: LC-MS/MS metode za simultano odreivanje viae Fusarium toksina Kristina Habschied1 ( HYPERLINK "mailto:kristina.habschied@ptfos.hr" kristina.habschied@ptfos.hr), Bojan `arkanj1, Sanja Zec Zrinuai1, Vinko Krstanovi1, }eljka Rup i1, Nikola Saka 2 1Prehrambeno-tehnoloaki fakultet Osijek, Sveu iliate Josipa Jurja Strossmayera u Osijeku, F. Kuha a 20, 31000 Osijek, Hrvatska 2Odjel za kemiju, Sveu iliate Jospia Jurja Strossmayera u Osijeku, Cara Hadrijana 8a, 31000 Osijek, Hrvatska. Sa~etak Paenica na polju prirodno je izlo~ena razli itim vrstama plijesni, ato za posljedicu mo~e imati kontaminaciju mikotoksinima. Analiza mikotoksina analiti ki je zahtjevna i najbolji rezultati dobivaju se analizama na LC-MS/MS ureajima. Cilj ovog rada bio je uhodati analiti ku metodu kojom bi se istovremeno odreivalo osam Fusarium mikotoksina koriatenjem LC-MS/MS ureaja. Usporeene su dvije metode na temelju validacijskih parametara. Validacija metoda provela se odreivanjem trihotecenskih mikotoksina te zearalenona u matriksu paenice. Uzorak paenice podvrgnut je dvjema razli itim ekstrakcijama te pro iaavanju na SPE koloni, prije analize na LC-MS/MS ureaju. Osim linearnosti, ispitivani validacijski parametri su bili repetabilnost, reproducibilnost, limit detekcije (LOD), limit kvantifikacije (LOQ) i iskoriatenje. Klju ne rije i: LC-MS/MS, validacija metode, trihoteceni, zearalenon, paenica Uvod Mikotoksini koje produciraju vrste iz roda Fusarium su skupina sekundarnih visokotoksi nih metabolita koji se biosintetiziraju kad se gljive nau u nepovoljnim uvjetima. Ovi mikotoksini predstavljaju zna ajnu opasnost u prehrambenom lancu (KBosovski i sur., 2010; Mankevi iene i sur., 2011), a najzna ajniji toksini koje proizvode plijesni roda Fusarium su nivalenol (NIV), deoksinivalenol (DON), zearalenon (ZEA), fuzarenon X (FUS X), diacetoksiscirpenol (DAS), 3-acetildeoksinivalenol (3-ADON), T-2 toksin (T-2) te HT-2 toksin (HT-2) (Slika 1). U prehrambenoj industriji predstavljaju opasnost zbog potencijalne karcinogenosti, imunotoksi nosti, reproduktivne i razvojne toksi nosti. Njihova pojavnost je naj eaa u ~itaricama i proizvodima od ~itarica, ato predstavlja problem s obzirom da su ~itarice najzastupljenije namirnice u ukupnom unosu hrane, te zbog toga postoji opravdana potreba za razvojem novih metoda za odreivanje mikotoksina (Frenich i sur., 2009). Obzirom na svjetske trendove u analitici mikotoksina, zahtjev koji se stavlja na takve metode su istovremena analiza razli itih mikotoksina, ato se posebno odnosi na trihotecene i zearalenon, budui da naj eae dolaze zajedno u namirnicama. Razli ita fizikalno-kemijska svojstva mikotoksina ote~avaju njihovo simultano odreivanje, odnosno ekstrakciju iz matriksa i samu analizu na ureaju (Cavaliere et al., 2005; Berthiller et al., 2007; Frenich et al., 2009). Kao ekstrakcijska otapala naj eae se koriste voda, acetonitril i metanol u razli itim omjerima. Otopina acetonitrila se pokazala kao dobra za ekstrakciju trihotecena (Soleimany et al., 2012). Takve metode moraju biti selektivne i specifi ne, te moraju osiguravati brzu i jednostavnu analizu. Danas se stoga sve viae primjenjuje LC-MS/MS metoda za analizu mikotoksina kao jedna od metoda s najveom osjetljivoau (Ren at al., 2007). Prije nego se metoda odobri za koriatenje u analizi mora joj se utvrditi valjanost. Validacijom analiti ke metode se osigurava da je odabrana metoda prikladna za kvantitativno mjerenje odreenih tvari u specifi nom matriksu. Osnovni parametri validacije su to nost, preciznost, selektivnost, osjetljivost, ponovljivost i stabilnost. Prihvatljivost analiti kih podataka dobivenih metodom je stoga u izravnoj vezi s kriterijima koriatenim u validaciji metode. Cilj ovog rada je bio ispitati prikladnost LC-MS/MS metode za simultano odreivanje viae Fusarium toksina.    SHAPE \* MERGEFORMAT  TrichotecenR1R2R3R4R5AHT-2-OH-OH-OAc-H-OCOCH2CH(CH3)2T-2-OH-OAc-OAc-H-OCOCH2CH(CH3)2DAS-OH-OAc-OAc-H-HBDON-OH-H-OH-OH=O3-AcDON-OAc-H-OH-OH=ONIV-OH-OH-OH-OH=OFUSX-OH-OAc-OH-OH=O Slika 1: Kemijske strukture odreivanih mikotoksina Materijali i metode Otopine standarda i kalibracijska krivulja Za kalibraciju je koriaten kalibracijski mix (Biopure, Romer Labs, Austria) koji se sastojao od slijedeih osam mikotoksina: NIV, DON, ZEA, FUSX, DAS, 3-ADON, T-2 te HT-2 pri koncentracijama u rasponu od 10,0 do 10,2 g/mL. Radne otopine su pripremljene razrjeivanjem kalibracijskog mixa sa odgovarajuom koli inom mobilne faze. Koncentracije standarda upotrebljene za kalibracijsku krivulju kretale su se u rasponu od 5 do 1020 ng/mL. Priprema uzorka Za prvu metodu priprema uzoraka je odraena prema protokolu Zearalatest (Romer Labs): paenica je samljevena i 25,00 0,1 g je ekstrahirano sa 100 mL otopine acetonitril: voda = 84:16 (V:V) u blenderu tokom 3 min nakon ega je izvraena filtracija preko naboranog filter papira. `est mL filtrata pro iaeno je preko Mycosep 226 Aflazon+ kolone nakon ega se 4 mL uzorka otparavalo pod vakumom na 70 C u termobloku. Otpareni ostatak rekonstituiran je s 400 L mobilne faze. Druga metoda raena je prema Frenich i sur. (2009): 5,00 0,1 g paenice je samljeveno i preneaeno u polipropilenske tube za centrifugu u koje je dodano 10 mL otpine acetonitril: voda = 80:20 (V:V). Smjesa je zatim vorteksirana 2 min i ekstrakcija je nastavljena na tresilici na 60 okretaja/min kroz 10 min. Nakon ekstrakcije separacija faza se provodi centrifugom na 4500 g kroz 5 min na sobnoj temperaturi, nakon ega se 5 mL supernatanta propusti kroz Mycosep 226 Aflazon+ kolonu. Nakon pro iaavanja na koloni, 2 mL uzorka se otpari na 80 C uz vakuum te se nakon uparavanja rekonstituiralo sa 400 L mobilne faze. Nakon rekonstitucije uzorci su preneaeni u vialice te analizirani na LC-MS/MS ureaju. LC-MS/MS analiza Za izvoenje analize upotrjebljen je API 2000 trostruki kvadrupol (Applied BioSystems MDS SCIEX) sa ionskim raspraiva em kao ionskim izvorom (ESI), dvije PerkinElmer Series 200 pumpe, grija em kolona, autosamplerom (PerkinElmer Series 200) te vakuum rasplinja em. Obrada podataka raena je pomou Analyst softwarea verzije 1.4.2. Trideset l uzorka je ubrizgano na Supelco Ascentis Express C18 kromatografsku kolonu (2,7 m, 150 x 2,1 mm) sa Supelco Ascentis Express C18 pretkolonom (5mm). Kolona je termostatirana pri 45 C. Protok pokretne faze A (voda) i B (metanol) bio je 200 l/min. Obje pokretne faze sadr~avale su 10 mmoL/L mravlje kiseline, te je pH podeaen na 3,8 s amonijakom. Za analizu je koriaten gradijent pokretne faze, pri emu je pokretna faza B linearno rasla od 20% do 50% u 10 minuta, zatim do 80% do 15-e minute, u minuti porasla do 100% i tako se zadr~ala kroz 10 minuta. Analiti su kvantificirani u pozitivnom i negativnom MRM modu, te su se pratile po dvije tranzicije masa za kvalitativnu i kvantitativnu analizu (za NIV 357/311, 281; DON 341/295, 265; ZEA 317/ 131, 175; FUSX 355/247, 175; 3-ADON 339/231, 203; DAS 384/ 307,247; HT-2 447/ 345, 285 te T-2 489/245, 215). Glavni MS parametri bili su slijedeih vrijednosti: IS -4500V (negativni mod) te 5500V (pozitivni mod), CUR 35, CAD 5, GS1 55, GS2 45. Temperatura izvora bila je 450 C. Ukupno vrijeme analize bilo je 30 minuta. Rezultati i rasprava Mjerenje mikotoksina klasi nim metodama postaje skupo, a i dugotrajno, ako se u obzir uzmu prednosti metoda za mjerenje viae mikotoksina odjednom (multimikotoksinske metode). Kako u podru ju isto ne Hrvatske prevladava vrsta plijesni Fusarium graminearum (Krstanovi et al., 2005; `pani et al., 2010), na uzorcima paenice detektirani su mikotoksini koje ta plijesan naj eae proizvodi, a navedeni su u Tablici 1. gdje su prikazani parametri: iskoriatenje, LOD i LOQ. Ovo istra~ivanje pokazalo je koja metoda je bolja s obzirom na promatrane parametre validacije. Tablica 1: rezultati validacije metoda MikotoksinMetoda 1 (Zearala test)Metoda 2 (Frenich i sur. 2009)Iskoritenje %LOD ng/mLLOQ ng/mLIskoritenje %LOD ng/mLLOQ ng/mLDON*1000,451,50196,437,91125,123-ADON87,50,341,12102,827,5490,87NIV10,20,240,8150,011,3637,50ZEA*62,90,020,0785,80,401,30T-2*39,60,040,14116,81,244,09HT-2*38,71,866,19121,86,2820,73DAS84,10,341,12313,58,6528,54FUSX61,70,652,1796,361.58203.20*zakonski regulirani mikotoksini (NN 146/12)  Slika 2: $&:PZ\nz|~ B D F ȻȮﻌ}m}b^bQC}jh\&0JCJUaJhp|h0JCJaJh\&jh!Fh\&Uhp|hCJH*OJQJaJhp|hCJOJQJaJhp|hM5CJOJQJaJ"hK\hK\56CJOJQJaJhv5CJOJQJaJh5CJOJQJaJhK\5CJOJQJaJhg5CJOJQJaJhp|5CJOJQJaJhp|h|5CJOJQJaJ~ ^&&2&3&4&A&D&G&$d$Ifa$gdr$ da$gdr $da$gdr $da$gdK $da$gdp|dgdS5dgdO $da$gdS5F J d f j Ʀ{o_PB6hp|h|6OJQJhp|h|6H*OJQJhIhp|CJOJQJaJhR~hR~CJH*OJQJaJhR~CJOJQJaJhICJOJQJaJhp|hp|CJH*OJQJaJhp|hp|CJOJQJaJhp|h|CJH*OJQJaJhhCJH*OJQJaJhp|h|CJOJQJaJhp|hR]CJH*OJQJaJhp|hR]CJOJQJaJhCJOJQJaJ v      0 F 2 4 X ǷzznbVJVbVhF|CJOJQJaJhVCJOJQJaJh*UCJOJQJaJhkCJOJQJaJhEyCJOJQJaJh7LCJOJQJaJh~CJOJQJaJh\CJOJQJaJhp|5CJOJQJaJhp|hp|5CJOJQJaJhp|h|CJOJQJaJhR~6OJQJhR~hR~6OJQJhR~6H*OJQJh|6OJQJ $`hj|LPbdprt;񣗊{{oc{{W{{hCJOJQJaJh_HCJOJQJaJhq CJOJQJaJhhp|CJOJQJaJh~6CJOJQJaJh7LCJOJQJaJhhCJOJQJaJh*UCJOJQJaJhh%A CJOJQJaJhCJOJQJaJhF|CJOJQJaJh4 CJOJQJaJhhCJOJQJaJ" V \^Z\lnʾzj[I<hTCJOJQJ]aJ"hThT6CJOJQJ]aJhW{6CJOJQJ]aJh`hW{CJOJQJ]aJhp|5CJOJQJaJhS5hS55CJOJQJaJh heCJOJQJaJh CJOJQJaJhQACJOJQJaJhp|CJOJQJaJhpCJOJQJaJhh%A CJOJQJaJhCJOJQJaJhhp|CJOJQJaJn &(VXZx.<JRlnȮաȃwwk_ShW{CJOJQJaJh`CJOJQJaJhrCJOJQJaJhMCJOJQJaJhTCJOJQJaJ"hOhO6CJOJQJ]aJhOCJOJQJ]aJhMCJOJQJ]aJhKCJOJQJ]aJhTCJOJQJ]aJhW{CJOJQJ]aJhA(CJOJQJ]aJhxvhKCJOJQJ]aJbdNPhzX|~ 68ZhjᩛqcqcUh!FCJOJPJQJaJhVCJOJPJQJaJh9CJOJPJQJaJhaxCJOJPJQJaJh gCJOJPJQJaJhJCJOJPJQJaJhS CJOJPJQJaJh4 CJOJPJQJaJhgECJOJPJQJaJhA(CJOJPJQJaJ hS5hS5CJOJPJQJaJhMoJCJOJPJQJaJ!  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Od ostalih, zakonski nereguliranih mikotoksina bolje rezultate iskoriatenja dala je metoda 1 za DAS (iskoriatenja za zakonski neregulirane mikotoksine se kreu izmeu 50 i 130%); dok je Metoda 2 dala bolje rezultate iskoriatenja za NIV i FUSX. Metoda 1 se pokazala boljom za znanstvena istra~ivanja poato ima dosta ni~i LOD; dok je metoda 2 imala dosta viai LOD, iako se i kao takva mo~e koristiti za slu~bena ispitivanja jer su grani ne vrijednosti unutar minimalno zakonom dozvoljenih propisanih koncentracija. U radu koji su objavili Frenich i sur. (2009) vrijednosti iskoriatenja kretale su se od 70  108,4% (nakon obogaivanja sa 5 i 50 g/kg), vrijednosti LOD-a bile su od 0,01  2,1 g/kg, a LOQ-a od 0, 03  6, 30 g/kg, dakle, postoji razlika u dobivenim vrijednostima u odnosu na vrijednosti koje su dobivene u ovom radu, ali to se pripisuje utjecaju matriksa s obzirom da je u radu Frenich i sur. (2009) koriaten kukuruz, a ovdje je koriatena paenica za analizu. Razlika u ekstrakcijskim otapalima pokazuje da se ekstrakcijom s viae vode (metoda 2) ekstrahira i viae matriksa koji onda ometa signal i uzrokuje poviaenje LOD-a, ali zato i poboljaava ekstrakciju polarnih mikotoksina. Da bi se obje metode mogle koristiti za mjerenje mikotoksina prema trenutno va~eim pravilnicima potrebna je dodatna modifikacija metoda, odnosno mala promjena u odnosu ekstrakcijskog otapala (AcN:W). Modifikacija tretiranja pro iaenog ekstrakta bi takoer mogla utjecati na kona no iskoriatenje, odnosno bla~i uvjeti otparavanja uz ni~u temperaturu i viai vakuum, ili dodatak struje duaika kako bi se sprije ila oksidacija mikotoksina. Poato je detekcija bila izvraena pomou MS/MS detektora oksidacija bilo koje grupe u strukturi detektiranih mikotoksina mo~e dovesti do la~no negativnih rezultata. Slika 2 prikazuje kromatogram sa ionskim tranzicijama detektiranih mikotoksina. Poato su neki od mikotoksina osjetljivi na ultraljubi asto svjetlo uvijek se mora obratiti pozornost na zaatitu gotovih ekstrakata koji se uvaju u zatamnjenim bo icama i kod pripreme iskoriatenja, uzorci moraju ostati u mra nom prostoru do ekstrakcije. Zaklju ci Uhodavanje LC-MS/MS metode za analizu viae mikotoksina odjednom slo~en je i zahtjevan posao, a bitna stavka je pravilna priprema uzorka, odnosno ekstrakcija mikotoksina iz uzorka. Kako bi se metoda validirala nu~no je provesti analizu odabranog uzorka kako bi se dokazala prikladnost metode za odreivanje odabranih trihotecena i zearalenona. Dobiveni rezultati ukazuju na to da se Metoda 2, (Frenich i sur. 2009), pokazala kao uspjeanija kod odreivanja viae tipova mikotoksina odjednom te da su podaci dobiveni prema toj metodi vjerodostojniji i precizniji, dok je s druge strane metoda 1 zanimljivija za znanstvena istra~ivanja zbog svojeg ni~eg LOD. Odreivanje viae tipova mikotoksina istovremeno komplicirano je zbog njihove razli ite topivosti tijekom ekstrakcije te u samoj mobilnoj fazi tijekom analize u ureaju. Ipak, optimiranjem otopina za ekstrakciju i temperatura otparavanja tijekom pripreme uzorka mogue je dobiti izvrsne rezultate pomou ove metode. Zahvale Zahvaljujemo se Hrvatskom ministarstvu za znanost, obrazovanje i sport (projekti 113-1130473-0334 i 113-1780691-0538). Literatura Berthiller, F., Sulyok, M., Krska, R., Schumacher, R., (2007): Chromatographic methods for the simultaneous determination of mycotoxins and their conjugates in cereals. International Journal of Food Microbiology, 119: 33-37. Cavaliere, C., Foglia, P., Pastorini, E., Samperi, R., Lagana, A., (2005): Development of multiresidue method for analysis of major Fusarium mycotoxins in corn meal using LC-MS. Rapid Communications in Mass Spectrometry, 19:2085-2093. Frenich, A.G., Vidal, J.L.M., Romero-Gonzlez, R., del Mar Aguilera-Luiz, M., (2009): Simple and high-throughput method for the multimycotoxin analysis in cereals and related foods by ultra-high performance liquid chromatograpy/tamdem mass spectrometry. Food Chemistry, 117: 705-712. KBosovski, G., Mikulski, D., (2010): The effect of raw material contamination with mycotoxin on composition of alcoholic fermentation volatile by-products in raw spirits. Bioresource Technology, 101: 9723-9727. Krstanovi, V., Klapec, T., Veli, N., Milakovi, Z., (2005): Contamination of malt barley and wheat by Fusarium graminearum and Fusarium culmorum from the crop years 2001-2003 in Eastern Croatia. Microbiological Research, 160: 353-359. Mankevi iene, A., Butkute, B., Gauril ikiene, I., Dabkevi ius, Z., Supreniene, S., (2011): Risk assesment of Fusarium mycotoxins in Lithuanian small cereal grains. FoodControl, 22: 970-976. Ren, Y., Zhang, Y., Shao, S., Cai, Z., Feng, L., Pan, H., Wang, Z., (2007): Simultaneous determination of multi-component mycotoxin contaminants in foods and feeds by ultra-performance liquid chromatography tandem mass spectrometry. Journal of Chromatography A, 1143:48-64. Soleimany, F., Jinap, S., Faridah, A., Khatib, A., (2012): A UPLS-MS/MS for simultaneous determination of aflatoxins, ochratoxin A, zearalenone, DON, fumonisins, T-2 toxin and HT-2 toxin, in cereals. Food Control, 25: 647-653. Spanic, V., Lemmens, M., Drezner, G., (2010): Morphological and molecular identification of Fusarium species associated with head blight on wheat in East Croatia. European Journal of Plant Pathology, 128: 511-516. LC-MS/MS methods for simultaneous determination of Fusarium mycotoxins Kristina Habschied1 ( HYPERLINK "mailto:kristina.habschied@ptfos.hr" kristina.habschied@ptfos.hr), Bojan `arkanj1, Sanja Zec Zrinuai1, Vinko Krstanovi1, }eljka Rup i1, Nikola Saka 2 1Faculty of Food Technology, University of Josip Juraj Strossmayer, Franje Kuha a 20, 31000 Osijek, Croatia 2Department of Chemistry of the University of Josip Juraj Strossmayer, Cara Hadrijana 8a, 31000 Osijek, Croatia Abstract In the field, wheat is naturally exposed to different species of fungi which, as a repercussion may results in mycotoxins (secondary, toxic, fungal metabolites) contamination of wheat grains. Mycotoxins analysis is very complex and the best results are obtained by LC-MS/MS. The aim of this research was to determine an analytical method for simultaneous analysis of eight mycotoxins by using LC-MS/MS. Two methods have been compared based on validation results. Validation was carried out by analysis of trichotecens and zearalenone in wheat samples. Mass spectrometer was adjusted to work in positive and negative ionization mode and optimization of other parameters was performed (declustering and focusing potential, entrance potential, collision energy and collision entrance and exit potential). Before the analysis, a calibration with standard solution of mycotoxins was performed to prove the acceptable method linearity. Wheat sample underwent two different extraction methods followed by SPE column purification before the LC-MS/MS analysis. Except linearity, also repeatability, reproducibility, limit of detection (LOD), limit of quantification (LOQ) and recovery were determined. 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