PRIMENA C18 KOLONA ZA PRECIŠCAVANJE SIROVOG EKSTRAKTA KUKURUZA U CILJU ODREÐIVANJA FUMONIZINA B1 I B2 TECNOM HROMATOGRAFIJOM

Sandra M. Jakšic', Biljana F. Abramovic2, Milica M. Živkov-Baloš', Željko A. Mihaljev', Zoran S. Mašic'

'Naucni institut za veterinarstvo «Novi Sad», Rumenacki put 20, 21000 Novi Sad, Srbija i Cma Gora, Sandra@niv.ns.ac.vu 2Prirodno-matematicki fakultet, Departman za hemiju, Trg Dositeja Obradovica 3, 21000 Novi Sad, Srbija i Crna Gora, abramovic@ih.ns.ac.vu

IZVOD

Ispitana je efikasnost precišcavanja sirovog ekstrakta kukuraza na hemijski modifikovanim C18 kolonama, kao i mogucnost njihove višestruke upotrebe u cilju odredivanja fumonizina BI (FB]) i B2 (FB 2) tecnom hromatografijom. Nadeno je da efikasnost odredivanja FB) i FB 2 u kukuruzu nakon precišcavanja sirovog ekstrakta na C? kolonama iznosi: 82,4±9,2% za FBi i 74,5±5,2% za FB2 i da ne postoji mogucnost njihovog višestrukog korišcenja. lako je postupak precišcavanja sirovog ekstrakta kukuruza na ovim kolonama komplikovan i dugotrajan, a odredivanje ometaju interference matriksa, rezultati odredivanja sadržaja FBi i FB2 u prirodno kontaminiranim uzorcima kukuruza se uglavnom dobro slazu sa onirna dobijenim upotrebom imunoafmitetnih kolona, kao i kolona sa jakim anjonskim izmenjivacem.

Kljucne reci: C|8 kolona, fumonizini, kukuraz, precišcavanje ekstrakta, tecna hromatografija.

UVOD

Fumonizini su grupa mikotoksina koja cesto kontaminira kukuruz i proizvode od kukuruza. Produkuju ih plesni Fusarium moniliforme Sheldon i Fusairum proliferation, a najrasprostranjeniji u prirodi i sa najvecim uticajem na zdravlje ljudi i životinja su fumonizini B -serije: fumonizin B] (FB,) i fumonizin B2 (FB 2).

U analizi fumonizina su najviše zastupljene hromatografske metode uz korišcenje razlicitih postupaka precišcavanja sirovog ekstrakta uzorka (Abramovic et al., 2002; Jakšic, 2004). U tu svrhu se koriste kolone (kertridži-cartridge) sa razlicitim sorbentima: kolona sa C18 reverznom fazom (Ci8), kolona sa jakim anjonskim izmenjivacem (SAX -Strong Anjon Exchange) ili imunoafinitetna (IMA - immunoaffinity) kolona.

XIMEÐUNARODNI S1MPOZ1JUM TEHNOLOGIJE HRANE ZA ŽIVOTINJE _________________________ 237

Upotreba ? )8 kolona za odredivanje fumonizina u razlicitim vrstama uzoraka je ispitivana u nekoliko radova. Tako su, na primer, Hopmans i Murphy (1993) koristili Ci8

kolone za precišcavanje sirovog ekstrakta iz kukuruza i razlicitih namirnica na bazi kukuruza i dali prvi izveštaj o prisustvu fumonizina B3 (FB3) i hidrolizovanih FB, (HFB,) u namirnicama. Efikasnost precišcavanja za FB, je iznosila 115%, a razlog za visok prinos je, po tumacenju autora, bilo obogacivanje uzorka preko ekstrakcionog rastvaraca, a ne direktno uzorka.

U svojim ispitivanjima, Rice i Ross (1994) koriste C\% kolone u cilju pripreme uzoraka urina i fecesa goveda, ovaca i pacova za analizu FBi, FB2, FB3 i hidrolizovanih formi FB,. Granica detekcije (100 ng/ml) i efikasnost metode (veca od 95%) za urin, feces i hranu za životmje su bile uporedive sa rezultatima obogacenih uzoraka kukuruza. C|g kolone su uspešno primenjene i za analizu HFBi u sirovom i pasterizovanom mleku sa efikasnošcu 69-83% i granicom detekcije 20-25 ng/ml (Scott et a/., 1994).

Bennett i Richard (1994) su poredili C|g i SAX kolone za odredivanje fumonizina BI i B2 u kukuruzu, stocnoj hrani i kulturi plesni. Prinos metode je bio 92-95% za SAX i 83-88% za Clg kolone, za 10 /*g svakog od FB, i FB2 standarda.

Nastavljajuci naša ranije zapoceta istraživanja (Abramovic et al., 2005a,b), cilj ovog rada je bio da se utvrdi efikasnost ? ,8 kolona za precišcavanje sirovog ekstrakta obogacenih uzoraka kukuruza i mogucnost njihove višestrake primene. Takode su prodiskutovani rezultati odredivanja fumonizina u prirodno kontaminiranim uzorcima sa aspekta precišcavanja na CI8 kolonama u odnosu na imunoafmitetne kolone, kao i kolone sa jakim anjonskim izmenjivacem.

MATERIJAL I METODE

Svi rastvaraci korišceni za ekstrakciju fumonizina iz uzoraka kukuruza, kao i za pripremu mobilne faze su bili cistoce za tecnu hromatografiju. Sve hemikalije su po kvalitetu bile cistoce reagent grade. Za pripremanje rasrvora je korišcena dejonizovana voda. osim gde je drugacije receno.

Standardni rastvori fumonizina. Osnovni rastvori fumonizina B| (Sigma, iz Fusarium moniliforme, oko 98% TLC) i 82 (Sigma, iz Fusarium moniliforme) su pripremljeni u smeši acetonitril-voda (50:50, v/v) u koncentraciji 100 ug/ml za FB) i FB2. odmeravanjem odgovarajuce mase na mikrovagi. Ovi rastvori su stabilni 6 meseci na 4°C (Visconti et al., 2001). Razblaženiji standardni rasrvor FB! koncentracije 10 ng/ul i FB2 koncentracije 5 ng/?! (radni rasrvor) je pripremljen odmeravanjem 500 ?! osnovnog rasrvora FB] i 250 ul osnovnog rasrvora FB2 u odmerni sud od 5 ml, koji je, zatim, dopunjen do oznake smešom acetonitril-voda (50:50, v/v). Odgovarajucim razblazivanjem radnog rasrvora istom smešom rastvaraca, pripremljena je serija standardnih rasrvora za tecnu hromatografiju. Standardni rastvori su cuvani na 4 °C.

Priprema o-ftaldialdehid-2-merkaptoetanol reagensa. 40 mg o-ftaldialdehida (OPA, Sigma, min. 99%) je rasrvoreno u 1 ml metanola, razblaženo sa 5 ml 0,1 mol/1 Na;B4O7 i zatim je dodato 50 ?! 2-merkaptoetanola (MCE, Serva). Reagens je cuvan u dobro zatvorenom vijalu zašticen od svetla, a stabilan je 8 dana.

XI MEÐUNARODNI SIMPOZUUM TEHNOLOGIJE HRANE ZA ŽIVOTINJE

LC mobilnafaza. Metanol-0,1 mol/1 NaH 2PO4 (Merck, extra pure) (78:22, v/v) ciji je pH podešen na 3,3 sa o-fosfornom kiselinom. Mobilna faza je filtrirana kroz membranski filter 0,45 urn (ISO-DISC™ Filters PTFE 25-4, Supelco).

Priprema uzoraka. Uzorci kukuraza su prikupljeni u toku jeseni 2002. godine, sa ceriri lokaliteta u Vojvodini. Odmah po uzorkovanju, lOOOg svakog uzorka je pnpremljeno mlevenjem u laboratorijskom mlinu, tako da >93% prolazi kroz sito velicine pora 0.8 mm, a zatim je uzorak homogenizovan mešanjem. Tako pripremljeni uzorci su pakovani u plasticne kese i skladišteni u zamrzivacu na -20 °C do analize. Pre svake analize, uzorci su temperirani do sobne temperature.

Priprema obogacenih uzoraka kukuruza. Odmereno je 20,0 g uzorka (sadržaj fumonizina BI <0,02 ug/g i Ba <0,06 ug/g), preneto u posudu za blendiranje i dodata odredena zapremina standardnog rastvora FB, i FB2. Uzorak je analiziran nakon 60 minuta stajanja na sobnoj temperaturi.

Princip odredivanja Fumonizini su ekstrahovani iz kukuraza smešom rastvaraca acetonitril-metanol-voda. Nakon filtriranja i razblaživanja, sirovi ekstrakt je precišcen na C|8 koloni. Finalni ekstrakt uzorka je derivatizovan sa o-ftaldialdehidom i 2-merkaptoetanolom i analiziran tecnom hromatografijom sa fluorescentnim detektorom.

Ekstrakcija. Odmereno je 20,0 g uzorka, a zatim ekstrahovano dva puta sa po 50 ml smeše acetonitril-metanol-voda (25:25:50, v/v/v) u laboratorijskom miksera u toku 2 minuta. Spojeni ekstrakti su centrifugirani na 3000 x g u toku 10 min, a zatim filtrirani kroz filter-papir (Macherey-Nagel, Type 751, Diiren, Nemacka).

Precišcavanje sirovog ekstrakta na C !8 kolonama. 5 ml profiltriranog ekstrakta je razblaženo sa 12 ml 1% rastvora KC1 i nakon filtriranja kroz 1,0-um mikrofiber filter-papir. 8.5ml je nanošeno na C !8 kolonu (LC-18 SPE Tubes Supelclean™, Supelco, Bellefonte. USA, -10% C, endcapped, kapaciteta 6ml, sa punjenjem od 1 g) koja je prethodno kondicionirana sa 5 ml metanola i 5 ml 1% rastvora KC1. Kolona je ispirana sa 10ml smeše acetonitril-1% KC1 (2:8, v/v). Nakon toga, fumonizini su eluirani sa kolone sa 12 mlsmeše acetonitril-voda (7:3, v/v). Brzina eluiranja je bila 1 kap u 10-15 sekundi. Eluat je uparen do suva na 60 °C. Uparen ostatak je reekstrahovan sa 200 ul smeše acetonitril-voda (50:50, v/v).

Derivatizacija i tecna hromatografija. Alikvot od 50 ul ekstrakta je pomešan sa 50 ul OP A—MCE reagensa, na sobnoj temperaturi, uz mešanje, u toku 1 minuta. 20 ul derivatizovanog rastvora je injektovano u LC sistem. Oprema za tecnu hromatografiju se sastojala od LC sistema - BioRad 2800 sa SupelCosil™ LC -18-DB kolonom (250 x 4,6 mm, velicina cestica 5 um) sa fluorescentnim detektorom Hewlett Packard 1046A, vreme odgovora 4 s, frekvencija lampe 220 Hz. Protok mobilne faze 1 ml/min. Talasna dužina pobudujuce svetlosti 220 nm i emitovane svetlosti 440 nm.

REZULTATII DISKUSIJA

Metode za odredivanje fumonizina sa precišcavanjem na C,8 kolonama nisu standardizovane od strane AOAC, ali su cesto korišcene pojedinacno ili u kombinaciji sa SAX kolonama (Holcomb et al., 1993; Moller i Gustavsson, 2000). Mogucnost odredivanja hidrolizovanih derivata fumonizina im daje prednost u odnosu na drage

XI MEÐUNARODNI SIMPOZIJUM TEHNOLOGUE HRANE ZA ŽIVOTINJE 239

kolone. Pošto u literatuii nije zabeleženo ispitivanje mogucnosti njihove višestruke primene, i ova ispitivanja su bila predmet ovoga rada.

Ekstrakt kukuraza je precišcavan na C,8 kolonama po modifikovanom postupku koji su koristili Rice i Ross (1994). Naime, obzirom da je naša kolona imala oko tri puta vece punjenje, zapremine metanola i 1% rastvora KC1 koji su korišceni za kondicioniranje kolone, kao i smeše acetonitril-1% KC1 za ispiranje kolone nakon nanošenja sirovog ekstrakta smo srazmerno tome povecali. Pri ispitivanju efikasnosti kolona propuštanjem standardnog rastvora koji sadrži smešu fumonizina B, i B2, zakljucili smo da je potrebno povecati i zapreminu rastvora za eluiranje, jer 2 ml 70% acetonitrila, koliko su koristili pomenuti autori nije dovoljno za efikasno eluiranje ramonizina. Uzastopnim eluiranjem, naaetih toksina na kolonu, u vise frakcija od po 2 ml, utvrdili smo da je za efikasno eluiranje potrebno 12 ml 70% acetonitrila.

Efikasnost Cig kolona za standardni rastvor fumonizina koji je sadržao 500 ng FB, i 250 ng FB2 (Slika 1A), dobijena ovim postupkorn, je iznosila 96,7% za FB) i 92,6% za FBi. Efikasnost C\g kolona za precišcavanje sirovog ekstrakta iz kukuruza u cilju odredivanja FB, i FB2 je ispitana pomocu obogacenog uzorka kukuruza (Slika IB).

240 XI MEÐUNARODNI SIMPOZIJUM TEHNOLOGIJE HRANE ZA ŽIVOTINJE

Analizom pnrodno kontaminiranog uzorka kukuru/a, konscenog za obogacivanje,

dobijen je sadržaj FB| od 0,06 (ig/g (Slika 2), što je nešto vise u odnosu na IMA i SAX kolone cijim korišcenjem je dobijeno da je sadržaj ispod granice detekcije, odnosno <0.02 ue/e (Abramovic etal, 2005b).

XI MEÐUNARODN1 SIMPOZUUM TEHNOLOGUE HRANE ZA Z1VOT1NJE_________________________ 241

lako hromatogrami dobijeni za ekstrakt iz kukuruza (Slike IB i 2) ne sadrže znacajne interferirajuce pikove, ekstrakt kukuruza precišcen na C|8 koloni je lošije precišcen u poredenju sa IMA i SAX kolonama (Abramovic et al., 2005a, b). Pored toga, dobijeni eluat je veoma mutan i mada se nakon rekonstituisanja dobija bistar uzorak, prilicno je obojen, te smo zato uveli dodatnu fazu u precišcavanju ekstrakta. Naime, sirovi ekstrakt uzorka kukuruza razblažen sa 1% rastvorom KC1 smo pre nanošenja na C|g kolonu profilrrirali kroz mikrofiber filter-papir. Medutim, i pored toga, kao i kod hromatograma dobijenih za uzorak kukuruza precišcenog na SAX koloni (Abramovic et al., 2005b), i ovde je primetan smetajuci uticaj matriksa kukuruza. Na osnovu svega izloženog se može zakljuciti da C)8 kolone, slicno SAX kolonama, imaju manju selektivnost za izdvajanje fumonizina iz sirovog ekstrakta uzorka kukuruza u poredenju sa IMA kolonama.

Povecanje efikasnosti i selektivnosti C!8 kolona se može postici pripremom modifikovanih kolona. Tako, na primer, Tracksess et al. (2000) predlažu za precišcavanje sirovog ekstrakta uzorka kineske šecerne trske i žitarica za dorucak, primenu CI8 kolone sa rrmogo vecim pakovanjem od komercijalne i dužim vremenom kondicioniranja. Problem kompleksnih matriksa može da se reši i primenom kombinacije kolona, npr. C,8 i SAX (Holcomb et al., 1993).

U Tabeli 1 su prikazani rezultati ispitivanja efikasnosti odredivanja FB, i FB2 u kukumzu nakon precišcavanja sirovog ekstrakta na Cj8 kolonama. U literaturi su zabeleženi i rezultati sa vecom efikasnošcu, ali su metode primenjene na druge vrste uzoraka (korn-fleks; Tracksess et al., 2000), ili je obogacivanje vršeno direktno u ekstrakcioni rastvarac, a ne na uzorak (Hopmans i Murphy, 1993).

Tabela 1. Efikasnost LC metode sa C|8 kolonama za precišcavanje ekstrakta iz kukuruza obogacenog sa 1 ug/g FB, i 0,5 jug/g FB 2

Efikasnost (%) Broj odredivanja

FB,a FB2b

1 83,8 76,0

2 72,6 78,8

3 90,9 68,7

Srednja vrednost 82,4 74,5

SD 9,2 5,2

RSD (%) 11,2 7,0

1500 ng standarda FB] propušteno kroz kolonu; ' 250 ng standarda FB2 propusteno kroz kolonu.

Nanošenjem i eluiranjem ekstrakta obogacenog kukuruza na istu C18 kolonu tri puta za redom ispitali smo mogucnost višestrukog korišcenja iste kolone. Kao što se iz Tabele 2 može videti, C]8 kolone se mogu koristiti samo jedanput, jer vec pri dragom nanošenju efikasnost odredivanja FB, opada za oko 60%. Efikasnost odredivanja FB2, nakon

242______________________XI MEDUNARODN1 S1MPOZI.1UM TEHNOLOGUE HRANE ZA ŽIVOTINJE

dnigog korišcenja kolone je cak nešto visa, ali je vec pri trecem korišcenju nedovoljna. Medutim, u svakom slucaju efikasnost odredivanja FB2 je znatno visa nego kod FB).

"abela 2. Efikasnost LC metode sa C]g kolonom pri sukcesivnom korišcenju za precišcavanje ekstrakta kukuraza obogacenog sa 1 ug/g FB, i 0,5 ug/g FB 2

Efikasnost (%) Broj odredivanja

FB, FB2 1 90,9 68,7

2 33,7 76,3

3 24,3 54,9

Regeneracija Qg kolona za precišcavanje sirovog ekstrakta kukuruza radi odredivanja fumonizina nije zabeležena u literaturi. Obzirom na to da smo pomocu ovih kolona vec nakon prvog korišcenja dobili relativno lose precišcen ekstrakt kukuruza, nismo ni ispitivali mogucnost njihove regeneracije.

Granice detekcije FB] i FB2 u kukuruzu nakon opisanog postupka precišcavanja na Cigkolonama, odredena kao odnos signal - sum 3:1, iznosi 0,02 /zg/g za FB, i 0,05 /ig/g zaFB 2.

Analiza prirodno kontaminiranih uzoraka je vršena tako što je isti sirovi ekstrakt kukuruza precišcavan uporedo na sve tri vrste kolona i analiziran metodom tecne hromatografije (Slika 3). Kao što se može videti rezultati odredivanja fumonizina u sva tri slucaja pokazuju zadovoljavajuce slaganje.

244______________________XI MEÐUNARODNl SIMPOZUUM TEHNOLOG1JE HRANE ZA Z1VOT1NJE

ZAKLJUCAK

Na osnovu prikazanih rezultata ispitivanja precišcavanja sirovog ekstrakta kukuruza na C]8 kolonama i detekcije OPA-MCE derivata fumonizina tecnom hromatografijom, može se zakljuciti da je efikasnost odredivanja FB, i FB 2 u kukuruzu 82,4±9,2% za FBi i "4.5=5.2°o za FB:. i da ne postoji mogucnost njihovog višestrukog korišcenja.

Nadeno je i da nema signifikantne razlike u dobijenim rezultatima sadržaja fumonizina u prirodno kontaminiranim uzorcima kukuruza nakon primene C !8 kolona, u odnosu na imunoafinitetne kolone, kao i kolone sa jakim anjonskim izmenjivacem.

Zahvalnost Rad je finansiran od strane Ministarstva nauke i zaštite životne sredine Republike Srbije (Projekat: "Razvoj novih i poboljšanje postojecih analitickih metoda i tehnika za pracenje kvaliteta životne sredine", No 1622).

LITERATURA

lAbramovic, B., Ðilas, S., Gaal, F., Mašic, Z. (2002) Metode odredivanja fumonizina,

Eko-konferencija 2002, Zdravstveno bezbedna hrana II, Novi Sad, 127-132. 2.Abramovic B.F., Jakšic S.M., Mašic Z.S. (2005a) Liquid chromatographic determination

of fumonisins B| and B2 in corn samples with reusable immunoaffinity column clean-up, J. Serb. Chem. Soc., 70, primljeno za štampu.

3-Abramovic B.F., Jakšic S.M., Mašic Z.S. (2005b) Efficiency of crude corn extract cleanup on different columns in fumonisins determination, Matica Srpska Proceeding for Natural Sciences, primljeno za štampu. 4.Bennett, G.A., Richard, J.L. (1994)

Liquid chromatographic method for analysis of the naphthalene dicarboxaldehyde derivate of fumonis ins, J. AOAC Int., 77: 501-506.

5.Holcomb, M., Thompson, H.C. Jr., Hankins, L.J. (1993) Analysis of fumonisin Bl in

rodent feed by gradient elution HPLC using precolumn derivatization with FMOC and fluorescence detection, J. Agric. Food Chem., 41: 764-767. 6.Hopmans, E.G.,

Murphy, P.A. (1993) Detection of fumonisins B,, B2, and B3 and hydrolyzed fumonisin B, in corn-containing foods, J. Agric. Food Chem., 41: 1655- 1658. "/Jakšic S. (2004) Prilog hromatografskom odredivanju fumonizina B, i B2

u kukuruzu, Magistarska teza, Univerzitet u Novom Sadu, Prirodno-matematicki fakultet, Novi

Sad. 8.M6ller, T.E., Gustavsson, H.F. (2000) Determination of fumonisins B, and B 2 in various

maize products by a combined SAX+C18 column and immunoaffinity column, J. AOAC Int., 83: 99-103. 9.Rice, L.G., Ross, P.P. (1994) Methods for detection and

quantitation of fumonisins in com, cereal products and animal excreta, J. Food Prot., 57: 536-540. 10.Scott,

P.M., Delgado, T., Prelusky, D.B., Trenholm, H.L., Miller, J.D. (1994) Determination of fumonisins in milk, J. Environ. Sci. Health, B29: 989-998.

ll.Trucksess, M.W., Cho, T-H., Ready, D.E. (2000) Liquid chromatographic method for

fumonisin B, in sorghum syrup and corn-based breakfast cereals, Food Addit. Contain., 17: 161-166. 12.Visconti A., Solfrizzo, M., De Girolamo, A. (2001) Determination

of fumonisins B, and B; in com and corn flakes by liquid chromatography with immunoaffinity column cleanup: collaborative study, J. AOAC Int., 84: 1828-1837.

APPLICATION OF C]8 COLUMNS FOR CRUDE CORN EXTRACT CLEANUP IN FUMONISINS BI AND B2

DETERMINATION BY LIQUID CHROMATOGRAPHY

Sandra M. Jakšic', Biljana F. Abramovic2, Milica M. Živkov -Baloš', Željko A. Mihaljev', Zoran S. Mašic'

'Scientific Veterinary Institute "Novi Sad", Rumenacki put 20, 21000 Novi Sad, Serbia and Montenegro, sandra@niv.ns.ac.yu 2Faculty of Science, Department of Chemistry, Trg Dositeja Obradovica 3, 21000 Novi Sad, Serbia and Montenegro, abramovic@ih.ns.ac.yu

ABSTRACT

Efficiency of crude com extract clean-up on chemically modified ? ]8 columns was studied, as well as the possibility of their multiple use in order to determine fumonisins B| (FB,) and J32 (FB 2) by liquid chromatography. It was found that FB, i FB2 recovery in com after crude extract clean-up on Ct8 columns is 82.4±9.2% for FB| and 74.5±5.2% for FB2. and that there is no possibility of their multiple use. Although the process of com extract clean-up on these columns is complex and lasting, and determination is distracted by matrix interferences, the results of determining content of FB, and FB2 in naturally contaminated corn samples mostly correspond to those obtained by immunoaffmity columns application, as well as strong anion exchanger.

Key words: C18 column, clean-up extract, corn, fumonisins, liquid chromatography.

INTRODUCTION

Fumonisins belong to the group of mycotoxins which often contaminate corn and com products. They are produced by Fusarium moniliforme Sheldon and Fusairum proliferation fungi. B-series fumonisins (fumonisin B,; FB! and fumonisin B2; FB2) are the most widespread in nature and they have the greatest influence on human and animal health.

Chromatography methods, after different crude sample extract clean-up procedures, are the most frequent methods used in fumonisin analysis (Abramovic et al., 2002; Jakšic, 2004). Because of that, columns (cartridges) with different sorbents are applied: reversed-phase C|g column (Cjg), column with strong anion exchange (SAX), and immunoaffinity column (IMA).

C|g column application for determining fumonisins in different sample types was examined in several papers. For example, Hopmans i Murphy (1993) used C18 columns

XI INTERNATIONAL SYMPOSIUM OF FEED TECHNOLOGY ___________________________________243

for clean-up of crude extract from corn and different corn-containing foodstuff, and they gave the first report of the presence of fumonisin B3 (FB,) and hydrolyzed FB] (HFB,) in human foods. Recovery of FB, was 115% and the reason for this high efficiency was, according to the authors', spiking of corn solvent extract, and not directly the sample.

In their examination. Rice and Ross (1994) used C|8 columns in order to prepare urine samples and cattle, sheep, and rat fecal samples for FB], FB2, FB3, and hydrolyzed FB] analysis. Detection limit (100 ng/ml) and recovery (higher than 95%) for urine, fecal samples and animal feed, were comparable to spiked corn samples. C|g columns were successfully applied for HFB] analysis in raw and pasteurized milk with 69-83% recovery and 20-25 ng/ml detection limit (Scott et al., 1994).

Bennett and Richard (1994) compared C,8 and SAX columns for determination of fumonisins B] and B2 in corn, animal feed and fungi cultures. Recovery of 10 jug standards of each FB, i FB 2 was: 92-95% for SAX, and 83-88% for C,8 columns.

Continuing our previous examination (Abramovic et al., 2005a,b), the objective of this paper was to determine efficiency of crude corn extract clean-up on C,8 columns, as well as possibility of their multiple use. The results of fumonisins determination in naturally contaminated samples, after ? ,8 columns clean-up was discussed, in comparison with immunoaffinity columns, as well as columns with strong anion exchanger.

MATERIAL AND METHODS

All solvents used for fumonisins extraction from corn samples, as well as for the mobile phase preparation were of LC grade. All chemicals, used in the investigation, were of reagent grade. Solutions were prepared in deionized water except when stated otherwise.

Fumonisins calibrant solutions. Fumonisins B, (Sigma, from Fusarium moniliforme. approx. 98% TLC) and B2 (Sigma, from Fusarium moniliforme) were purchased as analytical standards. Calibrant solutions were prepared in acetonitrile-water (50:50, v/v) at concentration of 100 ng/ml for FB, and FB2. Fumonisin calibrant solutions are stable up to 6 months when stored at 4 °C (Visconti et al.. 2001). Stock solution containing FB, at 10 ng/u,l and FB2 at 5 ng/u.1 was prepared by measuring 500 ?! calibrant solution FB, and 250 u.1 calibrant solution FB 2 into 5 ml volumetric flasks and diluting to volume with acetonitrile-water (50:50, v/v). Working calibrant solutions were prepared by appropriate dilution of stock solutions with acetonitrile-water (50:50, v/v). Standard solutions were stored at 4 °C.

Preparation of o-phtlialdialdehyde-2-mercaptoethanol reagent. 40 mg o-phthaldialdehyde (OPA, Sigma, min. 99%) were dissolved in 1 ml methanol, diluted with 5 ml 0.1 mol/1 Na2B4O7, and 50 ?! 2-mercaptoethanol (MCE, Serva) were added. This reagent is stable up to 8 days in a capped, aluminum foil-covered vial in the dark.

LC mobile phase. Methanol-0.1 mol/1 NaH2PO4 (Merck, extra pure) (78:22, v/v), with pH adjustment to 3.3 with o-phosphoric acid. The mobile phase was filtered through a 0.45 urn membrane (ISO-DISC™ Filters PTFE 25-4, Supelco).

Sample preparation. Corn samples were collected during autumn of 2002 from four localities in Vojvodina. Immediately after sampling, 1000 g of each sample was prepared

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by grinding in a laboratory mill in such a way that >93% passes through a sieve with pore diameter of 0.8 mm. After that, the samples were homogenized by mixing. The samples prepared in such a way were packed in plastic bags and stored in a freezer at -20 °C until the analysis. Prior to each analysis, the samples were allowed to reach room temperature.

Spiked corn samples preparation. 20.0 g of the sample (content of fumonisin BI <0,02 ng'g. and BZ <0,06 u.g/g), was transferred to a blender, and known volum e of standard solution of FB| and FB2 was added. The sample was kept 60 minutes at room temperature and then analyzed.

Principle of determination Fumonisins were extracted from corn with acetonitrile-methanol-water. After

filtration (and dilution), the crude extract was cleaned-up on one C!8 column. The final sample extract was derivatized with o-phthaldialdehyde and 2-mercaptoethanol and analyzed by reversed-phase liquid chromatography with fluorescence detector.

Extraction. 20.0 g of the sample was extracted twice with 50 ml of acetonitrile-methanol-water (25:25:50, v/v/v) in a laboratory blender for two minutes. The combined extracts were centrifuged at 3000 x g for 10 min, with subsequent supernatant filtration through filter paper (Macherey -Nagel, Type 751, Diiren, Germany).

?? column clean-up. 5 ml of the filtered extract was diluted with 12 ml of 1% KG solution and filtered through 1.0-u.m microfiber filter paper. After that, 8.5 ml of the diluted extract was applied to a C]8 column (LC-18 SPE Tubes Supelclean™, Supelco, Bellefonte, USA, ~10% C, endcapped, capacity 6ml, with a 1 g filling), previously conditioned with 5 ml of methanol and 5 ml 1% KC1 solution. The column was eluted with 10ml of acetonitrile-1% KCI (2:8, v/v) mixture. Fumonisins were subsequently eluted with 12 ml of mixture acetonitrile-water (7:3, v/v), the rate of elution being 1 drop per 10-15 seconds. The eluate was evaporated just to dryness at 60 °C. Purified residue was redissolved in 200 ?] acetonitrile-water (50:50, v/v).

Derivatization and liquid chromatography. A 50 uJ aliquot of the extract was mixed with 50 u,l of the OPA-MCE reagent at room temperature with the reaction time of one minute with stirring. 20 ?! of derived solution was injected into the LC system. The equipment consisted of an LC system - BioRad 2800 with Supelcosil™ LC-18-DB column (250 x 4.6 mm id, particle size 5 u,m) with a fluorescence detector Hewlett Packard 1046A, response time 4 s. flash frequency 220 Hz. LC pump delivered 1 ml/min constant flow rate. Wavelength of excitation radiation was 220 nm and emission 440 nm.

RESULTS AND DISCUSSION

Methods for fumonisins determination with C18 columns clean-up are not standardized by AOAC. In spite of that, these columns are often used individually or combined with SAX columns (Holcomb et a/., 1993; Moller and Gustavsson, 2000). Possibility of hydrolyzed derivatives fumonisin determination gives them the advantage over other columns. Since no examination of the possibility of their multiple use was recorded in literature, this research was a subject of the paper.

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Corn extract was cleaned-up with C,8 columns according to modified procedure used by Rise and Ross (1994). Since our column had threefold charge, volumes of methanol and 1% KCI solution used for column conditioning, as well as acetonitrile-1% KCI mixture for column washing after application of crude extract were proportionally increased. During the examination of column recovery by passing of standard solution, which contains fumonisins B] and B2 mixture, we concluded that it is also necessary to increase volume of elution solution, because 2 ml of 70% acetonitrile, used by above mentioned authors, is not sufficient for efficient fumonisin elution. By successive elution of toxins applied to column in several 2 ml fractions, we determined that 12 ml 70% acetomtrile is necessary for efficient elution.

C Ig columns efficiency for fumonisin standard solution which contained 500 ng FB, and 250 ng FB 2 (Figure 1 A), according to this procedure, was 96.7% for FB, and 92.6% for FB2. Spiked corn sample was used for the examination of C|g columns efficiency for raw corn extract clean-up in FB! and FB2 determination (Figure IB).

According to analysis of naturally contaminated corn sample used for spiking, content of FB, was 0.06 ug/g (Figure 2). That is higher than the results of IMA and SAX columns, which were below the detection limit (Abramovic et al., 2005b).

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Although chromatograms (Figures IB and 2) after C]g column corn extract cleaned-up do not contain significant interfering peaks, this extract is poorly cleaned up in comparison to IMA and SAX columns (Abramovic et al., 2005a,b). Besides, the received elute is unclear and although, after reconstruction, clear sample was received, it is quite colored, so we had to establish additional phase of extract clean-up. Namely, before application to ? ]8 column, we filtered the crude extract of corn sample, diluted with 1% KC1 solution, through micro fiber filter-paper. Nevertheless, as well as with chromatograms received from corn sample extracted on SAX columns (Abramovic et al., 2005b). interfering influence of corn matrix was noticeable. Therefore, we can conclude that C|8 columns, similar to SAX columns, have lesser selectivity of fumonisms from crude corn sample extract than IMA columns.

Enhancement of C]8 columns efficiency and selectivity can be achieved by modified columns preparation. For example, Trucksess et al. (2000) suggest the application of C,8

columns with significantly higher charge and with longer conditioning period than commercial columns for crude sample extract clean-up of sorghums and breakfast cereals. The problem of complex matrixes can be solved with combination of different columns, for example C,8 and SAX (Holcomb et a/., 1993).

In Table 1 are shown the results of FB, and FB 2 efficiency determination after C]8 columns crude corn extract clean-up. The results with higher efficiency are recorded in literature, but methods in these studies are applied to other types of samples (cornflakes; Trucksess et al., 2000), or spiking was done directly to extract solvent instead to sample (Hopmans and Murphy, 1993).

Table 1. Recovery of LC methods with C ]8 columns for clean-up of corn extract spiked with 1 ng/g FB, and 0.5 ug/g FB2

Recovery (%) Determination number

FB,a FB2b

1 83.8 76.0

2 72.6 78.8

3 90.9 68.7

Mean 82.4 74.5

SD 9.2 5.2

KSD (%) 11.2 7.0 a 500 ng FB] standard applied to the column; b 250 ng FB2 standard applied to the column.

We examined the possibility of multiple use of a column by application and elution of spiked corn extract on the same C]8 column three times in a row. As it is shown in Table 2, C )8 column can be used only once, because during the second application

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recovery of FB, is already reduced for about 60%. Recovery of FB 2, after the second column application is, even slightly higher, but during the third application it is insufficient. However, the recovery of FB2 is significantly higher than the recovery of FB,.

Table 2. Recovery of LC method with C,g column during the successive clean-up of com extract spiked with 1 ue/e FB, and 0.5 ue/e FB?

Recovery (%) Determination number FB, FB2

1 90.9 68.7 2 33.7 76.3 3 24.3 54.9

Regeneration of C,8 columns used for crude corn extract clean-up for determination of fumonisins has not been recorded in literature. Since we obtained relatively poorly cleaned-up extract after the first application of these columns, we did not study the possibility of their regeneration.

Detection limits for FB, and FB 2 in corn, after described C,8 columns clean-up procedure, determined as signal-to-noise ratio 3:1, are 0.02 jtg/g, and 0.05 jtg/g, respectively.

Analysis of naturally contaminated samples was done by applying the same crude corn extract to all three types of columns at the same time, and analyzed by liquid chromatography (Figure 3). As it can be seen, the results of fumonisin determination in all three cases show satisfactory agreement.

CONCLUSION

According to presented results of C|8 columns raw extract clean-up and detection of OPA-MCE fumonisins derivatives investigation by liquid chromatography, it can be concluded that recovery for FB, and FB 2 in corn is: 82.4±9.2%, and 74.5±5.2%, respectively, and that the possibility of their multiple use does not exist.

It was found that there is no significant difference in the obtained results for fumonisins content in naturally contaminated corn samples after C,g application, in comparison with immunoaffinity columns, as well as columns with strong anion exchanger. Acknowledgements: The work was financed by the Ministry of Science and Environment Protection of the Republic of Serbia (Project: "Development of New and Improvement of the Existing Analytical Methods and Techniques for Monitoring Quality of the Environment", No 1622).

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REFERENCES

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2. Abramovic B.F., Jakšic S.M., Mašic Z.S. (2005a) Liquid chromatographic determination of fumonisins B, and B2 in corn samples with reusable immunoaffmity column clean-up. J. Serb. Chem. Soc. 70, primljeno za štampu.

3. Abramovic B., Jakšic S.M., Mašic Z.S. (2005b) Efficiency of crude corn extract clean-up on different columns in fumonisins determination, Matica Srpska Proceeding for Natural Sciences, primljeno za štampu.

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