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METODO MULTIRESIDUO PER LA DETERMINAZIONE DI RESIDUI DI ESTRATTI VEGETALI IN ALIMENTI BIOLOGICI C.I.G. Tuberoso Dip. di Tossicologia Università di Cagliari Via Ospedale, 72 Cagliari Nell'agricoltura biologica si effettua il controllo dei pesticidi di sintesi, in quanto la normativa 2092/91 non ne consente l'uso, per cui la sola presenza di residui a livelli > di 0,01 mg/kg rende gli alimenti non conformi. I pesticidi naturali per i quali è consentito l'uso invece, di norma, non vengono controllati, principalmente per carenza di metodiche analitiche rapide, semplici e con buona sensibilità. Anche a livello di ricerca gli studi su questi pesticidi naturali sono molto limitati, per cui la conoscenza del loro livello di residui negli alimenti è quasi completamente sconosciuta. Gli insetticidi estratti da piante più utilizzati sono quelli ottenuti dalla Azadirachta indica, Derris elliptica, Ryania speciosa, e Chrysanthemum cinerariaefolium i cui principi attivi sono rispettivamente: azadirachtin, rotenone, rianodina con deidrorianodina e piretrina I e II. Il presente lavoro propone una metodica che consente la determinazione di questi antiparassitari naturali in maniera semplice e sensibile. La tecnica utilizzata è la cromatografia liquida accoppiata alla massa (HPLC-MS) Inizialmente si è ottenuta la separazione cromatografica dei principi attivi utilizzando una colonna C18, un gradiente di H2O e CH3CN al flusso di 0,2 ml/min ed un rivelatore a serie di diodi settato alle lunghezze d’onda ottimali per la determinazione dei singoli principi attivi. Nella determinazione col detector di massa è stata impiegata una sorgente APCI e tutti gli antiparassitari sono stati analizzati in SIM con due metodi (differenti per le temperature del probe, CDL e Block) che permettono di ottimizzare la risposta per la deidrorianodina, la rianodina e l’azadiractina (metodo 1) e per il rotenone e le piretrine (metodo 2). L’estrazione dei principi attivi da matrici vegetali è stata effettuata con acetato di etile, solvente che si è dimostrato ottimale per il recupero quantitativo di tutti i principi attivi. L'estratto non subiva alcuna purificazione in quanto non presentava alcun interferente. Nelle determinazioni quantitative la soluzione madre veniva preparata in estratto di matrice non trattata per eliminare l'effetto matrice. I limiti di determinazione ottenuti sono compresi tra 2 e 10 ppb. Il rotenone è l’antiparassitario che fornisce la risposta migliore permettendo di rilevare fino a 2 ppb. PDF creato con FinePrint pdfFactory versione dimostrativa http://www.secom.re.it/fineprint 24
PRESENCE OF OCHRATOXIN IN EXPERIMENTAL WINES RELATED TO PESTICIDES TREATMENTS EMPLOYED ON GRAPES R. Lo Curto*, F. Vilasi*, T. Pellicanò*, P. Munafò**, G.mo Dugo*, * Università degli Studi di Messina, Dip.to Chimica Organica e Biologica. ** Centro Analisi & Servizi S. r. l. via U. La Malfa, 18 - 98051 Barcellona P. G., Messina, Italia. Introduction Mycotoxins are a class of highly toxic chemical compounds produced, under particularly environmental conditions, by several moulds developing in many foodstuffs. Their presence is depending upon several factors such as: fungal strains, climate and geographical conditions, cultivation techniques and foodstuffs conservation (1). Mycotoxins may occur in various vegetal products as cereals, dried fruits, coffee beans, cocoa and beverages as beer and wine (2-4). Among mycotoxins very important are the ochratoxins. This term indicates a group of metabolites, having showing similar chemical structure, produced by strains of the genus Aspergillus (A. ochraceus) and Penicillium (P. ferrucosum) (1,5,6). The most studied, both for its diffusion and toxicological importance is Ochratoxin A (OTA) R-N-[(5-chloro-3,4dihydro-8-hydroxy-3-methyl-1-oxo-1H-2-benzopyran-7-yl)carbonyl]-phenylalanine. It has an oral DL50 level of 20 mg/kg in rats and pig (7). OTA is highly toxic and causes severe animal and human intoxications, for example “Porcine nephropathy” and “Balcan Endemic nephropathy” (8-10). OTA exhibits nephrotoxic and teratogen activites and moreover suppressive actions on immune system, causing a diminution of immune globulin level and of other humoral factors in mice and chicken and a reduction of cell immune response (10,11). Recently, more attention was focused on Ochratoxin A levels in commonly consumed foods, especially fruits and cereals (12,13) and in their fermentation products like beer (14-16) and wine (17-25). As far as wine is concerned, available data regarding the presence of OTA are very discordant, some Authors reported a high toxin concentration (up to 7.0 ng\ml) with considerable level of contamination (incidence up to 92%) in red wine produced in southern regions of Europe and in North Africa. Other Authors reported OTA contamination levels ranging 3.9% for white wines to 16.6% for red wines, even if these data were obtained by different analytical methods. Generally red wines contain a greater amount of OTA than white or rosè ones. These differences can be attributed to climatic factors, grape cultivation and storage conditions apart from wine-making techniques. The content of OTA in 23 samples of red and white wine, produced in the year 2000 by three experimental vite, situated in three different Italian regions treated with different pesticides see tables 1,2,3), is reported in this work (26). Materials and methods Chemicals and Reagents Standard of OTA, sodium chloride, polyethylene glycol (Peg 8000), sodium hydrogencarbonate, glacial acetic acid and toluene were obtained from Fluka (Milano-Italy). Immunoaffinity columns were purchased from Vicam (Waterton, MA, USA).Acetonitrile, methanol and water (HPLC grade) were supplied from Carlo Erba Reagents (Milano-Italy). Wine samples A total of 23 wine samples (16 white 7 red samples), produced in the year 2000 by three experimental vite, situated in three countries “Avellino”-Campania, “Catania”-Sicily and “Grosseto”-Tuscany, were analysed. Wine characteristics and pesticide treatments White wines came from Sicily and Campania in the crop year 2000. Sicilian wines were produced from 15 -20 years old plants, grown up on Etna (300 m asl, S. Venerina, Catania, Italy), in a vulcanic soil. Vines were grafted with Inzolia and Carricante varieties in 1:1 ratio. Wines from Campania were produced from 25 years old plants grown up on Montefredane hills (700 m asl, Avellino, Italy), in a clayey soil. Vines were grafted with Fiano d’Avellino variety. Red wines from Tuscany were produced from 25 years old plants, cultivated in Maremma Toscana coast (100 m asl, Grosseto, Italy), on a calcareous soil. Vines were grafted with Sangiovese variety, Morellino clone. Vinification process was run within 24 hours after grape harvesting and it was run using the following scheme: White Vinification Newly – cropped Vitis vinifera grapes from Sicily and Campania were crushed destemmed and then soft pressed by a pneumatic press. Must was treated with SO2 (30 g/hl), pectolitic enzymes (1.5 g/hl) and vitamin C (5 g/hl) to favour clarification before fermentation; temperature was maintained at 8°C for 24 hours. Clear must was spiked with 20 g/hl of thiamine and ammonium phosphate as fermentation coadjuvans and 30 g/hl of selected yeasts and fermentation was run at 15°C. In order to remove lees, after fermentation wine was decanted into a tank and spiked with SO2 (5 g/hl). Ten days later wine was decanted again and treated with SO2 (5 g/hl). Wine was finally filtered twice trough 1 µ and 0.45µ cardboards filters, spiked with SO2 (2-3 g/hl), bottled in dark bottles and maintained at 4°C for the duration of the experimentation. Each grape sample was separately vinificated, following the above mentioned protocol. Red vinification Newly – cropped Vitis vinifera grapes from Tuscany were crushed and destemmed, then spiked with SO2 (5 g/hl), added with selected yeasts (30 g/hl) and let to ferment for 10 days at 28°C, making three fullings a day. Wine was drawn from the vat and the vinasses pressed by a hydraulic press. After 4 weeks, lees were removed and the wine PDF creato con FinePrint pdfFactory versione dimostrativa http://www.secom.re.it/fineprint 25
Page 1 and 2: International Congress BIOLOGICAL P
Page 3 and 4: 15:40-16:20 G. Lozzia Università d
Page 5 and 6: MAIN SPEAKERS • V. Rotondo FIAO,
Page 7 and 8: Il settore oggi è regolamentato in
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Page 11 and 12: RELIABILITY AND PROSPECTIVES FOR TH
Page 13 and 14: Vineyard Passive diffusion Spontane
Page 15 and 16: of the host, a relatively limited n
Page 17 and 18: LOZZIA G.C., RIGAMONTI I.E., 1994 -
Page 19 and 20: DIFESA DELLE COLTURE DAI PATOGENI F
Page 21 and 22: THE FUNGAL PHYTOTOXINS AND THEIR PO
Page 23: NEED FOR RESEARCH AND APPROPRIATE A
Page 27 and 28: 5. Hohler, D. (1998). Ochratoxin A
Page 29 and 30: Table 3: Pesticide treatments on
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Page 39 and 40: PROTECTION OF GRAPEVINE FROM POWDER
Page 41 and 42: I MEZZI TECNICI PER L'AGRICOLTURA B
Page 43 and 44: POSTER 1. Agosteo G.E., Ambrosio M.
Page 45 and 46: 31. Pietri A., Bertuzzi T., Barbier
Page 47 and 48: 1 PROVE DI LOTTA ALL’OIDIO DELLO
Page 49 and 50: 2 LUPINUS ALBUS, AN ANCIENT EUROPEA
Page 51 and 52: Table 1. Benzo(a)pyrene and benzo(e
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- azadiractina nei confronti di F.
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Recovery and repeatabilty tests wer
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13 IMPIEGO DEL TIMOLO NEL CONTROLLO
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dello standard esterno. E’ stata
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Tab. III - Residui di timolo(mg/Kg)
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phytotoxicity index (%) Results In
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15 ORGANIC WHEAT QUALITY AND PRODUC
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16 IL CONTROLLO DELLA “ MOSCA DEL
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Nel primo anno si può notare un an
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17 Il contenimento della ticchiolat
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Obtained recoveries percent were: 8
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[Pb] (ng/g) 250,00 200,00 150,00 10
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Supelco Supelclean LC-18 SPE tubes
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12. Agullo, G.; Gamet, L.; Besson,
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Tab. 3 Flavonol content (µg/ml) in
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mg/L Fig.3 3,50 3,00 2,50 2,00 1,50
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Table 1 - Fungicides used during th
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Table 3 - Efficacy of different tre
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21 CONTAMINAZIONE DA MICOTOSSINE IN
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Ricerca: Aflatossine B1 B1+B2+G1+G2
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Campioni non Conformi 3,4 % B1 Camp
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22 PEPTIDATI DI RAME: PRODOTTI INNO
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23 CHARACTERIZATION OF ORGANIC VIRG
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24 CHARACTERIZATION OF ORGANIC VIRG
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25 EFFICACY EVALUATION OF BIOLOGICA
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Fig. 1 - Effects of Biological Cont
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26 IL CONTROLLO DELLE OPERAZIONI DI
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Results and Discussion S. carpocaps
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entompathogenic nematodes. CSC-EC E
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After this preliminary screening an
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FIGURE 3. Insecticidal activity of
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Several colonies of Lepidopteran, C
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M. Scribano research work was suppo
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Nematode isolate % Mortality 3 100
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In our trial organic wheat showed a
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per "strippare" i vapori nitrosi re
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L’ultima parte della ricerca è s
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33 INVESTIGATIONS ON THE BACTERICID
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Vengono qui riportati i risultati d
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while for dinocap is 10 pg/µL. For
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[Pb](ng/ml) Figure 1: Pb content of
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