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3 BENZO(a)PYRENE IN ORGANIC VIRGIN OLIVE OILS a Elena Barrese, b Cinzia Benincasa, a Nicola Lombardo, b Fabio Mazzotti, a Innocenzo Muzzalupo, a Massimiliano Pellegrino, a Enzo Perri, b Giovanni Sindona a) Istituto Sperimentale per l'Olivicoltura, 87030 Rende (CS)-Italy b) Dipartimento di Chimica, Università della Calabria, via P. Bucci, cubo 15/c I -87030 Arcavacata di Rende (CS)-Italy Introduction Polycyclic aromatic hydrocarbons (PAHs) are environmentally hazardous organic compounds because of their known or suspected carcinogenicity. PAHs are mostly formed during an incomplete combustion of organic material and they are known as highly stable contaminants present in many foods. This contamination can be a result of sorption from the environment or from food preparation methods. Air pollution is the main source of PAHs in the edible parts of plants. Hence, the determination of PAHs in foods such as olives and olive oil is important for human health. Benzo(a)pyrene (BaP) and Benzo(e)pyrene (BeP) are the best known PAHs and they have been often used as indicators of the presence of PAHs in foods. Several authors have reported the presence of Benzopyrenes (BP) in edible oils and fats. However, there are few reports on the BP content in olive oils (Mariani et al., 1984; Menichini et al., 1991; Fiume et al.2002) while there has been no reports on the BP content in organic virgin olive oils. In general, the detected levels of BP in the olive oils analyzed were in the range of a few µg/kg of olive oil (Pupin et al., 1996; Vazquez Troche et al., 2000). However, olive oil refining involving deodorization, bleaching and charchoal treatment reduces the content of PAHs. Therefore, paradoxically, higher levels of BP may be found in virgin olive oils, irrespective of farming system adopted, since they are, by definition, unrefined and untreated. Currently no legislation exists regarding BP levels in vegetable oils in European Union. However, the International Olive Council (IOC), the German food industries and some European nations (Italy, Spain and Portugal) recommend for olive-pomace oil and refined olive-pomace oil a BaP maximum level of 2 µg/kg. Therefore, there is a need to verify the level of BP in European olive oils before a regulation occur. The goal of this preliminary work was to assess the presence and determine the level of BP in organic virgin olive oils from the most important olive growing region of Italy: Apulia. Materials and methods Plant material. In 2000/2001 harvest, samples of organically cultivated olive drupes of Coratina cv from a farm at Andria (Bari, Italy) were collected 10 days before the traditional start of harvest, in the middle of the harvest period and 10 days after the end of harvest time. The olives were handpicked each time from 5 olive tree of the same cultivar. Work-up of plant material. Olive drupes (10 Kg) were crushed with a hammer mill and the oil was extracted by centrifugation after 20 minutes of malaxation at RT. Analytical procedures.Extraction and clean-up procedure were conducted by slightly modifying Vazquez Troche et al. (2000) protocol. The final extract was analyzed by a Hewlett Packard HP 5973 Mass Spectrometer interfaced with a HP 6890A GC (Agilent Technologies, Waldronn, GmbH) and BaP detected as molecular ion (m/z 252) by Selected Ion Monitoring (SIM) mode. Quantification was obtained by the external standard method. Results and discussion Detection limit for BP in the acetonitrile eluate was 0.3 µg/kg of olive oil. The standard calibration curves obtained with a linear coefficient of 0,999 generated from triplicate 1 µl injections at different concentration show a linear range from 0.3 µg/kg to 5.4 µg/kg with good-to-excellent R 2 values. For reproducibility test three concentrations were analyzed and the average coefficient of variation (CV%) was 5%. The recovery obtained for spiked samples with 4 µg/Kg of BaP was 66%. As expected, also the organic olive oils are affected with a PAHs. In fact, the levels of BaP and BeP in organic virgin olive oils from Coratina cv. at Andria (Apulia) ranged from 0.39 and 0.85 µg/kg, and 0.38 and 0.60 µg/kg , respectively, while the average content was 0.56 µg/Kg and 0.52 µg/Kg, respectively (table 1). Although the BP values were low, the determination of the other “heavy” PAHs is still required to assess the real intakes of PAHs from the total diet of apulian olive oil consumer. Therefore, the determination of the most important “heavy” PAHs in organic virgin olive oil by GC-MS is in progress. PDF creato con FinePrint pdfFactory versione dimostrativa http://www.secom.re.it/fineprint 50
Table 1. Benzo(a)pyrene and benzo(e)pyrene content in organic virgin olive oils from Coratina cv. at Andria (Apulia). Cultivar Origin Harvest time BaP (µg/Kg of olive oil) BeP (µg/Kg of olive oil) Coratina Andria (Apulia) 11/07/00 0.85 0,38 Coratina Andria (Apulia) 12/11/00 0.45 0,60 Coratina Andria (Apulia) 01/02/01 0.39 0,58 Mean + Std. dev. 0.56+ 0.20 0.52+ 0.10 Acknowledgements We thank Dr. Cosimo Cavallo (Regione Puglia), Dr. Nino Paparella (C.I.Bi., Bari), Dr. Nicola Panaro (C.I.Bi., Bari) and Drs. Edoardo and Giancarlo Ceci Ginistrelli for olive sampling, data collection on farming systems and olive grove management. We thank also Prof. Angelo Putignano and Prof. Francesco Prudentino (I.T.A.S. of Ostuni-BR) for olive oil extraction. References F. Fiume, F. Ferrieri, G. Froio, S. Spinello, O Lattarulo, G. Fanuzzi, 2002, Determinazione di idrocarburi policiclici aromatici in oli alimentari, Riv. Ital. Sostanze grasse, LXXIV, 151-155. C. Mariani, E. Fedeli,1984, Idrocarburi policiclici aromatici negli oli vegetali, Riv. Ital. Sostanze Grasse, 61, 305 -315. E. Menichini, A. Bocca, F. Merli F., Ianni, F. Monfredini, 1991, Polycyclic aromatic hydocarbons in olive oils on the italian market, Food Addit. Contam., 8(3), 363-369. A. M. Pupin and Maria Cecilia Figueiredo Toledo, Benzo(a)pyrene in olive oils on the brazilian market, Food Chemistry, 1996, 55(2), 185-188. S. Vazquez Troche, M.S. Garcìa Falcòn, S. Gonzales Amigo, M.A. Lage Yusty, J. Simal Lozano, Enrichment of benzo(a)pyrene in vegetable oils and determination by HPLC-FL, Talanta 2000, 51, 1069-1076. PDF creato con FinePrint pdfFactory versione dimostrativa http://www.secom.re.it/fineprint 51
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21 CONTAMINAZIONE DA MICOTOSSINE IN
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