Detection of the monoterpene carvacrol in mammal tissues by analytical pyrolysis (Py‐GC/MS)

Póster presentado en el la XVI Reunión Científica de la Sociedad Española de Cromatografía y Técnicas Afines (SECyTA2016) P‐NP‐1

Eds: González-Pérez, José Antonio.-- Almendros Martín, Gonzalo.-- González-Vila, Francisco Javier.-- Rosa Arranz, José M. de la

Responding to consumer demands on minimal processing and preservative‐free products, the use of essential oils (EOs) to extend shelf life of foods is on the spot in the food industry [1]. Carvacrol, main compound of Oregano EO, is registered as a flavouring in Europe; however, its use for other applications, such as active food packaging, may require higher concentrations and there is an increasing concern regarding exposure. Because of this, the European Food Safety Authority (EFSA) requires additional genotoxic studies data of substances which could be incorporated into food packaging like carvacrol. Here a detailed analytical pyrolysis (Py‐GC/MS) study is conducted as a complement to in vivo genotoxicity studies. Analytical pyrolysis was the technique chosen to search for carvacrol directly in viscera and to confirm that the compound effectively reached target tissues from orally exposed (0, 81, 256 or 810 mg carvacrol/kg bw, calculated according to carvacrol Maximum Tolerated Dose (MTD)) young adult male Wistar rats strain RjHan:WI*. Doses were prepared in corn oil at a final volume of 1 mL and during the treatment period, clinical signs, body weight, and food and water consumption were recorded daily. Rat stomach and liver composite samples were selected for pyrolysis and preserved at ‐80ºC until lyophilisation (Testal Cryodos, Madrid). Direct pyrolysis was performed in a double‐shot pyrolyzer (Frontier Lab 2020i) attached to a GC/MS system (Agilent 6890N + 973MSD). Detailed chromatographic conditions can be found in [2]. In short, lyophilized tissue (stomach and liver) were thoroughly homogenized and samples introduced (0.5 mg) into a preheated micro‐furnace at 500 ºC for 1 min and evolved gases transferred to the GC/MS for analysis. Compounds assignment was via single‐ion monitoring and by comparison with published and stored (NIST and Wiley libraries) data. In a previous study, it was determined that pyrolysis of carvacrol does not to produce major effects on its chemical structure and therefore was considered an adequate technique to detect the presence of the monoterpene in animal tissues. The analytical pyrolysis of target tissues of control rats was negative to any sign of carvacrol even when searching for the specific mass fragments (m/z 135 and 150). However, carvacrol was clearly detected in the tissues of rats treated at all doses. Furthermore, when normalizing the chromatograms to a common peak, a clear dose response was obtained. A conspicuous difference was found in the dose‐response curves between stomach and liver; whereas a direct lineal correlation could be drawn from the former, the response for the latter was best fit to a quadratic equation model. In this regard, the differences observed in those curves may be related to carvacrol metabolism in rats and the possible occurrence of saturation mechanisms limiting an excess of carvacrol metabolization/presence in liver. als received humane care.

[1] M. Llana‐Ruiz‐Cabello, S. Pichardo, A. Baños, C. Nuñez, J.M. Bermúdez, E. Guillamón, S. Aucejo, A.M. Cameán, Food Sci. Technol‐LEB 64 (2015) 1354‐1361 [2] M. Llana‐Ruíz‐Cabello, S. Pichardo, N.T. Jiménez‐Morillo, J.M. Bermúdez, S. Aucejo. F.J. González‐Vila, A.M. Cameán, J.A. González‐Pérez (2016). J. Sci. Food Agr. 96 (2016) 3207‐3212 (*) The Ethics Committee on Animal Experimentation of the University of Sevilla approved the in vivo experiments. Moreover, in compliance with the Directive 2010/63/EU for the protection of animals used for scientific purposes all anim

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