Università degli Studi di Napoli Federico II Dipartimento di Scienza degli Alimenti

1. Detection of biophenols from virgin olive oil in French-fried potatoes by high-performance liquid chromatography tandem electrospray ionization mass spectrometry (HPLC-ESI/MS) Università degli Studi di Napoli Federico II Dipartimento di Scienza degli Alimenti Savarese M.§, Parisini C.§, De Marco E.§, Sacchi R.§, §CRIOL-Centro Ricerche per l’Industria Olearia, Industria Olearia Biagio Mataluni, Montesarchio (BN), Italy Department of Food Science, University of Naples Federico II, Portici (Na), Italy; e-mail: criol@mataluni.com; sacchi@unina.it • RESULTS • Many different biophenols were identified in the frying oil samples, several of which were also revealed in fried potatoes. • Identification (Figure 2) of phenolic compounds was based on: • search for pseudomolecular [M-H]- ions, using extracted ion mass chromatograms • interpretation of collision-induced dissociation (CID) fragments. The structural information resulting from the deprotonated molecular ion was confirmed by modifying some MS parameters (interface voltage and Q-Array voltage) in order to obtain a moderate dissociation-fragmentation induced by the collisions in the ionic transport region. • comparison of retention times and mass spectra with those of standards, when available, or with those reported in literature [3; 4]. INTRODUCTION Deep frying is an important cooking technique employed in domestic, collective and industrial food preparation. During this process oil used as cooking medium is submitted to high temperatures (180°C) in presence of oxygen and moisture. These conditions promote a series of reactions determining deep modification in the frying oil as well as in the food being fried (Figure 1). Components already present in the frying medium or generated during frying may migrate from the oil into the food product, which in its turn may release substances into the frying medium. Extra virgin olive oil (EVOO) contains different phenolic compounds acting as antioxidants. These molecules may be partially transferred from oil to food during frying and may act as antioxidants also in fried food, extending the shelf-life and improving the nutritional quality of the products. EXTRA VIRGIN OLIVE OIL FRIED POTATOES Liquid Chromatography tandem MassSpectrometry (LC-MS) with ElectrosprayIonization (ESI) represents a very powerful tool for the analysis of natural phenolic extracts since the mass spectrometer is a universal detector, able to achieve very high sensitivity and to provide data on molecular weights as well as structural information. Figure 2. Total Ion Current (TIC) ESI-MS chomatograms of EVOO and French fried potatoes phenolic exctracts. Peaks Identification: 1.Hydroxytyrosol; 2.Tyrosol; 3.Decarboxymethyl elenolic acid oxidized;4.Not Identified.;5.Elenolic acid oxidized;6.Elenolic acid;7.Elenolic acid oxidized;8.Dialdehydic form of decarboxymethyl oleuropein aglycon;9.Oxidation product of decarboxymethyl oleuropein aglycon;10.Aldehydic form of oleuropein aglycon;11.Aldehydic form of ligtroside aglycon;12.Oxidation product ofdecarboxymethyl ligstroside aglycon;13. Hydroxytyrosol derivative;14.Aldehydic form of oleuropein aglycon;15.Aldehydic form of oleuropein aglycon;16.Oxidation productof oleuropein aglycon;17.Luteolin;18.Aldehydic form of oleuropein aglycon;19.Aldehydic form of oleuropein aglycon;20. Aldehydic form of oleuropein ligstroside;21.Oxidation productof oleuropein ligstroside;22. Apigenin;23. Methoxyluteolin. Figure 1.Changes occurring during deep-fat frying. OBJECTIVE Aim of the present study was the application of ESI-MS to evaluate the possible migration of antioxidant compounds from extra virgin olive oil to the fried food during a deep-frying process. The frying process proved to determine a partial loss of all antioxidants in the fried oils paralleled by their well as their enrichment in the fried food (Table 1). Oxidation forms of elenolic acid and of secoiridoid derivatives were observed in both frying oils and fried potatoes (Figure 2; Table 1). MATERIALS AND METHODS Frying trials An electrical deep fat fryer (Tefal, Milan), operating at a temperature of 180 °C, was used for frying frozen pre-fried French fries (Orogel, Cesena, Italy). The trial was carried out with an extra virgin olive oil, purchased in a local market, and with a refined olive oil, as control. The fryer was filled with 1 l of oil and 12 frying cycles were carried out with 30 min interval between each other. During each cycle a French fries sample (100 g) was fried. Both oil samples (50 ml) and French fried potato sample were collected every hour of frying and stored at -20 °C until analysis. Table 1.Presence/absence of identified phenolic compounds in EVOO and French fried potatoes during deep-frying (- undetected  detected) Peak 9 [M-A]- (m/z 199); [M-A-B-C]- (m/z 111); [M-D]- (m/z 235) Extraction of phenolic compounds from French fried potatoes. Extraction of phenolic compounds from French fried potatoes was carried out following the procedure described by Brenes et al. [1], with some modifications. Extraction of phenolic compounds from oils Extraction of EVOO phenolic compounds was carried out according to the method described by Sacchi et al. [2], to which some modifications have been applied. Peak 12 [M-A]- (m/z 199); [M-A-B]- (m/z 155); [M-C]- (m/z 249) Peak 21 [M-A]- (m/z 333); [M-B]- (m/z 249) Figure 3.Full scan MS spectra of oxidation form of decarboxymethyl oleuropein aglycon (peak 9), oxidation form of decarboxymethyl ligstroside (peak 12) and oxidationform of ligstroside aglycon (peak 21) detected in EVOO and in fried potatoes after deep-frying. Liquid chromatography tandem electrospray ionization mass spectrometry The LC system consisted of two Shimadzu LC-10AD VP pumps (Shimadzu, Milan, Italy). The presence of these compounds, which proposed structures are shown in Figure 3, suggests that further investigation is required in order to verify their properties in terms of antioxidant power and nutritional impact. Since oxidation regards only the elenolic group, these important properties may be preserved as they are due to the phenolic moiety. • Column: Discovery HS C18 column (5mm, 150 mm x 2.1 mm i.d., Supelco, USA), • Flow rate: 0.35 ml min-1, • Solvent system: solvent A (water + formic acid 0.25%), solvent B (methanol + formic acid 0.25%), with a step gradient from 5 to 55% B (45 min). • Injected volume: 20 ml of methanol extract Peak 17 Peak 22 • The mass spectrometry system consisted of a mass spectrometer Shimadzu LCMS-2010EV (Shimadzu). • interface voltage: 4 kV; • nebulizer gas flow: 1.5 l min-1; • block heater temperature: 250 °C; • curved desolvation line (CDL) temperature and voltage: 300 °C and -5 V; • Q-Array voltage: 0 V DC and 150 V RF; • detector voltage: 1.5 kV; • scan range: m/z 60-900. Figure 4.Full scan MS spectra of luteolin (peak 17) andapigenin (peak 22) detected in EVOO and in fried potatoes after 6h of deep-frying. Tyrosol and hydroxytyrosol were revealed only in fried potatoes, confirming the previously reported hydrolysis and diffusion mechanisms and consequent partition between oil-water phases [2, 5, 6]. Flavonoids (luteolin, apigenin, methoxyluteolin) proved to be particularly resistant to frying process (Figure 4), being present in EVOO even after 6h of deep-frying, as well as in fried potatoes. Although phenolic compounds give peaks of lower intensity in negative than in positive ion mode, in the present study analyses were performed in negative mode because cleaner spectra were obtained. References [1] Brenes M., Rejano L., Garcia P., Sanchez H.A., Garrido A. J. Agric. Food Chem., 1995, 43, 2702-2706. [2] Sacchi R., Paduano A., Fiore F., Della Medaglia D., Ambrosino M.L., Medina I. J. Agric. Food Chem., 2002, 50, 2830-2835. [3] Rovellini P., Cortesi N. Riv. Ital. Sost. Grasse, 2002, 69, 1-14. [4] Ríos J.J., Gil M.J., Gutiérrez-Rosales F. J. Chromatogr. A, 2005, 1093, 167-176. [5] Kalogeropoulos N., Chiou A., Mylona A., Ioannou M.S., Andrikopoulos N.K. Food Chem., 2007, 100, 509-517. [6] Brenes M., Garcia A., Dobarganes M.C., Velasco J., Romero C. J. Agric. Food Chem., 2002, 50, 5962-5967. Acknowledgements This work was supported by Italian Ministry of University and Research (MIUR) and by Industria Olearia Biagio Mataluni s.r.l. within the project “Controllo Qualità ed Innovazione Tecnologica nell’Industria olearia” (DM 593- 08/08/2000, Prot. MIUR 1866 – 18/02/2002).