1802 J. Agric. Food zyxwvuts Chem. 1995, zyxwvut 43, 1802-1807 GC-MS Evaluation of Phenolic Compounds in Virgin Olive Oil Franca Angerosa," Nicola d'Alessandro,* Panajotis Konstantinou, and Luciana Di Giacinto Istituto Sperimentale per la Elaiotecnica, Contrada Fonte Umano 37, 65013 Citta Sant'Angelo, Pescara, Italy A new gas chromatographic method for detection of phenolic compounds in virgin olive oils was developed. Identification of chromatographic peaks was made by mass selective detection. The presence of a main peak at zyxwvuts mlz 192 or at mlz 280, related only to tyrosol and hydroxytyrosol, evident in the mass spectra of linked phenols, was very profitable for assigning the phenolic nature to minor polar compounds extracted by methanol from virgin olive oil. Twelve structures are possible, and some of them are deemed more likely on the basis of the chemical behavior of the compounds studied. The presence of a ligstrosid aglycon containing no carbomethoxy group and of oleuropein aglycon derivatives was evidenced. Keywords: Phenolic compounds; virgin olive oil; mass spectroscopy INTRODUCTION Phenolic compounds are of great importance in bio- logical systems and, in particular, in the vegetable kingdom. They are present principally in the fruits but also, in a minor amount, in leaves, flowers, and other vegetable organs (Vazquez Roncero et al., 1974; Gut- finger, 1981; Bianchi and Pozzi, 1994). Virgin olive oil contains phenolic derivatives, in contrast to edible seed oils that lose this important group of compounds in the various refining stages (Cantarelli, 1961; Montedoro and Cantarelli, 1969). Their content is heavily affected by the variety, location, maturity degree, and oil extraction procedures (Solinas et al., 1978; Montedoro and Garofolo, 1984; h iot et al., 1986; Solinas, 1987). The level of these substances is a very important parameter in the evaluation of virgin olive oil quality since phenols are strictly related both to the oil's resistance to oxidation because of their antioxidative properties (Montedoro, 1972; Papadoupoulos and Bosk- ou, 1991) and to the typical bitter taste of olive oil (Angerosa and Solinas, 1990; Olias, 1992). Further- more, some studies showed that the amount of phe- nolic substances present, just as the fatty acid com- position, is related to the health beneficial effects that make virgin olive oil a very valuable and appreci- ated commodity (Panizzi et al., 1960; Galli et al., 1992). Therefore, it is very important to assay phenols in oil both qualitatively and quantitatively. Several methods were proposed: their direct quantification on the meth- anolic fraction by means of Folin-Ciocalteu reagent had been earlier modified by adding to the procedure a purification by paper chromatography or TLC on silica gel or cellulose, before spectrophotometric analysis (Ragazzi and Veronese, 1973). Subsequently, GLC (Janer del Valle and Vazquez Roncero, 1980; Solinas and Cichelli, 1982; Solinas, 1987) and HPLC (Solinas and Cichelli, 1982; Amiot et al., 1986; Montedoro et al., 1992; Tsimidou et al., 1992;Akasbi et al., 1993) methods were developed. HPLC methods, which generally use UV detection, are more successful, since they, in con- trast to GLC methods, do not require * Authors to whom correspondence should be ad- dressed. 0021 zyxwvutsrq -8561/95/1443-1802$09.00/0 derivatization prior to the quantitative analysis. An excellent series of experiments was performed by Mon- tedoro and his group. By using HPLC and NMR techniques, they separated and identified some aglycon derivatives present as the dialdehyde forms of elenolic acid linked to both hydroxytyrosol and tyrosol in the olive oil (Montedoro et al., 1993). Recently HPLC was coupled with an amperometric detector with interesting results (Mannino et al., 1993); phenols in olive oils were also measured by using organic phase biosensors (Wang et al., 1993). Neverthe- less, these new methods for the determination of phenolic substances did not allow their identification. In 1987 an HRGC method was developed by Solinas for the quantitative evaluation of phenolic substances. The methodology needs (i) purification of the methanolic extract by absorption of the mixture on Celite-Polyclar AT; (ii) removal of, first, the largest part of methanol and then the last traces of solvent by means of pentane- acetone-methanol azeotropic distillation; (iii) GC analy- sis after BSTFA derivatization. Although Solinas was able to characterize the sim- plest compounds such as tyrosol and hydroxytyro- sol, using this method he could only offer specula- tions about the identities of the other linked phenols occurring in large quantities in the second part of chromatogram. Therefore, our aims were, first, to simplify the extrac- tion and purification procedures to have at our disposal a rapid, more effective, and accurate HRGC method of phenol dosage, which could be suitable for routine determinations; and, second, to characterize the more complex molecules by means of GC-MS and spectro- scopic techniques. EXPERIMENTAL PROCEDURES Materials and Reference Compounds. Virgin olive oil extracted from olives of Coratina variety cultivated in Abruzzo was used. 4-Hydroxyphenyl acetic acid, p-coumaric acid, vanillic acid, 4-hydroxybenzoic acid, syringic acid, caffeic acid, 3,4-dihy- droxyphenylacetic acid, protocatechuic acid, ferulic acid, /3-glu- cosidase from almonds, and 2-(4-hydroxyphenyl)ethanol (Ty) were purchased from Fluka (Buchs, Switzerland); and resorcin, cinnamic acid, omovanillic acid, and bis(trimethylsily1)trifluo- racetamide (BSTFA) from E. Merck (Darmstadt, Germany). 0 1995 American Chemical Society