INTRODUCTION

1. Characterization of biophenols from olive oil mill wastewaters by high-performance liquid chromatography tandem electrospray ionization mass spectrometry (HPLC-ESI/MS) University of Naples Federico II Department of Food Science De Marco E.*, Savarese M.*, Parisini C.*, Sacchi R.§* * CRIOL, Centro Ricerche per l’Industria Olearia, Industria Olearia Biagio Mataluni, zona industriale, 82016 Montesarchio (BN), Italy § University of Naples Federico II, Department of Food Science, via Università 100, 80055 Portici (NA), Italy e-mail: criol@mataluni.com; sacchi@unina.it INTRODUCTION OBJECTIVES Olive oil mill wastewaters (OMWW) represent the main environmental problem of olive oil production process, being they characterized by a high organic load due to a high concentration of sugars, tannins, phenolic compounds, polyalcohols, pectins and lipids. The phenolic fraction, in particular, is the main responsible for the OMWW antimicrobial activity and the consequent difficult biological degradation; on the other hand, the phenolic compounds are characterized by a strong antioxidant activity and by several important biological properties. The extraction of these biologically active compounds from OMWW, in which they are very abundant, may then turn a polluting residue into a source of natural antioxidants, object of growing interest in pharmaceutical and food industries since reactive oxygen species are involved in the onset of several human diseases and in the oxidative degradation of food. Several factors influence the occurrence of specific biophenols in OMWW, such as olive cultivar, fruit ripeness degree, climatic and agronomic conditions, storage conditions, extraction process. Such great complexity and variability make it difficult to characterize OMWW phenolic fraction, which composition is still under investigation. • characterization of OMWW phenolic fraction directly on the residue, without previous extraction • development of an analytical procedure based on electrospray ionization tandem mass spectrometry (ESI-MS) able to characterize and identify phenolic compounds and flavonoids RESULTS Identification of phenolic compounds was based on the search for pseudomolecular [M-H]- ions, using ion chromatograms extracted from the total ion current chromatogram, together with the interpretation of mass spectra and with the comparison of retention times and mass spectra with those reported in the literature or shown by authentic standards, when available. HPLC-ESI/MS proved to be a suitable technique to analyze phenolic compounds and flavonoids in wastewaters, giving clear extracted ion chromatograms and easy interpretable mass spectra, in which the pseudomolecular ion is often the most abundant ion. Total Ion Current (TIC) chromatogram, extracted ion current chromatograms and mass spectra of some phenolic compounds identified in an OMWW Hydroxytyrosol-glucoside MW=316 OMWW samples collected during October-November 2005 from a continuous three phases olive processing plant (Industria Olearia Biagio Mataluni, Montesarchio, BN, Italy). MATERIALS AND METHODS TIC m/z 191 Caffeic acid MW=180 m/z 219 m/z 315 m/z 317 m/z 153 b-hydroxy-acteoside MW=640 Acidification to pH 2 with hydrochloric acid. TIC m/z 407 m/z 461 m/z 447 m/z 343 Centrifugation (5000 rpm for 20’) and removal of the deposit. m/z 305 Demethyl oleuropein MW=526 TIC m/z 377 Verbascoside (R1=caffeic acid; R2=H) Isoacteoside (R1=H; R2=caffeic acid) MW=624 Filtration on cellulose acetate syringe filter. m/z 639 m/z 525 m/z 623 m/z 523 Injection Rutin MW=610 TIC m/z 319 m/z 551 m/z 301 m/z 285 m/z 555 Luteolin MW=286 HPLC-UV system ESI-MS system Retention time, main peaks found in the mass spectra (m/z) and suggested identification for OMWW phenolic compounds Many phenolic compounds were identified in OMWW samples, several of which were never identified in this residue. OHTy-glucoside was previously reported for olive leaf, pulp, oil and pomace (Bianco et al., 1998; De Nino et al., 1999; Cardoso et al., 2005), but never for OMWW. Total Ion Current (TIC) chromatogram of an OMWW HPLC-UV chromatograms of 4 different OMWWs Chrysoeriol-7-glucoside was previously identified only in olive pulp (Bouaziz et al., 2005). The two diastereoisomers of b-hydroxyacteoside, differing respect to verbascoside for the presence of a hydroxyl group in the b-position of OHTy, were reported only for solid olive residue by Mulinacci et al. (2005). HPLC analysis, performed on a system made up of a DGU-14A degasser, a LC-10AT VP pump, a SPD-10AV VP UV-detector and a SCL-10A VP interface (Shimadzu, Japan). Column: Discovery HS C18 (5 m, 250 x 4,6 mm i.d., Supelco, St. Louis, MO, USA). Flow rate: 1.2 ml min-1 Elution gradient: from 98% solvent A (water + formic acid 0.25%) to 55% solvent B (methanol + formic acid 0.25%) in 45 min. LC-MS analysis, performed on a LC-10AD VP (Shimadzu, Japan) liquid chromatograph on-line with a LCMS-2010EV (Shimadzu, Japan) mass spectrometer, equipped with an electrospray ionization (ESI) interface. Column: Discovery HS C18 (5mm, 150 mm x 2,1 mm i.d., Supelco, St. Louis, MO, USA) Flow rate: 0.35 ml min-1 Elution gradient: from 95% solvent A (water + formic acid 0.25%) to 55% solvent B (methanol + formic acid 0.25%) in 45 min. MS conditions: m/z 60-900; negative ion mode; interface voltage 4 kV; nebulizer gas flow 1.5 l min-1; block heater temperature 250 °C; curved desolvation line temperature and voltage 300°C and -5 V, respectively; Q-Array voltage 0 V DC and 150 V RF; detector voltage 1.5 kV. Isoacteoside, a verbascoside isomer, differing for the position of the caffeoyl molecule on the central glucose, was previously identified in solid olive residue and in olive drupes (Mulinacci et al., 2005; Owen et al., 2003). 6’-glucopyranosil oleoside and 6’-rhamnopyranosil oleoside structures were proposed by Cardoso et al. (2005) for two compounds present in olive pulp and olive pomace methanolic extracts. It was possible to submit to HPLC analysis crude OMWW samples, after only simple pre-treatments such as centrifugation and filtration. Analyzing the phenolic fraction of natural matrices directly, without previous extraction, is a great advantage, saving time and solvents and avoiding the problem of low extraction recovery. REFERENCES A rapid and effective analysis of OMWW phenolic fraction is of great interest as this residue is characterized by wide variability in phenolic composition, which needs further investigation, and by high concentration in phenolic antioxidants, which recovery would turn a polluting residue into a valuable source of natural antioxidants. Bianco A., Mazzei R.A., Melchioni C., Romeo G., Scarpati M.L., Soriero A., Uccella N. 1998. Microcomponents of olive oil - III. Glucosides of 2(3,4-dihydroxy-phenyl)ethanol. Food Chem., 63, 461-464. Bouaziz M., Grayer R.J., Simmonds M.S.J., Damak M., Sayadi S. 2005. 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Isolation, structure elucidation and antioxidant potential of the major phenolic and flavonoid compounds in brined olive drupes. Food Chem. Toxicol., 41, 703-717. 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” (Ministerial Decree no. 593 of 8/08/2000, Italian Ministry of University and Research reference number 1866 of 18/02/2002).