Characterization of biophenols from olive oil mill wastewaters by high-performance liquid

chromatography tandem electrospray ionization mass spectrometry (HPLC-ESI/MS)

Characterization of biophenols from olive oil mill wastewaters by high-performance liquid

chromatography tandem electrospray ionization mass spectrometry (HPLC-ESI/MS)

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.

INTRODUCTIONINTRODUCTION

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

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

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

OBJECTIVESOBJECTIVES

RESULTRESULTSS

REFERENCESREFERENCES

criol Centro Ricerche per l’Industria Olearia

University of Naples Federico II

Department of Food Science

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).

ACKNOWLEDGEMENTACKNOWLEDGEMENTSS

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MATERIALS MATERIALS AND AND

METHODSMETHODS

OMWW samples collected during October-November 2005 from a continuous three phases olive processing plant (Industria Olearia Biagio Mataluni, Montesarchio, BN, Italy).

Acidification to pH 2 with hydrochloric acid.

Centrifugation (5000 rpm for 20’) and removal of the deposit.

Filtration on cellulose acetate syringe filter.

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 (5m, 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.

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.

HPLC-UV chromatograms of 4 different OMWWs

Total Ion Current (TIC) chromatogram of an OMWW

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

Retention time, main peaks found in the mass spectra (m/z) and suggested identification for OMWW phenolic compounds

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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.

Chrysoeriol-7-glucoside was previously identified only in olive pulp (Bouaziz et al., 2005).

The two diastereoisomers of -hydroxyacteoside, differing respect to verbascoside for the presence of a hydroxyl group in the -position of OHTy, were reported only for solid olive residue by Mulinacci et al. (2005).

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.

Many phenolic compounds were identified in OMWW samples, several of which were never identified in this residue.

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.

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.

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. Identification and antioxidant potential of flavonoids and low molecular weight phenols in olive cultivar Chemlali growing in Tunisia. J. Agric. Food Chem., 53, 236-241.

Cardoso S.M., Guyot S., Marnet N., Lopes-da-Silva J.A., Renard C.M.G.C., Coimbra M.A. 2005. Characterisation of phenolic extracts from olive pulp and olive pomace by electrospray mass spectrometry. J. Sci. Food Agric., 85, 21-32.

De Nino A., Mazzotti F., Morrone S.P., Perri E., Raffaelli A, Sindona G., 1999. Characterization of Cassanese olive cultivar through the identification of new trace components by ionspray tandem mass spectrometry. J. Mass Spectrom., 34, 10-16.

Mulinacci N., Innocenti M., La Marca G., Mercalli E., Giaccherini C., Romani A., Saracini E., Vincieri F.F., 2005. Solid Olive Residue: insight into their phenolic composition. J. Agric. Food Chem., 53, 8963-8969.

Owen R.W., Haubner R., Mier W., Giacosa A., Hull W.E., Spiegelhalder B., Bartsch H., 2003. Isolation, structure elucidation and antioxidant potential of the major phenolic and flavonoid compounds in brined olive drupes. Food Chem. Toxicol., 41, 703-717.

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.

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.

TIC

m/z 191

m/z 219m/z 315

m/z 317m/z 153

TIC

m/z 407

m/z 461m/z 447

m/z 343m/z 305

TIC

m/z 377

m/z 639m/z 525

m/z 623m/z 523

TIC

m/z 319

m/z 551m/z 301

m/z 285m/z 555

Injection

HPLC-UV system

ESI-MS system

peak number

R.T. main fragments (m/z) compound

1 1.4 191-421-179 caffeoyl quinic acid derivative 2 2.0 219-337 3 2.5 191 quinic acid derivative 4 3.3 331 5 3.9 217-285 6 4.5 315 7 5.2 317 8 6.0 245-183 9 10.4 153-123 OHTy (hydroxytyrosol) 10 11.3 324-280 11 11.6 324-280 12 11.9 315-153-123 OHTy-glucoside 13 12.9 349-245-375 14 13.5 407-389 15 13.8 407-389 16 14.2 461-299 chrysoeriol-7-glucoside 17 14.4 447 18 14.8 345-241-183-299 elenolic acid derivative 19 17.2 343-431-315-153-123 OHTy-glucoside derivative 20 17.6 353-191 quinic acid derivative 21 18.5 294-389-241 22 18.8 343 23 19.0 179-135 caffeic acid 24 19.3 565-377 25 20.4 447 26 20.9 305 27 21.4 447 28 22.7 335-481 29 24.2 377-197 syringic acid derivative 30 25.0 639 -hydroxy-acteoside 31 25.3 639 -hydroxy-acteoside 32 29.0 525 demethyloleuropein 33 30.7 623 verbascoside 34 31.2 523-453 ligstroside isomer

35 32.4 319-183 dialdehydic form of decarboxymethyl

oleuropein aglycon 36 33.4 623 isoacteoside 37 34.0 609 rutin 38 35.2 539-377 oleuropein 39 36.5 523-453 ligstroside aglycon 40 37.4 551-507 6’-glucopyranosil oleoside 41 38.4 301-539 oleuroside 42 40.8 535-491 6’-rhamnopyranosil oleoside 43 41.9 523 ligstroside 44 43.0 301 quercetin 45 44.0 285 luteolin 46 47.0 555-623 10-hydroxy-oleuropein 47 50.9 557 48 51.3 409

Hydroxytyrosol-glucoside

MW=316

Caffeic acid

MW=180

-hydroxy-acteoside

MW=640

Demethyl oleuropein

MW=526

Verbascoside (R1=caffeic acid; R2=H)

Isoacteoside (R1=H; R2=caffeic acid)

MW=624

Rutin

MW=610

Luteolin

MW=286