Hydroxytyrosol and tyrosol were purchased from Extrasyntese (Genay Cedex, France). Methanol, n-hexane, ethyl acetate, and acetonitrile were obtained from VWR (Milan, Italy). All solvents used were of analytical grade. Four different OMWW samples (5 L) were collected from four different continuous three-phase olive processing mills located in southern and western Sicily (Sciacca and Suvarelli, respectively) and eastern Sicily (Mount Etna) immediately after milling on October 2015. In detail, OMWW samples were obtained from milling Cerasuola (from Sciacca mill), Biancolilla (Suvarelli), Tonda Iblea (Mount Etna, 1000 m above sea level), and Nocellara Etnea (Mount Etna, 200 m above sea level) olive cultivars. Olives were in each case grown according to organic (pesticide-free) farming protocols. To avoid decomposition all samples were stored at −20 °C until use. No stabilizing agents were added to avoid chemical alteration of the crude matrices.
Extraction of OMWW
All OMWW samples were subjected to liquid–liquid and solid–liquid solvent extraction.
Liquid–liquid extraction
Typically, one sample of raw OMWW (500 mL) was centrifuged twice at 9000 rpm (Beckman allegra X-22R centrifuge with a fix-angle rotor F0630) in 30 mL vials for 10 min in order to remove pulp and any other suspended solid residues. The resulting water phase was then filtered through Whatman filter paper to get rid of any residual solids. The resulting mildly acidic (pH ≈ 5) green-black aqueous phase was further acidified to pH ≈ 2 using concentrated HCl (2 M). The color of the mixture quickly turned into deep red. The acidified water phase was thus defatted in a separatory funnel using n-hexane (3 × 25 mL). The aqueous layer was further extracted with EtOAc (4 × 40 mL) to recover all phenolic compounds, after which the extract was dried over anhydrous Na2SO4 and evaporated in a rotary evaporator at 40 °C under reduced pressure (180 mbar). A yellow–brown crude oil sample was obtained, depending on the cultivar. To eliminate the resins, each crude sample was separately dissolved in EtOAc and 2 g of silica gel added to the resulting mixture. The solvent was evaporated in a rotary evaporator and the resulting oil adsorbed on silica loaded on a silica gel column (silica gel 60, particle size 0.063–0.200 mm, 70–230 mesh ASTM; 11 g) packed in n-hexane. The silica column was then eluted with n-hexane (100 mL) to remove the residual non-active apolar components. EtOAc (100 mL) was then added in order to recover the polyphenol fraction. The eluate was evaporated in a rotary evaporator, affording a liquid–liquid polyphenol mixture (LLPM) isolated as yellow-orange oil (Fig. 1).
Solid–liquid extraction
In a typical solid–liquid extraction the crude OMWW was first centrifuged and defatted as reported above. Then a sample (50 mL) of the defatted OMWW was lyophilized using a freeze dry system (Freezone 4.5, Labconco corporation) operated at 0.018 mbar and −51 °C. The resulting powder was suspended in MeOH (4 mL) and sonicated for 10 min in an ultrasonic bath (Elma Transsonic 460/H) kept at 30 °C. The methanol extract was filtered through Whatman filter paper, dried over anhydrous Na2SO4, and evaporated using a rotary evaporator at 40 °C under reduced pressure (150 mbar). Once again, a yellow–orange crude oil was obtained, whose color depended on the cultivar. These oils were separately mixed with silica in a mortar, loaded onto a silica gel septum and purified using the optimized methodology reported above. The four different biophenol extracts obtained from the different cultivars were labelled solid–liquid polyphenol mixtures (SLPM).
Carbon analysis
Total organic carbon (TOC), total carbon (TC) and inorganic carbon (IC) analyses of each OMWW sample were performed on the defatted and on the centrifuged samples. In detail, a small (500 µL) sample was dissolved in 100 mL of highly pure (milli-Q) water, and the resulting solution filtered through a 0.2 µm Whatman Teflon syringe filter. The resulting solution was analyzed using a Shimadzu TOC-5000A analyzer.
HPLC–MS analysis
SLPM and LLPM extraction fractions were dissolved, respectively, in 5 and 10 mL of EtOAc. An aliquot (1 μL) of each solution was qualitatively monitored by HPLC and LC–MS by comparison and combination of retention times and mass spectral data (Agilent 6130 Series Quadrupole LC/MS Systems, equipped with G1329A High Performance Autosampler, G1316A Thermostated Column Compartment and G1315D Diode Array Detector). The UV detector was operated at 280 nm. Separation was carried out using an Agilent Eclipse XBD-C18 (4.6 × 150 mm, 5 lm) column maintained at 30 °C. Polyphenol compounds were identified and assessed using a G6120B Single Quadrupole LC/MS system equipped with an electrospray ionization source (ESI). For target compound analysis, a flow injection analysis (FIA) was carried out to determine the fragmentor setting to improve the compound response. The potential chosen was 200 V. Selected ESI work conditions were capillary voltage 5000 V, gas flow rate 13 L/min, gas temperature 300 °C and nebulizer pressure 60 Psi. To obtain the best sensitivity, the quadrupole was used in SIM mode. Optimum separation was achieved with a binary mobile phase gradient at a flow rate 0.5 mL/m. The mobile phase consisted of a binary solvent system using (A) water/formic acid (pH 3.1) and (B) acetonitrile previously degassed.