4.5.1. Polyphenol Extraction
The solid fraction and ingredients used before cooking (0.5 g) were extracted with 5 mL of methanol:ultra-pure water (80:20, v
) with 0.1% of formic acid for 1 min, sonicated for 10 min in an ice bath, and centrifuged at 4000 rpm for 15 min at 4 °C (Sigma 1-16KL, Sigma, Osterode am Harz, Germany). The supernatant was transferred into a tube and the extraction was repeated. After that, the supernatants were combined and evaporated using a vacuum evaporator (miVac DNA concentrator, Genevac LTD, Warminster, England). The residue was suspended in up to 2 mL of ultra-pure water with 0.1% of formic acid [7
The oil fraction (1 g) was diluted with 1 mL of n
-hexane and after the addition of 2 mL of methanol, it was homogenized and centrifuged at 3000 rpm for 3 min at 4 °C. The nonpolar and polar phases were separated and extracted again with 1 mL of n
-hexane and 2 mL of methanol, respectively. The polar phases were combined, cleaned up with 50 mg of C18 to remove the polar fatty acids, homogenized for 1 min and centrifuged. In order to check for the presence of residual lipids, the extract was placed in an ultra-freezer for 15 min and a visual inspection was performed. The clean liquid was evaporated until dryness using a vacuum evaporator and the residue was suspended in up to 2 mL of ultra-pure water with 0.1% of formic acid [48
The water fraction (1 g) was weighed and centrifuged at 4000 rpm for 15 min at 4 °C. The supernatant was transferred to a 2 mL volumetric flask to which ultra-pure water with 0.1% of formic acid was added. All the extracts were filtered thought a 0.22 µm polytetrafluoroethylene (PTFE) filter into a 2 mL amber vial for HPLC and stored at −80 °C until analysis.
4.5.2. Polyphenol Analysis by UPLC-ESI-QqQ-MS/MS
The polyphenols typically found in tomato, onion and garlic were identified and quantified by a UHPLC-MS/MS method validated for tomato polyphenols (Tomato method) [49
] and EVOO polyphenols were analyzed using a another method more approprieate for them (Olive oil method) [50
]. A UPLC Acquity system equipped with a binary pump, autosampler, and oven from Waters (Milford, MA, United States) with a BEH C18 column (50 mm × 2.1 mm) i.d., 1.7 µm (Waters, Milford, MA, United States) was used. The injection volume was 10 µL, the samples were maintained at 4 °C and the column at 30 °C.
The separation of tomato polyphenols (Tomato method) was carried out with a phase A consisting of acetonitrile (0.1% formic acid) and phase B of water (0.1% formic acid). A gradient elution was applied as follows: 0 min, 10%A; 0.5 min, 10%A, 1.5 min, 15% A; 2.0 min, 20% A; 2.5 min, 50%A; 3.0 min, 100% A; 3.5 min 100% A, and 4.5 min, 10% A. For the EVOO polyphenols (Olive oil method), the separation was performed with a phase A consisting of acetonitrile (0.2% acetic acid) and phase B of water (0.2% acetic acid). The gradient elution was: 0 min, 5%A; 2.5 min, 5%A; 12.5 min, 40%A; 12.6 min, 100%A; 13.5 min, 100%A; 13.6 min, 5%A, and 15.0 min, 5%A. A flow rate of 400 µL/min was applied for both methods.
An API 3000 triple quadrupole mass spectrometer (ABSciex, Framingham, MA, USA), coupled with a Turbo Ionspray source in negative ion mode was used for the MS/MS analysis. The settings of the Turbo Ionspray were: capillary voltage, -3500 V; nebulizer gas (N2), 10 (arbitrary units); curtain gas (N2
), 12 (arbitrary units); collision gas (N2
), 4 (arbitrary units); and drying gas (N2
) heated at 400 °C introduced at a flow rate of 8000 cm3
/min. To improve the detection, the declustering potential, focusing potential, and collision energy were optimized for each compound by direct infusion experiments: 10 ppm individual standard solutions, dissolved in 1:1 (v
) mobile phase, were infused at a flow rate of 5 µL/min with a model syringe pump (Harvard Apparatus, Holliston, MA, USA). A full-scan data acquisition in profile mode, scanning from 100 to 800 m
, was used in cycles of 2 s with a step size of 0.1 u and a pause between each scan of 0.002 s (Table 1
The polyphenols were quantified using multiple reaction monitoring mode (MRM), tracking the transition of parent ion and product ions specific for each compound. The quantification was performed using the internal standard method, applying ethyl gallate as the internal standard, and quantified with calibration curves related to the corresponding standard. The limit of quantification (LoQ) was: 1.13 ng/mL for chlorogenic acid; 9.75 ng/mL for caffeic acid, protocatechuic acid, ferulic acid and verbascoside; 27.0 ng/mL for naringenin-7-O-glucoside, p-coumaric, isolariciresinol, lariciresinol; 75.0 ng/mL naringenin, quercetin, rutin, apigenin, luteolin, hydroxytyrosol, and oleuropein. When commercial standards were not available, the compound was quantified using a standard of the same class of polyphenol. The results were expressed as µg/g of fraction.