*3.7. HPLC and Gas Chromatography/Mass Spectrometry (GC/MS) Analysis of Volatile Organic Compounds*

Frozen onion samples (1 g) were incubated overnight in absolute methanol at 4 ◦C. Then, the methanol was separated from the pieces of onion and collected in a balloon. The onion pieces were homogenized with absolute methanol (10 mL) in a mortar and stirred 30 min at room temperature (25 ◦C). Samples were then centrifuged, and each supernatant was mixed with the other methanol. The precipitates were resuspended in methanol (10 mL), and the above operations were repeated twice. The methanolic phases were combined, reduced to a volume of 10 mL in a rotary evaporator, and stored at −18 ◦C until use. Methanolic extracts (1 mL) were diluted with dimethylformamide (1 mL) and filtered through an Iso-Disk P-34, 3 mm in diameter poly(tetrafluoroethylene) (PTFE) membrane, and 0.45 μm pore size supplied by Supelco. Diode array detection

(DAD)-HPLC (Shimadzu, Kyoto, Japan) separation of onion flavonoids was performed according to the method described by [49].

Reverse phase-diode array detector-high-performance liquid chromatography (RP-DAD-HPLC) analyses of the samples were carried out with a Shimadzu system (Kyoto, Japan) consisting of a LC-10AD pump system, a vacuum degasser, a quaternary solvent mixing, a SPD-M10 diode array detector, and a Rheodyne 7725i injector (Merck KGaA, Darmstadt, Germany). Separation of each compound was done on a 250 × 4.6 mm i.d., 5-μm Discovery C18 column, supplied by Supelco Park (Bellefonte, PA, USA) equipped with a 4.0 × 20-mm guard column. The column was placed in a column oven set at 25 ◦C. The injection loop was 20 μL, and the flow rate was 1.0 mL/min. The mobile phase consisted of a linear gradient of solvent A (acetonitrile) in 2% acidified water (acetic acid:H2O, 2:98) as follows: 0–80% (0–55 min), 90% (55–70 min), 95% (70–80 min), 100% (80–90 min), and 0% (90–110 min). UV-Vis spectra were measured between 200 and 600 nm and simultaneous detection using a diode array at 278 and 325 nm. Compounds were identified using their retention time and UV spectra through comparisons with purified standards (Sigma Chemical Co., St. Louis, MO, USA). Anthocyanins were extracted from frozen onion tissues (0.5 g) homogenized in a mortar with 10 mL of methanol containing 1 mL L−<sup>1</sup> HCl at room temperature for 2 h. The extracts were filtered through an Iso-Disk P-34, 3 mm in diameter PTFE membrane of 0.45-μm pore size (Supelco), and utilized for HPLC analysis. HPLC separation was carried out using a Spherisorb S5 ODS2, Merck KGaA, Darmstadt, Germany (250 mm × 4.6 mm i.d., 5 μm), as described by [51]. To detect S-methyl-L-cysteine sulfoxide (SMCSO), small pieces of frozen red onion (250 mg) were homogenized with 5 mL of distilled water and filtered through filter paper. SMCSO was quantified by HPLC after derivatization with o-phthalaldehyde, as reported in [52].

A red onion volatile organic compound (VOC) analysis was performed using a Thermo Fisher gas chromatograph (TRACE 1310, Thermo Fisher Scientific, Waltham, MA, USA) equipped with a single-quadrupole mass spectrometer (ISQ LT, Thermo Fisher Scientific, Waltham, MA, USA), as reported in [17].

#### *3.8. Red Onion Sample Preparation for Cell Culture Treatments*

The lyophilized onion samples were dissolved in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (*v*/*v*) fetal bovine serum (FBS), 1% (*v*/*v*) L-glutamine, and 1% (*v*/*v*) penicillin/streptomycin and incubated for 1 h at 37 ◦C. After this time, samples were centrifuged at 2600× *g* for 15 min, and the supernatant was filtered and sterilized through a 0.22-μm membrane filter. The final concentration of the stock solution was 10 mg/mL in DMEM. The onion extract (OE) concentrations tested for the cell culture treatments were: 0.5, 1, 2, 5 and 10 mg/mL.
