*2.7. Carotenoids Extraction and HPLC Analysis*

Tomato carotenoids were extracted using the method of Siracusa et al. [31]. An aliquot of the cherry tomato puree sample (0.5 g) was transferred into a vial and 5 mL of a n-hexane/acetone/ethanol (2:1:1) solution were added. The vial was left shaking for 40 min. in the dark at room temperature. Subsequently 1 mL of H2O (HPLC grade) was added and a further 2 min. agitation was applied. The resulting heterogeneous mixtures were left decanting until phases separation. The apolar coloured layers were transferred into an amber vial and analysed.

Quantitative analyses were carried out on an HPLC (Shimadzu USA Manufacturing Company Inc., Class VPLC-10 Dvp, Canby, OR, USA) equipped with a DAD (Shimadzu SPD-M10Avp). The column was a Gemini NX C18 (150 × 4.6 mm; 3 µm particle size; Phenomenex, Italy), fitted with a guard cartridge packed with the same stationary phase. The flow rate was 0.7 mL/min. and the injector volume was 20 µL. Carotenoids were eluted with the following gradient of A (Methanol: H2O 75:25) and B (Ethyl acetate): T0 30% B; T15 82% B; T25 30% B. All reagents used were HPLC purity grade: water, methanol and Ethyl acetate were obtained from Merck. The wavelength range was 220–660 nm, and the chromatograms were monitored at 473 nm for lycopene; at 453 nm for β-carotene; at 348 nm for phytofluene and at 288 nm for phytoene. Carotenoids were identified by splitting the peak of the carotenoids from the tomato-solution sample with a standard of β-carotene and lycopene; (*p* ≥ 95% and *p* ≥ 98%, Sigma-Aldrich, St. Louis, MO, USA) and by comparing retention times and UV spectra with those of standards. Quantification of β-carotene and lycopene was performed using external calibration curves; for phytofluene and phytoene the calibration curve of β-carotene was used. Linearity was checked for β-carotene between 3.36 and 21 mg kg−<sup>1</sup> (*R* <sup>2</sup> = 1.00) and lycopene between 2.56 and 40.0 mg kg−<sup>1</sup> (*R* <sup>2</sup> = 1.00). All analyses were performer in triplicate, including the extraction procedure, and the results were expressed as mg kg−<sup>1</sup> DW.
