3.2. Carotenoid Extraction

To prevent the degradation and oxidation of carotenoids, all the experiments were conducted under limited light conditions. For carotenoid extraction, the method of extraction described by Vrebalov et al. [25] was used with some modifications and a schematic diagram for the same is presented in Scheme 2. All the solvents used were of HPLC grade. Frozen tomatoes were ground and about 100 mg of the tissue powder was placed in a 2 mL tube with two glass beads (6 mm). The frozen tissues were homogenized with 15 mg of magnesium carbonate and 300 μL of tetrahydrofuran (THF) for 60 s using FastPrep-24™ instrument (MP Biomedicals, Santa Ana, CA, USA). The extracts were homogenized again after adding 300 μL of methanol (MeOH) containing 5% butylated hydroxyl-toluene (BHT). The homogenate was transferred to Spin-X centrifuge filter tube (0.45-mm nylon filter, Corning Incorporated, Corning, NY, USA) and centrifuged for 1 min at 4000 rpm at 4 °C. The original 2 mL tube was kept on ice and 150 μL of THF and 150 μL of MeOH, containing 5% BHT, were added to the tube. The tissue debris in the original tube was transferred using a cut tip to Spin-X centrifuge filter tube and then centrifuged for 1 min at 4000 rpm and 4 °C. The filtered extract was transferred to a new 2 mL tube. For the complete extraction of carotenoid, 350 μL of THF was added in Spin-X centrifuge filter tube, incubated on ice for 15 min, and then centrifuged at 4000 rpm for 5 min at 4 °C. This step was repeated. The filtered extract was combined with the previous extract in a 2 mL tube. To separate the carotenoid/nonpolar phase from the aqueous phase, 375 μL of petroleum ether and 150 μL of 25% sodium chloride were added into the filtered extract, vortexed vigorously, and centrifuged for 3 min. The upper phase was separated from the lower phase or inter-phase into a new 2 mL tube. For re-extraction, 500 μL of petroleum ether was added and the upper phase was separated as described for the previous step. The petroleum ether extract was dried using MICRO-CENVAC machine (NB-503CIR, N-BIOTEK, Bucheon, Korea) for 2 h at 45 °C. If HPLC was not performed immediately, the carotenoid extract was stored at −80 °C.