Ascorbic acid quantification

Ascorbic acid (AsA) content was determined as reported by Stevens et al. [28] with slight modifications reported by Rigano et al. [2]. In brief, 500 mg of tomato frozen powder from fruits at different ripening stages were added to 300 μL of ice-cold 6% trichloroacetic acid (TCA). Samples were mixed and left on ice for 15 min, then centrifuged for 15 min at 25,000×g at 4 °C. Twenty μL of supernatant were transferred to a clean tube with 20 μl of 0.4 M phosphate buffer (pH 7.4) and 10 μl of double distilled (dd) H₂O. Afterwards, 80 μl of reagent solution, prepared by mixing solution A [31% H₃PO₄, 4.6% (w/v) TCA and 0.6% (w/v) FeCl₃] with solution B [4% 2,20-dipyridil (w/v) made in 70% ethanol] at a proportion of 2.75:1 (v/v), were added. The mixture was incubated at 37 °C for 40 min and measured at 525 nm by using a NanoPhotometerTM (Implen). Three separated biological replicates for each sample and three technical assays for each biological repetition were measured. Values were expressed as mg/100 g of fresh weight (FW).