Carotenoid Extraction and Quantification

Extraction procedure and saponification were performed from 10 mg of ground, freeze-dried leaf tissue according to the recently described protocol (Demurtas et al. 2023 (link)). LC separation was performed using an LC-PhotoDiode Array (PDA) system (Accela, Thermo Fisher Scientific, Thermo Fisher Scientific, Waltham, MA, USWaltham, MA, US) and a carotenoid C30 column (100 × 3 mm², 3-µm particle size) (YMC, Dinslaken, Germany), the column temperature was 25 °C. Elution system was A, MeOH; B, MeOH/water (4:1 v/v) with 0.2% ammonium acetate; and C, tert-Butyl methyl ether (C), and gradient was 0 to 1.2 min 95% A, 5% B; 3.5 min 80% A, 5% B; 6.8 min 30% A, 5% B, 65% C; 16 min 95%, 5%. Injection volume was 10 µL, chromatographic flux after equilibration was 0.8 ml/min and the total run time 18 min. PDA data were recorded in the 200–700 nm range. Carotenoids were measured based on their absorption spectra as detected by the PDA, integrated at their individual λmax and normalized to DL-α-tocopherol acetate (Sigma–Aldrich, Cat. No. T3376-5G) at 285 nm, as recently described (Demurtas et al. 2023 (link)).