Melatonin Extraction and Quantification

Melatonin and melatonin-derivatives extraction was done through a modification of the methods described by Riga et al [28 (link)] and Li et al. [33 (link)]. Briefly, 1 g of frozen leaves were ground into powder with liquid nitrogen and homogenized in a mix of acetone:methanol:water (89:10:1) containing 2.5 mM trichloroacetic acid. The homogenates were shaken for 30 min at RT and centrifuged at 10,000× g at 4 °C for 15 min. The supernatants were centrifuged again and subsequently filtered with Whatman filter paper (0.4 µm). The filtered supernatants were purified using an SPE cartridge (Waters, Milford, MA, USA). The cartridge was then washed with 10 mL 5% methanol, and melatonin was finally eluted at a natural flow rate with 2 mL 80% methanol. The extracts were subsequently filtered through a Whatman filter paper (0.20 μm) before UHPLC-ESI-MS/MS analysis. Melatonin, 3OH-Mel, AFMK and AMK determination and quantification was analyzed using a UHPLC-ESI-MS/MS (UHPLC-1290 Series and a 6460 QqQ-MS/MS; Agilent Technologies, Waldbronn, Germany) with an Agilent SB-C18 column (4.6 × 50 mm; 1.8 μm; Agilent Technologies, Santa Clara, CA, USA). The data reported are the mean ± SE of 3 biological replicates per treatment.