Quantifying Melatonin Metabolites in Broccoli

About 1 g of fresh samples of broccoli seedlings were collected and ground. Melatonin and its precursors, tryptamine, serotonin, and N-acetylserotonin, were then extracted using methanol and quantified with LC-MS/MS (API 4000), as described in Yu et al. [27 (link)]. After centrifugation for 5 min at 8000× g and 4 °C, the supernatants were dried with a Termovap Sample Concentrator and dissolved in 200 μL of methanol. Melatonin and its precursors in the extraction solutions were separated using an Agilent Poroshell 120 EC-C18 column (150 mm × 2.1 mm, 2.7 μm, Agilent Technologies). Solvent A consisted of water with 0.1% (v/v) formic acid, while solvent B consisted of methanol. The LC gradient program was set as follows: 0–2 min, 20% of solvent A; 2–5 min, 20% of solvent A; and 5.1–12 min, 80% of solvent A; the flow rate was 0.3 mL min⁻¹. The ion modes and m/z values of the precursor and product ions for each metabolite are listed in Table S1. The method for the calculation of melatonin and its precursors was adopted from Kang et al. [28 (link)].