Ultimate 3000 hplc

Production strains were inoculated in 0.5 ml SC media for initial growth (24 h, 30 °C, 250 RPM). Production cultures were then set up by replacing culture media by pelleting (2500 × g, 3 min) and resuspension in 500 µl fresh SC. Cultures were then incubated for production in 96-deep-well plates, which for HPLC was done with an initial OD₆₀₀ = 0.2 (24 h, 30 °C, 250 RPM) and for biosensing an initial OD₆₀₀ = 2.0 (5 h, 30 °C, 250 RPM). To acquire the pure media supernatant for quantification of ligand production, cells were removed by pelleting (5000 × g, 5 min) and extraction of supernatant by pipetting twice. HPLC analysis of serotonin and melatonin amounts produced by yeast strains was done on the Thermo Scientific™ UltiMate™ 3000 HPLC using the Agilent Zorbax C18 4.6 × 100 mm 30l5-Micron column with a Phenomenex AFO-8497 filter. Solvent A was 0.05% acetic acid, solvent B Acetonitrile. Data analysis was done using Chromeleon™ Chromatography Data System (CDS) Software. Serotonin and melatonin values of samples were determined according to standard curves of serotonin hydrochloride and melatonin, in the range of 10 to 150 uM and 2 to 20 uM, respectively. Serotonin peaks were detected at 1.920 min, while melatonin peaks were seen at 7.037 min.

The qualitative analysis of three studied actinomycetes extracts was performed by high performance liquid chromatography-mass spectrometry (HPLC-MS) using an LTQ-XL Ion Trap mass spectrometer (Thermo Fischer Scientific Spa, Rodano, Italy) equipped with an Ultimate 3000 HPLC (Agilent Technology, Cernusco sul Naviglio, Italy). Chromatographic separation was obtained using a Kinetex Polar C₁₈ column (100 × 3.0 mm, 100 Å, 2.6 µm) (Phenomenex, Torrance, CA, USA). The injection volume was 0.5 mL/min and a mobile phase consisting of a combination of A (0.1% formic acid in water, v/v) and B (0.1% formic acid in acetonitrile MeCN); a linear gradient, which ranged between 5 and 60% B in 25 min, from 60 to 95% B in 10 min, and held at 95% B for 5 min, was used. The mass spectrometer was set in positive ion mode. ESI source conditions were the following: capillary voltage −48 V; tube lens voltage −176.47; capillary temperature 280 °C; sheath 15 and auxiliary gas flow (N₂) 5; sweep gas 0; and spray voltage 5. MS spectra were obtained at 30,000 resolutions by full-range acquisition with a scan range between 150 and 1500 m/z.