Biphenyl column

The UHPLC-HR-MS system was composed of a Vanquish Flex Binary pump LC and a Q Exactive Plus MS equipped with an electrospray ionization (ESI) source, the Orbitrap-based FT-MS system (Thermo Fischer Scientific Inc., Dreieich, Germany). The chromatography was performed on a Kinetex Biphenyl column (100 × 2.1 mm, 2.6 µm particle size) composed of SecurityGuardTM Ultra Cartridges (Phenomenex, Bologna, Italy). Elution was carried out with formic acid in H₂O 0.1% v/v (solvent A) and formic acid in acetonitrile 0.1% v/v (solvent B) with a linear solvent gradient (5 to 60% B within 16 min). Samples (5 µL) were injected into the LC system at a flow rate of 0.5 mL/min, maintaining autosampler and column oven temperatures at 4 and 35 °C, respectively. MS parameters were applied as previously reported [8 (link)].

Prior to measuring roseoflavin or other flavins by HPLC, proteins were precipitated from the culture supernatants or cell-free extract samples by mixing with trichloroacetic acid (TCA) to a final concentration of 2.5%. The samples were kept on ice for 5–10 min, centrifuged at 15,000×g for 2 min and filtrated through 0.22 µm cellulose-acetate filters. When culture supernatants of S. davaonensis were analyzed, TCA treatment was preceded by treatment with α-amylase (Sigma-Aldrich) to degrade starch present in YS. Determination of flavin levels was performed using an Agilent 1260 Infinity system and a 6130 Quadrupole ESI/MS from Agilent Technologies (Waldbronn, Germany) and a biphenyl column (2.6 μm particle size, 150 mm × 2.1 mm) from Phenomenex (Aschaffenburg, Germany). The injection volume was 15 µl and a running buffer containing 35% (v/v) methanol as well as 10 mM ammonium formate (pH 3.7) was used at a flow rate of 0.2 ml/min. Detection of riboflavin, RP, AFP and roseoflavin was carried out using a photometer set to different wavelengths. Riboflavin and RP were detected at 480 nm. AFP and roseoflavin were detected at 480 nm and 509 nm, respectively.

Aromatic waters were diluted in MeOH (1:1, v/v), centrifuged (4000 rpm) and injected (5 μL injection volume) into the LC system composed by a Vanquish Flex Binary UHPLC coupled with a Vanquish DAD and a Q Exactive Plus mass spectrometer, Orbitrap-based FT-MS system, equipped by an ESI source (Thermo Fischer Scientific Inc., Bremem, Germany). Elution was performed on a Kinetex^® Biphenyl column (100 × 2.1 mm, 2.6 μm) provided of a SecurityGuard^TM Ultra Cartridges (Phenomenex, Bologna, Italy), using formic acid in MeOH 0.1% v/v (solvent A) and formic acid in H₂O 0.1% v/v (solvent B) as eluent and developing a linear solvent gradient of increasing 5 to 55% A within 15 min, at a flow rate 0.3 mL/min. During analysis, autosampler and column oven temperatures were maintained at 4 and 35 °C, respectively. UV data were registered using 254, 280 and 325 nm as preferential channels. A positive ion mode was used for ESI interface in a scan range of m/z 150–1200 and spectra were acquired both in full (70,000 resolution, 220 ms maximum injection time) and data dependent-MS/MS scan (17,500 resolution, 60 ms maximum injection time). The following ionization parameters were used: spray voltage 3500 V, capillary temperature 300 °C, sheath gas (N₂) 20 arbitrary unit, auxiliary gas (N₂) 3 arbitrary unit, collisionally activated dissociation (HCD) 18 eV. Data were elaborated with Xcalibur software.