Masshunter workstation b 06

For the chromatographic separation, 6530 Q-TOF LC/MS (Agilent Technologies) equipped with an autosampler (G7129A), a Quaternary Pump (G7104C), and a Column Compartment (G7116A) was used. The injection volume was 5 μL. The analytes were separated on a Zorbax RP-18 column (150 mm × 3 mm, 2.7 μm) in a flow rate of 0.3 mL/min. The mobile phase was composed of solvent A (aqueous formic acid, 0.1% v/v) and solvent B (0.1% formic acid/acetonitile). A gradient mode was implemented as follows; at t = 0–2 min, solvent A/solvent B (90/10); at t = 10 min, solvent A/solvent B, (80/20); at t = 52–80 min, 100% solvent B. Mass spectra were acquired using ESI in negative ionization mode with a capillary voltage of 4500 V. The mass spectra were recorded in the m/z range of 50–3000 m/z. The gas temperature and drying gas flow rate were 200 °C and 8 L/min, respectively. The skimmer and fragmentation voltages were set at 65 and 130 V, respectively, and collision energy was 10 V. The nebulization pressure was 58 psi. Data processing was performed using MassHunter workstation B.06.00 (Agilent Technologies, 2012) and compounds were tentatively identified according to their mass spectra, accurate mass and retention time, in comparison with literature.

Reversed-phase chromatographic analysis was performed using an Agilent 1200 Series HPLC system (Agilent Technologies, Inc., Waldbronn, Germany) equipped with an Eclipse Plus C18 (ZORBAX, 3.5 µm, 2.1 × 150 mm, Agilent Technologies, Inc., Waldbronn, Germany) as described previously (Voigt et al. 2021 ). Briefly, a gradient of the eluent system acetonitrile/water, both acidified with formic acid, was applied.
An Agilent 6530 Q-ToF mass spectrometer (Agilent Technologies, Santa Clara, USA) equipped with a Jet-Stream Electrospray Ion Source (ESI) was coupled to the HPLC system and used in the positive ion mode. The fragmentation voltage was set to 125 V. For MS/MS spectra, the collision energy was set to 30 eV. The HPLC–MS was controlled using Mass-Hunter Workstation B.06.00 (Agilent Technologies, Santa Clara, USA).

Reversed-phase chromatographic analysis was performed using a Agilent 1200 Series HPLC system (Agilent Technologies, Inc., Waldbronn, Germany) equipped with an Eclipse Plus C18 (ZORBAX, 3.5 µm, 2.1 × 150 mm, Agilent Technologies, Inc., Waldbronn, Germany). A flow rate of 0.3 mL min⁻¹ and a column temperature of 40 °C were used. Elution started with eluents A and B going from 99:1 to 70:30 within 1 min, followed by isocratic conditions of A and B 25:75 during the next 10 min. At 11.1 min, the solvent composition was set to 1:99 and hold for 0.1 min. At minute 15, the gradient was reset to starting conditions and held for further 15 min. For structure identification and recording concentration-time (c-t) curves, the HPLC system was coupled to an electrospray ionization (ESI) quadrupole time-of-flight mass spectrometer Agilent 6530 (Q-TOF-MS, Agilent Technologies, Inc., Waldbronn, Germany) with a Dual AJS electrospray ionization interface. The interface capillary temperature was set to 300 °C and the gas flow to 8 L∙min⁻¹. The fragmentor voltage was set to 125 V. Spectra were recorded in the positive ion mode with a mass range from 100 to 1000 m/z at a scan rate of 1 spectrum s⁻¹. Both instruments were controlled using MassHunter Workstation B.06.00 (Agilent Technologies, Inc., Waldbronn, Germany).