3.4. HPLC-DAD and LC-HRMS Analysis of the Extracts

HPLC analysis was conducted on a Thermo Finnigan HPLC system (Ontario, Canada) equipped with a SpectraSystem P4000 pump, a SpectraSystem 1000 degasser, a SpectraSystem AS3000 automated injector, and a UV SpectraSystem UV6000LP detector. Data acquisition was controlled by the ChromQuest™ 5.0 software (ThermoScientific™). HPLC-DAD experiments were run on a Supelco Analytical (Sigma-Aldrich) HS C18 column, with dimensions 25 cm × 4.6 mm, 5 μm, at room temperature. As elution solvent system, acidified H₂O (0.1% v/v of formic acid) and acetonitrile were used. Gradient was started with 98% of H₂O and reached 2% after 60 min. At 61^st min, the elution system returned to the initial conditions and stayed for 4 min. Flow rate was set at 1 mL/min and injection volume at 10 μL. Chromatograms of 254 nm, 280 nm and 366 nm, were recorded.

All the produced extracts were analyzed with UPLC-HRMS/MS. The experiments were accomplished on an H class Acquity UPLC system (Waters, Milford, CT, USA) coupled to an LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Fisher Scientific, Waltham, CT, USA). For the chromatographic separation, a Fortis C-18 column (1.7 µm, 150 × 2.1 mm) was used, and temperature was set at 40 °C. The elution solvent system was consisted of acidified water with 0.1% formic acid (A) and acetonitrile (B). Gradient started with 2% B for 2 min, which reached 100% at 18 min and stayed for 2 more min. At the 21st minute, system returned to the initial conditions and stayed for 4 min for system equilibration. The flow rate was at 400 μL/min, the injection volume was 10 µL and samples were kept at 7 °C. Ionization was carried out in negative and positive ion mode (ESI±). The mass spectrometric parameters were: capillary temperature 350 °C; sheath gas 40 units; aux gas 10 units; capillary voltage 30 V; and tube lens 100 V for the positive mode. For negative ionization capillary voltage was adjusted at −20 V and tube lens of −80 V, while all the other parameters remained stable. Data were recorded in full scan from 113 to 1000 m/z and HRMS/MS experiments were carried out with data dependent method with collision energy 35.0% (q = 0.25).