2.4.3. Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-DAD-HRMS)

Aliquots of 3 μL of plant material were analyzed on a UHPLC Elute system coupled on-line with a quadrupole time-of-flight Impact II mass spectrometer equipped with an ESI source (Bruker Daltoniks, Bremen, Germany). Chromatographic separation was carried out on an RF-C18 Halo column (150 mm × 2.1 mm, 2.7 μm particle size, Advanced Material Technology). The mobile phase consisted of water (A) and acetonitrile (B), containing 0.1% formic acid, at a flow rate of 600 μL/min. The elution conditions were as follows: 0–18 min, linear gradient to 50% B; 18–20 min, linear gradient to 90% B; 20–23 min, isocratic 90% B; and 23–24 min, linear gradient to 0% B (followed by 11 min re-equilibration time). The column and the autosampler were maintained at 45 ◦C and 8 ◦C, respectively. High-resolution mass spectra were acquired in the ESI negative mode. Internal calibration was achieved with an ammonium formate 10 mM solution introduced to the ion source via a 20 μL loop at the beginning of each analysis, using a six-port valve. The mass spectrometric parameters were set as follows: end-plate offset: 500 V; capillary voltage: −2.5 kV; nebulizer: 4 bars; dry gas: 8 L/min; heater temperature: 200 ◦C; m/z range 100–1000 Da; acquisition mode: data-dependent analysis (Auto MS/MS), acquisition rate of 3 Hz, and using a dynamic method with a fixed cycle time of 3, and an isolation window of 0.03 Da. Data acquisition and processing were performed using Data Analysis 4.2 software.
