4.4. Untargeted Metabolomics by LC-Q-Orbitrap HRMS

The Salvia hispanica hydromethanolic extracts were investigated using a Dionex Ultimate 3000 UHPLC system (Thermo ScientificTM, Dionex, San Jose, CA, USA). Chromatography separations were performed using SynergiTM Hydro-RP A (150 × 4.5 mm, 4 µm, Phenomenex) column. Mobile phases A (water) and B (acetonitrile), both acidified with formic acid (0.1% v/v), were pumped at a flow rate of 0.8 mL/min¹, according to the following gradient pattern: 0 min, 5% B; 20 min, 50% B; 25 min, 100% B; 27 min, 100% B and finally, the initial conditions were held for 8 min as a re-equilibration step. The injection volume was 4 μL. The chromatographic unit was coupled to a Q ExactiveTM Focus quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) with a heated electrospray ionization source (HESI II). The HESI parameters in negative polarity included: sheath gas flow rate, 35 arb; auxiliary gas flow rate, 15 arb; sweep gas flow rate, 3 arb; spray voltage, 2.5 kV; capillary temperature, 350 °C; S-lens RF level, 50; heater temperature, 300 °C. Full scan data in the negative mode was acquired at a resolving power of 70,000 FWHM; AGC target, 1e6; max IT, auto. A scan range of m/z 100–1200 was chosen for the compounds of interest. The parameters of data-dependent MS2 were as follows: resolution, 17,500; isolation window, 3.0 m/z; normalized collision energy, 30; AGC target, 1e6; max IT, auto. Mass calibration was performed once a week, in both positive and negative modes, using mixture containing n-butylamine, caffeine, Met-Arg-Phe-Ala (MRFA) and Ultramark 1621.

Raw data from high-resolution mass spectrometry were elaborated with Compound Discoverer (v. 2.1, Thermo, Waltham, MA, USA). Major metabolite identification was based on accurate mass and mass fragmentation pattern spectra against MS-MS spectra of compounds available on customized database of different classes of phytochemicals created on the basis of literature data on the Salvia species and implemented in the software. Raw data from three experimental replicates and a blank sample were processed using a workflow presented in Kusznierewicz et al. [87].