Polaris q

Volatile compounds were analyzed using a gas chromatograph (Trace CG Ultra) coupled to the mass spectrometer (Polaris Q) with an ion-trap analyzer (Thermo Scientific, San Jose, CA, USA). Compounds were separated using a capillary column HP−5 MS (5% phenyl and 95% methylpolysiloxane) of 30 m long, 0.25 mm internal diameter, 0.25 μm film thickness, and helium gas with a constant flow rate of 1 mL min⁻¹ (Agilent Techonolgies Inc., Waldbronn, Germany). The injector (splitless mode) was maintained for 5 min at a temperature of 250 °C, the ion source at 200 °C, and the interface at 270 °C. The oven was programmed at 40 °C for 1 min, followed by an increase in temperature at a rate of 12 °C min⁻¹ until it reached 120 °C, maintaining it for 2 min. Then, at 15 °C min⁻¹ to 150 °C and finally, at 20 °C min⁻¹ to 245 °C, maintaining for 2 min.
The identification of volatile compounds was carried out using the National Institute of Standards and Technology Research Library (NIST). This identification was also based on articles that determined volatile compounds in Eugenia klotzschiana O. Berg. The total peak area was obtained in Xcalibur1.4 from Thermo Electron Corporation (Thermo Electron, San Jose, CA, USA) and analyzed in Microsoft Office Excel 2010^®.

1D and 2D NMR spectra were recorded on a Bruker AVANCE™ III HD-400, equipped with a CryoProbe™ Prodigy or on a Bruker DRX-600 equipped with TXI CryoProbe^TM in CDCl₃ or CDCl₃/CD₃OD (δ_H values reported refer to CHCl₃ or CHD₂OD protons at 7.26 or 3.34 ppm, respectively; δ_C values refer to CDCl₃ or CD₃OD carbon at 77.0 or 49.0 ppm, respectively). High-resolution mass analyses were performed on a Q-Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific, Milan, Italy) equipped with a HESI source on-line with a UHPLC Infinity 1290 (Agilent Technologies, Santa Clara, CA, USA). GC-MS analysis was performed on an ion-trap MS instrument in EI mode (70 eV) (Thermo, Polaris Q) connected with a GC system (Thermo, GCQ) by a 5% phenyl/methyl polysiloxane column (30 m × 0.25 mm × 0.25 µm, Agilent, VF-5ms) using helium as the gas carrier. HPLC purifications were carried out on a Shimadzu high-performance liquid chromatography system (Shimadzu, Kyoto, Japan) LC-20ADXR equipped with a Diode Array Detector SPDM-20A and a LUNA C-18(2) column 250 × 10 mm, 5 µm, 100 A (Phenomenex, Castel Maggiore, Italy). TLC plates (KieselGel 60 F254) and silica gel powder (Kieselgel 60 0.063–0.200 mm) were from Merck (Darmstadt, Germany). Chemicals were of analytical reagent grade and solvents of HPLC/LC-MS grade (Sigma-Aldrich, Milan, Italy) and were used without any further purification.

The separation and identification of the main chemical compounds of each essential oil were carried out by gas chromatography (Trace GC ultra, Thermo, MN, USA), coupled to a mass spectrometer (Polaris-Q, Thermo, USA). Helium was used as carrier gas (1 mL/min) and the column used was a Rxi-5Sil MS (Restek, France) (40 m × 0.18 mm; thickness 0.18 µm). The injector temperature was 240 °C while the detector’s temperature was 200 °C. In total, 0.8 µL of the solution prepared in hexane at 0.5% was injected for each essential oil (splitless mode). The temperature program was set at 50 °C during 6 min, then increased at a rate of 2 °C/minute until reach 250 °C and held for 10 min. The essential oil components were identified based on their retention indices (comparing to the major compounds also analyzed) and peak area percents were used to obtain quantitative data (n = 2).