Orbitrap exactive ms

Mass spectrometry was performed on an Orbitrap Exactive MS (Thermo Fisher Scientific, USA) acquiring data simultaneously in full scan ion mode (m/z 100–1200, resolution 25,000, AGC 1e⁶) in both positive and negative modes. The capillary voltage was maintained at 25 V in the positive ion mode and at 27 V in the negative ion mode. All other interface settings used were same for both positive and negative modes. The voltages of tube lens and skimmer in positive mode were set to 115 and 22 V respectively. Negative mode voltages of tube lens and skimmer were set to 140 and 28 V respectively. The flow rates of sheath gas, auxillary gas and sweep gas for both positive negative modes were adjusted to 30, 15 and 5 (arbitrary units). The capillary temperature and heater temperature were maintained at 350 and 300 °C respectively in both positive and negative modes.

The dried ethanol extracts of the propolis samples were dissolved in methanol (HPLC grade) to obtain a concentration of 1 mg/mL, the samples were filtered using a syringe filter (Acrodisc 0.45 μm) and 10 µL samples were injected into an UltiMate-3000 HPLC system connected to an Orbitrap Exactive MS (Thermo Fisher Scientific, Hemel Hempstead, UK). The mobile phase was composed of 0.1% formic acid in acetonitrile as solvent A and 0.1% formic acid in water as solvent B and set at a flow rate of 300 µL/min (Table 5). The high-resolution mass spectra were obtained using with a needle voltage of 4.5 kV in positive and −4kV in negative mode. The liquid chromatography was performed on an ACE-C18 column (150 × 3 mm, 3 µm) from HiChrom UK. The MS detection range was from m/z 100 to 1500 and the scanning was performed under ESI polarity switching mode to permit acquisition of positive and negative ion data in a single experiment. Samples containing the purified compounds were dissolved (100 μg/mL) in methanol.

LC-MS analysis and data processing was performed according to a previously described procedure (Alazzo et al., 2019 (link)). Briefly, LC was performed on a ZIC-pHILIC 5 μm, 4.6 × 150 mm column from Merck Sequant (Watford, UK), using an Accela LC system with a mobile phase consisting of A: 20 mM ammonium carbonate and B: 100% acetonitrile as previously described (Creek et al., 2011 (link), Surrati et al., 2016 (link)). Chromatographic separation was carried out using the following linear gradient: 20% A (0 min) to 95% A at 15 min to 20% A at 17 min and held to 24 min. The flow rate was 300 μL min⁻¹ and the injection volume was 10 μL. Samples were maintained at 4 °C, and the column was maintained at 45 °C.
MS was performed on an Orbitrap Exactive MS (Thermo Fisher Scientific, Hemel Hempstead, UK) with ESI running in positive and negative ionisation modes. Spectra were acquired in full MS scan in the range of m/z 70–1400. The capillary and probe temperatures were maintained at 275 and 150 °C, respectively. The instrument calibration was performed by modified Thermo calibration mixture masses with inclusion of C₂H₆NO₂ (m/z 76.0393) for positive ion electrospray ionisation and C₃H₅O₃ (m/z 89.0244) for negative ion electrospray ionisation in order to extend the calibration mass range to small metabolites.