Q exactive orbitrap lc ms ms

One hundred mg of each lyophilized sample was used to determine the polyphenols concentration, as described in Kyriacou et al. [15 (link)]. The samples were analyzed with a UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA), equipped with a quaternary pump (Ultimate 3000 Dionex, Sunnyvale, CA, USA) and a thermostated column (100 × 2.1 mm, Kinetex 1.7 µm biphenyl, Phenomenex, Torrance, CA, USA). Mass spectrometry analysis was performed using a Q Exactive Orbitrap LC–MS/MS (Thermo Fisher Scientific, Waltham, MA, USA). The determination of the polyphenol concentrations was possible thanks to a reference standard mixture (Thermo Fisher Scientific, Waltham, MA, USA). Data analysis was performed with Xcalibur software, version 3.0.63 (Thermo Fisher Scientific, Waltham, MA, USA). All results were expressed as µg 100 g⁻¹ fw.

All mass experiments were conducted at a Q Exactive Orbitrap LC-MS/MS (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an ESI source (HESI II, Thermo Fisher Scientific, Waltham, MA, USA) operating in negative ion mode (ESI-). (Table 2) The accuracy and calibration of the Q Exactive Orbitrap LC-MS/MS were checked weekly. A reference standard mixture was purchased by Thermo Fisher Scientific (Waltham, MA, USA). The Xcalibur software v. 3.1.66.10 (Xcalibur, Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze and process data.

Mass experiments were done with a Q Exactive Orbitrap LC-MS/MS (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an ESI source (HESI II, Thermo Fisher Scientific, Waltham, MA, USA) operating in negative ion mode (ESI−). The ion source parameters were spray voltage −3.0 kV, sheath gas (N₂ > 95%) 30, auxiliary gas (N₂ > 95%) 15, capillary temperature 200 °C, S-lens RF level 50, auxiliary gas heater temperature 305 °C. The MS (Mass) detection was conducted in two acquisition modes: Full scan (negative-ion modes) and targeted selected ion monitoring. The parameters of the full scan acquisition mode were: Mass resolving power 35,000 full width at half maximum (at m/z 200), scan range 100–1500 m/z, scan rate 2s⁻¹, and the automatic gain control target 1 × 10⁵ ions for a maximum injection time of 200 ms. The parameters of the targeted selected ion monitoring acquisition mode were: 15s-time window, resolution power 35,000 full width at half maximum (at m/z 200), and quadrupole isolation window 1.2 m/z. The mass list containing exact masses and expected retention times of the target polyphenolic compounds was taken in.

A UHPLC system (UHPLC, Thermo Fisher Scientific, Waltham, MA, USA) was used for quantification and separation of polyphenolic compounds. A Q Exactive Orbitrap LC–MS/MS (Thermo Fisher Scientific, Waltham, MA, USA) was employed to facilitate the analysis of the mass spectrometry. The details of UHPLC-high-resolution mass spectrometry analysis are as described by Graziani et al. [20 (link)].

The leaf polyphenolic assay was performed analogously to the protocol followed by Kyriacou and collaborators [46 (link)]. Extraction was carried out on 100 mg of lyophilized leaf sample in 5 mL of a 60:40 v/v methanol/water solution. Phenolics separation was obtained via UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA), equipped with 1.7 µm Biphenyl (100 × 2.1 mm) column (Phenomenex, Waltham, CA, USA). Mass spectrometry data were obtained via a Q Exactive Orbitrap LC-MS/MS (Thermo Fisher Scientific, Waltham, MA, USA). All polyphenolic data are expressed as mg kg⁻¹ DW.

A Q Exactive Orbitrap^® LC-MS/MS (Thermo Fisher Scientific, San Jose, CA, USA) mass spectrometer coupled with a Vanquish UPLC system (Thermo Fisher Scientific, San Jose, CA, USA) was employed for the LC-MS analysis, which was equipped with an ESI source. The separation of the EL-EA fraction was acquired with a Hypersil Gold column (C18) column (150 × 2.1 mm, 5 μm) at 25 °C. The injection volume of the sample was 1 µL. Scan range m/z 70-1050; ESI Spray Voltage: 3.2 kV; Sheath gas flow rate: 35 (arbitrary units); Aux Gas flow rate: 10 (arbitrary units); Capillary Temp: 320 °C. Polarity: negative; MS/MS data-dependent scans. The mobile phase B and the used gradient condition are shown in Table 3.
Compounds in EL-EA were identified by comparing the retention time, parent ion, and mass fragments with available standards in databases (ChemSpider, mzCloud, mzVault, HMDB, and PubChem) and/or literature data [9 (link),10 (link),11 (link),12 (link),13 (link),14 (link),15 (link),16 (link),17 (link),18 (link),19 (link),20 (link),21 (link)].