Luna omega c18

An UltiMate™ 3000 (Thermo Fisher Scientific, Waltham, MA, USA) high-performance liquid chromatograph was combined with a Thermo Scientific PAD detector. Separation was performed on a Phenomenex Luna Omega C18 reversed-phase analytical column (100 × 2.1 mm, 1.6 µm). The gradient elution was carried out using a mixture of water (eluent A) and acetonitrile (eluent B), according to the following program: 0 min: 30% B; 21.0 min: 62.7% B; 21.3 min: 97.5% B; 28.8 min: 97.5% B; 29.0 min: 30% B. The wavelength range of 190–800 nm was scanned and the chromatograms of BAs were recorded at 340 nm, which was identified as the optimal wavelength for the analysis.

The UHPLC-Q-TOF-MS/MS was provided in our previously published article [14 (link)]. Chromatographic separation was conducted on a Luna Omega C18 (100 × 2.1mm, 1.6 μm, Phenomenex Inc., CA, USA) keeping at 40°C. 0.1% aqueous formic acid (v/v, A) and acetonitrile (B) were used as mobile phases. The gradient elution with the flow rate of 0.3 mL/min was performed as follows: 0-15 min, 25% B; 15-18 min, 25%-55% B; 18-40 min, 55%-95% B; 40-42 min, 95% B for column cleaning, and a conditioning cycle time of 3 min with the same initial conditions of 5% B. The sample inject volume was 3 μL.

Chromatographic analyses were performed using a Thermo Scientific Ultimate 3000 RSLC system equipped with an autosampler, a binary pump, a thermostated column compartment, a DAD detector (Dionex, Germering, Germany) and an ELSD Sedex 100LT detector (Sedere, Olivet, France) in dynamic gain. The mobile phase was composed of water (A) and acetonitrile (B) with both acidified with 0.1% of formic acid. Elution was performed in the gradient mode at a flow rate of 500 μL·min⁻¹ and with the following binary gradient program: starting with 3% of solvent B during 0.2 min, 3–45%B from 0.2 to 12 min, 45–90%B from 12 to 14 min, and 90–3%B from 15 to 15.5 min. Then, the column was re-equilibrated with 3% of solvent B during 3 min. The column was a Luna^® Omega C18 (150 × 2.1 mm; 1.6 µm) (Phenomenex, Le Pecq, France,) heated at 40 °C and then 10 µL of the oak extracts and fractions were injected.

The chromatographic analysis of EFVF was performed using a 1290 infinity UHPLC- DAD system (Agilent Technologies, Santa Clara, CA, USA) equipped with an analytical column (Luna Omega C₁₈, 2.1 × 50 mm, 1.6 μm, Phenomenex, Torrance, CA, USA). The extract or STDs were analyzed using a sequential gradient mobile phase system from a 95:5 mixture to a 40:60 mixture of 0.1% (v/v) formic acid (mobile phase A) and acetonitrile (mobile phase B) within 40 min at a flow rate of 0.2 mL/min. The DAD signals of EFVF and STDs were detected at 280 nm, and the spectral scanning was performed at a wavelength range of 200–400 nm. The column and auto-sampler temperature were set at 40 and 4 °C, respectively. Each constituent from EFVF was identified by comparing the retention time (tR) and specific DAD spectrum pattern of each peak in the EFVF with those of the corresponding STD. The chromatographic data were processed by the Agilent OpenLAB CDS software

All extracts were solubilized at concentration of 1 mg/ml, in isopropanol for hexane extracts or MeOH for the others extract. Pure compounds were prepared at a concentration of 0.2 mg/ml in MeOH. Ultra-high Performance Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS) was performed on a Dionex Ultimate 3000 RS equipped with a diode array detector and coupled to Bruker QTOF Maxis with an electrospray ionization source. The analyses (injection volume: 2 µL) were performed on a reversed-phase column (Phenomenex Luna Omega C-18, 150 × 2.1 mm, 1.6 µm) employing a gradient of 10–100% CH₃CN in water over 10 min followed by 3 min at 100% CH₃CN (all solvents buffered with 0.1% formic acid) with a flow rate of 0.5 ml/min. Analyses were performed in positive mode.

Analysis was performed according to Becker et al. (2017) [46 (link)], and further details are given in Supplementary Materials, Section S1. Briefly, liquid chromatography (LC) separations were performed on a Luna Omega C₁₈ analytical column (100 × 2.1 mm, 1.6 µm particle size) from Phenomenex (Torrance, CA, USA). The LC system comprised an Accela 4 Open AS autosampler and a quaternary pump from Thermo Fisher Scientific (San Jose, CA, USA). Mass spectrometry was performed using a Thermo TSQ Vantage triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source operating in separate runs in positive or negative ionization mode. The acquisition of the selected compounds was achieved in multiple reaction monitoring (MRM) modes, where two transitions were selected for ion confirmation, with the most abundant transition used for quantification and the other one for qualification/confirmation purposes. MRM transitions can be observed in Table S4 (Section S1) for both negative and positive ESI polarities. Data acquisition and processing were performed using Xcalibur v4.3 software (Thermo Fisher Scientific, San Jose, CA, USA).