Luna omega ps c18 column

GLSs were analyzed using the same UHPLC system used for the determination of PAs. The separation was performed using a Luna Omega PS-C18 column (Phenomenex Inc., Torrance, CA, USA, 50 mm × 2.1 mm ID) at 40 °C and a flow rate of 0.4 mL min⁻¹. Phase A was 0.005 M TBAHS and phase B was methanol. Chromatographic separation was achieved using the following elution gradient: mobile phase A 70% (0 min), 60% (3 min), 60% (5 min), 70% (6 min), and 70% (8 min). The wavelengths of the four channels used for detection were 210, 220, 225, 237, and 280 nm. Clarity Chromatography software (8.6 version) for Windows (DataApex, Prague, Czech Republic) was used for data acquisition and elaboration. The external standard method was used for the calibration and a 100 µg mL⁻¹ stock solution of a mixture of the GLSs in methanol/phase A (30:70, v:v) was used to prepare the working solutions. Calibration curves were constructed for each standard compound, with a linearity between 1 and 20 μg mL⁻¹.

The polyphenols characterization was carried out by a reversed-phase liquid chromatography coupled with diode array detection and electrospray ion trap mass spectrometry (RP-LC-DAD-ESI-MS) analysis. At this purpose a Luna Omega PS C18 column (150 × 2.1 mm, 5 µm; Phenomenex, Torrance, CA, United States) at room temperature (RT) and with a flow rate of 0.4 mL/min was used. The mobile phase and elution program was the same reported in Smeriglio et al. (2018b) (link). The injection volume was 5 µl. The UV-Vis spectra were recorded in the 190–600 nm range and chromatograms were acquired at different wavelength (260, 292, 330 and 370 nm) to identified all polyphenol classes. The experimental parameters of the mass spectrometer (ion trap, Agilent model 6320) operating in both positive (ESI+) and negative (ESI−) ionization mode were set as follows: the capillary voltage was 3.5 kV, the nebulizer (N₂) pressure was 40 psi, the drying gas temperature was 350°C, the drying gas flow was 9 L/min and the skimmer voltage was 40 V. The mass spectrometer was operated in full-scan mode in the m/z range 90–1000. Data were acquired by Agilent ChemStation version B.01.03 and Software Trap control version 6.2.

The phytochemical profile of AGE was investigated by liquid chromatography coupled with diode array detection and electrospray/ion-trap tandem mass spectrometry (LC-DAD-ESI-MS/MS) analysis. Separation was carried out by a Luna Omega PS C18 column (150 × 2.1 mm, 5 μm; Phenomenex, Torrance, CA, USA) at RT and with a flow rate of 0.4 mL/min using 0.1% formic acid (Solvent A) and methanol (solvent B) as mobile phase, according to the elution program and mass spectrometer parameters reported in Smeriglio et al. [27 (link)]. Mass spectra were acquired using a fragmentation energy of 1.2 V (MS/MS). The peaks were identified by comparing retention times, UV–VIS and mass spectra of the analytes with those of commercially available HPLC-grade reference standards (purity ≥ 95%), as well as by comparison with those reported in literature, and quantified by using external calibration curves of oregonin (1–50 μg/mL) for diarylheptanoids and quercetin-3-O-glucoside for quercetin derivatives.

Polyphenols characterization of LE, FE, FrE, and BE was carried out according to Smeriglio et al. [29 ] by RP-LC-DAD-ESI-MS analysis. Separation was carried out by a Luna Omega PS C18 column (150 mm × 2.1 mm, 5 μm; Phenomenex, Torrance, CA, United States) at 25 °C by using a mobile phase consisting of solvent A (0.1% formic acid) and solvent B (methanol) according to the following elution program: 0–3 min, 0% B; 3–9 min, 3% B; 9–24 min, 12% B; 24–30 min, 20% B; 30–33 min, 20% B; 33–43 min, 30% B; 43–63 min, 50% B; 63–66 min, 50% B; 66–76 min, 60% B; 76–81 min, 60% B; 81–86 min, 0% B and equilibrated 4 min for a total run time of 90 min. The injection volume was 5 µL. The UV-Vis spectra were recorded ranging from 190 to 600 nm and chromatograms were acquired at different wavelengths (260, 292, 330, and 370 nm) to identified all polyphenol classes. The experimental parameters of the mass spectrometer (ion trap, model 6320, Agilent Technologies, Santa Clara, CA, USA) operating in negative (ESI−) ionization mode were set as follows: capillary voltage 3.5 kV, nebulizer (N₂) pressure 40 psi, drying gas temperature 350 °C, drying gas flow 9 L/min and skimmer voltage 40 V. Acquisition was carried out in full-scan mode (90–1000 m/z). Data were acquired by Agilent ChemStation software version B.01.03 and Agilent trap control software version 6.2.

The phytochemical profile of EE and HGE was elucidated by LC-DAD-ESI-MS analysis. Separation was carried out by a Luna Omega PS C18 column (150 mm × 2.1 mm, 5 µm; Phenomenex, Torrance, CA, USA) at 25 °C by using 0.1% formic acid (Solvent A) and acetonitrile (Solvent B) as a mobile phase according to the elution program reported in Danna et al. [71 (link)]. Five microliters of each extract were injected, and the UV–Vis spectra of analytes were recorded in the range of 190 to 600 nm. Chromatograms were acquired at 260, 292, 330, 370, and 520 nm, to detect all polyphenol classes, whereas the ion trap (model 6320, Agilent Technologies, Santa Clara, CA, USA) was carried out in full-scan mode (90–1000 m/z) following both positive and negative electrospray ionizaton (ESI) according to Danna et al. [71 (link)]. Identification was carried out by comparing the retention times and UV–Vis and MS spectra of each analyte with those of commercially available HPLC-grade standards (see Table 2), as well as with literature data and UV–Vis and mass spectra databases. Quantification was carried out by using external calibration curves of the authentic reference standard, when commercially available, or structurally similar compounds (see Table 2 footnotes). Results were expressed as mg of each compound/100 mL of LE ± standard deviation of three independent analyses in triplicate (n = 3).

To identify the product formed, 75μM FAICAR in 0.050M Tris buffer at pH 7.3 was incubated with purified enzyme (0.22 μM TK0430 or 0.28 μM AF1811) or a buffer blank at 60°C for 30 minutes, followed by HPLC on a Phenomenex Luna Omega PS C18 column (250 x 4.6 mm) with 5mM tetrabutylammonium phosphate and 60mM ammonium phosphate (95% aqueous, 5% methanol) as the mobile phase at pH 6.0, with a flow rate of 1 mL/min. Monitoring of absorbance was a 248nm. Retention times were compared to authentic IMP (75 μM in 0.050M Tris buffer at pH 7.3).