Zorbax eclipse xdb phenyl column

As per the method described by Xiao et al. (10 (link)), samples were extracted with 80% methanol (1:40) at 50 °C for 4 h followed by cooling to room temperature. After centrifuging the extracts at 
15 000×g (model BS-SP-70BL; Biogenix System, Delhi, DL, India) for 15 min, the solvent was evaporated under reduced pressure and the obtained residue was dissolved in HPLC grade methanol (80%). The final solution was filtered through syringe filter (0.45 µm PVDF membrane) and injected to HPLC (Agilent Technologies, Wilmington, DE, USA) equipped with a reverse-phased ZORBAX Eclipse XDB-phenyl column (4.6 mm×250 mm, 5 µm particle size; Agilent Technologies) with the gradient elution solution A containing 80% acetonitrile, 2% acetic acid, 18% water and solution B containing 8% acetonitrile, 2% acetic acid and 90% water. The other parameters were adjusted as follows: solvent flow rate 1 mL/min, column oven temperature 25 ºC and injection volume 20 µL. Detection of gallic, syringic and cinnamic acids was performed at 276 nm, of chlorogenic, caffeic and ferulic acids at 325 nm, and of p-hydroxybenzoic and vanillic acids at 257 nm (model Spectro-4; Laby Instruments Industry). Results were expressed as mass fractions (g per 100 g of sample).

LC-MS analysis was conducted on lipid nanodiscs using Agilent Technologies 1200 Series Instrument with a G1316A variable wavelength detector set at λ = 210 nm, 1200 Series ELSD, 6110 quadrupole ESI-MS, using an Agilent Zorbax Eclipse XDB-Phenyl column (3 × 100 mm, 3.5 μm particle size, flow rate 1 mL/min, the mobile phases 0.05% formic acid in water and 0.05% formic acid in acetonitrile).

Three types of LC-MS/MS systems were used as follows: Surveyor plus LC/TSQ Quantum ultra EMR system (Thermo Fisher Scientific, San Jose, CA, USA), 1290 Infinity II LC /6495 Triple quad MS system (Agilent Technologies Palo Alto, CA, USA), and 1200 LC/6460 Triple quad MS system (Agilent Technologies, Palo Alto, CA, USA). Chromatographic separation was achieved using a Zorbax Eclipse XDB-Phenyl column (Agilent, 150 x 3.0 mm, 3.5 um). The mobile phase was a binary mixture of 0.1% formic acid in water (A) and acetonitrile containing 0.1% formic acid (B) in a gradient elution mode at a flow rate of 300 μL /min. The gradient elution profile was 10–90% (B) for 10 min, 90% (B) for 2 min, and 10% (B) for 8 min to condition and the injection volume is 10 μL. The samples were analyzed in positive ion electrospray ionization mode with a spray voltage of 4 kV under an N₂ sheath gas flow rate of 30 arbitrary units. The capillary temperature was maintained at 300°C. The multiple-reaction monitoring (MRM) transitions monitored were as follows: for ENR, 360.2 m/z → 316.2 m/z (quantifier ion) and 360.2 m/z → 342.2 m/z (qualifier ion); and for ENR-d₅ (IS): 365.2 m/z → 321.2 m/z (quantifier ion) and 360.2 m/z → 347.2 m/z (qualifier ion).