Synergi fusion rp 80a

The analysis of flavan-3-ols and dihydrochalcones at 280 nm, phenolic acid at 320 nm, flavonols at 360 nm and anthocyanin at 520 nm were carried out on a liquid chromatography model L-7455 with a quaternary pump model L-7100 equipped with a Multisolvent Delivery System model D-7000 HSM cooperated with a diode array detector (DAD), and an autosampler model L-7200 (Merck-Hitachi, Tokyo, Japan). The separation was performed on a Synergi Fusion RP-80A (150 × 4.6 mm, 4 μm; Phenomenex Torrance, California, USA) column at 30 °C and the flow rate was 1.0 mL/min. The mobile phase was composed of solvent A (2.5% formic acid in water, v/v) and solvent B (100% of acetonitrile). Elution was the same as described in Section 2.3. The retention times and spectra were compared with those of pure standards within 200–600 nm. The calibration curves were made from (−)-epicatechin, (+)-catechin, procyanidin B1, B2 and C1, chlorogenic acid, p-coumaric acid, phloretin-2′-O-glucoside, quercetin-3-O-glucoside, -3-O-galactoside, and -3-O-arabinoside as standards. Thus, phloretin-2′-xyloglucoside was quantified as phloretin-2′-O-glucoside, and p-coumaroylquinic acid was quantified as p-coumaric acid. All the samples were analyzed in triplicate and the results were expressed as g per kg dry weight (dw).

Unless otherwise stated, reagents, starting materials, and solvents were from Sigma-Aldrich (St. Louis, MO, USA). Reactions were carried out with magnetic stirring in round-bottomed flasks. Analytical thin layer chromatography (TLC) was performed on pre-coated glass silica gel plates 60 (F254, 0.25 mm, VWR International). Purifications were performed by flash column chromatography on silica gel (230−400 mesh, Merck Millipore). 1D and 2D NMR spectra were recorded with Bruker Avance (400 MHz) spectrometer, at room temperature. Chemical shifts are reported in δ values (ppm) relative to internal Me₄Si, and J values are reported in hertz (Hz). The following abbreviations are used to describe peaks: s (singlet), d (doublet), dd (double doublet), t (triplet), q (quartet), and m (multiplet). HR-MS experiments were performed using an LTQ-Orbitrap-XL-ETD mass spectrometer (Thermo Scientific, Bremen, Germany), using electrospray ionization. Analytical RP-HPLC was performed on a Phenomenex Synergi Fusion RP-80A (75 mm × 4.6 mm, 4 μm), with a flow rate of 1 mL/min, using a tunable UV detector at 254 nm. Mixtures of CH₃CN and 0.05% TFA in H2O were used as mobile phase. All compounds showed a purity ≥ 95%. The synthetic androgen R1881 was used at 1 or 10 nM. Enzalutamide (Selleckchem, Munich, Germany) was used at 10 μM.

The extraction procedure and both the separation and detection for ABA with tandem mass spectrometry (UPLC-MS/MS) were carried out according to [57 (link)]. Briefly, leaf or root samples were homogenized in liquid N₂ and extracted with methanol:water (2:1) to a final sample ratio of 100 mg FW mL⁻¹. UPLC-MS/MS analysis was performed on a Waters Acquity I class UPLC system coupled to a Waters Xevo TQ-XS (Milford, MA, USA), equipped with a UniSpray ion source (US) operated in timed MRM mode, with argon (Gruppo SIAD, Bergamo, Italy) as a collision gas. Separation was performed on a Waters Acquity HSS T3 column (1.8 μm, 100 mm × 2.1 mm) at 40 °C. For gradient elution, water and acetonitrile containing 0.1 v/v% formic acid were used. Data processing was performed using Waters MassLynx 4.2 and Target-Lynx software (Milford, MA, USA).
SA extraction was performed according to Pál et al. [76 (link)]. After separation on a reverse-phase column (Synergi Fusion-RP, 80A, 150 × 4.6 mm, 4μm, Phenomenex, Torrance, CA, USA) SA was quantified fluorometrically (W474 scanning fluorescence detector, Waters, Milford, CT, USA), with excitation at 305 nm and emission at 407 nm for SA.