Lc 6ad pump

Isolation of the alkaloids was performed on a semi-preparative scale on a Shimadzu chromatograph composed of a CBM-20A communication module, an SPD-20AV UV detector, a DGU-20A5 degasser, an LC-6AD pump, a 200-μL Rheodyne injection valve, and a Luna C18 column (250 × 15.00 mm, 5 μm) (Phenomenex, Torrance, CA, USA) with a flow rate of 3 mL/min. The mobile phase was composed of B (methanol) and A (formic acid 1% v/v in H₂O) with a linear elution gradient (1): 0–14 min 20–80% B, 20–30 min 80% B, and a linear elution gradient (2): 0–20 min 20–80% B, 20–35 min 80% B, 35–45 min 20–80% B. The UV detector was set to monitoring at 260 nm and 280 nm. The gradient (1): fractions containing lindoldhamine isomer (3.6 mg), stepharine (22.1 mg), and palmatine (8.7 mg); the gradient (2): fractions containing 5-N-methylmaytenine (11.2 mg) and N-trans-feruloyltyramine (3.0 mg) were collected and analyzed by high-resolution mass spectrometry (HRMS) and NMR spectroscopy.

Isolation of the alkaloids was performed on a semi-preparative scale on a Shimadzu chromatograph composed of a CBM-20A communication module, SPD-20AV UV detector, DGU-20A5 degasser, LC-6AD pump, 200 μL Rheodyne injection valve, and Luna C18 column (250 x 15.00mm, 5μm) (Phenomenex–Torrance, CA, USA) with a flow rate of 3 mL/min. The mobile phase was composed of B (methanol) and A (formic acid 1% v/v in H₂O), with a linear elution gradient: 0–20 min 20–80% B, 20–35 min 80% B, 35–45 min 20–80% B. The UV detector was set to monitoring at 260nm and 280nm. Fractions containing 5-N-methylmaytenine (11.2mg—1) and stepharine (22.1mg—2) were collected and analyzed by high-resolution mass spectrometry (HRMS) and NMR spectroscopy.

HRESIMS data were acquired with electrospray ionization in negative ion mode on an UltrOTOF-Q instrument (Bruker Daltonics, Billerica, MA, USA). NMR spectroscopic data were recorded at room temperature in CDCl₃, acetone-d₆, CD₃OD, and/or pyridine-d₅ (Cambridge Isotope Laboratories, Andover, MA, USA) on a Bruker DPX-300 spectrometer (Bruker, Karlhue, Germany) operating at 300.13 MHz (¹H)/75.47 MHz (¹³C). Standard pulse sequences were used for homo- and heteronuclear correlation experiments. Optical rotation was determined on a Perkin Elmer 341 polarimeter (λ = 589 nm, PerkinElmer Inc., Waltham, MA, USA). Column chromatography procedures were performed on silica gel 60 (70–230 mesh, Merck, Darmstadt, Germany), silica gel 60 RP-18 (230–400 mesh, Merck, Darmstadt, Germany) and Sephadex LH-20 (Amersham Biosciences, Buckinghamshire, UK). Reversed-phase semipreparative HPLC separations were carried out with a Shimadzu (Shimadzu, Kyoto, Japan) LC-6AD pump using a Phenomenex Luna RP-18 column (5 µm, 21.6 × 250 mm) at flow rates of 12 or 14 mL/min, with monitoring at 210, 230 or 254 nm.