2545 pump system

LCMS data were obtained using a Waters LCMS system comprising of a Waters 2767 autosampler, Waters 2545 pump system and a Phenomenex Kinetex column (2.6 μ, C₁₈, 100 Å 4.6 × 100 mm) equipped with a Phenomenex Security Guard precolumn (Luna C₅ 300 Å) eluted at 1 mL/min. Detection was performed by Waters 2998 diode array detector between 200 and 600 nm; Waters 2424 ELSD and Waters SQD-2 mass detector operating simultaneously in ES⁺ and ES⁻ modes between 100 m/z and 650 m/z. Solvents were A, HPLC-grade H₂O containing 0.05% formic acid; and B, HPLC-grade CH₃CN containing 0.045% formic acid. Gradients were as follows: Method 1 (optimised for non-polar compounds): 0 min, 10% B; 10 min, 90% B; 12 min, 90% B; 13 min, 10% C and 15 min, 10% B. Method 2 (optimised for polar compounds): 0 min, 10% B; 10 min, 40% B; 12 min, 90% B; 13 min, 10% B and 15 min, 10% B. HRMS was obtained using a UPLC system (Waters Acquity Ultraperformance, running the same method and column as above) connected to a Q-TOF Premier mass spectrometer.

The crude extract was dissolved in HPLC grade methanol and subjected to mass-directed preparative HPLC. Purification of compounds was achieved using a Waters mass-directed auto-purification system comprising of a Waters 2767 autosampler, Waters 2545 pump system, a Phenomenex LUNA column (5 μ, C₁₈, 100 Å, 10 × 250 mm) equipped with a Phenomenex Security Guard precolumn (Luna C₅ 300 Å) eluted at 16 mL/min. Solvents used were: A, HPLC grade H₂O + 0.05% formic acid; B, HPLC grade CH₃CN + 0.045% formic acid. The post-column flow was split (100:1) and the minority flow was made up with solvent A to 1 mL/min for simultaneous analysis by diode array detector (Waters 2998; Milford, MA, USA), evaporative light scattering (Waters 2424; Milford, MA, USA) and ESI mass spectrometry in positive and negative modes (Waters Quatro Micro; Milford, MA, USA). The gradient started with 40% to 80% B over 15 min, continued for 0.5 min to 95% and ended with 1 min isocratic conditions. Fractions of 24 consecutive runs were combined to yield 9.5 mg of 3 and 9.0 mg of 4.