Synergi fusion c18 column

Oleic acid, hydrolysis product of 2-OG, was analysed in negative ion mode (NIM) employing a TSQ Quantum Access MAX triple quadrupole mass spectrometer (Thermo) equipped with a heated electrospray ion source (H-ESI). The following parent-product ion transitions were selected: oleic acid: m/z 281.2 → 281.2 ([M-H]-; Tube Lens 78 V; Collision Energy: 5 eV), HDA (IS): m/z 269.2 → 269.2 ([M-H]-; Tube Lens 78 V; Collision Energy: 5 eV). A Phenomenex Synergi Fusion C18 column (100 × 2.0 mM, 4 μm) and the following gradient conditions were employed: A, 0.1% ammonium hydroxide in water; B, 0.1% ammonium hydroxide in methanol (B) at a flow rate of 350 μl/min. After 1 min at 60%A, a linear gradient was applied to 5%A in 7 min; then, after further 3 min at 5%A, it returned to 60%A, followed by 3 min reconditioning. Total run time was 14 min. H-ESI parameters were set as follows: probe middle (D) position; capillary temperature: 270 °C; ion spray voltage: −2.5 kV, vaporizer temperature: 250 °C. Nitrogen was used as nebulizing gas at the following pressure: sheath gas: 35 psi; auxiliary gas: 15 arbitrary units (a.u.). Argon was used as the collision gas at a pressure of approximately 1.5 mtorr (1 torr = 133.3 Pa). Data acquisition and processing were performed using Thermo Xcalibur software (version 2.1).

A portion of CCM (2.8 g) was fractionated on a Sephadex LH-20 column (1 m × 5 cm) using MeOH as eluent, at a flow rate of 1 mL/min. The fractions, 7 mL each, were collected and combined into four main groups (I–IV) after TLC analysis (Si-gel, n-BuOH AcOH-H₂O (60:15:25)). The effect of fractions I (580.36 mg), II (450.24 mg), III (543.18 mg), and IV (474.34 mg) on AGS cell viability was investigated.
Bioactive fractions II and III were purified via RP-HPLC with a refractive index detector on a Synergi Fusion C₁₈ column (250 × 10 mm i.d., 4 μm, Phenomenex, Torrance, CA, USA), at a flow rate of 2 mL/min.
Fraction II (318.8 mg) was purified with the elution solvent MeOH/H₂O 3.5:6.5 v/v, yielding the following compounds 1 (0.6 mg, t_R = 35 min) and 2 (0.6 mg, t_R = 50 min). Fraction II (20.0 mg) was also purified using a mixture of MeOH/H₂O 5:5 v/v as a mobile phase, isolating the compounds 3 (0.7 mg, t_R = 19 min) and 4 (0.9 mg, t_R = 23 min).
Fraction III (358.2 mg) was purified with the elution solvent MeOH/H₂O, 3.5:6.5 v/v to obtain the following compounds: 5 (1.0 mg, t_R = 35 min), 6 (0.9 mg, t_R = 43 min), and 7 (1.2 mg, t_R = 62 min). Fraction III (58.3 mg) was also purified using a mixture of MeOH/H₂O 5:5 v/v as a mobile phase, isolating compounds 8 (0.2 mg, t_R = 25 min), 9 (1.2 mg, t_R = 20 min), and 10 (2.0 mg, t_R = 23 min).