Agilent 1260 2 uv

Sample fractionation was carried out on an Agilent 1100 G1312A binary pump coupled to an Agilent 1260 II fraction collector (Agilent Technologies, Waldbronn, Germany) with an Agilent 1260 II UV detector (Agilent Technologies, Waldbronn, Germany). Kinetex^® LC-C18 (4.6 × 250 mm, 5 µm, 100 A, Phenomenex) column was used for sample fractionation. The mobile phase for analysis consisted of: 5 mM ammonium formate and 0.1% formic acid in water (H₂O) (A) and MeOH (B). Chromatographic separation was performed by a slow linear gradient elution: Starting with 60% B to 100% B over 85 min. The mobile phase flow rate was 1 mL·min⁻¹ and the injection volume was 100 µL. Previous studies allowed to determine the chromatographic region where the CTX-like compounds elute optimizing the collection of the fractions in this region (data not shown). A total of 49 fractions were collected removing solvents under nitrogen. Cytotoxicity of each fraction was determined by N2a assay.

HPLC-C18 fractionation was carried out according to the conditions described in [35 (link)]. Briefly: The fractionation of extracts of dinoflagellates was carried out on an Agilent 1100 G1312A LC system coupled to an Agilent 1260 II automatic fraction collector with an Agilent 1260 II UV detector (Agilent Technologies, Waldbronn, Germany). A Kinetex® LC-C18 column (4.6 × 250 mm, 5 µm, 100 Å, Phenomenex) was used for the fractionation. Water with 5 mM of ammonium formate and 0.1% of formic acid (A) or methanol (B) constituted each mobile phase. The mobile phase gradient started from 60% B to 100% B, taking 85 min at a flow rate of 1 mL/min. The injection volume was 100 µL. A total of 49 fractions were collected. The solvent was removed by evaporation under N₂ at 50 °C and reconstituted in 1 mL of MeOH LC-MS.
The initial methanolic extract of the G. australes (IRTA-SMN-17-271) was fractionated under the above-described conditions. A portion (1 mL) of the initial methanol extract, containing the equivalent of 609,011 cells, was evaporated to dryness under N₂ at 50 °C and reconstituted in 100 µL of methanol and filtered through 0.22 µm (Syringe Driver filter Unit, Millex®-CV 0.22 um, 13 mm, Millipore, Billerica, MA, USA) before its injection into the HPLC system.