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.
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.
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