Fractionation of lipid extract

Lipid extracts were fractionated according to da Costa et al. method⁴¹. Fractionation was performed by solid-phase extraction using a Supelclean™ LC–Si SPE Tube (bed wt. 500 mg, volume 3 mL cartridges, SUPELCO). Column was activated with 6 mL of n-hexane and then the sample of lipid extract (110 and 4 mg of macroalgae and sea slug, respectively) was applied after to be dissolved on 300 μL of chloroform. Subsequently, the following sequential elution was performed to separate lipid fractions: 5 mL (C. tomentosum)/4 mL (E. viridis) of chloroform, 8 mL (C. tomentosum)/3 mL (E. viridis) of diethyl ether:acetic acid (98:2, v/v), 5 mL of acetone:methanol (9:1, v/v) and 6 mL of methanol. Total lipid extracts were fractionated in four lipid fractions: fraction 1 (rich in neutral lipids), fraction 2 (rich in pigments), fraction 3 (rich in glycolipids) and fraction 4 (rich in betaine lipids and phospholipids). Fractions 3 and 4 were recovered separated and dried under nitrogen stream and stored at −20 °C prior to analysis by HILIC-LC-MS.