*4.3. Total Lipid Extraction*

Lipid extraction was performed by adding 3.75 mL of a mixture of methanol/chloroform (2:1, *v*/*v*) to 150 mg of ascidians and 250 mg of seaweeds (five biological replicates per biological matrix tested) in a glass test tube with a Teflon-lined screw cap. Samples were then homogenized and incubated in ice on a rocking platform shaker (Stuart Scientific STR6, Bibby, UK) for 2 h and 30 min. The mixture was centrifuged at 2000 rpm for 10 min., and after the organic phase was collected. The biomass residue was re-extracted two times with 2 mL of methanol and 1 mL of chloroform. Afterward, water was added (2.3 mL) to the total collected organic phase, centrifuged at 2000 rpm for 10 min and the organic (lower) phase was recovered. Solvents were dried under a stream of nitrogen gas. Total lipid extract was estimated by gravimetry. Lipid extracts were stored in dark vials and stored at −20 ◦C before analysis by gas chromatography-mass spectrometry (GC–MS). Reagents were purchased from Fisher Scientific Ltd. (Loughborough, UK). All other reagents were purchased from major commercial suppliers. Milli-Q water (Synergy, Millipore Corporation, Billerica, MA, USA) was used.
