2.1. Transesterification Procedure and Gas Chromatography (GC) Analysis

The transesterification protocol optimized for bovine hearth cardiolipin and applied to the phospholipids extracted from liposome suspensions was performed taking care of dry conditions, as follows: 0.5 mg of CL were dissolved in 0.5 mL of a 22 mM NaOMe solution in MeOH corresponding to 32 equiv. in respect of CL, i.e., 8 equiv. in respect of each FA chain (see in Supporting Materials the details of this preparation) and the resulting mixture was stirred for 2 h at room temperature in a close vessel, under nitrogen atmosphere. Thin-layer chromatography was used to monitor the progress of CL transformation. Eluent: CHCl₃/MeOH/H₂O 5:2:0.2, and R_f (lyso-CL) = 0.32, R_f (CL) = 0.65, R_f (18:2 acid) = 0.88, R_f (18:2 methyl ester) = 1, to also evidence the hydrolysis of the starting material. After the elapsed time, the reaction mixture was extracted with n-hexane (3 × 2 mL) containing 0.1 mL of a standard solution of 17:0 methyl ester; n-hexane was then evaporated, the residue dissolved in again in 1 mL of n-hexane and 1 µL was injected in GC.

Fatty acid methyl esters (FAME) were analyzed by GC (Agilent 6850, Agilent, Cernusco sul Naviglio, Milan, Italy), using the split mode (50: 1), equipped with a 60 m × 0.25 mm × 0.25 µm Zebron column (Phenomenex, Torrence CA, USA) and a flame ionization detector with the following oven program: temperature started from 165 °C, held for 3 min, followed by an increase of 1 °C /min up to 195 °C, held for 40 min, followed by a second increase of 10 °C /min up to 240 °C, and held for 10 min. A constant pressure mode (29 psi) was chosen with hydrogen as the carrier gas. FAMEs were identified by comparison with authentic samples and chromatograms were examined as described previously. Quantitative studies of the performed reactions were done by multiple-points calibration curves of the standard references (5 points). The GC peak areas were adjusted using a correction factor obtained by the recovery of the 17:0 methyl ester used as standard.