4.5. Analysis of Glycosphingolipids

Lipids were extracted by the tetrahydrofuran/phosphate buffer method [50], using 4 × 75 cm² dishes, for each sample. The aqueous phase obtained, containing gangliosides, was evaporated to dryness, resuspended, and exhaustively dialyzed against distilled water before drying. The gangliosides, resuspended in chloroform/methanol, 2/1, by vol, were analyzed using HPTLC (Silica gel 60 TLC) plates developed in chloroform/methanol/0.2% acqueous CaCl₂ 55/42/11, by vol (solvent system B). Visualization was performed with p-dimethylaminobenzaldehyde spray reagent. The organic phase obtained, containing neutral glycosphingolipids, was evaporated to dryness. Lipids, after mild alkaline hydrolysis, were dialyzed, collected and finally dried. The neutral glycosphingolipids were resuspended in chloroform/methanol, 4/1, by vol, and analyzed using HPTLC (Silica gel 60 TLC) plates developed in chloroform/methanol/formic acid/water, 65/25/8.9/1.1, by vol (solvent system C). Visualization was performed with a 10% CuSO₄/8% phosphoric acid aqueous spraying solution.