FIGURE 3.

Procedure of sphingolipid extraction. Prior to extraction, fungal cell extracts or lyophilized media are spiked with suitable internal standards. First extraction is performed using Mandala extraction buffer (ethanol: dH₂O: diethyl ether: pyridine: 4.2N NH₄OH; 15: 15: 5: 1: 0.018; v/v). Subsequently, a second Bligh and Dyer extraction is performed using methanol and chloroform. Samples at this stage can be used to determine the Pi content, lipid dry weight or to purify specific sphingolipid components using solid phase extraction. Finally, samples are base hydrolyzed using mild alkaline base hydrolysis (0.6 M KOH in methanol) to remove the glycerol backbone containing lipid contaminants. For certain fungal masses it is required to first crush the sample using glass beads in the lipid extraction buffer itself or to make the sample into powder using pestle and mortar in liquid N₂ prior to extraction.