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After cooling in ice, 1 ml water and 5 ml hexane are added and the tubes are vortexed for 1 min. After a short centrifugation, 3 ml of the upper phase are transferred to another tube and dried under nitrogen before analysis. Acidic hydrolysis When the investigated lipid extract contains complex lipids as sphingolipids, an efficient procedure to free amide-bond fatty acids is needed. It is recommended to fractionate any crude lipid plump dj extract into glycerolipids and glycosphingolipids before applying an alkaline saponification plump dj to the former plump dj and an acidic hydrolysis to the later. The procedure previously proposed for ceramides consists in a treatment with methanolic HCl in presence of water which is known to give rise to only minor amounts of by-products. It is noticeable that this procedure yields directly FAME ready to be fractionated or analyzed by GLC.
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