Determination of free sphingosine in biological systems by High Performance Liquid Chromatography

A procedure is described for the extraction, purification and determination of sphingosine from biological matrixes. After being frozen and pulverized, the lipidic fraction of these matrixes is isolated by liquid-liquid extraction. The lipidic extract is then purified by solid phase extraction technique with an LC-18 cartridge and, after derivatization with orthophthaladehyde, the sphingosine is separated and determined by HPLC. This technique has been applied to the study of sphingosine on cultures of human lymphocytes transformed using Epstein-Barr virus. It is also been applied to the evaluation of sphingosine in rat brain, which has a high sphingolipid and sphingoid-bases cerebrosides content, for the purpose of evaluating any variations in the ageing process. The recoveries obtained were > 77.9 +/- 4.7 %, and > 77.5 +/- 4.6 %, respectively for cell cultures and rat brains. An evaluation was made of possible interferences represented by compounds having a structure similar to that sphingosine and present in the lipidic fraction of the tissues used by us.