Comparative quantification of sphingolipids and analogs in biological samples by high-performance liquid chromatography after chloroform extraction

Sphingosine 1-phosphate (S1P) is an extra- and intracellular messenger that specifically activates five G-protein-coupled cell surface receptors designated S1P_1-5. The S1P₁ receptor is particularly important for the maintenance of immune surveillance by regulating egress of lymphocytes from thymus and secondary lymphoid organs. S1P is generated through phosphorylation of sphingosine which is catalyzed by sphingosine kinase types 1 and 2. The immunosuppressant and sphingosine analog Fingolimod (2-amino-2-(2-4-octylphenyl]ethyl)-1,3-propanediol, FTY720) can also be phosphorylated and induces lymphopenia by downregulating cell surface expression of the S1P₁ receptor on lymphocytes. To analyze the role of S1P in lymphocyte circulation and distribution we established a high-performance-liquid-chromatography-based method for parallel detection and quantification of Fingolimod, sphingosine, and dihydrosphingosine together with their phosphorylated derivatives Fingolimod-phosphate, S1P, and dihydrosphingosine 1-phosphate. Phosphorylated and nonphosphorylated lipids were efficiently isolated from biological samples such as cells, tissues, serum, plasma, and media by simple chloroform extraction. Fluorescence labeling with 9-fluorenylmethyl chloroformiate ensured high selectivity and enhanced sensitivity for sphingolipid detection. The described method provides an accurate approach to investigate phosphorylation, dephosphorylation, hydrolyzation, and dehydrolyzation of sphingolipids and analogs. In addition it works independently from enzymatic conversions, measuring actual concentrations rather than enzymatic activities.