Quantitative analysis of sphingoid base-1-phosphates as bisacetylated derivatives by liquid chromatography-tandem...

Sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (DHS1P) are important signaling sphingolipids. The presence of nanomolar levels of S1P and DHS1P in tissues, cells, and biological fluids requires a highly sensitive and selective assay method for their reliable detection and quantitation. Preliminary findings employing positive ion electrospray ionization (ESI) liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis indicated significant sample carryover from previous injections of authentic standards of S1P and DHS1P. This article details a negative ion ESI LC-MS/MS technique following modification of the zwitterionic nature of S1P and DHS1P via derivatization. A highly selective and sensitive LC-MS/MS technique capable of reliable detection of less than 50 fmol of the derivatives of S1P and DHS1P without significant sample carryover was developed. Standard curves for S1P and DHS1P are linear over wide ranges (0-300 pmol) of analyte concentrations with correlation coefficients (r2) greater than 0.995. The levels of S1P and DHS1P in human platelet poor plasma were 590.8+/-42.1 and 130.7+/-20.7 pmol/ml, respectively. The levels of S1P and DHS1P in fetal bovine serum were 141.7+/-4.6 and 0.6+/-0.2 pmol/ml, respectively. The addition of sphingosine (1 microM) to human pulmonary artery endothelial cells in culture resulted in a more than 20-fold increase in the cellular level of S1P, whereas the level of DHS1P was unchanged.