Quantitative analysis of sphingosine-1-phosphate by HPLC after napthalene-2,3-dicarboxaldehyde (NDA) derivatization

Sphingosine-1-phosphate (SIP) is an important sphingolipid signaling molecule that regulates numerous cellular processes. In this paper we report anew method to quantify the levels of SIP in biological samples that relies on derivatization with napthalene-2,3-dicarboxaldehyde (NDA) and quantification by reverse-phase high performance liquid chromatography (HPLC). The limit of detection (LOD) using S1P standards was 20.9 fmol (12.6 nM), and the limit of quantification (LOQ) was 69.6 fmol (41.7 nM). The recovery of SI P standards was up to 97.5%. The mean relative standard deviations (RSD) for the intra- and inter-day assay were 4.1% and 7.7%, respectively. To validate this procedure, we quantified the SI P levels in plasma from human, horse, and mouse (mean values of 0.45, 0.25, and 1.23 mu M, respectively). We also used this technique to evaluate the SI P content in mouse tissues, as well as in rat neuronal cell cultures before and after sphingosine treatment. The results demonstrate that this new procedure can provide fast, sensitive, and reproducible S1P quantification, and offers several advantages over existing methods. The technique also may be used for determining the activity, as well as the inhibition, of sphingosine kinase. In the future it could be an important tool for investigators studying the role of SI P in signal transduction, cell growth and differentiation, and disease pathogenesis and treatment. (C) 2009 Published by Elsevier B.V.