Simultaneous determination of sphingosine and sphingosine 1-phosphate in biological samples by liquid chromatography–tandem mass spectrometry

d-erythro-sphingosine (Sph) and its phosphorylated product, d-erythro-sphingosine 1-phosphate (S1P) are sphingolipids mediating numerous cellular processes. Imbalance of Sph/S1P levels contributes to many diseases. Given the interconversion of these two opposing signaling molecules, it is essential to examine their levels simultaneously. In the present study, we developed a rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to simultaneously quantify the levels of Sph and S1P in biological samples using C17-Sph and C17-S1P as internal standards. With one step of methanol-induced protein precipitation, each sample was subjected to LC–MS/MS analysis using positive electrospray ionization under selected reaction monitoring mode. The running time was within 4 min with a simple mobile phase consisting of methanol–0.1% formic acid (95:5, v/v) at a flow rate of 0.2 mL/min. Standard curves were linear over ranges of 1–100 ng/mL for Sph and 0.1–10 ng/mL for S1P with correlation coefficient (r2) greater than 0.997. The lower limit of quantifications (LLOQs) were 1 ng/mL for Sph and 0.1 ng/mL for S1P. The intra-batch and inter-batch precision was less than 15% for all quality control samples. The recoveries of the method were found to be 76.36–89.84%. The method was applied to simultaneously determine the Sph and S1P levels in mouse kidney, human plasma, and HEK 293 cells treated with tumor necrosis factor-α (TNF-α) and N,N-dimethylsphingosine (DMS). The S1P levels increased in cells treated with TNF-α whereas decreased in cells treated with DMS. These results indicated that this new LC–MS/MS method was rapid, sensitive, specific and reliable to quantify Sph and S1P levels in biological samples simultaneously.