UPLC–MS/MS method for analysis of sphingosine 1-phosphate in biological samples

A simple and sensitive liquid chromatography–tandem mass spectrometry (negative ion-electrospray ionization) methodology to determine sphingosine 1-phosphate (S1P) and sphinganine 1-phosphate (DH-S1P) in biological samples is described. The method has been validated over the linearity range of 2–100 ng/ml (r > 0.999) using synthetic C17-sphingosine 1-phosphate (C17-S1P) as an internal standard. In multiple reaction monitoring analysis (378.2 > 79.2), the lower limit of quantification for S1P was 5.0 ng/ml but the detection limit for the bioactive lipid was below 5 pg (12 fmol). Chromatographic separation was achieved on a UPLC BEH Hilic column with a binary mobile phase consisting of 30 mM ammonium acetate (pH 4.0) and acetonitrile/MeOH/30 mM ammonium acetate buffer (pH 4.0). The methodology detected 176.7 ± 54.0 ng/ml of S1P and 81.2 ± 23.3 ng/ml of DH-S1P in human plasma, as well as 201.0 ± 72.0 ng/ml of S1P and 96.5 ± 20.1 ng/ml of DH-S1P in mice plasma.

Research highlights▶ A simple LC-MS/MS procedure for detection and quantitation of phosphosphingolipids. ▶ HPLC separation uses ionic strength buffer eluants to reduce carry-over effects. ▶ Negative mode detection reduces interference from other molecules in MS analysis. ▶ MRM quantitation provides sensitive and specific response for the bioactive lipids. ▶ Method response is linear at the physiological range for S1P in mammalian tissues. ▶ Method sensitivity (LD below 5 pg) allows analysis of small-amount samples.