Sphingolipid analyses by HPLC–MS/MS

For the measurement of Sph, dhSph, S1P and dhS1P, the serum samples collected from the study subjects were removed from − 80 °C storage just before the measurement. Sphingolipids were measured in duplicate of each sample (100 μL each), using a High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC–MS/MS) methodology as previously described with some modifications[9, 17]. Briefly, the samples were mixed with 10 μL of internal standards (S1P-d7; Avanti, Alabama, USA) and 500 μL of extraction solution consisting of isopropanol, methanol and formic acid (45:45:10 v/v). Then, the mixtures were vortexed, sonicated and centrifuged at 1500×g for 10 min at 4 °C. Supernatants were diluted with 30% methanol before analysis. The samples were analyzed by positive-ion Ultrahigh-Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (UPLC-ESI–MS) in a multiple reaction monitoring mode performed on a Shimadzu LC-20AD liquid chromatography instrument (Shimadzu, Japan) and coupled to an QTrap5500 mass spectrometer (Applied Biosystems Sciex, Canada). Chromatographic separations were performed under a gradient elution on an Eclipse XDB-C8 (2.1 × 100 mm, 1.8 μm) column (Agilent Technologies) using a binary solvent system at a flow rate of 300 μl/min. Prior to sample injection, the column was equilibrated for 2 min with a solvent mixture of 70% mobile phase A (methanol/H2O/formic acid, 69/29/2/, v/v/v, with 2 mmol ammonium formate) and 30% phase B (methanol/tetrahydrofuran/formic acid, 97/2/1, v/v/v, with 2 mmol ammonium formate) as previously described[18]. After injection, the mobile phase A/B ratio was maintained at 70/30 for 2 min, followed by a linear gradient to 100% B over 5 min. The flow was then maintained for 10 min at 100% B, followed by a wash of the column with 70:30 A/B for 0.5 min before the next run. The mass transitions were m/z 300.2 → 282.2 for Sph, 302.2 → 254.2 for dhSph, 380.2 → 264.2 for S1P, 382.2 → 266.2 for dhS1P, 387.3 → 271.3 for S1P-d7, respectively. Levels of Sph, dhSph, S1P and dhS1P were quantified using external standard curves ranging from 0.1 to 1000 nmol, and normalized to each the synthetic internal standard (Avanti, Alabama, USA). All of the data were acquired and processed using Analyst 1.6 software from Sciex Inc., Concord, ON, Canada.