Sphingolipid extraction and ultraperformance liquid chromatography-MS/MS analysis

Lipid extractions were carried out as previously described (17) using a single-phase system to maximize recovery (18). Briefly, plasma (50 μl) was added to ice-cold ethyl acetate:isopropanol:water (6:3:1, v/v/v) and spiked with 4 ng each of deuterated internal standards for sphingosine (sphingosine-d7; Avanti Polar Lipids, Alabaster, AL) and S1P (S1P-d7; Avanti Polar Lipids). Samples were then incubated on ice for 30 min, centrifuged to pellet out the denatured proteins, and the supernatant was collected and dried down under a gentle stream of nitrogen. The resulting lipid residues were resuspended in methanol containing 0.1% (v/v) formic acid (mobile phase B) and stored at −20˚C until analysis. Assay recoveries for sphingosine-d7 and S1P-d7 were 87.7% and 90.1%, respectively. Sphingosine and S1P were analyzed by ultraperformance liquid chromatography coupled to electrospray ionization MS/MS, as previously described (19), using an Acquity ultraperformance liquid chromatography system (Waters Corporation, Wilmslow, UK) paired with a triple quadrupole mass spectrometer (Xevo TQ-S; Waters Corporation); data acquisition was carried out using MassLynx software (Waters Corporation). Separation was performed using a reverse-phase Acquity BEH C8 column (1.7 μm 2.1 × 100 mm) at a flow rate of 0.3 ml/min and a column temperature of 30˚C. Separation was performed using a gradient system of mobile phase A (water containing 0.1% [v/v] formic acid) and mobile phase B (methanol containing 0.1% [v/v] formic acid). Electrospray ionization was performed in positive-ion mode using the following settings: capillary voltage, 3.5 kV; source temperature, 100˚C; and desolvation gas temperature, 450˚C. Analytes were quantitated using multiple reaction monitoring: S1P m/z 380.249 > 264.278; S1P-d7 m/z 387.292 > 271.25; and sphingosine m/z 300.332 > 282.205; sphingosine-d7 m/z 307.300 > 289.300.

Calibration lines were constructed using synthetic standards (Avanti Polar Lipids) to allow accurate quantitation. Results are expressed as nanogram/milliliter plasma and have been converted to nanomoles/milliliter to aid comparison with previously published results.