Measurement of sphingolipids using liquid chromatography tandem mass spectrometry (LC-MS/MS)

Sphingolipids were measured by a LC-MS/MS method modified based on previous publications [27, 28]. Analyses were performed using the Agilent 6460 triple quadrupole mass spectrometer coupled with the Agilent 1200 Rapid Resolution HPLC (Agilent Technologies, Santa Clara, CA) with detection of sphingolipids in positive mode by multiple reaction monitoring (MRM) technique. The HPLC mobile phases consisted of methanol-H₂O-formic acid (74:25:1, v/v/v; RA) and methanol-formic acid (99:1, v/v; RB); both RA and RB contain 5 mM ammonium formate. For measurement of Cers and sphingoid bases, Agilent column XDB-C18 (4.6 × 50 mm) with particle size of 1.8 μm, was used with isocratic run (100% B) or gradient (0–1 min, 20% B, 10–13 min, 100% B and 15–20 min at 20% B), respectively. For measurement of SMs, Agilent Zorbax XDB-C8 (2.1 × 50 mm) with particle size of 3.4 μm, was used with gradient (0–1 min, 20% B, 10–20 min, 100% B, 22–30 min, 20% B). The MS/MS parameters were as follows: gas temperature, 325–350 °C; gas flow rate, 7–10 L/min; nebulizer pressure, 45–50 psi; capillary voltage, 3500 V; The fragmentor voltage was 100 V and collision energy was 12–20 V. Precursor-to-product ion transitions for each sphingolipid were used according to the method of Merrill et al. [27].