Simultaneous Sphingolipid and Ceramide Quantification

The concentration of the 10 sphingolipids and isotopic enrichment of [¹³C₁₆]16:0-ceramide in muscle were simultaneously measured against an extracted concentration standard curve as well as an enrichment standard curve on a Thermo TSQ Quantum Ultra mass spectrometer (Waltham, MA) coupled with a Waters Acquity UPLC system (Milford, MA). The sphingolipids were separated on the UPLC with a Waters Acquity C8 UPLC BEH column 2.1 × 150 mm, 1.7 μm at 43°C using two buffers. Buffer A was methanol, 2 mM ammonium formate, 0.1% formic acid; buffer B was water, 1 mM ammonium formate, 0.1% formic acid. The flow rate was 0.4 ml/min, and the gradient conditions were as follows: 0 min at 20% B, 0–1.5 min 20-10% B, 1.5–2.3 min isocratic at 10% B, 2.3–9.3 min 10-1%B, 9.3–11 min isocratic at 1%B, 11–11.3 min 1–20%B, 11.3–13 min isocratic at 20%B. Standards and samples were re-suspended in 50 μl buffer A prior to injecting 5 μl onto the UPLC/MS/MS. Figure 1 shows the separation of all species in the standards (panel A) and muscle (panel B).
The mass spectrometer was equipped with an electrospray ionization interface. The following conditions were used: the spray voltage set at 4000V, sheath gas at 0.675 L/min, ion sweep gas at 0.6 L/min, aux gas at 1.2 L/min, and transfer capillary at 275°C. The collision gas was set at 1.2 mTorr. All sphingolipids, except C16:0-Cer, were monitored as [M+H]⁺ in positive mode. The C16:0-Cer and [¹³C₁₆]16:0-Cer were monitored as [M+2+H]⁺ and [M+16+H]⁺ respectively. Transition of masses and collision energy are shown in Table 1. The entire analysis was performed in SRM mode.