Quantification of Circulating Sphingolipids

CS levels in mouse and human plasma were determined using liquid chromatography tandem-mass spectrometry (LC-MS/MS) as previously described (Bandaru and Haughey 2014 (link)). Briefly, 30 μL of plasma was mixed with 120 μL of honokiol internal control solution (honokiol dissolved in methanol, 200 ng/ml). After centrifugation at 10,000 g for 15 min, 0.2 μL of the supernatant was injected into a Shimadzu LC-20AD HPLC. Chromatography was conducted using an Agilent Extend 5 μM C18 column (Agilent Technologies, Inc., Santa Clara CA, United States) with solvent A (pure methanol) and solvent B (0.01% ammonia) under the gradient conditions of 60% A at 0–1 min and gradient to 95% A at 1–2.2 min at a flow rate of 0.5 ml/min. Quantification of CS was performed on an AB SCIEX Triple Quad 5500 LC/MS/MS system (AB SCIEX, United States) by multiple reaction monitoring (MRM) using Analyst 1.6.2 software (Applied Biosystems). CS stock solution with a concentration range of 0.5 ∼3.0 μg/ml was used as an internal standard, and calibration curves were plotted using the peak area ratios of CS to honokiol (internal control).