Liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements of plasma sphingolipids

All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) and of reagent grade unless otherwise specified. d16:1 S1P was custom-synthesized as described in the Supplementary Information. An internal standard solution (ISTD) consisting of 20 ng/mL ¹³C₂D₂–d18:1 S1P in methanol was prepared. Ten microliters of each plasma sample was used for S1P extraction. Plasma samples were mixed with 100 μL of ISTD and sonicated for 30 min at 23 °C. Samples were then centrifuged at 16,000g for 10 min at 23 °C, after which the supernatant was aliquoted and vacuum-dried (Savant SpeedVac® vacuum concentrator, Thermo Fisher Scientific, Waltham, MA, USA). Dried lipid extracts were resuspended in 100 μL methanol and sonicated for 10 min at 23 °C. Derivatization of S1P was then carried out by adding 10 μL of TMS-diazomethane and shaking on thermomixer at 700 rpm for 20 min at 23 °C. After 20 min, 1 μL of 100% acetic acid was added to stop the derivatization reaction. Samples were vortexed, then centrifuged at 16,000g for 10 min to remove potential debris before transferring into a 96-well plate. Plates were stored at 4 °C until LC-MS/MS analyses using an Agilent 1290 UPLC system connected to an Agilent 6495 Triple Quadrupole mass spectrometer operated in positive ion MRM mode (Agilent Technologies, Santa Clara, CA, USA). The column utilized was an Acquity® hydrophilic interaction chromatography (HILIC) column (100 × 2.1 mm, 1.7 μm particle size) (Waters Corporation, Milford, MA, USA). MS source parameters used gas temperature of 200 °C with gas flow of 12 L/min and nebulizer at 25 psi. Sheath gas temperature of 400 °C with gas flow of 12 L/min. Solvents used for the HILIC are 50% acetonitrile in water containing 25 mM ammonium formate pH 4.7 (solvent A) and 95% acetonitrile in water containing 25 mM ammonium formate pH 4.7 (solvent B). Analytes were eluted with the following gradient: 0.1% solvent A and 99.9% solvent B from 0 to 5 min, 60% solvent A and 40% solvent B from 5 to 5.5 min, 90% solvent A and 10% solvent B from 5.5 to 6.6 min, 0.1% solvent A, and 99.9% solvent B from 6.6 to 9 min with a constant flow rate of 0.4 mL/min. After collision-induced dissociation of the S1P precursors, two productions were produced and monitored: m/z 60 was used as a “quantifier” and m/z 113 was used as the “qualifier.” These two ions were present after fragmentation of all S1P molecular species. The ion with m/z 60 represents the trimethylated amine fragment while the ion with m/z 113 represents the mono-methylated phosphate as previously described [24]. Quantification of the four S1P isoforms (Table 1) was performed based on the internal standard method, calibrating peak areas of the sample to the ISTD. Data were extracted and analyzed using Agilent MassHunter Qualitative and Quantitative software (Santa Clara, CA, USA). Principle Components Analysis (PCA) was carried out to ensure the results’ quality and validity.

Nomenclature and probable molecular structures of S1P isoforms quantified in this study