The sphingolipids content were measured with the use of UHPLC/MS/MS according to Blachnio-Zabielska et al. [23 (link)] with minor modifications. Briefly, muscle samples (~20 mg) were pulverized and then homogenized in a buffer consisting of 0.25 M sucrose, 25 mM KCl, 50 mM Tris, and 0.5 mM EDTA, pH 7.4. Immediately afterwards, a mixture of internal standards (Sph-d7, SPA-d7, S1P-d7, C15:0-d7-Cer, C16:0-d7-Cer, C18:1-d7-Cer, C18:0-d7-Cer, 17C/20:0-Cer, C24:1-d7-Cer, C24-d7-Cer Avanti Polar Lipids, Alabaster, Al) and extraction mixture (isopropanol:water:ethyl acetate, 30:10:60); v:v:v) have been added to each sample. After extraction, the samples were evaporated under a stream of nitrogen and suspended in LC Solvent B (2 mM ammonium formate, 0.1% formic acid in methanol) for UHPLC/MS/MS analysis. The chromatographic separation was performed using a reverse-phase Zorbax SB-C8 column 2.1 × 150 mm, 1.8 μm (Agilent Technologies, Santa Clara, CA, USA) in binary gradient using 1 mM ammonium formate, 0.1% formic acid in water as solvent A, and 2 mM ammonium formate, 0.1% formic acid in methanol as solvent B at the flow rate of 0.4 mL/min. Sphingolipids were analyzed using Sciex QTRAP 6500 + triple quadrupole mass spectrometer (AB Sciex Germany GmbH, Darmstadt, Germany) with multiple reaction monitoring (MRM) against standard curves constructed for each compound.
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