2.16. Sphingolipid Quantification by HPLC–MS/MS

Cells were incubated with 250 μM palmitic acid (16,16,16-d3) from Cortecnet (Voisins-le-Bretonneux, France) applied as a BSA complex for 16 h at 37 °C. Afterward, cells were pelleted, washed, and lysed in an aqueous buffered solution. Aliquots were subjected to lipid extraction using 1.5 mL methanol/chloroform (2:1, v:v), as described [36]. The extraction solvent contained d7-dihydrosphingosine (d7-dhSph), d7-sphingosine (d7-Sph), d7-sphingosine 1-phosphate (d7-S1P), C17-ceramide (C17:0 Cer) and C16-d31-sphingomyelin (C16:0 d31-SM) (all Avanti Polar Lipids, Alabaster, USA) as internal standards. Chromatographic separations were achieved on a 1260 Infinity HPLC (Agilent Technologies, Waldbronn, Germany) equipped with a Poroshell 120 EC-C8 column (3.0 × 150 mm, 2.7 µm; Agilent Technologies). MS/MS analyses were carried out using a 6490 triple-quadrupole mass spectrometer (Agilent Technologies) operating in the positive electrospray ionization mode (ESI+) [37]. MS/MS parameters for detection of canonical and deuterated sphingolipids are given in Supplementary Table S2. Quantification was performed with MassHunter Software (Agilent Technologies). Determined lipid amounts were normalized to the actual protein content (determined via Bradford assay) of the cell lysate aliquot used for extraction.