Xcalibur processing software

Lipid concentrations were determined using an Accela1250 HPLC system coupled with an ESI–MS Orbitrap Exactive (ThermoFisher Scientific) as described by de Kok et al. (33 ). In short, samples were prepared by applying 150 µl distilled 1-butanol, mixing by vortex and separating by centrifugation (16.000 RCF, 2 min), and this process was repeated once more. The lipid layer containing 1-butanol was collected in glass vials and dried under a nitrogen gas stream. Lipid films were subsequently dissolved in methanol and injected on a Waters ACQUITY Premier CSH C18 (1.7 µm, 2.1 × 150 mm) column. Lipid separation was achieved by applying a changing gradient of eluent A: MQ:MeCN (40:60) containing 5 mM ammonium formate and eluent B: MQ:MeCN:1-BuOH (0.5:10:90) also containing 5 mM ammonium formate. Mass spectrometry specifications and settings have been described previously (34 (link)). Thermo Scientific XCalibur processing software was used to analyze spectral data by applying the Genesis algorithm-based automated peak area detection and integration. Total ion counts for each extracted lipid were normalized for the internal standard (10:0 PG).