Acquity liquid chromatography system

The concentrations of acylcarnitines in the plasma and PBMC samples were determined with a UPLC MS/MS method using a Waters ACQUITY liquid chromatography system and a Waters Quattro Micro or Waters Xevo TQ‐S mass spectrometer, as previously described (Liepinsh et al., 2017).

Sim‐H and Sim‐L were prepared as 10 mM solutions in water and DMSO, respectively, and stored at −78°C, and warmed to room temperature immediately prior to injection; 1 μL of each solution was added to 1 mL of water or Tris/HEPES (250 mM HEPES, 80 mM Tris, 10 μM free Ca²⁺, pH 7.2) buffer, respectively, and sampling by LC‐MS was begun immediately. Samples were kept at room temperature for the duration of the analysis. Analyses were performed using a Thermo Exactive mass spectrometer equipped with Waters Acquity liquid chromatography system. Instrument control was performed using Thermo Xcalibur Software. Electrospray source conditions were adjusted to maximize sensitivity; 3 μL of each sample was injected onto the system. The column was a 2.1 × 50 mm (3 μm) ACE equivalence C18. A mixed eluent gradient was employed and is described in Supporting Information Table S3. The four sample types were analysed sequentially; the 5 min analysis time for each sample provided a 20 min sampling frequency for each sample. Mass analysis was performed using MestReNova MS software (2016, V11.0), where the peaks were integrated to determine relative percentages of Sim‐L and Sim‐H over time.