4.5. Lipid Extraction

Lipid extraction was performed in 15 mL glass centrifuge tubes. First, 50 µL of a prepared internal standard stock solution (Table 1) was added to the tubes. The solvent was removed by vacuum centrifugation. Afterwards, an amount of 2 mg liver tissue (wet weight) homogenized in either H₂O, H₂O/MeOH = 1/1 (v/v), H₂O/MeOH = 1/1 +1% SDS, or MeOH was added. Sample material from pellet resuspension (Figure 3 and Figure 6) and recovery of residues (Figure 4) was suspended in MeOH/CHCl₃ (2:1 v/v). The concentration of the liver homogenates was adjusted to 0.05 mg/µL resulting in a sample volume of 40 µL. Data presented in Figure 1 were generated from liver homogenates adjusted to 0.2, 0.1, 0.05, 0.02, and 0.01 mg/µL and the corresponding sample volume used for extraction was 10, 20, 40, 100, and 200 µL, respectively. Of note, for volume spikes >40 µL, the solvent composition of the extraction (H₂O, MeOH, and CHCl₃) was reduced accordingly.

The lipid extraction was performed according to the protocol described by Bligh and Dyer [18]. A volume of 0.8 mL H₂O and 3 mL MeOH/CHCl₃ (2:1 v/v) was added to each sample. The suspension was mixed by vortexing and incubated for 1 h at room temperature. Subsequently, 1 mL H₂O and 1 mL CHCl₃ was added to obtain a final solvent ratio of 1.8:2:2 for H₂O:MeOH:CHCl₃. Samples were mixed and centrifuged for 10 min at 4000 rpm (17,860× g) inducing phase separation. A volume of 500 μL of the chloroform phase was transferred into a sample vial by a pipetting robot (Tecan Genesis RSP 150, Männedorf, Switzerland) and vacuum dried. The residues were dissolved in 1 mL of 7.5 mM ammonium formate in chloroform/methanol/2-propanol (1:2:4 v/v/v).