Round bottom culture tubes

AT was homogenized in 1 mL of MeOH by FastPrep24™ 5G (3x30s, Lysing Matrix D mode) with cooling on ice after each homogenization round. Homogenate were transferred into glass tubes (11.5 mL, round bottom culture tubes, VWR) using glass Pasteur pipettes. Beads and lysing tubes were washed with MTBE (1000 μL), and the solution was transferred to a glass tube. MTBE (3.95 mL) and MeOH (500 μL) were added (to reconstitute ratio MTBE/MeOH = 3.3:1, v/v). Mixture was incubated on roller mixer (4°C, 1 h, 210 rpm), and H₂O (1240 μL) was added (to reconstitute ratio MTBE/MeOH/H₂O = 3.3:1:0.8, v/v), incubated on roller mixer (4°C, 10 min, 210 rpm), and centrifuged to achieve phase separation (4°C, 10 min, 2000 × g). Upper organic phase with collected with Pasteur pipette. Re-extraction was done by adding MTBE/MeOH/H₂O (1.95 mL; 10:3.3:2.5, v/v) with subsequent incubation on the roller mixer (4°C, 10 min, 210 rpm) and centrifugation (4°C, 10 min, 2000 × g) to achieve phase separation. Organic phases were combined and dried in vacuo (Eppendorf concentrator 5301, 1 mbar).

AT was homogenized in 1 mL of MeOH by FastPrep24™ 5G (3x30s, Lysing Matrix D mode) with cooling on ice after each homogenization round. Homogenate were transferred into glass tubes (11.5 mL, round bottom culture tubes, VWR) using glass Pasteur pipettes. Beads and lysing tubes were washed with hexane (Hex; 1000 μL), solution was transfer to glass tube. Hex (350 μL), i-PrOH (350 μL), HOAc (105 μL; 100%, Roth), and H₂O (1.125 mL) were added to glass tube (to reconstitute ratio HOAc/i-PrOH/Hex = 2:20:30, v/v; Mix/H₂O = 2.5:1, v/v). Mixture was incubated on a roller mixer (4°C, 1 h, 210 rpm), Hex (3.75 mL) was added, incubate on the roller mixer (4°C, 10 min, 210 rpm), and centrifuged to achieve phase separation (4°C, 10 min, 2000 x g). Upper organic phase was collected with Pasteur pipette. Re-extraction was done by adding Hex (3.75 mL) with subsequent incubation on the roller mixer (4°C, 10 min, 210 rpm), and centrifugation (4°C, 10 min, 2000 x g) to achieve phase separation. Organic phases were combined and dried in vacuo (Eppendorf concentrator 5301, 1 mbar).

AT lipid extract corresponding to 20 (for workflow optimization) or 45 mg (for LC-MS analysis) of AT was dried in a glass tube (11.5 mL, round bottom culture tubes, VWR). Hex (4.5 mL) and EtOH/H₂O (1.5 mL, 87:13, v/v) were added, tubes were vigorously vortexed (Tube A). Separated EtOH/H₂O phase was transferred into second glass tube containing 4.5 mL Hex (Tube B), vigorously vortexed, and the resulting EtOH/H₂O phase was transferred into an empty glass tube (Tube C). New portion of EtOH/H₂O (1.5 mL, 87:13, v/v) was added to the remaining Hex phase in Tube A, vortexed and transferred to Tube B, vortexed again and transferred to Tube C. This procedure was repeated six more times (in total eight times of EtOH/H₂O extraction from the original Hexane fraction). All collected EtOH/H₂O phases were combined and dried in vacuo (Eppendorf concentrator 5301, 1 mbar).