Briefly, five milligrams of tissue from each brain region were homogenized in 225 µL of −20 °C cold, internal standard-containing methanol using a GenoGrinder 2010 (SPEX SamplePrep) for 2 min at 1,350 rpm. The extraction methanol contained the following internal standards for quality control and retention time normalization: sphingosine (d17:1), LPE (17:1), LPC (17:0), MG (17:0/0:0/0:0), DG (12:0/12:0/0:0), PC (12:0/13:0), cholesterol-d7, SM (18:1/17:1), ceramide (d18:1/17:0), PE (17:0/17:0), TG (14:0/16:1/14:0)-d5, TG (17:0/17:1/17:0)-d5, acylcarnitine (18:1)-d3, fatty acid (16:0)-d3, MAG (17:0/0:0/0:0), PI (15:0–18:1)-d7, PG (17:0/17:0), PS (15:0-18:1)-d7, glucosylceramide(d18:1/17:0), mono-sulfo galactosylceramide(d18:1/17:0), and 5-PAHSA-d9. The homogenate was vortexed for 10 s. 750 µL of −20 °C cold, internal standard-containing methyl tertiary-butyl ether (MTBE) was added, and the mixture was vortexed for 10 s and shaken at 4 °C for 5 min with an Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments). MTBE contained cholesteryl ester 22:1 as internal standard. Next, 188 µL room temperature water was added and vortexed for 20 s to induce phase separation. After centrifugation for 2 min at 14,000×g, two 350 µL aliquots of the upper non-polar phase and two 125 µL aliquots of the bottom polar phase were collected and dried down. The remaining fractions were combined to form QC pools and were injected after every set of 10 biological samples.
The non-polar phase employed for lipidomics was resuspended in a mixture of methanol/toluene (60 µL, 9:1, v/v) containing an internal standard [12-[(cyclohexylamine) carbonyl]amino]-dodecanoic acid (CUDA)] before injection. Resuspension of dried polar phases for HILIC analysis was performed in a mixture of acetonitrile/water (90 µL, 4:1, v/v) containing the following internal standards: CUDA, caffeine-d9, acetylcholine-d4, TMAO-d9, 1-methylnicotinamide-d3, Val-Tyr-Val, betaine-d9, acyl carnitine (2:0)-d3, N-methyl-histamine-d3,