Masshunter quantitative analysis

For GC-MS, the alignment was carried out with Agilent Mass Profiler Professional version 15.1 and exported into Agilent MassHunter Quantitative Analysis version 10.0 to assign target ions and obtain the compound abundances.
For CE-MS, raw data were processed with MassHunter Profiler software (version 10.0) applying the molecular feature extraction (MFE). All features extracted by MFE were aligned across all samples with the batch recursive feature extraction. The continuously infused references masses (m/z 121.0509 and m/z 922.0098) and those features found in blanks were excluded from the final list.
For both GC-MS and CE-MS analysis, metabolites with poor reproducibility (coefficient of variation (CV) in the QCs greater than 30%) and those features not presented in 70% of samples in at least one sample group were discarded. Regarding GC-MS, the final concentration of each metabolite was also normalized according to the IS abundance. Finally, the QC intensity drop was corrected, and the matrixes were further used for statistical analysis (full description in Supplementary Data 2).

The cerebral organoid extract was analyzed using a 1290 Infinity II UHPLC (Agilent) system coupled with the 6469 Triple Quadrupole mass spectrometer (Agilent). 1 µl of lipid extract was injected twice on the reverse phase microbore column (CSH, 1 mm *100 mm, 1.7 μm, Waters), separated at 100 µl/min flow rate over 15 min. For the gradient elution, mobile phase A was 10 mM ammonium formate in acetonitrile: water (60:40), and mobile phase B was 10 mM ammonium formate in isopropanol: acetonitrile (90:10). The gradient elution program was: 0 min 15% B, 1.86 min 30% B, 2.32 min 48%, 9.5 min 82% B, 12.5 to 13.5 min 99% B and 13.5 to 15 min column re-equilibration. The positive ion mode source parameters were: gas temp 200 °C, gas flow 14 l/min, nebulizer pressure 45 psi, sheath gas temp 400 °C, and sheath gas flow 8 l/min capillary voltage 4 kV, nozzle voltage 500 V, and unit resolution for Q1 and Q3. Data were acquired using the dynamic SRM mode [79 (link)–81 (link)], with a 2 min retention time window per transition. Raw data files were processed using Mass Hunter Quantitative analysis (B.07.00, Agilent Technologies) software. The concentrations of lipid species were calculated from the respective internal standard and further normalized to total lipid content to account for the variable sample amount.

All experiments were carried out on an Agilent 1200 HPLC system coupled to a 6460 Triple Quadrupole mass spectrometer equipped with a Jet stream ESI-source (Agilent Technologies, Waldbronn, Germany). The chromatographic separation was achieved temperature controlled at 25 °C on a Synergi Fusion-RP column (2.5 μm, 50 × 2.0 mm) equipped with a pre-column of the same material (4 × 2.0 mm) both from Phenomenex (Aschaffenburg, Germany). A gradient of mobile phase A (0.1 % formic acid) and mobile phase B (acetonitrile) was used as shown in Table S2 in the Supplementary Material (flow rate of 0.5 mL/min, injection volume of 10 μL). The MS was operated in positive ion mode with multi-reaction monitoring (MRM). The MS/MS fragmentation pattern of 6dEB and the internal standard compounds were determined and the MS parameters were optimized. The optimized source parameters are displayed in Table S3 in the Supplementary Material.
6dEB was monitored with transitions m/z 409.1 to m/z 311.2 for relative quantification and m/z 409.1 to m/z 391.2 and m/z 293.2 for identification. Collision energies for the transitions of 6dEB were 25, 21, and 25 V, respectively. Data was acquired and evaluated using the Mass Hunter software; quantitative analysis was done using Mass Hunter Quantitative Analysis (version B03.02, Agilent Technologies, Waldbronn, Germany).