In addition to the samples to be analyzed, each sequence containe

In addition to the samples to be analyzed, each sequence contained several control runs including blank, chloroform and derivatized amino acid and organic acid standard mix samples before and after the biological samples to detect and potentially NSC683864 clinical trial correct for instrumental variation during the sample series. GC-MS was performed using an Agilent 7890GC-5975MS system, EI source operated at 70 eV, equipped with a 30 m × 250 µm × 0.25 µm Agilent 122-5532G DB-5MS+DG capillary column. The data acquisition

method Inhibitors,research,lifescience,medical was run in constant pressure mode with an operating pressure of 1 bar. D5-glutamate was used for retention time locking, which enabled retention time correction as columns were cut during maintenance operations. 2 µL sample was injected in pulsed split-less mode, and Inhibitors,research,lifescience,medical the metabolites were separated by using a 10 °C/min temperature gradient from 45 °C to 300 °C. The MS was operated in scan mode (start after 6 min, mass range 50–550 a.m.u. at 2.5 scans/s). The GC-MS data were analyzed semi-automatically using the Agilent ChemStation DRS (Deconvolution Reporting Software) and the AMDIS (NIST) deconvolution software using an in-house DRS library containing Inhibitors,research,lifescience,medical fifty metabolites. After automatic peak identification and integration, all compound

peaks were inspected visually for the correct peak selection Inhibitors,research,lifescience,medical (retention time, qualifier ions) and the consistent peak integration, and manual correction was performed if necessary. To further assess the resulting dataset, the average, standard deviation, minima and maxima in retention time for respective compound peaks found in the 32 to

36 GC-MS runs (one time-series distributed over up to three sequences) were calculated. By that means, potential errors concerning peak Inhibitors,research,lifescience,medical choice were identified and corrected. 3.5. LC-MS/MS Analysis LC-MS/MS analysis was based on the method introduced by Luo and co-workers [41] and performed on an Agilent 1200 series Amisulpride LC connected via an electrospray ion source to an Agilent 6410 triple quadrupole MS instrument. Forty-two common phosphorous containing metabolites were included in this MS/MS method, and collision energies were optimized for each individual metabolite. For the LC-MS/MS analysis, sequence variability was evaluated by quantification of the internal standards added to the samples prior to metabolite extraction. 3.6. Data Processing and Visualization LC-MS/MS determined absolute metabolite concentrations in extracts were normalized to the CDW to give concentrations in µmol/g CDW. The processed LC-MS/MS data are given in Supplementary Tables 1–3.

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