Tracegold tg 5silms column

The dried samples, QC sample, and processing blank sample, were derivatized using methoxyamine hydrochloride (MOX) and N,O-Bis(trimethylsilyl)trifluoroacetamide (TMS) (Sigma). Briefly, dried extracts were reconstituted in 30 μL of 11.4 mg/ml MOX in anhydrous pyridine (VWR), vortexed for 10 minutes, and heated at 60°C for 1 hour. Next, 20 μL TMS was added to each reconstituted extract, vortexed for 1 minute, and heated at 60°C for 30 min. The derivatized samples, QC samples and processing blank samples were immediately analyzed using GC/MS.
GC chromatographic separation was conducted on a Thermo Trace 1300 GC with a TraceGold TG-5SilMS column (0.25 μM film thickness; 0.25mm ID; 30 m length). 1 μL of derivatized sample, QC, or blank was injected. The GC was operated in split mode with the following settings: 20:1 split ratio; split flow: 24 μL/min, purge flow: 5 mL/min, Carrier mode: Constant Flow, Carrier flow rate: 1.2 mL/min). The GC inlet temperature was 250°C. The GC oven temperature gradient was as follows: 80°C for 3 min, ramped at 20°C/min to a maximum temperature of 280°C, which was held for 8 min. The injection syringe was washed 3 times with pyridine between each sample. Metabolites were detected using a Thermo ISQ single quadrupole mass spectrometer. The data was acquired from 3.90 to 21.00 min in EI mode (70eV) by single ion monitoring (SIM).

Gas chromatographic separation was conducted on a ThermoFisher Scientific Trace 1300 GC with a TraceGold TG-5SilMS column (0.25 μm film thickness; 0.25 mm ID; 30 m length; ThermoFisher Scientific), as described (Tompkins et al., 2019 (link)). The injection volume of 1 μL was used for all samples. The gas chromatograph was operated in split mode with the following settings: 20:1 split ratio; split flow: 24 μL/min, purge flow: 5 mL/min, Carrier mode: Constant Flow, Carrier flow rate: 1.2 mL/min). The inlet temperature was 250°C. The oven temperature gradient was: 80°C for 3 minutes, ramped at 20°C/minute to a maximum temperature of 280°C, held for 8 minutes. The injection syringe was washed 3 times with methanol and 3 times with pyridine between samples. Metabolites were detected using a ThermoFisher Scientific Q-Exactive Plus mass spectrometer. The data were acquired from 3.90 to 21.00 minutes in EI mode (−70eV) in full scan (m/z 56.7–850) at 60K resolution. Metabolite profiling data were analyzed using Tracefinder 4.1, utilizing standard verified peaks and retention times.