Enhanced chemstation software

An amount of 1 μL of each extract was injected into a split/splitless injector held at 280 °C in splitless mode. A fused-silica capillary column (30 m × 0.32 mm ID × 0.5 μm film thickness) coated with a stationary phase DB-5MS (J&W Scientific, Folsom, CA, USA) was used. Helium was used as a carrier gas at a velocity of 43 cm.s⁻¹ in constant flow mode.
For simulants S1, S2, S3, and S5. The oven temperature program was as follows: The initial oven temperature was 55 °C. It was raised to 320 °C at a rate of 15 °C/min⁻¹. Finally, the temperature was maintained at 320 °C for 5 min.
For simulants S4 and S6, the oven temperature program was as follows: The initial oven temperature was 45 °C. It was raised to 320 °C at a rate of 5 °C/min⁻¹. Finally, the temperature was maintained at 320 °C for 5 min.
The mass detector was a quadrupole mass spectrometer, an MSD 5973 from Agilent, using electron impact (70 eV) in full scan mode (mass range 29–450 uma). The MS source temperature was 230 °C, and the MS Quad temperature was 190 °C. Enhanced ChemStation software (Agilent Technologies, Santa Clara, CA, USA) was used for data treatment. Compounds were identified by comparing their mass spectra with mass spectra in the Wiley and NIST databases and verified using the linear retention index.

Volatiles were sampled by SPME with a 2 cm × 50/30-micron DVB/Carboxen/PDMS Stable Flex fiber (Sigma, Milano, Italy). Extraction and desorption of the volatiles were performed automatically by a CombiPAL autosampler (CTC Analytics, Zwingen, Switzerland) as described (Zorrilla-Fontanesi et al., 2012 (link)). Chromatography was performed on a DB-5 ms (30 m × 0.25 mm × 1 mm) column (Sigma, Milano, Italy) with Helium at a constant flow of 1.2 mL/min, accordingly to Zorrilla-Fontanesi et al. (2012 (link)). Mass spectra were recorded in scan mode in the 35 to 220 mass-to-charge ratio range by a 5975B mass spectrometer (Agilent Technologies, Cernusco sul Naviglio, Italy) (ionization energy 70 eV; scanning speed 7 scans/s). The Enhanced ChemStation software (Agilent Technologies, Cernusco sul Naviglio, Italy) was used for recording and processing of chromatograms and spectra. Three technical replicas were conducted for each sample.

The raw gas chromatography mass spectrometry (GC/MS) data were processed with the AMDIS (v.2.1) software of NIST (National Institute of Standards and Technology, Gaithersburg, USA). Thereafter, the metabolites were identified with the Agilent Enhanced ChemStation software (Waldbronn, Germany). Generally, all metabolome analysis experiments were performed with three biological replicates and two technical replicates of each biological replicate. Metabolite concentrations were scaled to the internal standard ribitol and the optical density of cell culture and then calibrated to the total signal intensity of the GC spectra. Metabolite concentrations below the detection limit were set to the lowest measured signal. The median intensity values of the six replicates were applied to statistical analysis. Principal component analysis was performed with the software package PAST 4, version 4.13 (May 2023) (Hammer et al., 2001 ).