5975c inert xl msd mass spectrometer

The headspace volume of the tubes was sampled by means of needle trap microextraction (NTME) and analyzed by GC-MS as described elsewhere (14 (link), 15 (link)). The GC-MS system consisted of an Agilent 7890A gas chromatograph and an Agilent 5975C inert XL MSD mass spectrometer. In order to identify unknown VOCs from the mass spectra, first, a mass spectral library search (NIST 2005 Gatesburg, PA, USA) was carried out and, subsequently, compounds were verified and quantified by measurements of pure reference substances. Altogether, more than 100 volatile substances were detected in the headspace volumes. VOCs which could not be identified unequivocally, which could not be quantified or which were assigned to contamination from room air were excluded from the VOC panel in a pre-processing screening of the GC-MS spectra.

To analyze the compounds produced by the dual culture in SFM or R5A media, 250 mg of activated charcoal (Norit^®) was placed on top of the chamber´s central piece, aiming to capture the emitted VOCs. This methodology was also applied to analyze VOCs produced by a single culture of E. weberi and a single culture of Streptomyces spp. in SFM or R5A media (only for strains CS057, CS131, CS014, and CS147). A VOC environmental control, an empty plate with active carbon, was also arranged. After 5 days of incubation at 28 °C, the activated charcoal was removed, and the captured volatiles were extracted with 750 µL of ethyl acetate. Also, an ethyl acetate sample was analyzed as a control to evaluate the volatile compounds that could be present in the solvent. Afterward, these samples were analyzed via gas chromatography–mass spectrometry (GC-MS) using an Agilent Technologies 7890A GC System coupled with a 5975C Inert XL MSD mass spectrometer in SCAN mode. Gas chromatography was carried out on an Agilent DB-5MS column (30 m × 0.25 mm × 0.25 µm with helium as carrier gas at 1 mL/min). The initial oven temperature was 50 °C, held for 5 min, ramped at 5 °C/min up to 300 °C, and held for 20 min. MassHunter Unknowns Analysis software and the NIST20 mass spectral library were used to identify compounds with a high percentage of reliability.

Organic acids were analyzed using standard techniques. Briefly, to 0.5 mL of dialysate sample (72 h) 2.5 mL SETH buffer [0.25 mol/L sucrose; 2 mmol/L (K)EDTA; 10 mmol/L Tris; 5 × 10⁴ U heparin (pH 7.4)] was added. The mixture was acidified to pH2 with concentrated HCl (10%), after which the organic acids were extracted by ethylacetate twice, derivatized with trimethylsilyl (TMS), and analyzed on an Agilent 7890A gas chromatograph (GC), coupled to a flame ionization detector (FID) and an Agilent 5975C inert XL MSD mass spectrometer. Quantification of organic acids was done by calculation of peak areas and comparison with an internal standard (4-phenylbutyric acid). Relative concentrations were expressed relative to the lowFe condition which was set at 100%.