Volatile compounds analysis

The volatile compounds released by cell cultures were sampled and then analyzed with a gas-chromatography mass-spectrometer (GC-MS) equipment (GCMS-QP 2010 Shimadzu). To collect the volatile compounds special lids in polymethylmethacrylate (PMMA) to fit with Petri dishes were designed. The lids were endowed with a fixture suitable to be connected with the holder of a solid phase micro-extraction (SPME) fiber. SPME is a standard sampling method in VOCs analysis that allows for an efficient collection and storage of volatile compounds. The fibers used in this experiment were coated with 50/30 μm Divinylbenzene/Carboxen/PDMS (SUPELCO, Bellefonte, PA, USA). The fibers were exposed to the cells headspace for 1 h. During the exposure the cultures were kept in the incubator at a temperature of 37°C and at a CO₂ concentration of 5%.

The samples collected in the SPMEs were analyzed the same day of collection with a GCMS-QP 2010 Shimadzu series gas chromatograph mass spectrometer, equipped with an EQUITY-5 capillary column (poly(5% diphenyl/95% dimethyl siloxane) phase (SUPELCO, Bellefonte, PA, USA). The column was 30 m length × 0.25 mm I.D.× 0.25 μm thickness.

The volatile compounds adsorbed in the SPME were desorbed from the fiber at 250°C for 3 minutes in the GC injection port. The volatile organic compounds were separated on the GC column using an initial oven temperature of 40°C for 5 minutes, then increased by 7°C/min to 220°C, a second temperature increase by 15°C/min to 300°C that was held for 3min (total runtime: 39 min). The analyses were performed in splitless mode, using ultra-high purity helium as carrier gas.

The instrument was operated in linear velocity constant mode, with a carrier gas pressure of 24.9 kPa, total flow of 5.9 mL/min, column flow of 0.7 mL/min and linear velocity of 30.2 cm/s.

The mass spectrometer was a single quadrupole analyzer in electronic ionization mode, scanned over a mass range of m/z 40-450 amu in the full scan mode. The detector voltage was fixed at 0.7 kV. The temperature of interface and ion source was kept constant at 250°C. The GC-MS data were analyzed using the section GCMS post-run analysis of the GCMS solutions software (version 2.4, Shimadzu Corporation). Tentative identification of compounds was done using both NIST 127 and NIST 147 libraries. For the identification of a specific compound butyric anhydride (purum, ≥97% (NE), Fluka Analytical, Italy) was used as reference standard, spiking the DLD1 cell culture medium.

For investigating butyric anhydride in more detail, it has been dissolved in culture medium at a final concentration of 0.1%. The solution, kept at 37°C in a thermal bath was analyzed by VOCs in the headspace sampled by SPME with an exposure time of 1h.