Stableflex

Two types of sensors were tested: i—fibres coated with divinylbenzene (DVB), carboxen (CAR), and polydimethylsiloxane (PDMS); StableFlex 1 cm–50/30 μm, (Supelco Inc., Bellefonte, PA, USA), and ii—2-cm-long Stir Bar Sorptive Extraction (SBSE) (Gerstel GmbH, Co.KG, Mülheim an der Ruhr, Germany) coated with 1-mm-thick polydimethylsiloxane (PDMS). The stability of the collected volatiles on the stir bar was previously tested under various transportation temperatures and storage conditions until analysis (data not shown). Finally, stir bars were stored at room temperature, sent for analysis 2 weeks after VOC collection at most, and immediately analysed by GC-MS. Before sampling, each stir bar was conditioned in the injector of a gas chromatographer under a stream of helium according to the manufacturer’s recommendations.

Volatiles from the formulated microparticles were extracted by solid-phase microextraction (SPME). A total of 0.3 g of the sample, 4.7 g of water, and 1 g of NaCl were weighed into a 10 mL glass vial. For the extraction of volatiles, SPME fiber coated with a divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) sorbent (50/30 µm, StableFlex™, Supelco, Bellefonte, PA, USA) was used. The method was described by Vukoja et al. [18 (link)]. Compounds were confirmed by matching their mass spectra with the National Institute of Standards and Technology mass spectral database (NIST, East Amwell Township, NJ, USA) and through their retention time (RT) and retention index (RI). Two repetitions were made for each sample. Quantification was conducted with myrtenol as an internal standard, and results were expressed as µg/kg.

Headspace volatiles from 15 g fresh phase II and phase III composts were collected in glass cylinders (I.D. 80 mm, length 200 mm) with quick-fit connections on both ends. The incoming air was filtered with charcoal (10 g) air-purification system using PTFE tubing (I.D. 5 mm). Continuous, 1 l min^-1 airflow was drawn through the setup with a vacuum pump (Thomas G 12/02 EB, Garder Denver Thomas GmbH, Fürstenfeldbruck, Germany). Volatiles were trapped on 5 mg activated charcoal adsorbents (Brechbühler AG, Schlieren, Switzerland), purified as described by Molnár et al. (2015 (link)). Each collection lasted for 4 h and was replicated 3 times. The adsorbed volatiles were eluted with 100 μl of dichloromethane (purity 99.9%, VWR Chemicals) and kept at −40 °C. The extracts were subsequently used for electrophysiological recordings (GC-FID/EAD) and chemical identification (GC-MS).
Solid-phase microextraction (SPME) was also implemented with DVB/PDMS/CAR coated fibers (StableFlex, 50/30 μm, Supelco, Sigma-Aldrich, Bellefonte, PA, USA) to further examine the volatile profile of phase III compost with GC-MS and to estimate the headspace ratio of antennally active compounds. The SPME fibers were exposed into the sampling vials filled with 200 g cultivation materials for 5 min at room temperature and the extraction was repeated five times.

Volatile compounds from milk samples were extracted by static headspace solid-phase microextraction using Car/PDMS fiber (StableFlex, 10 mm length, 85 μm film thickness; Supelco, Bellefonte, PA, USA). The fiber was placed into the headspace of the vial for 1 h at 30 °C with stirring (400 rpm). Extraction was processed without heat incubation to preserve the extraction of compounds that could be degraded in a short-term period.

Volatile compounds produced by inoculated bacteria were analyzed using a gas chromatography device coupled with a mass spectrometer. Briefly, 1 g of vanilla sample was weighed into a 20 ml vial, with 3.80 ml of distilled water, 1.60 g of NaCl, and 0p.01 g of Ethyl nonanoate standard. The vial was heated at 40 C for 50 min to equilibrate the system. The SPME fiber, 85 μm carboxen/polydimethylsiloxane Stable Flex™ (Supelco), was inserted through the septum and exposed in the headspace of the vial for 25 min, to allow absorption of the volatile compounds onto the SPME fiber. The SPME fiber was then introduced into the injector port of the gas chromatograph for 5 min in splitless mode, set at 280°C, to desorb the volatile compounds. The desorbed components were analyzed on a capillary column. Compounds identification was based on a comparison of retention indices (RI), and mass spectra. GC data were analyzed by PCA using the package factoextra on R (R version 4.0.5).

In a first method application, ten SPME fibers (Car/PDMS, StableFlex, 10 mm length, 85 μm film thickness; Supelco) were simultaneously placed into the headspace of a milk sample for 1 h at 30 °C with stirring (400 rpm). After extraction, the fibers were placed into an empty glass tube and blocked with a glass wool plug and a tension spring to be further desorbed into the thermal desorption unit.

Plant material (pooled samples of 0.5–1 g fresh mass of either several fully developed, i.e., mature leaves, or young leaves and shoot tips or cotyledons as indicated) was put into vials (20 mL headspace) sealed with a silicon/polytetrafluoroethylene septum prior to the SPME gas chromatography/mass spectrometry (SPME-GC/MS) analysis. Sample preparation using the static headspace solid phase microextraction (sHS-SPME) technique was carried out with a CTC Combi PAL (CTC Analytics AG, Switzerland) automatic multipurpose sampler using a 65 μM StableFlex polydimethyl siloxane/carboxene/divinyl benzene (CAR/PDMS/DVB) SPME fiber (Supelco, United States). After an incubation period of 5 min at 100°C, extraction was performed by exposing the fiber to the headspace of a 20 mL vial containing the sample for 10 min at 100°C. The fiber was then immediately transferred to the injector port of the GC/MS, and desorbed for 1 min at 250°C. Injections were made in splitless mode. The SPME fiber was cleaned and conditioned in a Fiber Bakeout Station in pure nitrogen atmosphere at 250°C for 15 min.