Divinylbenzene carboxen polydimethylsiloxane fiber

Infested and uninfested apple seedlings (ca. 30 cm high, cv. Royal Gala) were enclosed for 24 h in individual 14 × 50 cm glass chambers (five replicates). No air flow was allowed into these chambers and both inlet and outlet were closed. SPME (solid phase microextraction) analysis used a 50/30-μm divinylbenzene/carboxen/polydimethylsiloxane fiber (Supelco Inc., Bellefonte, PA) for headspace sampling around the foliage. The SPME fiber was inserted from the top of the chamber and was exposed to each sample for 5 min at ambient temperature (23–27 °C), after which each sample was injected into a GC/MS injection unit. SPME fibers were conditioned prior to sample collection by injecting the fiber in the GC inlet for 1 hour at 220 °C.

The identification of volatile organic compounds (VOCs) was determined by gas chromatography coupled to a mass spectrometer (GC/MS; Agilent 6890 N; Agilent Technologies Italia, Milan, Italy) using a solid phase micro-extraction technique (SPME), as described in detail by Ciriello et al. [60 (link)]. Briefly, a divinylbenzene/carboxen/polydimethylsiloxane fiber (Supelco^®, Bellefonte, PA, USA) was introduced into the headspace of the vial containing the previously heated sample (30 °C for 10 min) for the adsorption of VOCs and then inserted into the split-splitless injection system of GC/MS (250 °C; 7.64 PSI; flow rate 24 mL min⁻¹; 10 min desorption step). The chromatographic run lasted 30 min and 67 s (temperature gradient mode 50–280 °C; helium as carrier gas with a flow rate of 1 mL min⁻¹; mass spectrum at 70 eV). VOCs were identified by comparison with the NIST spectral database and expressed as a percentage of total area normalization. Each treatment was analyzed in triplicate.

Volatile organic compounds of a 10 mL sample of watermelon juice with 1 μL internal standard (2-methyl-3-heptanone, 0.816 μg/μL) added in a 40 mL headspace vial (Agilent, Inc., Santa Clara, CA, USA) were extracted manually with SPME using a 2 cm, 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane fiber (Supelco, Inc., Bellefonte, PA, USA). Samples were allowed to equilibrate for 20 min at 40 °C before collection for 40 min with continuous stirring at 100 rpm (J&K Scientific Ltd., Beijing, China) [14 (link)].

The 20 mg combined sample was weighed into a 20 mL headspace vial, sealed, and incubated at 40 °C for 10 min. For SPME analysis, a 1 cm 30/50 divinylbenzene-carboxen polydimethylsiloxane fiber (Supelco, Bellefonte, PA, USA) was incubated in the sample headspace for 30 min and desorbed in the GC inlet for 3 min. The fiber was reconditioned between samples.
For static headspace analysis, 1 mL of the gas phase was removed using a gas-tight headspace syringe and injected directly. Different incubation temperatures from 40–145 °C were trialed to maximize recovery of higher-boiling sesquiterpenes. The syringe was kept at 150 °C and flushed with high-purity nitrogen between samples to avoid carryover.

One gram of the leaves’ powder was placed into a 20 mL head-space vial. The vials were sealed using crimp-top caps with TFE-silicone headspace septa (Agilent). Each vial was placed at 60°C for 10 min, and then a 65 μm divinylbenzene/carboxen/polydimethylsiloxane fiber (Supelco, Bellefonte, PA, United States) was exposed to the headspace of the sample for 20 min at 60°C. The volatile compound identification and quantification were carried out using an Agilent Model 7890 BGC and a 7000D mass spectrometer (Agilent), equipped with a 30 m × 0.25 mm × 1.0 μm DB-5 ms capillary column. Helium was used as the carrier gas at a flow rate of 1.0 ml/min. The injector temperature and detector temperature were 250°C and 280°C, respectively. The oven temperature was programmed at 40°C for 5 min, increased by 6°C/min to 280°C, and held for 5 min. The ionization voltage was 70 eV. Mass spectra were scanned from 30 to 350 amu. The compounds were identified by comparing the mass spectra with the MWGC database (Metware Biotechnology Co., Ltd., Wuhan, China) and a linear retention index.

Determination of VOCs in the juices and peels was carried out by solid-phase micro extraction (SPME) and analyzed with a gas chromatography-mass spectrometry (GC-MS). An automatic injection autosampler CombiPal (CTC-CombiPal Analytics, Zwingen, Switzerland) was used for SPME sampling. The experiments were performed using a 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane fiber (Supelco, Bellefonte, PA, USA). The fiber was conditioned according to the manufacturer’s recommendation to remove contaminants. Before analysis, a fiber blank was run to confirm no contamination peak.
VOCs of juices and peels: An aliquot (8 g) of each juice diluted 20 times was weighed into a 20 mL vial and spiked with 80 µL of internal standard (camphor 3000 mg/L). Each sample was equilibrated at 40 °C for 10 min under stirring (500 rpm). After equilibration, the juice or peel were extracted by exposing the SPME fiber at 40 °C for 10 min (juice) and 2 min (peel). The analytes were desorbed at 250 °C for 15 min in the GC injection port. Measurements were always repeated at least in triplicates.

To detect volatile organic compounds (VOCs) of bread samples, gas chromatography/mass spectrometry (GC/MS) analyses were performed after extraction of VOCs by headspace solid-phase micro-extraction (HS-SPME) sampling technique. First, 0.75 g of ground bread were placed in 20 mL glass vials and 10 μL of internal standard solution (4-methyl-2-pentanol, 0.033 g/L) were added. A COMBIPAL-xt autosampler (CTC Analysis AG, Zwingen, Switzerland) was used to standardize the extraction procedure. Bread samples were equilibrated at 60 °C for 10 min. After equilibration, a divinylbenzene/carboxen/polydimethylsiloxane fiber (Supelco, Bellefonte, PA, USA) was exposed to headspace for 50 min at 60 °C. The extracted VOCs were subjected to 3 min-long desorption in splitless mode at 230 °C and then injected into a Clarus 680 (Perkin Elmer, Waltham, MA, USA) gas-chromatography system equipped with a Rtx-WAX (30 m × 0.25 mm i.d., 0.25 μm film thickness) capillary column (Restek Superchrom, Milan, Italy), using the temperature diagram and carrier gas conditions reported by Vitellio et al. [26 (link)]. A single-quadrupole mass spectrometer Clarus SQ8MS (Perkin Elmer), coupled to the gas-chromatography system, was used to detect the VOCs. Peaks were identified and VOCs were quantified as reported previously [26 (link)].