Spme fibre

Enzymatic assays for isoprene synthase were performed as previously described (Sasaki et al., 2005) using DMAPP as the substrate. After enzyme assay incubation, headspace products were collected with a 100 μm solid‐phase micro extraction (SPME) fibre (Supelco, St Louis, MO, USA) at 42°C for 15 min and analysed by GC–MS.

We collected (commonly discoloured) droplets of honeydew from plants heavily infested with pea aphids, using a 10-µL glass capillary fitted with a rubber bulb. We collected a total of 50 µL of honeydew and expelled it into a 4-mL glass vial with a rubber septum lid. Through this lid, we inserted a carboxen-polydimethylsiloxene-coated solid-phase micro extraction (SPME) fibre (75 µm; Supelco Inc., Bellefonte, PA, USA), allowing absorption of honeydew odorants on this fibre for 24 h at room temperature. Prior to each odorant collection, we conditioned the fibre at 280 °C for 5 min in a gas chromatograph (GC) injection port. We desorbed odorants from the fibre in the hot (250 °C) injection port of the GC, and analyzed odorants by GC-mass spectrometry (MS) using a Saturn 2000 Ion Trap GC-MS fitted with a DB-5 GC-MS column (30 m × 0.25 mm i.d.; Agilent Technologies Inc., Santa Clara, CA, USA) in full-scan electron impact mode. We used a flow of helium (35 cm s⁻¹) as the carrier gas with the following temperature program: 40 °C (5 min), 10 °C min⁻¹ to 280 °C (held for 10 min). We identified volatiles by comparing their retention indices (RI) relative to n-alkane standards [47 (link)] and their mass spectra with those reported in the literature [48 ] and with those of authentic standards.

The lemongrass EO sample and the white spots from the developed TLC-based bioautography plates were analysed with HS-SPME. In the case of lemongrass EO the liquid EO was analysed, while in the case of the TLC spot the cut-out silica plate was the analysed sample. The samples were put into vials (20 mL) and sealed with a silicon/PTFE septum prior to HS-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, Zwingen, Switzerland) automatic multipurpose sampler using a 65 μM StableFlex carboxen/polydimethylsiloxane/divinylbenzene (CAR/PDMS/DVB) SPME fibre (Supelco, Bellefonte, PA, USA). After an incubation period of 5 min at 100 °C, extraction was performed by exposing the fibre to the headspace of a 20 mL vial containing the sample for 10 min at 100 °C. The fibre 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 fibre was cleaned and conditioned in a fibre bakeout station in a pure nitrogen atmosphere at 250 °C for 15 min.