2.2 | Volatile collections and chemical analyses
The volatiles emitted from M. humile seeds were collected using solid phase micro extraction (SPME) using a 66-µm PDMS/DVB fiber (Supelco, Bellefonte, PA, USA). The seeds with pulp were placed in 4-mL glass vials and sealed with aluminum foils. The SPME fiber was inserted into the covered vial and volatiles were sampled for 1 h at room temperature. Blank assays with empty vials were performed before each volatile collection session. Three independent samples were collected and analyzed.
Immediately after sampling, the SPME fiber was injected into the split/splitless injector of a gas chromatograph (GC17A; Shimadzu, Japan) equipped with a DB-WAX column (30 m length × 0.25 mm inner diameter × 0.25 µm film thickness; J & W Scientific Inc., Folsom, CA, USA) and mass spectrometry detectors (QP5000; Shimadzu, Japan) with electron impact ionization (70 eV). The oven temperature was maintained at 40 °C for 5 mins, programmed to increase to 220 °C at a rate of 10 °C min-1, and held isothermal for 10 min. The SPME fiber was desorbed in splitless mode for 1 min, while the injector and interface temperatures were 220 °C. Helium was used as carrier gas, and the column head pressure was 100 kPa. Volatile compounds were tentatively identified by the matches with the NIS mass spectral database, and identifications were confirmed by matching mass spectra and Kovat’s retention index with those of commercially available authentic standards.