E 2 hexenal

Authentic samples of the compounds tentatively identified by GC‐MS were purchased for structure confirmation by GC peak enhancement and for behavioural assays. (Z)‐3‐Hexenal (50% solution in triacetin), (E)‐2‐hexenal (99%), p‐cymene (99%), 1,8‐cineole (99%), γ‐terpinene (95%), linalool oxide [2‐(5‐methyl‐5‐vinyltetrahydro‐1‐furyl)‐2‐propanol] (>97%) and (R/S)‐camphor (96%) were obtained from Sigma‐Aldrich (Gillingham, Dorset, UK). (Z)‐3‐Hexen‐1‐ol (98%) and (Z)‐3‐hexenyl acetate (99%) were purchased from Alfa Aesar (Heysham, Lancs, UK), and (Z)‐ocimene (90%) was obtained from Bush Boake Allen (London). 3‐Ethylacetophenone (95%) was obtained from Maybridge (Tintagel, Cornwall, UK). (E)‐Ocimene (95%) was synthesised according to Chou et al.13 (E)‐4,8‐Dimethyl‐1,3,7‐nonatriene (95.6%) was synthesised from geraniol by oxidation to the corresponding aldehyde, followed by Wittig methylenation.14

All reagents utilized in this assay were of analytical quality. Sodium chloride (NaCl, 99.5%), and calcium chloride (CaCl₂, >99.0%) were obtained from Panreac (Spain, Barcelona). Ultrapure water was supplied from a Milli-Q^® system (Millipore); the 3-octanol used as internal standard and other VOCs for identification, namely, 1-butanol, 1-heptanol, 1-octanol, 1-propanol, (E)-2-hexenal, 2-methyl-butanal, 2-ethyl-1-hexanol, 2-methyl-1-propanol, 2-phenylethanol, acetaldehyde, benzaldehyde, ethanol, ethyl acetate, ethyl butanoate, ethyl decanoate, ethyl hexanoate, ethyl octanoate, ethyl propanoate, ethyl 3-hydroxybutanoate, hexyl acetate, and pentanal with purity up to 98% were acquired from Sigma Aldrich (Madrid, Spain). Helium gas purity of 5.0 (Air Liquide, Portugal) was utilized as the GC carrier gas. Solid-phase microextraction holder and divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber were supplied from Supelco (Bellefonte, PA, USA). The Kovats index (KI) was calculated by the injection of a series of C₈ to C₂₀ straight-chain n-alkanes (at 40 mg L⁻¹ in n-hexane) produced by Fluka (Buchs, Switzerland).

A mixture of n-alkanes (C₈—C₂₀) dissolved in hexane for retention index determination was purchased from Fluka (Buchs, Switzerland). Reference compounds, (E)-2-hexen-1-ol (> 96%), (E)-2-octen-1-ol (> 97%), 1-decyne (> 98%), 1-dodecene (≥ 99%), (E,E)-2,4-hexadienal (≥ 97%), 2-cyclohexen-1-ol (> 95%), (E)-2-hexenal (≥ 99%), (E)-2-octenal (≥ 95%), 4-tert-butylcyclohexyl acetate (mixture of E and Z, > 98%), allyl cyclohexanepropionate (≥ 98%), cyclopentanemethanol (≥ 98%), cyclopentanol (≥ 99%), cyclopentanone (≥ 99%), decanal (≥ 98%), 1-dodecanol (≥ 98%), dodecyl acetate (> 97%), hexyl acetate (≥ 99%), hexyl salicylate (≥ 99%), limonene (> 97%), nonanal (≥ 97%), octyl acetate (≥ 99%), p-cymene (> 99%), 1-tridecanol (≥ 99%), and α-hexylcinnamaldehyde (≥ 95%) were supplied by Sigma Aldrich (Munich, Germany).

Citral, carvacrol and (E)-2-hexenal were obtained from Sigma-Aldrich (Milano, Italy), while thyme EO was purchased from Flora s.r.l. (Pisa, Italy). Stock solutions of the natural antimicrobials were diluted in absolute ethanol (Sigma-Aldrich, Milano, Italy) and stored for up to a month at 4°C until use.

After sampling for 10 min the fiber was desorbed for 1 min in the injection port which was constantly kept at 250°C. Compounds were separated on HP-5 ms column (30 m × 250 μm, 0.25 μm film thickness; Agilent) in an Agilent 7890A gas chromatograph with a temperature program set to 40°C for 5 min, increasing to 140°C at a rate of 5°C per min, followed by increasing temperature to 250°C at a rate of 15°C per min and an additional 5 min at 250°C. Helium was used as the carrier gas with the transfer column flow set to 3 mL per minute and a flow rate of 1 mL per min thereafter. Mass spectra were generated by an Agilent 7200 accurate-mass quadrupole time-of-flight mass spectrometer, operating in electron ionization mode (70 eV) at 230°C and collected with an acquisition rate of 20 scans per second. volatiles were identified and quantified using standard solutions of Z-3-hexenal, Z-3-hexenol, E-2-hexenal, E-2-hexenol and (E,Z)-2,6-nonadienal (Sigma-Aldrich). (Z,Z)-3,6-nonadienal was synthesized from (Z,Z)-3,6-nonadienol (Ventos) as described below.

For the determination of the retention times of the main volatile compounds, the standards of Z-3-hexen-1-ol (≥98%), 1-hexanol (≥99%), hexanal (≥97%), E-2-hexenal (≥95%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). For the HS-SPME procedure, amber glass vial of 20 mL, a screw thread cap with microcenter PTFE/silicone septa and SPME manual injection kit were obtained from Restek Corporation (Bellefonte, PA, USA). For the HS-SPME procedure, an amber glass vial of 20 mL and screw thread cap with microcenter PTFE/silicone septa and SPME manual injection kit were obtained from Restek Corporation (Bellefonte, PA, USA), as were SPME fibers coated with 65 um Divinylbenzene/Polydimethylsiloxane (DVB/PDMS). A heater from Fisher Scientific and a balance from Sartorius also were used.