Volatile Organic Compounds Profiling of Antirrhinum and Petunia Flowers

We used completely developed 3–4 days old flowers of the Antirrhinum majus inbred line 165E [8 (link), 24 (link)] in order to generate the raw data which were then used to compare semi-quantification methods. Additionally, a flower scent profile was generated for Petunia x hybrida line Mitchell. The sampling system consisted in flowers placed inside a beaker with 4 ml of 5% sucrose in distilled water, supported by a glass slide, and a stir bar was attached to the border of the beaker with a stainless-steel paperclip. The beaker was then placed in a 2-l desiccator (Fig. 1). Fig. 1

Sampling system for VOCs in HSSE

Flowers for CG-MS analysis were kept under conditions of 12 h light and 12 h dark at 23 °C and 18 °C, respectively, in a growth chamber (Sanyo MRL 350). In case of A. majus, stir bars sampled the floral volatiles of 3 flowers in 3 different desiccators during 12 h of light or 12 h of dark periods. In case of Petunia x hybrida, stir bars sampled floral volatiles for 4 or 24 h, sampling times applied in circadian rhythm studies [25 (link)]. The VOC profile of A. majus, is based on compounds which appeared unanimously in the day and night replicas (Table 1). Contaminants were identified and omitted in subsequent analyses.Table 1

Chromatographic parameters for A. majus VOCs analysed in column HP5 MSVi

Retention time	Compound	CAS	LRI	LRI bibliography	Reference
2.23	Methyl 2-methylbutanoate	868-57-5	805	774	[26 ]
5.58	β-myrcene	123-35-3	996	991	[27 ]
6.62	Ocimene	6874-10-8	1044	1038	[28 ]
7.19	Acetophenone	98-86-2	1071	1065	[27 ]
7.77	Methyl benzoate	93-58-3	1099	1091	[27 ]
7.96	Nonanal	124-19-6	1107	1104	[29 (link)]
9.03	Acetophenone, 2’-hydroxy	118-93-4	1167	1160	[26 ]

We used 10 mm long Twisters™ (Gerstel, Mülheim an der Ruhr, Germany) (stir bars), covered with a 0.5 mm film of polydimethylsiloxane (PDMS). We also tested dual-phase stir bars (ethylene glycol and silicone) (Gerstel, Mülheim an der Ruhr, Germany). Both types of stir bars were conditioned for adsorption according to manufacturer indications.
Compounds adsorbed by the stir bars were analysed by GC–MS in a gas chromatograph HP-6890N coupled to a 5975 mass spectrometer (Agilent Technologies, Palo Alto, USA) combined with a TDU and cooling injector system (CIS4) (Gerstel, Mülheim an der Ruhr, Germany).
Desorption was carried out by heating from an initial temperature of 40° to 250 °C at 100 °C min⁻¹ with 5 min hold time on splitless mode. Desorbed compounds were captured in a cool trap at − 100 °C. This process was automated by using a multipurpose sampler MPS2XL (Gerstel, Mülheim an der Ruhr, Germany).
Chromatographic separation was done in a HP5MS-UI column (Agilent Technologies, Palo Alto, USA) with helium as gas carrier in constant pressure mode and split ratio 1:50. Initial temperature was 50 °C, increasing at a ratio of 5 °C min⁻¹ until 70 °C held 1 min. In the next step, temperature was increased until 240 °C at 10 °C min⁻¹ held for 15 min.
The mass spectrometer operated at 70 eV ionization voltage. Source and quadrupole temperatures were 230 and 150 °C, respectively. Mass range was 30.0 to 450.0 uma at 4 scan/s. MSD transfer line was maintained at 280 °C.
We used ChemStation software (version E.02.02 SP1, Agilent Technologies, Palo Alto, USA) to acquire chromatograms. Compounds were qualitatively identified by comparison with mass spectral database Willey10th-NIST11b (Agilent Technologies, Wilmington, USA), considering match qualities above 90%. We used ocimene, acetophenone, methyl benzoate and methyl cinnamate (Sigma-Aldrich, W353901, 42163, 18344 and 96410, respectively) as standards. Methanol was used as solvent for dilution of standards (Panreac, 361091). Linear retention indexes (LRI) were calculated as a parameter for identifying compounds by comparing with retention times (RT) of C8-C20 alkanes (Sigma Aldrich, 04070), analysed under the same chromatographic conditions (Table 1) [30 (link)].