# Floral Scent Evaluation of Three Cut Flowers Through Sensorial and Gas Chromatography Analysis
## 2 Materials and Methods
### Evaluation of VOCs Using GC-MS  
The collection of VOCs from oriental lily, freesia, and chrysanthemum was performed as previously described [3; 21] using three replicates for each species. Briefly, samples were presented as previously described (Figure 1B), enclosed in 1-L glass jars, with 100 mL of distilled water. The collection of VOCs from the headspaces over the flowers was performed using solid phase microextraction (SPME). A 50/30 \(\upmu\)m divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS)-coated solid-phase microextraction (SPME) silica fiber (StableFlex fibre, Sigma Aldrich, Gillingham, UK) was exposed to the headspace for 30 min. This exposure time was established after testing 20, 30, and 40 min of exposure time, with 30 min being the best exposure time of the fiber to the sample for the higher recovery of volatile compounds (data not shown). Desorption of the collected VOCs was performed using the injection port of the gas chromatograph (GC 6890, Agilent, California, CA, USA) and exposing the fiber at 240 \({}^{\circ}\)C for 2 min. VOCs were separated using a 30 m, 0.25 mm ID capillary column over 0.25 \(\upmu\)m HP-5 MS (Agilent) and using the following temperature program: Initial temperature 40 \({}^{\circ}\)C for 5 min, first step increase 6 \({}^{\circ}\)C/min to 80 \({}^{\circ}\)C, holding this temperature for 5 min, and second step increase 4 \({}^{\circ}\)C/min up to 170 \({}^{\circ}\)C. Electron Impact mass spectra were recorded in full scan mode from 35 to 500 m/z (70 eV, MSD 5975, Agilent) coupled to a GC (GC 6890, Agilent). Ten microliters of an internal standard composed of 2 \(\upmu\)L of 4-methyl-2-pentanol in 10 mL of ethanol were injected regularly to ensure that the conditions of the instrument were consistent in time. Tentative identification was achieved by comparison of the mass spectra with the NIST mass spectra library (v. 2.0, 2011), and also comparing them with the Kovats found in the Flavornet (www.flavornet.org) and VCF (www.vcf-online.nl) databases. For this purpose, an n-alkanes solution (C6-C30) (Sigma Aldrich, Gillingham, UK) was injected in the same conditions as previously described to calculate Kovats retention indices.  
Data were expressed as the relative area, calculated by dividing the peak area of the target ion (major ion) of each compound by the peak area of the target ion of the internal standard (major ion). Moreover, the major compound of each species, \(\upbeta\)-cis-ocimene (lily), \(\upbeta\)-pinene (chrysanthemum), and linalool (freesia) was quantified by constructing calibration curves with 5 points (known concentrations) (Appendix A). Calibration was performed using the same conditions previously described for the GC-MS analysis, collecting the headspace of increasing volumes of each pure compound diluted in ethanol. The volume of flowers was calculated and substituted by adding the same volume of water to the sampling jars. Data in Table 3 were expressed in the concentration (\(\upmu\)g-L\({}^{-1}\)) obtained from calibration curves with reference standards (relative area vs. concentration).