# Propolis Hydroalcoholic Extracts: Biochemical Characterization and Antifungal Efficacy
## 2 Materials and Methods
### Biochemical Characterization of Propolis Metabolites  
#### 2.7.1 Volatile Compounds  
The propolis samples' volatile compounds were extracted by HS-SPME with a divinylbenzene/carboxen/polydimethyl siloxane (DVB/CAR/PDMS) fibre (df 50/30 \(\mu\)m; 2 cm length) (Supelco, Bellefonte, PA, USA), chosen on the basis of previous studies [25]. The SPME fibre was conditioned before use as recommended by the manufacturer. Sampling was carried out on 1.00 \(\pm\) 0.03 g of propolis extract, precisely weighed in a 20 mL headspace vial and kept at 40 \({}^{\circ}\)C for 60 min under constant agitation. An Internal Standard IS 2-methyloctynoate was used to monitor sampling repeatability (i.e., response fluctuation \(\leq\) 20% Relative Standard Deviation). An IS working solution was prepared at 0.100 g/L in dibutyl phthalate and stored at \(-18\)\({}^{\circ}\)C in sealed vials. To preload the IS onto the SPME fibre, 5 \(\mu\)L of the working solution was placed in a 20 mL glass sealed vial and exposed to the SPME device for 5 min at 40 \({}^{\circ}\)C. After extraction, the SPME device was automatically transferred to the split/splitless injection port of the GC \(\times\) GC system, kept at 270 \({}^{\circ}\)C, and thermal desorption lasted for 5 min. Samples were analysed in four replicates randomly distributed. Comprehensive two-dimensional GC analyses were carried out using an Agilent 7890B GC chromatograph (Agilent Technologies, Wilmington, DE, USA) coupled with a Markes BenchTOF Select(tm) mass spectrometer featuring Tandem Ionization(tm) (Markes International, Llantrisant, UK). The GC transfer line was set at 270 \({}^{\circ}\)C. The TOFMS was tuned for single ionization at 70 eV, and the scan range was set between 35 and 350 \(m/z\) with a spectrum acquisition frequency of 100 Hz. The thermal modulator was a loop-type, two-stage KT 2004 (Zoex Corporation, Houston, TX, USA) cooled with liquid nitrogen and controlled by Optimode, v2.0 (SRA Instruments, Cernusco sul Navigilio, Milan, Italy). The modulation period (PM) was set at 4 s, while the hot-jet pulse duration was set at 350 ms. The cold-jet stream at the mass flow controller (MFC) was programmed to linearly reduce the total flow (i.e., 20 L/min) from 35% to 5% along the analytical run.  
The column set consisted of a 1D HeavyWax(tm) column (100% polyethylene glycol--PEG; 30 m \(\times\) 0.25 mm dc \(\times\) 0.25 \(\mu\)m df) coupled with a 2D DB17 column (50% phenylmethylpolysiloxane; 1.0 m \(\times\) 0.10 mm dc \(\times\) 0.10 \(\mu\)m df), both supplied by Agilent Technologies (Wilmington, DE, USA). A fused-silica capillary loop (1.0 m \(\times\) 0.1 mm dc) was used in the modulator slit. SiITitle(tm) \(\mu\)-unions (Trajan Scientific and Medical, Melbourne, Australia) were used to connect the columns with the capillaries. The GC split/splitlessinjector port was set at 270 \({}^{\circ}\)C and operated in split mode with a 1:5 split ratio. A specially design liner for SPME thermal desorption was used to improve the transfer of the analytes to the 1D column and to limit band broadening in space. Helium was used as the carrier gas at a nominal flow of 1.0 mL/min. The oven temperature programme was set as follows: from 50 \({}^{\circ}\)C (1 min) to 260 \({}^{\circ}\)C (10 min) at 3.5 \({}^{\circ}\)C min\({}^{-1}\). The n-alkanes solution for \(l^{T}s\) determination was analysed under the following conditions: split/splitless injector in split mode, split ratio: 1:50, injector temperature: 270 \({}^{\circ}\)C, and injection volume: 1 \(\upmu\)L. Analyte identification was performed by combining retention data (carried out using experimental \(l^{T}\) with \(\pm\) 10 unit tolerance vs. tabulated ones) and comparing electron ionization (EI)-MS spectral signature with reference spectra in commercial and in-house databases by using the NIST identity search algorithm with direct match factor (DMF) and reverse match factor (RMF) scores threshold \(\geq\) 900.