An automated SPME sampling PD-L1 inhibitor unit (CombiPal. Zwingen, Switzerland) was used with a SPME StableFlex fibre with 50/30 μm divinylbenzene/carboxen on polydimethylsiloxane coating (DVB/CAR/PDMS) purchased from Supelco (Sigma Aldrich, UK). Five mL of juice sample was transferred to a 30 mL vial crimp-sealed with 23 mm diameter aluminium seal and a Teflon septum. In addition, pure aqueous systems of cis-3-hexenol (25 μL/L) were prepared and analysed together with apple juice samples in a fully randomised order. After 10 min
equilibration at 20 °C, the SPME fibre was exposed to the sample headspace for 15 min. The fibre was then removed from the vial and immediately inserted into the injector port of the GC–MS system for thermal desorption at 220 °C for 10 min. Analysis of the aroma components were performed on a Trace GC Ultra (Thermo Scientific, USA) that was attached to a DSQ series mass spectrometer (Thermo Scientific, USA). The gas chromatograph was equipped with a low bleed/fused-silica ZB-Wax capillary column (100% polyethylene glycol phase, Smad inhibitor 30 m × 0.25 mm × 1.0 μm) (Phenomenex, UK). Helium was the carrier gas
with a constant flow rate of 1.5 ml/min into the GC–MS. The GC oven was held for 2 min at 40 °C and heated to 220 °C at a rate of 8 °C/min. The GC to MS transfer line was maintained at 250 °C. Analysis was carried out in the electron impact mode with a source temperature of 230 °C, ionising voltage of 70 eV, and a scanned mass range Thalidomide of m/z = 50–200. Pure apple juices were run in triplicate. Compounds were identified by comparison to NIST Library and the retention time of authentic standards. A MS Nose interface (Micromass, Manchester, UK) fitted to a Quattro Ultima mass spectrometer (Milford,
Waters) was used for the static headspace analysis of apple juice samples. Fifty mL aliquots of samples were placed in 100 mL flasks fitted with a one port lid. After a 30 min equilibration period at room temperature (20 °C), the headspace was drawn into the APCI-MS source at a rate of 5 mL/min. The samples were analysed in full scan mode, monitoring ions of mass to charge (m/z) ratios from 40 to 200. The intensity of these ions was measured at cone voltage of 20 V, source temperature of 75 °C and dwell time of 0.5 s. Moreover, headspace analysis was carried out in the splitless injection mode, at a flow of 20 mL/min, splitless valve time of 1.5 min and constant pressure of 124 kPa. All analyses were run in triplicate. The chromatographic data was subject to one-way ANOVA followed by Duncan’s post hoc means comparison test. Moreover, principal components analysis (PCA) was also performed on the chromatographic dataset (36 samples, 16 variables) after standardization in order to explore the clustering of the apple juices in terms of their flavour volatile compounds composition. All analysis were performed using MINITAB release 16 (Minitab Inc., Pennsylvania, US).