Ctc combipal autosampler

A volume of 1 μL of each derivatised sample was injected splitless by a CTC Combi Pal autosampler (CTC Analytics AG, Zwingen, Switzerland) into an Agilent 6980 GC equipped with a 10 m X 0.18 mm i.d. fused-silica capillary column chemically bonded with 0.18-um DB 5-MS stationary phase (J&W Scientific Folsom, CA). The injector temperature was set to 270°C. Helium was used as the carrier gas at a constant flow rate of 1 mL min^-1 through the column. For every analysis, the purge time was set to 60 s at a purge flow rate of 20 mL min^-1 and an equilibrium time of 1 min. The column temperature was held initially at 70°C for 2 min, then increased to 320°C at a rate of 30°C min^-1, where it was held for 2 min. The column effluent was introduced into the ion source of a Pegasus III time-of-flight mass spectrometer (Leco Corp., St Joseph, MI). The ion source and transfer line temperatures were set to 200°C and 250°C, respectively. Ions were generated by a 70-ev electron beam at a current of 2.0 mA. Masses were acquired in the mass range 50–800 m/z at a rate of 30 spectra s^-1. The acceleration voltage was turned on after a solvent delay of 150 s.

All chemicals and reagents used in this study were of analytical grade. The EPA 524.2 fortification solution (20 μg/mL of fluorobenzene, 4-bromofluorobenzene, and 1,2-dichlorobenzene-d₄) (Sigma-Aldrich, Tokyo Japan) was used as an internal standard (IS). The n-alkane standard solution purchased from Fluka Chemical (Tokyo, Japan) was used for the determination of the RI (C8–C20). The EDTA solution in a concentration of 100 mM (pH 7.5) was used to inhibit enzymatic activity [1 (link)].
The 10 mg of frozen powder of each sample was weighed and dissolved in 1 mL of distilled water to prepare a stock solution. The stock solution of 10 mg/mL concentration was diluted up to 50 µg/mL of working solution and stored in −30 °C up to date of analysis. The working aliquots were sonicated for 10 min using the Branson sonicator (Branson Ultrasonics, CT, USA) before analytes for VOC extraction. The samples were prepared in 20 mL headspace Supelco vials (Missouri, USA) consisted of 1 mL of EDTA solution, 1 mL of the sample solution, and 10 μL of IS.
The preconditioned 50/30 μm solid-phase microextraction (SPME) fiber assembly DVB/CAR/PDMS purchased from Supelco (Cassopolis, MI, USA) was used to extract VOCs from samples HS. The extracted analytes were sampled through CTC CombiPAL auto-sampler (CTC Analytics, Zwingen, Switzerland), purchased from AMR (Tokyo, Japan).

Berries collected at seven developmental stages as described above were extracted for the analysis of volatile compounds. The extraction of volatile compounds followed the method described by Lan et al. [41 (link)]. For each replicate, sub-samples of 100 g de-seeded berries were grounded and blended with 1 g polyvinylpolypyrrolidone and 0.5 g d-gluconic acid lactone in liquid nitrogen. The blended powder was macerated at 4 °C for 4 h and then centrifuged at 8000 rpm at 4 °C for 10 min. Obtained clear juice was used for headspace solid phase microextraction (HS-SPME). HS-SPME was automatically conducted by a CTC-CombiPAL autosampler (CTC Analytics, Zwingen, Switzerland) equipped with a 2 cm DVB/CAR/PDMS 50/30 μm SPME fiber (Supelco, Bellefonete, PA., USA). Briefly, the mixture of 5 mL clear juice and 1 g NaCl, as well as 10 μL 4-methyl-2-pentanol (internal standard), was prepared in a 20 mL vial tightly capped with a PTFE-silicon septum. Samples were agitated at 500 rpm for 30 min at 40 °C, and then the SPME fiber, which was pre-conditioned at 250 °C for 1 h, was inserted into the headspace of the vial to absorb volatiles at 40 °C for 30 min. Afterwards, the SPME fiber was inserted into a gas chromatography injector for 8 min to thermally desorb volatile compounds.