2. Materials and methods2.1. Raw material and preparationThe plant sample was freshly harvested, with commercialmaturity (2–4 months) obtained from high land, Chiangmai, Thailand.Upon arrival at the laboratory, the sample was washed withrunning tap water to remove debris and damaged portions. Theleaves and petioles of pennywort were stripped from the plantand extracted with water (1:1 w/v) by grinding at room temperaturefor 15 min. This juice was filled into the double layer of anLDPE Stomacher pouch (Seward Limited, UK) before HPP and filledin a retort pouch (PET/nylon/aluminium/PP) (Royal Can IndustriesCo., Ltd, Thailand) before pasteurisation and sterilisation. The sampleswere subjected to pasteurisation (90 C for 3 min), sterilisation(121 C for 4 min) and HPP (400 MPa for 20 min at <30 C).2.2. Chemicals1,2-Dichlorobenzene (99% from Sigma–Aldrich Company, Ltd,Poole, UK) was used as an internal standard (IS) for GC–MS analysis,as this compound was not found in the pennywort juice.2.3. Headspace concentrationThe sample (20 ml) was transferred to a 40 ml amber vial whichwas capped with a PTFE septum. 1,2-Dichlorobenzene (10 ll) wasadded and the vial was incubated for 5 min at 35 C, before a75 lm Supleco SPME fibre Carboxen/Polydimethylsiloxane (PDMS)was introduced and exposed to the sample headspace for 30 min.The SPME fibre was then inserted into the GC–MS injection portand held for 15 min with the adsorbed components being effectively
desorbed. The isolation and GC–MS analysis of volatiles from
each sample of pennywort juice was repeated 4 times.
2.4. GC–MS analysis
Analysis was carried out with a Hewlett–Packard 5890 series G
GC connected to a 5972 series MS and a 60 m VF-5 ms capillary
column (i.d. 0.25 mm, 0.25 lm film thickness, Varian). The injector
port was in splitless mode, and split flow was programmed to turn
on after 0.5 min. The temperature of the injector was 250 C. The
oven temperature was kept at 50 C for 3 min and programmed
to increase at 4 C min1. The final temperature was set at 240 C
and held for 5 min
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