Team:Valencia UPV/Project/modules/methodology

Headspace solid-phase microextraction is one of the most convenient techniques for analysing Volatile Organic Compounds (VOCs). It is a very sensitive, inexpensive, robust and easy-to-use technique since it does not require the use of solvents and is able to detect metabolites up to ppt (parts per trillion) with a low sample quantity.

In our case, we chose this technique coupled to a Gas Chromatography-Mass Spectrometry detection (see GC-MS) to analyse the presence of pheromones ((Z)-11 hexadec-1-ol, (Z)-11 hexadecenal, (Z)-11 hexadecenyl acetate)[see biosynthesis] in our samples, Nicotiana benthamiana leaves [see agroinfiltration].

This technique is based on the detection of volatiles present in the headspace of a vial. The volatiles diffuse from the sample (Solid phase) to the headspace (Gas phase) and are captured by an absorbent, a polymer-coated fiber with high affinity for them. After desorption from the fiber, volatiles are analysed by GC-MS.

In order to perform an accurate analysis of the sample, this technique must follow 3 simple steps:

• Sample preparation: it is vital for a proper analysis that samples are unaltered during the whole process.
• Therefore the biological material, leaves in this case, must be immediately kept on liquid Nitrogen after the removal from the plant.
• Then, leaves are ground to a fine powder with mortar and pestle and introduced in a screw cap headspace vial.
• Afterwards, EDTA and saturated solution of CaCl2 are added to inhibit enzymatic activity and stabilize the sample.
• Finally, they are sonicated to induce the release of volatiles from the solid phase to the gas phase in the headspace of the vial.
• Trapping of volatiles: volatiles present in the gas phase must be extracted for further analysis. Therefore a fiber coated with an adsorbent polymer is introduced in the headspace, and ‘traps’ the volatiles from the sample.
• Desorption: release of the volatiles from the fiber by thermal desorption which takes place in the insertion port of the Gas chromatograph.