Harvesting of plant tissue (leaves or fruits)
1) Harvest the tissue (500mg-1.0g) from plant under the desired experimental condition.
2) Immediately freeze the tissue by immersing in liquid nitrogen.
3) Frozen tissues can be freeze dried. Lyophilized tissue can be stored at -80°C for several weeks or processed immediately.
1) Transfer a known amount of lyophilized tissue, typically 300-500mg in a glass vial with cap.
2) Add 10µl of internal standard 5α -cholest 7en-3β-ol (1mg/ml stock) to the vial.
3) Add 3.75ml of CHCl3: methanol (1:2) and vortex vigorously.
4) Add 1.25 ml of CHCl3 and vortex well.
5) Add 1.25 ml of dH2O and vortex once again.
6) Transfer bottom organic phase to a fresh vial with the help of a pipette. The solvent should be allowed to evaporate completely at 35⁰C.
Critical step: Weighing of the tissue should be done immediately to avoid thawing.
1) Add 500 µl of 6% methanolic KOH (w/v) to the dried residue and incubate at 85⁰C for ½ -1 hrs.
2) To it, add half the volume i.e. 250 µl of dH2O and then equal volume i.e. 750 µl of n-heptane and vortex well.
3) Allow it to stand for sometimes till the layers get separated. Transfer upper phase to a fresh vial. Repeat the above step twice.
4) Allow heptane to evaporate completely (16- 24hrs).
To the dried residue, add 100 µl of derivatization reagent (80 µl BFSTA+20 µl TMCS) and incubate at 65⁰C for 1 hrs and inject in to GC-MS.
Critical step: The most critical point is to avoid any water or moisture during derivatization especially the silylating step is highly vulnerable.
For GC-MS analysis 1µl of the sample is injected in split mode in the instrument. Use a Rtx5MS- 30m column with 0.25-mm ID and 0.25µm df. Following are the parameters standardized for GC-MS run:
• Injection temperature: 300°C,
• Interface temperature: 300°C,
• Ion source should be adjusted to 250°C.
• Carrier gas: Helium (flow rate of 1 ml min-1).
• Perform the analysis using the following temperature program:
1 min. of isothermal heating at 100⁰C followed by heating at 300⁰C for 20 mins.
• Mass spectra were recorded at 2 scan sec-1 with a scanning range of 40 to 850 m/z. Quantify each component based on peak areas and normalization based on the internal standard.