Sample plant material in 2ml Eppendorf tube (round bottom-shaped). Define the exact mass of plant sample (should be 57-64 mg fresh weight for leafs or roots, ~30mg fresh weight for potato tubers or fruits, document the freshweight of each sample). Shock-freeze sample in liquid N2. Shock-freezing is best done before determination of sample mass. Also sample empty micro vials and use for non-sample controls. Make sure to include a set of non-treated control samples in order to screen for relative changes of metabolite levels. Provide 6 (better 8-16) replicate samples of each condition to be analysed. Do replications at the level of individual plants. Pooling of samples from a set of plants and replicate analyses of this pool is possible but less informative.

Homogenize with a Retsch ball mill, 3 min at 15 1/s. Take care that sample remains frozen.

Add 300 µl 100 % Methanol (100%), pre-cooled at –20°C, vortex. Most enzymatic activity stops.

Add 30 µl stock nonadecanoic acid methylester (2 mg/ml Stock in CHCl3) for quantitative internal standardization for the lipid phase

Add 30 µl of a pre-mixture made from ribitol (used for quantitative internal standardization for the polar phase), d4-alanine, D-(-)-isoascorbic acid and any stable isotope labelled internal standard of special interest, vortex. The internal standards may also be included in the methanol solvent used for extraction (refer to 3).

Shake 15 min at 70°C. After approx. 1 min of incubation, open the micro vials for a short moment and relieve built-up gas pressure. Let cool down to room temperature.

Add 200 µl CHCl3.

Shake 5 min at 37°C.

Add 400 µl H2O, vortex.

Centrifuge 5 min at 14000 rpm.

Transfer at least two 160µl-aliquots (for potato tubers two additional 20µl-aliquots!) from the upper polar phase to clean micro vials (1.5ml).

Dry in the Speed Vac overnight, without heating. Small volumes (< 80 µl) may be dried within 2-3 h.

Process one replicate dried aliquot. Store the remaining dried aliquot of the polar phase under argon. Close caps of micro vials after gentle flushing with argon, better flood the Speed Vac with argon or nitrogen before opening. Put micro vials into a plastic bag add Silica Gel to the bag, flush with argon and heat seal the bag. These bags may be transported at room temperature but are best stored at –80°C. Surplus aliquots are used as back up samples in case of processing failures. Make sure that reference and treated samples are stored in the same bag. When processing bags from the freezer wait for temperature equilibration and remove moisture before opening the bag.

Add 40 µl Methoxyaminhydrochlorid (20 mg/ml in Pyridin) to the dried aliquot.

Shake 1.5 h at 30°C (incubation in a 28°C cabinet or room may be used).

Spin down shortly the drops on the snap cap.

Add 10 µl alkanemixture to the polar phase.

Add 70 µl MSTFA. Optional for step 5-6: Make a premix from Alkanmix and MSTFA.

Shake 30 min at 37°C (incubation in a 37°C cabinet or room may be used).

Spin down shortly the drops on the cover.

Transfer 80µl into GC sample vials.

Also prepare vials with 110 µl MSTFA + 10 µl alkane mixture. These samples are used to per-condition the GC-MS system.

Check machine: Tuning, Mass calibration, quick leak check, syringe (sometimes plugged), amount of wash buffer, liner

Prepare the samplelist in the following order:

1. Mstfa+Alkane
2. Mstfa+Alkane
3. Non-sample control (blank)
4. Non-sample control (blank)
5. Your samples in an appropriate order

After Starting of the machine check, if the machine is running. Check the chromatogram, peakshape, sensitivity, performace of alkanes, baseline, mass spectral properties.