NSF Project: Protocols

Preparation and extraction of secondary metabolites

1. Frozen tissue was lyophilized for three days and homogenized
2. 20.00-20.06 mg of tissue was weighed into 1-dram glass vials for each sample
3. 40 ng of 7-Hydroxycoumarin was added to each sample as an internal standard
4. 1.8 mL of 80% methanol was added to each sample Sample was extracted overnight at room temperature with constant agitation from an orbital shaker
5. The vials were spun at 2900xg for 30 minutes so supernatant could be collected
6. 1.4 mL of supernatant was collected and dried using rotary evaporator.
7. Samples were stored -20° C until processing and then resuspended in 100uL water prior to injection.
8. 30 uL of sample was injected onto column.

LC/MS
Analysis was carried out on Bruker Esquire-LC-ion trap mass spectrometer equipped with an electrospray. HPLC separation was achieved using a reversed-phase, C18, 5 ?m, 4.6X250 mm column (J.T. Baker). Samples were eluted with 0.1% acetic acid and acetonitrile; 5-70%B over 65 min. Negative-ion mass spectra were recorded over the range of 50-2200 m/z.