Phytochemical Extraction and Analysis
Desiccated leaves were homogenized under liquid nitrogen and stored at
-80°C prior to extraction. To extract a broad range of polar and
nonpolar metabolites, the homogenized samples were mixed with 1 mL of a
1:1 methanol-chloroform solution in conjunction with 1 µL of
ethyl-decanoate (Sigma-Aldrich, Inc.) as an internal standard. Sample
extracts were vortexed in an orbital shaker for 2 hours to expedite
extraction at 160 rotations min-1. Post extraction,
each sample was filtered through a 0.2 μm nylon filter into a 2 mL amber
glass vial, where 1 µL of the sample extract was used for direct
injection into a single quadrupole GCMS-QP2020 NX gas chromatograph-mass
spectrometer (Shimadzu, Inc.). Each extract was injected at 230°C in
splitless mode with helium carrier gas flow set to 2 mL
min-1 by an AOC-6000 autosampler (Shimadzu, INC.). The
oven was held isothermal at 40°C for 1 min, ramped at 15°C
min-1 to 330°C where it was held isothermal for 1 min.
Electron impact mass spectra were recorded in full scan mode from m/z 50
to 450 in the single quadrupole mass spectrometer. Data were initially
preprocessed using GCMS solutions v1.4 (Shimadzu, Inc.). Signals were
integrated using total ion count and measurements such as the area and
height of chromatogram peaks were normalized to the internal standard.
Compounds were putatively identified by comparing observed mass spectra
with the NIST mass spectra library using a 75% significance index
cutoff.