Hplc grade

D r a f t 1 One sample from each of the two packets was analyzed. An aliquot of the dried leaf product was placed in a mortar and pulverized to a fine powder. For the extraction, 0.3 g of each sample was placed in a tube and 2 mL of a methanol-chloroform mixture (1:1) was added (HPLC-grade; JT Baker (USA)). The tubes containing the samples were shaken in a vortex for 1 minute. The samples were filtered and injected in a GC-MS. To perform the GC/MS analysis, an Agilent 6890N GC equipped with an RXi-1MS capillary column (25 m × 200 μm × 0.33 μm) was employed. Helium was used as the carrier gas; the injection port was maintained at a temperature of 250 °C, and a split ratio of 10:1 was used. The column oven temperature program was as follows: initial temperature of 100 °C for 2.0 min, followed by linear 12 °C/min increments up to 300 °C, and hold for 10.0 min. Total chromatographic run time was 28.7 min.
Detection was performed with a quadrupole mass spectrometer (Agilent 5973 inert). The m/z range was 30-550 amu. Identification of substances was performed by comparison of the mass spectra obtained with the National Institute of Standards and Technology (NIST) electronic library.

For the measurement of proposed plant-derived amino acids in the BCD fraction, liquid chromatographic separation was conducted using a Kinetex HILIC column (100-mm length, 4.6-mm internal diameter, 2.6-μm particle size; Phenomenex, Torrance, CA) using a 1200 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA) as described previously [39] . The injection volume for each measurement was 2 μL. The sample tray and column compartment were set to 6°C and 40°C, respectively. The mobile phase was composed of 20 mM ammonium acetate in water (solvent A) and 10 mM ammonium acetate in 90% acetonitrile and 10% water (solvent B) (HPLC grade, Honeywell Burdick & Jackson, CA, USA). Ammonium acetate was prepared from a stock solution of 100 mM ammonium acetate and 0.7 % formic acid (98-100% chemical purity, from Sigma-Aldrich, St. Louis, MO, USA) in water. Amino acids were separated with the following gradient: 90% to 70%B in 4 min, held at 70%B for 1.5 min, 70% to 40%B in 0.5 min, held at 40%B for 2.5 min, 40% to 90%B in 0.5 min, held at 90%B for 2 min. The flow rate was varied as follows: held at 0.6 mL/min for 6.5 min, linearly increased from 0.6 mL/min to 1 mL/min in 0.5 min, and held at 1 mL/min for 4 min. The total run time was 11 min. The mass spectrometry parameters have been previously described [69] .