Analysis of Stevia Leaf Metabolites

Leaves harvested from the 4th and 5th nodes of Stevia plants grown in the greenhouse for 3 weeks were homogenized in liquid nitrogen. Three hundred fifty milligrams of leaf powder was extracted with 350 μL of ethyl acetate containing 20 μg/mL of camphor (Sigma-Aldrich) as an internal standard. After 3 h incubation at room temperature with constant shaking, the ethyl acetate fraction was transferred into a new tube and treated with anhydrous Na₂SO₄. The treated extracts were then filtered through a 0.45 μm nylon centrifuge tube (Corning, USA). The GC-MS analysis was performed on Agilent 7890A GC (Agilent Technologies, USA) system as described previously [12 (link)].