2.6. Determination of Tetrahydrocannabinol (THC), Tetrahydrocannabinolic Acid (THCA), Cannabidiol (CBD), and Cannabidiolic Acid (CBDA)

The freeze-dried (100 mg) leaf sample was dissolved in 5 mL of methanol (100%) and sonicated at room temperature for 20 min. After filtration through a syringe filter (0.45 µM, Millipore, Bedford, MA, USA), the solution was kept in a refrigerator at 4 °C. The HPLC system (Shimadzu LC-20 AT, Shimadzu Co., Ltd., Kyoto, Japan) with a UV-VIS detector and a reverse phase Zorbax SB-C18 column (4.6 mm × 100 mm, 3.5 µm, Agilent Technologies, Inc., Santa Clara, CA, USA) was used. The mobile phase was 70% acetonitrile containing 0.1% phosphoric acid with isocratic elution mode. The retention times of standard CBDA, CBD, Δ9-THC, and Δ9-THCA were 3.60, 4.34, 9.60, and 13.00 min, respectively (Supplementary Figure S1). A 10 µL sample was injected where the flow rate and oven temperature was 1.5 mL min⁻¹ and 27 °C, respectively. The detection wavelength was used 275 nm with three biological replications.