*3.3. Cannabinoid Extraction*

To extract cannabinoids, an aliquot of powder sample, about 25 mg, was weighed using an analytical balance; 10 mL of methanol-chloroform extraction solvent 9:1 (*v*/*v*) was added as reported by De Backer et al. (2009) [32], Jin et al. (2017) [33], and was placed first for 10 min on an oscillating oscillator set at 350 oscillations per minute and then for 10 min in an ultrasonic bath. The sample was centrifuged for 10 min at 1125 g, and the supernatant was removed. The extraction was performed twice. The two fractions containing cannabinoids were collected in a 25 mL volumetric flask and were brought to volume with methanol/chloroform (9:1, *v*/*v*). The samples were filtered with a 45 μm nylon filter. Two mL of the filtered extract was transferred to a glass tube. The solvent was removed, leading to dryness with the help of a weak nitrogen flow, and recovered with 500 μL acetonitrile. The solution was injected into an HPLC-UV.

#### *3.4. Preparation of Standard Solution*

Appropriate aliquots of a standard mixture of cannabinoids are diluted with acetonitrile to obtain solutions of known concentration, in particular eight points in a concentration range between 0.05 and 100 μg/mL (0.05, 0.50, 4.17, 8.33, 16.70, 25.00, 50.00, 100.00 μg/mL). The standard solutions were prepared to construct calibration curves for the 10 cannabinoids considered: CBDA, CBGA, CBG, CBD, THCV, CBN, Δ9-THC, Δ8-THC, CBC, and THCA. The standard solutions were stored away from light at a temperature of −20 ◦C. The stability of standard solutions stored at −20 ◦C was evaluated every week for 3 months with the HPLC-UV system, and no degradation of cannabinoids was found.
