## 4 Materials and Methods
### LC-UV Method Validation and Quantitative Analyses  
The analytical system for cannabinoid quantitative analysis consisted of an Agilent 1200 Series Gradient HPLC System equipped with a quaternary gradient pump unit, a DAD (diode array detector, 190 nm-400 nm), and a standard autosampler (0.1-100 \(\mu\)L) (Agilent Technologies, Santa Clara, CA, USA). The autosampler was set to inject 20 \(\mu\)L. All the experiments were performed at room temperature (25 \({}^{\circ}\)C).  
The separation was attained on a reversed-phase Luna C18, 5 \(\mu\)m (150 \(\times\) 4.6 mm, 100 A) analytical column, preceded by a security guard cartridge. The linear gradient was between eluent A (water containing 0.1% formic acid) and eluent B (acetonitrile). The column temperature was 25 \({}^{\circ}\)C and the flow rate was 1 mL/min. The elution gradient was set as below: 0-17 min (35-5% A), 17-22 min (5% A), 22-24 min (5-35% A), and 24-28 min (35% A). The wavelength value employed for UV detection was 220 nm for THCA, CBDA e CBN, and 210 nm per THC and CBD.  
The LC-UV validation protocol included parameters such as linearity, precision (for both peak area and retention time), accuracy, limits of detection (LOD), and limits of quantification (LOQ). The stock solutions of CBD, THC, CBDA, THCA, and CBN were prepared by diluting concentrated standard solutions in MeOH by using 50:50 methanol/acetonitrile (\(v/v\)). All samples were analyzed in triplicate using the optimized method described above. The linearity was investigated in the range 0.1-50 mg/L for each compound, according to the regression line by the method of least squares and expressed by the coefficient of correlation (R\({}^{2}\)). Accuracy, expressed as recovery, was calculated from the spikes of 1 mg/L and 10 mg/L standard solutions to cannabis extracts. Precision was measured as percentage relative standard deviation (%RSD) for two levels (k = 2), 1 mg/L and 10 mg/L. The repeatability was calculated in the same day for six replicates (\(n=6\)); instead, the intermediate precision was obtained within several days (\(p=3\)) for the ten replicates (\(n=10\)). A %RSD below 15% and 30% for repeatability and intermediate precision, respectively, were considered suitable [29]. The detection limit (LOD) is the lowest quantity or concentration of analyte in the sample that can be reliably distinguished from zero [22]. It was calculated as follows: LOD = (3.3 \(\sigma\))/m, where \(\sigma\) is the residual standard deviation of the calibration line and m is the slope of the calibration line. The quantification limit (LOQ) is the concentration of analyte, below which it is determinable with a level of precision that is too low with inaccurate results. The LOQ can be determined by using the following formula: LOQ = (10 \(\sigma\))/m, where \(\sigma\) is the residual standard deviation of the calibration line and m is the slope of the calibration line. All tests were performed in triplicate.  
Quantification of cannabinoids, and thus chemotypes definition, was carried out by usingthe external standard method. According to official methods, the peak area ratio of each standard cannabinoid was plotted versus the analyte concentration [30; 31; 32; 33]. Values are provided as percent analyte per 100 g dry weight (%), the standard deviation (SD) being estimated for three replicates.  
Data acquisition and analyses were accomplished using the HPLC 1200 offline (Agilent Technologies, Santa Clara, CA, USA). The chromatographic raw data were imported, elaborated, and plotted by SigmaPlot 11.0 (Systat Software, Inc., London, UK).