## 2 Analytical Methods for Phytocannabinoid Profiling
### Sample Preparation Techniques
In the next step, thermal processing occurs, which aims to remove moisture, usually to 8-13% residual humidity, as recommended by UNODC [5], achieved by drying at room temperature for several days or at 70 \({}^{\circ}\)C until the leaves become brittle or, according to EC, within 48 h using any method below 70 \({}^{\circ}\)C [70]. Lower drying temperatures should be avoided, as they result in mould growth [71]. Despite this, drying is frequently performed at variety of temperatures and durations, such as at 135 \({}^{\circ}\)C, 2 h, 120 \({}^{\circ}\)C [72], 103 \({}^{\circ}\)C, 4 h [73], 65 \({}^{\circ}\)C, 16 h [74] or 48 h [75; 76], 60 \({}^{\circ}\)C, 12 h [77], 40-50 \({}^{\circ}\)C [54], 40 \({}^{\circ}\)C,72 h [78], 48 h [17; 79] or 24 h [74; 80; 81], 38 \({}^{\circ}\)C, 4-8 h [82], 35 \({}^{\circ}\)C, 24 h [83], 30-40 \({}^{\circ}\)C, 1-2 days [46], 30 \({}^{\circ}\)C [57] on forced ventilation oven, by natural ventilation at 32 \({}^{\circ}\)C for 60 h [84], at 30 \({}^{\circ}\)C overnight [52] or for 4 h [80] or air-dried at room temperature (20-22 \({}^{\circ}\)C) for 24 h [81], for 3 days [44; 85], 6 days after harvesting [86], for 4 weeks [87] or until a residual humidity \(\leq\)12% is achieved [47]. Dried and mechanically processed samples are extracted using maceration, LLE, PLE, HS-SPME, SFE or FUSE. Other extraction techniques, including ultrasonic assisted extraction (UAE), microwave assisted extraction (MAE), dynamic maceration (DM) and accelerated solvent extraction (ASE), are faster and use less extraction fluids than the \"classic\" maceration. The number of the consecutive extractions did not have significant effects on total phytocannabinoid yield. Yet, phytocannabinoids yield after sonication was found to be slightly lower than the yield obtained by one-day DM [50]. Phytocannabinoid extraction was omitted in only one study, which, consequently, reported low sensitivity (Table S1); thus difficulties occurred during quantification of trace phytocannabinoidsin cannabis plant tissues [88]. The summary of the properties of the most frequently used sample preparation techniques are given in Table 2.  
The conventional sample preparation methods for cannabis plant material are maceration and LLE using versatile organic solvents with great affinity towards phytocannabinoids. Although universal and simple, they are time-consuming and not environment-friendly, as they require large quantity of organic solvents. UNODC recommends maceration prior to GC-flame ionization detector (FID) analysis; 0.2 g dried and homogenized herbal cannabis, 0.1 g cannabis resin or 0.05 g cannabis oil is extracted with internal standard (IS) solution of tribenzylamine in 96% EtOH (0.5 mg/mL) for 15 min in an ultrasonic bath [5]. DAB's _Camabis flos_ monograph proposes extraction with EtOH (96%, _v/v_), while the AHP's Cannabis inflorescence monograph proposes extraction with MeOH/CHCl\({}_{3}\) (9:1, _v/v_) [33]. EC recommends maceration of 0.1 g semi-fine powdered herbal cannabis with IS solution of 35 mg of squalane/100 mL hexane [70]. The most commonly used solvent and solvent mixtures for extraction of phytocannabinoids from cannabis and cannabis-based products are given in Table 3. \begin{table}
\begin{tabular}{l l l l} \hline \hline \multicolumn{1}{c}{**Sample Preparation Technique**} & \multicolumn{1}{c}{**Advantages**} & \multicolumn{1}{c}{**Disadvantages**} \\ \hline \multirow{6}{*}{LLE} & \multirow{6}{*}{-} & variety of solvents and solvent \\  & & mixtures with appropriate \\  & & extraction efficiencies; \\  & - & appropriate for all matrices \\  & - & low price \\ \hline \multirow{6}{*}{PLE} & \multirow{6}{*}{-} & possibility to perform \\  & & decarboxylation in situ \\ \cline{1-1}  & & greatreproducibility \\ \cline{1-1}  & & low price \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Sample preparation techniques for phytocannabinoid profiling of cannabis and cannabis-based products.