## 2 Analytical Methods for Phytocannabinoid Profiling
### Sample Preparation Techniques
Prior to maceration/LLE, lactose-containing cannabis products (cream cheese, butter, coffee beverages with milk) require lactase pre-treatment/lactose hydrolysis in order to avoid matrix interferences [90]. For high sugar and carbohydrate matrices including hard candies, honey and fruit preserves, a matrix trapping effect occurs, resulting  
\begin{table}
\begin{tabular}{l l} \hline \hline
**Solvent/Solvent Mixture** & **References** \\ \hline CCl\({}_{4}\) (later evaporated and extracts reconstituted in chloroform) & [131] \\ \hline MeCN/MeOH (8:2, _v/e_) & [132] \\ \hline hexane/isopropanol (9:1, _v/e_) & [57; 94; 106; 133] \\ \hline hexane/ELAe (9:1, _v/e_), (7:3, _v/e_), (6:4, _v/e_) & [54; 57; 66; 94; 104] \\ \hline hexane/CHCl\({}_{3}\) (1:1, _v/e_) & [134; 135] \\ \hline MeOH/CHCl\({}_{3}\) (4:1, _v/e_) & [48; 136] \\ \hline MeOH/CHCl\({}_{3}\) (9:1, _v/e_), (9:1, _v/e_) & [57; 67; 86; 106; 137] \\ \hline MeOH/hexane (9:1, _v/e_) & [138] \\ \hline petroleum ether/MeOH (9:1, _v/e_) & [45] \\ \hline EtOH/H\({}_{2}\)O (1:1, _v/e_) & [133] \\ \hline KOH in MeOH and hexane/EtAc (9:1, _v/e_) & [139] \\ \hline IS (tribenzylamine) in 96\% EtOH & [57] \\ \hline IS (tribenzylamine) in MeCN & [140] \\ \hline IS (nonadecane) in EtOH & [138] \\ \hline IS (diphenylhydramine) in EtOH & [74] \\ \hline IS (4-androstene-3,17-dione) in EtOH & [9; 137] \\ \hline IS (docosane) in petroleum ether & [128; 141] \\ \hline IS (nonadecane) in MeOH/CHCl\({}_{3}\) (9:1, _v/v_) & [67] \\ \hline IS (squalane) in hexane & [76; 89; 142] \\ \hline IS (chrysene-d\({}_{12}\)) in hexane & [71] \\ \hline IS (Retamine hydrochloride) in MeCN & [124] \\ \hline IS (4-androstene-3,17-dione) in MeOH/CHCl\({}_{3}\) (9:1, _v/e_) & [9; 75; 86; 143; 144; 145; 146; 147; 148; 149; 150; 143; 144; 146; 147; 148; 149; 151; 152; 153; 154; 155; 156; 157; 158; 159; 160; 161; 162; 163; 164; 165; 166; 167; 168; 169; 170; 17  
in the formation of glassy, impervious precipitates which are hard to extract. To avoid this, the aqueous portion of the extractant is added first, followed by warming the matrix in order to dissolve the sugars and carbohydrates, and addition of ACN (to 83-91% final proportion) [90]. Maceration and LLE of cannabis-based products is further aggravated by the presence of glycerine and propylene glycol, especially in oral supplements and vape products. They interfere cannabinoid profiling significantly. High levels of co-extracted glycerine or propylene glycol may swamp the silylating derivatization agents, disabling complete derivatization of CBD to CBD-2 TMS, along with undesired side conversion of \(\Delta^{8}\)-THC and \(\Delta^{9}\)-THC. The problem can be avoided with CAN extraction, as sugars and glycerine have much lower solubility in ACN than in EtOH [90]. While most of the methods include vortexing, ultrasonication in bath and centrifugation immediately after solvent addition, older methods include immersion, e.g., for 1 h [95; 121; 143; 144], overnight [121], 10-days soaking [123], several hours' maceration [87] or heating near boiling for few hours in solvent, followed by separation of the liquid extract by filtration [108]. Only one study evaluated the shaking time during extraction with MeOH/CHCl\({}_{3}\) (9:1, _v/v_) at room temperature in a range from 10 min to overnight shaking and showed that 20 min of shaking gained sufficient extraction efficiency for \(\Delta^{9}\)-THC, CBD and CBN [101]. Finally, exhaustive extractions in Soxhlet apparatus are rarely performed [95; 117]. In terms of temperature conditions, extraction is performed with highest efficiency at room temperature, with rare exemptions (e.g., at 4 \({}^{\circ}\)C) [77], in order to avoid conversion of phytocannabinoids from plant material [1]. If additional heating is required (e.g., when no preliminary decarboxylation was performed), hot extraction (e.g., at 70-78 \({}^{\circ}\)C) is performed by sonication [92] or in Soxhlet apparatus [95; 117].