## 2 Analytical Methods for Phytocannabinoid Profiling
### Sample Preparation Techniques
After extraction of cannabis plant material, additional filtrationis performed, using sintered glass disc [44], sterile cotton plugs [9; 75; 143; 144], PTFE syringe filters, with 0.22 \(\upmu\)m [68; 111] or 0.45 \(\upmu\)m pore size [56; 94; 95; 108], millipore filter (0.45 \(\upmu\)m) [50; 84], nylon filters (0.45 \(\upmu\)m) [133], membrane filter [49], PVD filter (0.22 \(\upmu\)m) [113] or, alternatively, drying on MgSO\({}_{4}\) and filtration [67; 138]. The insoluble plant material can also be removed by vacuum filtration. The filtered extract is usually dried under nitrogen and dissolved in IS (androst-4-ene-3,17-dione) in EtOH [58; 77], or in anhydrous pyridine [119]. Alternatively, IS can be added directly to the supernatant after filtration [44] or centrifugation [10; 103; 106; 111]. As filtration cannot be performed efficiently for cannabis resin extracts due to filter clogging by felt of trichomes and other vegetable debris [44], centrifugation is only performed. Solvent exchange may also be included as final step, by diluting extracts with solvents and solvent mixtures more similar to the mobile phases employed, in case of (HP)LC analysis. For example, cannabis-infused chocolate is first soaked in IS solutions and isopropyl alcohol, extracted with MeCN + 1% acetic acid and finally diluted with MeCN prior to LC-MS/MS or MeCN/H\({}_{2}\)O (75:25, _v/v_) prior to HPLC-UV analysis [65]. Solvent exchange is also performed prior to GC analysis, especially when derivatization is performed [7; 80; 87; 104; 121; 123; 133]. Extracts are reduced to dryness usually under gentle N\({}_{2}\) steam, causing least damage to total extracted amounts of phytocannabinoids and terpen than drying in rotary evaporator or in a speedvac, the latter reducing the concentrations of \(\Delta^{9}\)-THC and CBG for two-thirds [66]. Reduced extracts can be dissolved in solvents (e.g., pyridine and benzene [116], CHCl\({}_{3}\)[5], MeCN [116], dry EtAc [55]), in a mixture of derivatization agent and solvent (e.g., toluene and BSTFA [94], pyridine and BSTFA + 1% TMCS [118], pyridine, isooctane and MSTFA [99], pyridine and BSTFA [90], pyridine and MSTFA + 1% TMCS [84]) or directly in the derivatization agent [80; 99; 100; 133]. As silylation agents are harmful for GC injection port and column, additional evaporation to dryness is frequently performed, followed by dissolution in solvent, e.g., _n_-hexane [84] or MeCN [95]. If no derivatization is performed, EtAc [120; 126; 127] and EtOH [66; 123] are the most commonly used solvents for reconstitution prior to GC analysis. Before instrumental analysis, decarboxylation may be also introduced, initiating thermal degradation of phytocannabinoid acids to neutral counterparts for the purpose of accurate phytocannabinoid profiling and potency examination. Dried extracts are most commonly heated at 150-210 \({}^{\circ}\)C for 10-30 min and reconstituted in the same solvent or solvent mixtures [7; 52; 60; 101; 128], or, alternatively, at 50 \({}^{\circ}\)C for 180 min and then 145 \({}^{\circ}\)C for 15 min [69]. A total 15 min of decarboxylation at a temperature range of 120-180 \({}^{\circ}\)C showed that maximum yield is achieved at 140-160 \({}^{\circ}\)C, with no significant within-range differences [115]. Decarboxylation temperatures higher than 160 \({}^{\circ}\)C should be avoided, as \(\Delta^{9}\)-THC is oxidized to CBN [115] and isomerised to \(\Delta^{8}\)-THC [87]. However, it is almost impossible for decarboxylation to yield 100%, which initiates significant discrepancies in potency data. Laboratories quantifying the total \(\Delta^{9}\)-THC as the sum of the \(\Delta^{9}\)-THC already present in the plant and \(\Delta^{9}\)-THCA get higher values than laboratories that perform decarboxylation prior to instrumental analysis [115]. Once prepared, cannabis extract should be stored in light-protected conditions, at room temperature, refrigerated at 4 \({}^{\circ}\)C [17; 48; 84; 126], at \(-20\)\({}^{\circ}\)C [53; 103] or at \(-80\)\({}^{\circ}\)C [78] prior to instrumental analysis.