## 2 Analytical Methods for Phytocannabinoid Profiling
### Sample Preparation Techniques
When compared to DM, UAE and MAE with same extraction solvent (EtOH) in same _w/v_ ratio to sample, no significant difference existed between SFE and UAE, with the lowest extraction yields for CBD, CBDA and CBGA. DM and MAE showed higher yield for CBD and CBGA, but DM was selected as the optimal sample preparation technique (EtOH, room \({}^{\circ}\)C, 45 min). In case of MAE, increased CBD yield was accompanied by decreased CBDA yield, suggesting partial decarboxylation due to high extraction temperature [142]. FUSE is used for phytocannabinoid extraction from herbal cannabis, employing cyclohexane/isopropanol (1:1, _v/v_) in an ice-water media in order to avoid degradation and solvent evaporation, followed by centrifugation and filtration through 0.45 \(\upmu\)m nylon filter [43]. FUSE is slightly more efficient than SFE for extraction of \(\Delta^{9}\)-THC, CBD and CBN, as 80% of the phytocannabinoids are extracted at the first extraction, while <40% are extracted after the third extraction with pure SC-CO\({}_{2}\) and more than 90% are extracted with the first extraction in the presence of co-solvent. Therefore, adding the ability of SFE to separate terpen from phytocannabinoids, and therefore minimize matrix interferences, SFE was selected as more optimal sample preparation technique [43]. PLE is one of the fastest and most efficient extraction techniques for plant metabolites. High extraction yields are achieved under pressure, using the extractant at a temperature above its normal boiling point, thus increasing its diffusion into the plant matrix. PLE on herbal cannabis is performed using _n_-hexane as extraction solvent at 100 \({}^{\circ}\)C and 40 bar for 15 min. MeOH and _n_-hexane are found equally efficient for \(\Delta^{9}\)-THC and CBN, but not for \(\Delta^{9}\)-THCA, as it is less soluble in _n_-hexane [95]. PLE with hot water, i.e., pressurized hot water extraction (PHWE) is used to yield CBD-rich extracts while supressing the THC and CBN content. Here, decarboxylation is performed in situ, i.e., in the extraction cell, heated in the oven prior to the dynamic extraction [80]. SPE with QuEchERS is used for purification of honey extracts, with high recoveries for CBDA, CBGA, \(\Delta^{9}\)-THCA, CBG, CBD and \(\Delta^{9}\)-THC and low intra- and inter-day variability. The method was more efficient than UAE with H\({}_{2}\)O at 40 \({}^{\circ}\)C, yielding homogeneous solution with no phase separation or solid residues, followed by extraction of phytocannabinoids from the aqueous phase through LLE with _n_-hexane or EtAc, observing higher phytocannabinoid yield in _n_-hexane extracts [40]. QuEchERS is also used for purification of MeCN extracts of hemp seeds, hempseed oil, hemp proteins, raw and skimmed milk, coffee and chocolate, using reaction mixture of MgSO\({}_{4}\)/NaCl/C\({}_{6}\)H\({}_{5}\)Na\({}_{3}\)O\({}_{7}\)\(\times\) 2H\({}_{2}\)O/disodium hydrogen citrate sesquihydrate (4:1:1:0.5, _w/w_); with further supernatant dilution with MeCN/H\({}_{2}\)O (1:1) or with H\({}_{2}\)O. d-SPE with MgSO\({}_{4}\), different combinations of C\({}_{18}\), primary secondary amines and zirconia-coated silica sorbents were also evaluated. When PSA is used, cannabinoids were trapped by interactions with amines, resulting in low recoveries; other combinations achieved satisfactory extraction [144]. Cloud point extraction (CPE) involves employment of non-ionic surfactant, salt (Na\({}_{2}\)SO\({}_{4}\)) and deionized water to extract \(\Delta^{9}\)-THC from cannabis resin using heating (40-90 \({}^{\circ}\)C) and centrifugation. Despite the low extraction efficiency (60%), CPE offers many advantages, such as the possibility of extraction and pre-concentration of analytes in a single, solvent-free step and avoidance of analyte loss during solvent evaporation [145]. Centrifugal partition chromatography (CPC) is a liquid-liquid partitioning technique in which the stationary phase is immobilized by centrifugation force, while the mobile phase is pumped through at high flow rates.