## 2 Analytical Methods for Phytocannabinoid Profiling
### Sample Preparation Techniques
HS-SPME is a solvent-free sample preparation method used for analysis of phytocannabinoids in the headspace over solutions or solid samples [5]. Multiple factors affect the extraction efficiency during HS-SPME, including SPME fiber coating, exposure temperature, extraction time and desorption time. Evaluation of the effect of the fiber coating on extraction efficiencies of \(\Delta^{9}\)-THC, CBD and CBN from herbal cannabis samples showed that among polymethylsiloxane (PDMS) 100 \(\upmu\)m, PDMS/divinylbenzene (PDMS/DVB) 65 \(\upmu\)m, Carboxen(r)/PDMS and divinylbenzene/Carboxen(r)/PDMS (DVB/Carboxen(r)/PDMS) 50/30 \(\upmu\)m, PDMS 100 \(\upmu\)m performed optimally in general, although the PDMS/DVB fiber provided higher extraction efficiency for CBD, due to its higher polarity and affinity to PDMS/DVB. Among the three exposure temperatures (80 \({}^{\circ}\)C, 90 \({}^{\circ}\)C, 150 \({}^{\circ}\)C), 150 \({}^{\circ}\)C was optimal, simultaneously promoting volatilization and decarboxylation [59; 148]. Extraction time depends upon matrix viscosity and lipophilicity that define the speed of diffusion of analytes from the liquid to the gas phase and, as a result, HS-SPME rate and efficiency. HS-SPME is more appropriate for simpler matrices (e.g., cannabis tea), as the extraction recoveries are proportional to the sample amount [99], while complex liquid- and protein-containing matrices cause significant matrix retention and lower recoveries, with higher LODs and lower method precision. For fatty/oily matrices, such as versatile hemp foods, alkaline hydrolysis with NaOH and Na\({}_{2}\)CO\({}_{3}\) is performed prior to HS-SPME in order to sapoirify the matrix lipids and reduce lipid matrix interferences [99]. Therefore, extraction time varies depending upon the sample matrix, from 10 min for herbal cannabis [59; 148] to 25 min for different hemp food products [99]. Finally, desorption time depends upon analytes' lipophilicities [148]. It is superior to LLE (_n_-hexane/EtAc (9:1, _v/v_)) in terms of chromatographic peak shape and matrix interferences, despite the good agreement of achieved LODs in food samples [99]. SFE uses supercritical fluids (SCFs) and liquefied gases as green solvents for extraction and fractionation of complex samples. SFE offers low solvent consumption and ensures stability of thermolabile and light-sensitive compounds. For the purpose of phytocannabinoid profiling, SFE is rarely used in sample preparation; its main purpose is to separate the aromatic fraction for further analysis. It is usually performed using supercritical CO\({}_{2}\) (SC-CO\({}_{2}\)) as attractive SCF with solvent strength tuned by sensitive changes in temperature and pressure above the critical point (31.1 \({}^{\circ}\)C, 73.7 bar), conditions that are experimentally easy to reach [4], along with the low cost, short processing time and low environmental impact [149]. However, SC-CO\({}_{2}\) is a low polarity solvent that poorly dissolves phytocannabinoids; therefore, employment of co-solvent, usually H\({}_{2}\)O, alcohols and acids, to improve the overall extraction rate of phytocannabinoids is required. EtOH (5-20% in CO\({}_{2}\)) is most commonly used co-solvent, added in constant flow [40; 43; 143; 150; 151] or in pulses [143]. Higher SC-CO\({}_{2}\) pressures offer lower extraction selectivity, but high initial extraction rate, apparent solubility and total yield, which are also a function of temperature, exposure time and phytocannabinoid content of the plant material [143]. A 90-94% extraction yields for \(\Delta^{9}\)-THC, CBD and CBN are achieved at 100 bar, 35 \({}^{\circ}\)C and 1 mL/min flow during 10 min [43; 150]; at 340 bar, 55 \({}^{\circ}\)C and 200 g/min maximal yields up to 92%are reported [143], along with satisfactory yields at milder conditions, (37 \({}^{\circ}\)C, 250 bar). Extraction efficiency was further improved by washing of the extract with fresh SC-CO\({}_{2}\) and addition of a cold separator (separating chamber) immediately after the sample containing chamber [149].