## 2 Analytical Methods for Phytocannabinoid Profiling
### Sample Preparation Techniques  
Rapid and simple extraction methods are essential for time- and material-efficient high-throughput phytocannabinoid profiling. Optimization of the three key parameters during extraction is crucial for the overall analytical method [40], which are: (1) the _granulometry_ of the solid sample; (2) the _system temperature_ and (3) the _affinity_ of the extraction liquid towards compounds of interest [41]. _C. sativa_ and cannabis-based products are very complex and inhomogeneous matrices, as their different parts may have different cannabinoid profiles due to the variety of phytocannabinoids, terpen and other volatile compounds and high sugar and fat content. Thus, extraction of phytocannabinoids from plant material and cannabis-based products in an efficient and consistent manner, with acquisition of accurate and reliable potency data can be a challenging task. In addition, there are no standardized preparation procedures for the hemp-based infusions (hemp leaf, hemp-based tea mixtures), and the cannabinoid content could be significantly affected by the infusion preparation procedure [42]. Finally, there is large and unpredictable variability of the average composition as a result of genetic and environmental differences, making the efforts for standardization of sample preparation techniques an ultimatum [43]. Initial steps of sample preparation include mechanical preparation aiming to increase the contact surface between the solvent and the active ingredients. It is of special importance for cannabis plant material, since, despite that most of the active resin is claimed to occur in the superficial glandular trichomes, significant amounts are found in non-glandular tissues. Consequently, immersion of unbroken fresh plant material would give unsatisfactory extraction [44]. Cannabis or its resins are reduced to small pieces by a grater [5] or spatula [45], grinded or pulverized, while cannabis oils are directly proceeded to instrumental analysis. Manual pulverization and homogenization of the dried plant material can be performed using mortar and pestle [46; 47; 48; 49; 50], metal spoon [51] or glass rod [52], by cutting the plant material [53] or crushing and riddling (0.5 mm) [54] or by manual grinder [50; 55]. According to the UNODC [5], dried herbal cannabis material and cannabis should be pulverized by a cutter (at high revolution speed, i.e., 100 rps) and sieved (mesh size 1 mm). Plant samples can also be homogenized in a crucible [56], in laboratory blender, usually to 60-80 mesh (177-250 \(\upmu\)m) particle size, but sometimes a larger (\(\leq\)335 \(\upmu\)m) or smaller (100-150 \(\upmu\)m) particle size is required [57; 58; 59]. Unlike classical, mechanical hand grinders and electrical grinders, some sample preparation methods employ superfine grinding of cannabis plant material [60; 61; 62]. However, manual grinding with a handheld herb grinder resulted in higher yield of total phytocannabinoids (17.5 \(\pm\) 0.5%) than with electric blender (12.0 \(\pm\) 0.3%). The minimization of analyte loss using manual grinder is attributed to the adhesion of cannabis resin to the blades and plastic housing surface of a plastic blender during the high-speed pulverization [50]. Mechanical grinding-activation in an intensity planetary vibrational mill [62; 63], ball mill [55], knife mill [64] or freeze mill [65] are also applied. Instead of drying, fresh cannabis plant material can be frozen with liquid nitrogen  
Figure 2: Analytical methods used in phytocannabinoid profiling of cannabis and cannabis-based products. and crushed [66; 67] or frozen, lyophil at \(-50\)\({}^{\circ}\)C and grounded by hand [68] or in a mill [69]. Comparison of coarse homogenization by sieving through a 1-mm mesh and fine homogenization with a ball mill revealed better extraction efficiency for CBDA and THCA for the finely powdered plant material, and no difference for the neutral phytocannabinoids (CBD, A\({}^{9}\)-THC) [55].