## 2 Analytical Methods for Phytocannabinoid Profiling
### Instrumental Analysis
#### 2.2.2 LC-Based Methods
Raptor ARC-18 is the C18 column with the widest applicability in phytocannabinoid profiling, offering the most appropriate separation of 17 phytocannabinoids (CBG, CBD, CBN, \(\Delta^{9}\)-THC, A\({}^{8}\)-THC, THCA, THCV, THCVA, CBC, CBGA, CBDA, CBL, CBLA, CBDV, CBDVA, CBLA) at a runtime suitable for commercial environment. It improves peak resolution issue of some of the aforementioned columns, such as Kinetex C18, Luna C18, Luna Polar C18 and Raptor C18 [100] and has been shown to, together with Raptor ARC-18 EXP guard column, be suitable for analysis of pesticides, mycotoxins and cannabinoids [65]. The latest research points out the importance of core-shell technology columns (e.g., Poroshell) for separation of 96 phytocannabinoids by ESI-LC/MS [19]. C18 columns with advanced bonding of the trifunctional C18 phase and end-capping process, such as Acquity UPLC\({}^{\copyright}\) HSS T3, are also used [183,184]. RP-C18 columns, most commonly Synergi Hydro RP C18 [63,65,66], RP-C18 Hydro [61,62], Mediterranean RP-C18 [78], LiChrospher 60, RP-Select B [191], RP-C18 [165], with [60, 61, 62, 63, 191] or without [78,165] C18 guard column are also used. Other columns, such as Ascentis Express RP-amide are also used [64]. A better chromatographic performance (in terms of both resolution and sensitivity), a shorter analysis time (10 min vs. 12 min) and a considerable saving of solvent consumed while working at a flow rate of 0.5 mL/min instead of 1.5 mL/min, was observed while working on a fuse-core stable bond (SB) RP-C18 column rather than fully porous RP-C18 column [182]. Another study evaluated three different columns, RP-C18, fused-core stable bond (SB) RP-C18 and fused-core end-capped (EC) RP-C18 with numerous mobile phases and gradient conditions in an attempt to shorten the run time and to increase the separation of 8 phytocannabinoids (CBDA, CBGA, CBD, CBG, CBN, \(\Delta^{9}\)-THCA, \(\Delta^{9}\)-THC and \(\Delta^{8}\)-THCA). The SB RP-C18 core shell column provided the best performance due to significant improvement in separation and symmetry of chromatographic peaks with a baseline separation between CBD and CBG within 20 min shorter run time [176]. * HPLC Detectors For the purpose of phytocannabinoid profiling, (U)HPLC analytical platforms are coupled to UV, DAD, PAD or MS. Phytocannabinoids have low molar absorptivity, which results in relatively low sensitivity of LC methods employing UV and DAD. This restricts employment of DAD detection to low wavelengths where there is often strong background absorbance from the eluent components, especially during gradient elution experiments [176]. Additionally, UV/DAD methods often have low specificity for some phytocannabinoids, e.g., CBDA and CBGA, due to similar UV/DAD spectra [178]. Phytocannabinoids have different UV behaviour on the basis of their chemical structure. Cannabinoid acids (CBDA and CBGA) are characterized by three absorption maxima (\(\lambda_{\text{max}}\)), one stronger at 220-223 nm, the second at 266-270 nm and the third one around 305 nm, while neutral phytocannabinoids (CBD and CBG) show a first \(\lambda_{\text{max}}\) at 210-215 nm and an additional one at 270 nm. Generally, the ranges 190-600 nm and 200-650 nm are most commonly used for UV acquisition, while two wavelengths are selected-210 nm for neutral phytocannabinoids and 220 nm for cananbinolic acids [43; 72; 185]. For DAD, a narrower range is selected (i.e., 200-400 nm [83], 190-500 nm [182]). Single wavelength can be selected for evaluation of multiple phytocannabinoids in hemp seed oils [179], in plant material (230 nm) [68], 214 nm [188; 189] or 220 nm in plant material and resins [69; 165] or cannabis extracts [27], in commercial veterinary supplements (225 nm) [192] or for quantification of \(\Delta^{9}\)-THC, \(\Delta^{9}\)-THCA, CBN and CBD (210 nm) [191] or \(\Delta^{9}\)-THC and \(\Delta^{9}\)-THCA in plant material (211 nm and 220 nm) [185].