## 2 Analytical Methods for Phytocannabinoid Profiling
### Instrumental Analysis
#### 2.2.2 LC-Based Methods
for assay of _Cannabis__fos_ and _Cannabis__extractum__normatum_ focus on the five main phytocannabinoids: CBDA, CBD, CBN, \(\Delta^{9}\)-THC and \(\Delta^{9}\)-THCA using aqueous solution of 85% _o_-phosphoric acid and MeCN as mobile phases [34; 35; 36; 38]. AHP's _Cannabis__fos_ monography recommends identical mobile phases for quantification of the major phytocannabinoids (\(\Delta^{9}\)-THCA, \(\Delta^{9}\)-THC, CBD, CBDA, CBG, CBGA and CBN) (Table S2) [33]. Binary mobile phase system consisted of H\({}_{2}\)O/MeOH (10:90, _v/v_) [186], H\({}_{2}\)O/MeOH (17/83, _v/v_) [187] and H\({}_{2}\)O + 0.1% formic acid/MeOH + 0.1%; formic acid is most commonly used for phytocannabinoid profiling [11; 63; 147; 185; 188]. Other binary systems that provide good peak shape and improved resolution are also used, such as H\({}_{2}\)O + 0.1% formic acid/MeCN + 0.1% formic acid [40; 55; 64; 80; 193; 144; 182; 183; 185; 188; 189], H\({}_{2}\)O + 0.1% TFA/MeOH + 0.1% TFA [68], MeCN/H\({}_{2}\)O (75:25, + 0.05% formic acid, _v/v_)/ isopropanol:MeCN (80:20 + 0.05% formic acid, _v/v_) [183; 184], 5% MeCN + 0.1% formic acid/MeCN + 5% H\({}_{2}\)O + 0.1% formic acid [78], H\({}_{2}\)O + 0.1% formic acid/MeCN [27; 63; 185], 5% MeCN + 0.1% formic acid/80% MeCN + 0.1% formic acid [69] and MeCN/H\({}_{2}\)O + 0.85% phosphoric acid [42]. Other buffering solutions are less frequently used, such as 0.1% acetic acid in a tertiary system, e.g., H\({}_{2}\)O + 0.1% acetic acid/MeCN + 0.1% acetic acid/MeOH [19], MeCN/0.5% acetic acid (66:34, _v/v_) [190], ammonium formate in a binary system MeOH/H\({}_{2}\)O + 50 mM ammonium formate (pH 5.19) [83], 5 mM ammonium formate + 0.1% HCOOH/MeCN + 0.1% HCOOH [65; 100] or 5 mM ammonium formate/MeCN + 0.1% HCOOH [92]. Decrease in buffer concentration from 50 mM to 25 mM eliminated baseline drifting, thus avoiding decrease in UV absorption. As ammonium formate causes co-elution of \(\Delta^{9}\)-THCA with CBG, it is preferably replaced by ammonium acetate (25 mM, pH 4.75), which offers more reproducible, reliable and rugged chromatographic separation, especially between CBG and \(\Delta^{9}\)-THCA with improved peak shape and, thus, sensitivity. Better reproducibility is achieved using H\({}_{2}\)O/MeCN (15:85, _v/v_) + 50 mM phosphoric acid than with 0.1% formic acid as buffering solution [185]. Fast separation of 10 phytocannabinoids in less than 8.5 min using binary system H2O + 0.085% phosphoric acid/MeCN + 0.085% phosphoric acid as mobile phase was achieved, that, together with employment of RP-C18 column prevented co-elution of CBD and THCV and both isomers, \(\Delta^{9}\)-THC and \(\Delta^{8}\)-THC [165]. Another, less frequently used buffer is triethylammonium phosphate (TMAP) in MilliQ, with MeCN in isocratic elution programme [191]. * HPLC Columns The multitude of LC-based methods for phytocannabinoid profiling use similar columns; it is the variation of the instrumental conditions that produces superior quantification approaches. Only one methods use direct injection [184], bypassing the column. Most of the published methods employ columns with normal phase C18 stationary phase (Ascentis Express C18 [40,178], Luna C18 [69,177], Kinetex C18 [11,19,71], Luna Omega Polar C18 [58,114,118,139], Luna Omega PS C18 [27], XTerra MS C18 [83], Acquity UPLC BEH C18 [181], Acquity BEH Shield RP18 [144], MacMod ACE5 C18-AR [190], ACE 3 C18-PFP [187], ACE Excel 3 C18 [111], Poroshell 120 SB-C18 [182], Poroshell 120 EC-C18 [188,189] or 120 SB-C18 [34, 35, 36, 38], Shim-pack XR-ODSII RP C18 [149], Nucleodur\({}^{8}\) C18 Gravity [185], Zorbax Eclipse Plus C18 [92,183], Zorbax Eclipse XDBC18 [186], Zorbax SB-C18 [133], Atlantis T3 C18 [42], with [11,19,34,35,36,38,38,188,189] or without [43,184,185,188,189,190] guard column or C18 guard cartridge [177] that allow reliable separation and quantification of a wide range of phytocannabinoids (focused on, but not limited to, CBDV, CBDA, CBGA, CBG, CBD, THCV, CBN, \(\Delta^{9}\)-THC, A\({}^{8}\)-THC, CBC, \(\Delta^{9}\)-THC).