## 2 Analytical Methods for Phytocannabinoid Profiling
### Instrumental Analysis
#### 2.2.2 LC-Based Methods  
LC-based methods are recently becoming methods of choice for qualitative and quantitative phytocannabinoid profiling. The simplified sample preparation and the low temperatures, high pressure and high flow rates used during TLC, HPTLC, HPLC and UHPLC, and the recently emerging supercritical fluid chromatography (SFC) analysis allow sample preservation without decarboxylation and decomposition, reliable separation of neutral and acidic phytocannabinoid species and, thus, direct identification and quantification of both neutral and acidic forms of phytocannabinoids in the extracted samples [18; 165]. The simplified sample preparation methods, along with avoidance of analytes loss favourized LC over GC in cannabinoid profiling [66]. \(\bullet\) TLC and HPTLC Methods  
TLC is an attractive method for analyses of herbal drug constituents [168], and especially suitable method for the purpose of preliminary semi-quantitative screening of cannabinoid content in routine tests [164]. It is method of choice for identification of cannabis flowers in all _Cannabis fos_ monographs (DAB, AHP, Ph.Helv., Ph.Eur) [33; 34; 35; 36] and DAB's _Cannabis extractum normatum_ monograph [38]. Using TLC, cannabinoid identification is performed by comparing retardation factors (R\({}_{\text{F}}\)s) of analytes with R\({}_{\text{F}}\)s of standards on a TLC plate developed with appropriate mobile phase, whereas visual evaluation is obtained by dipping or spraying the TLC plate into/with the appropriate visualization reagent under UV light, or under daylight. In DAB [36] and Ph.Eur. [35] analytical monographs of cannabis flower, a MeOH extract of 0.1 g pulverized drug is identified by comparison of R\({}_{\text{F}}\)s of analytes to reference solutions of CBD and \(\Delta^{9}\)-THCA (5 mg each) in MeOH. Solutions are applied on TLC C\({}_{18}\) silica gel F\({}_{254}\) plate (2 to 10 \(\upmu\)m) and developed with H\({}_{2}\)O/glacial acetic acid/MeOH (15:15:70, _v/v/v_). After air drying, the TLC plate is sprayed with vanillin reagent, dried at 100-105 \({}^{\circ}\)C for 15 min and examined on daylight. Identical procedure applies to _Cannabis extractum normatum_ according to DAB [38]. Ph.Helv. includes filtration of MeOH extract of cannabis flower through membrane filter (0.45 \(\upmu\)m) as an additional step prior to application to the C\({}_{18}\) silica gel F\({}_{254}\) plate and reference solution of CBDA in MeCN and \(\Delta^{9}\)-THCA in 2-isopropanol, following the identical procedure for development and detection [34]. AHP's cannabis flower monograph employs TLC C\({}_{18}\) F\({}_{254}\) plate with MeOH/H\({}_{2}\)O with 1% glacial acetic acid (75:25, _v/v_) as a mobile phase for identification of CBC, \(\Delta^{9}\)-THCA, CBN, CBG, CBD THCV, \(\Delta^{9}\)-THCA and CBDA in dichloromethane extract of 0.1 g pulverized drug. Visualization is performed using Fast Blue reagent and vanillin/H\({}_{2}\)SO\({}_{4}\) under UV (254 nm) [33]. UNODC suggests maceration in ultrasound bath with 10 mL of solvent (MeOH, petroleum ether, _n_-hexane, toluene, CHCl\({}_{3}\) or solvent combinations-MeOH:CHCl\({}_{3}\) (9:1, _v/v_) for 15 min at room \({}^{\circ}\)C, using three systems for elution of HPTLC silica gel plates (A: petroleum ether 60/90/diethyl ether (80:20, _v/v_); B: cyclohexane/di-isopropyl ether/diethylamine (52:40:8, _v/v_) and C (for cannabinoid acids): _n_-hexane:dioxane/MeOH (70:20:10, _v/v_)). Fast Blue reagent BB or RR in MeOH or MeOH:H\({}_{2}\)O is used as spraying reagent using visualization method 1 or method 2 [5]. The accuracy, repeatability and the acceptable LODs and LOQs in the linear dynamic range of this methodology makes TLC methods attractive for fingerprinting cannabis [4]; however, such parameters are fairly low compared to the more sophisticated LC analytical platforms.