## 2 Analytical Methods for Phytocannabinoid Profiling
### Instrumental Analysis
#### 2.2.1 GC-Based Methods  
GC coupled to versatile detectors and mass analyzers is one of the oldest, but still the most preferred and researched analytical platforms for phytocannabinoid profiling in both plant material and biological matrices due to its robustness, reproducibility, sensitivity and speed [1; 42; 58; 153]. As such, GC methods are officially employed by authorities for phytocannabinoid profiling [1], including the predominant phytocannabinoids (\(\Lambda^{9}\)-THC, CBD and CBN) and quantification of \(\Lambda^{9}\)-THC/CBD ratio. This analytical platform is also used for terpene profiling, pesticide screening and residual solvents analysis, which affords potential benefits to regulatory bodies and cannabis industry [39]. By combining short columns, fast oven temperature ramps, high carrier gas linear velocities, narrow columns, hydrogen carrier gas and low film thickness, fast and robust GC methods are generated, appropriate for phytocannabinoid profiling in both research and monitoring purposes [39]. The access to the well-established MSLs, such as the National Institute of Standards and Technology (NIST) Mass Spectral Library and the Wiley Registry Mass Spectral Library eases compound identification through GC-MS analysis. Here, phytocannabinoids identification is performed by comparison of acquired MS or MS/MS spectrum to spectra present MSLs, and further confirmed by analysis of analytical standards. Although the main employment of GC-based platforms is for profiling of terpen, it has been extensively used for phytocannabinoid profiling as well (Table S1). * Derivatizationof Phytocannabinoids First action, after injection of the sample in the injector port of the GC (regardless of employed detector), is vaporization that is achieved at temperature ranges 250-290 \({}^{\circ}\)C and causes in situ decarboxylation of acidic phytocannabinoids (\(\Lambda^{9}\)-THC, CBDA, CBGA) to the corresponding neutral phytocannabinoids (\(\Lambda^{9}\)-THC, CBD, CBN) prior to chromatographic separation. Thus, acidic and neutral phytocannabinoids are not distinguishable, but rather the result is the sum of neutral cannabinoid present in the extract and neutral cannabinoid generated during decarboxylation. The issue is of no concern for studies aiming to quantify total THC (\(\Lambda^{9}\)-THC and \(\Lambda^{9}\)-THCA) levels, as it is the case with most of the GC-based studies included in this review without prior derivatization [9; 17; 18; 45; 47; 48; 49; 52; 54; 56; 58; 59; 63; 67; 72; 73; 74; 75; 76; 79; 81; 82; 85; 86; 89; 93; 96; 98; 101; 102; 103; 105; 108; 102; 101; 103; 105; 108; 115; 121; 122; 123; 124; 128; 138; 139; 141; 148; 150; 152; 154; 155; 156; 157; 158; 159; 160] (Table S1). However, derivatization is of great importance for studies aiming more thorough phytocannabinoid profiling. In order to prevent their degradation and achieve profiling of the native chemical constitution of the cannabis material, a reaction of derivatization has to be performed prior to GC analysis. In such a way, derivatization improves the limited volatility and thermal stability of phytocannabinoids, and thus their amenability to GC analysis, which further improves peak shape, peak resolution (especially for CBC and CBD) and sensitivity [161]. This comes at the price of increased analyses cost, duration [2] and measurement uncertainty, as derivatization yields are sometimes highly variable and seldomly obtain 100% for phytocannabinoids [23; 91; 122], making quantification results speculative. Silylation is the most common derivatization reaction performed. It involves substitution of a hydrogen atom that is bound to a hetero atom (such as -OH, -COOH, -NH\({}_{2}\), =NH, and -SH) by a silyl group, i.e., a trimethylsilyl (TMS) or _tert_-butyldimethylsilyl group (TBDMS). The resulting TMS/TBDMS derivatives have lower polarity and increased thermal and catalytic stability and GC amenability. However, they can be thermally degraded in injector port and/or column system [162].