## 2 Analytical Methods for Phytocannabinoid Profiling
### Instrumental Analysis
#### 2.2.2 LC-Based Methods
For wide range methods, such as cannabis-based medical extracts [182], cannabis-infused cholcate [65] and 17 phytocannabinoids in cannabis inflorescences and oils [100], 228 nm has been shown to be the most suitable. Multiple detection wavelengths, e.g., 220 nm, 240 nm, 270 nm and 307 nm are also used for phytocannabinoids profiling [111; 190]. Evaluation of these detection wavelengths for five phytocannabinoids (CBD, CBDA, CBN, \(\Delta^{9}\)-THC and \(\Delta^{9}\)-THCA) in versatile cannabis-based products was performed. While none of the phytocannabinoids showed \(\lambda_{\text{max}}\) at 240 nm, this wavelength tended to equalize the response (slope) across the five phytocannabinoids, except for CBN, which has the highest response. The highest response for all five components was found with 220 nm and was useful for low level quantification (Table S2). 270 nm and 307 nm provided high selectivity for CBDA and \(\Delta^{9}\)-THCA, which was used to minimize or eliminate detection of matrix interferences, as needed. Detection wavelengths in the range of 270-280 nm are inappropriate for CBD, CBN, and \(\Delta^{9}\)-THC due to retention time (R\({}_{\text{t}}\)) interferences [90]. Although many studies employ UV/DAD for separation of the major phytocannabinoids (CBDA, CBD, CBN, \(\Delta^{9}\)-THC and \(\Delta^{9}\)-THCA), most do not account for interference from minor phytocannabinoids (e.g, CBNA). Such interference is of special importance during profiling of concentrates where minor phytocannabinoids can be enriched to detectable levels. Additionally, some terpen absorb UV light at the same wavelength as phytocannabinoids. All these issues decrease sensitivity, specificity and selectivity of UV/DAD methods, which are easily overcome by employment of MS detection. Detection of neutral phytocannabinoids based on their ability to absorb fluorescence under the acidic conditions used in RP-LC is only recently reported [111] as a fast, low-cost and selective alternative, but without the ability to detect phytocannabinoid acids and a somewhat narrower linear range than DAD due to saturation at high concentrations. Most commonly used MS analyzers include simple Q spectrometers [181], QQQ analyzers [11; 58; 68; 114; 147; 193; 194], IT analyzers [28; 43; 72; 185], QTRAP [67; 159; 191; 195] or high resolution-accurate mass MS (HRAM-MS) analysers, such as Q-TOF [83; 88; 139; 189] and Q-ExactiveTM Orbitrap [19; 63; 65; 66]. P phytocannabinoid profiling is most commonly performed using ESI, rarely using variations such as dual source [78] or heated ESI source [19; 63]. Acquisitions are performed in positive (+) [88; 147; 189; 191; 193; 194], negative (-) [134] or both (+) and (-) modes [62; 63; 64; 69; 144; 178; 181]. In fact, all major phytocannabinoids are detected in both ionization modes, except for THCV [78]. However, it is noted that acidic phytocannabinoids (CBDA, THCA, CBCA, CBGA, CBNA, CBCVA) give better signals in the (-) mode, while neutral phytocannabinoids are better ionized in (+) mode (CBDV, CBG, CBD, CBDA, CBGA, \(\Delta^{9}\)-THCA, \(\Delta^{9}\)-THC and \(\Delta^{8}\)-THC) [43; 58; 83]. ESI (-) provides identification and quantification of additional minor phytocannabinoids, such as CBGA methyl ester (CBGMA) [40] and improves identification accuracy for two neutral phytocannabinoids (CBL and CBN) [78]. Atmospheric pressure chemical ionization (APCI) is also used in (+) [11] and (-) mode [69] for phytocannabinoid profiling in plant material. Formation of (+) chargedsodium adducts [M+Na]\({}^{+}\) instead of the precursor ion (M+ H)\({}^{+}\) is observed in (+) ionization mode. This feature increases sensitivity and allows accurate identification of 7 phyto-cannabinoids (cannabicoumaric acid, CBCA, CBGA, CBGAM, 10-ethoxy-9-hydroxy-\(\Delta^{6}\alpha\)-THC, 4-acetoxycannabichromeand \(\Delta^{9}\)-THCA-C4). APCI (\(-\)) is shown to be suitable for CBD, CBG and CBGA [69].