## 2 Results and Discussion
### LC-ESI--LTQ-MS\({}^{n}\) Cannabinoids Identification  
Despite tandem mass spectrometry (MS) not being routinely available to most laboratories, MS is confirmed as a powerful technique for the identification of compounds in complex mixtures [9]. Since a few recent works outline the main cannabinoid fragmentation patterns by using positive and negative ion electrospray ionization [6; 23], here we performed the identification of THC, CBD, THCA, CBDA, and CBN in 34 cannabis light preparations by comparing the obtained CID-MS\({}^{n}\) spectra with literature data. Positive electrospray ionization (ESI) was used for neutral cannabinoids analysis, while negative ion mode was chosen for acid derivatives, since they can be easily deprotonated in the ESI source [24]. The obtained results are shown in Table 2. We also reported as an example the HPLC-UV chromatographic profile of the three selected samples chosen for toxicity studies (See Section 2.4). THC and CBD were both identified as protonated ions ([M + H]\({}^{+}\): C\({}_{21}\)H\({}_{31}\)O\({}_{2}\)\({}^{+}\)) at \(m/z\) 315. As they showed a very similar MS/MS spectrum in positive ion mode, retention times were used to confirm their identity by comparing the retention time of the analogous commercial standards. As reported in Table 2, THC and CBD MS/MS fragmentation patterns, together with MS\({}^{3}\), MS\({}^{4}\), and MS\({}^{5}\) experiments, allowed for confirmation of their identity, as already reported in literature [6]. All fragment ions were assigned as follows: ion at \(m/z\) 259 was caused by the loss of four C units of the terpenic portion; the \(m/z\) 235 ion was consistent with terpene breakage, with only four carbon units of this group left; ion at \(m/z\) 193 corresponded to olivetol moiety linked to C2 of the benzene ring; ion at \(m/z\) 181 was attributed to the olivetol moiety; ion at \(m/z\) 135 was obtained after the bond cleavage of the aromatic portion with the cyclohexenyl group, togheter with a hydrogen shift; ions at \(m/z\) 273, 245, 233, 231, and 207 were attributable to the olivetol derivatives; ion at \(m/z\) 227 was assigned to the loss of alkyl chain from dehydrated ion at \(m/z\) 297; and ions at \(m/z\) 175, 123, and 111 were assigned to structures containing the more stable aromatic group of CBD, retaining both oxygen atoms. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \multirow{2}{*}{**Compounds**} & **Linearity Range (mg/L)** & \multicolumn{2}{c}{**Calibration Equations**} & \multirow{2}{*}{**LOD (mg/L)**} & \multirow{2}{*}{**LOQ (mg/L)**} & \multicolumn{2}{c}{**Recovery (\%)**} & \multicolumn{2}{c}{**Repeatibility/Intermediate Precision (\%RSD)**} \\ \cline{3-3} \cline{5-10}  & & **y = ax (mg/L) + b** & & & & **R\({}^{2}\)** & **1 mg/L** & **1 mg/L** & **1 mg/L** & **10 mg/L** \\ \hline
**THC** & 0.2–25 &
\begin{tabular}{c} y = (129.27 \(\pm\) 0.40)x + \\ (0.78 \(\pm\) 2.69) \\ \end{tabular} & 0.9998 & 0.07 & 0.20 & 94.0 & 95.0 & 0.36/2.37 & 0.84/1.59 \\ \hline
**CBD** & 0.15–25 &
\begin{tabular}{c} y = (162.80 \(\pm\) 0.39)x + \\ (2.84 \(\pm\) 2.62) \\ \end{tabular} & 0.9999 & 0.05 & 0.15 & 94.4 & 91.1 & 0.23/3.49 & 0.08/1.86 \\ \hline
**THCA** & 0.25–25 &
\begin{tabular}{c} y = (75.63 \(\pm\) 0.29)x + \\ (11.83 \(\pm\) 1.93) \\ \end{tabular} & 0.9999 & 0.07 & 0.25 & 90.0 & 106.6 & 0.14/1.18 & 0.10/0.34 \\ \hline
**CBDA** & 0.25–25 &
\begin{tabular}{c} y = (89.37 \(\pm\) 0.33)x + \\ (7.60 \(\pm\) 2.20) \\ \end{tabular} & 0.9999 & 0.08 & 0.25 & 97.5 & 93.8 & 0.04/0.31 & 0.05/0.07 \\ \hline
**CBN** & 0.15–25 &
\begin{tabular}{c} y = (128.92 \(\pm\) 0.30)x + \\ (6.59 \(\pm\) 2.02) \\ \end{tabular} & 0.9998 & 0.05 & 0.15 & 87.2 & 89.0 & 1.51/1.50 & 0.19/1.72 \\ \hline \end{tabular}
\end{table} 
Table 1: Validation parameters of the LC-UV method used for the quantitative analysis of cannabinoids understudy, THC (\(\Delta^{9}\)-tetrahydrocannabinol), CBD (cannabidiol), THCA (\(\Delta^{9}\)-tetrahydrocannabinolic acid), CBDA (cannabidiolic acid), and CBN (cannabinol).