## 5 Analytical Methods for Detection, Identification, and Quantification of C-CTXs
### Analysis of C-CTXs in Fish by HPLC-MS/MS (Low-Resolution)
#### 5.2.1 First Quantification of C-CTX-1 in Caribbean Fish Using HPLC-MS/MS
All the identified C-CTXs analogues \begin{table}
\begin{tabular}{c c c} \hline \hline
**References \(\rightarrow\)** & [110] & [175] & [176] \\ \hline \multicolumn{3}{c}{**LC conditions**} \\ \hline LC column & Agilent Poroshell 120-EC C18 (100 \(\times\) 2.1 mm, 2.7 \(\mu\)m) \\ Mobile phases & (A) 5 mM ammonium formate and 0.1\% formic acid in H2O; (B) acetonitrile \\ \hline \multicolumn{3}{c}{**Precursor and Product ions for confirmation of C-CTX-1 \({}^{\#}\) and isomers**} \\ \hline
[M+H–H2O]+ & C-CTX-1 [M+H–nH2O]+ \(m/z\) 1105.6; 1087.6; 1069.6 \\ \(m/z\) 1123.6 & specific fragments \(m/z\) 191.1; 108.9 \\ \hline \multicolumn{3}{c}{**Precursor and Product ions for confirmation of C-CTX-1157**} \\ \hline
[M+Na]+ & u.m. & [M+Na]+ \(m/z\) 1179.6 \\
[M+H–H2O]+ & u.m. & [M+H–nH2O]+ \(m/z\) 1121.6; 1103.6; 1085.6 \\ \(m/z\) 1139.6 & & specific fragments \(m/z\) 191.1; 108.9 \\ \hline \multicolumn{3}{c}{**Precursor and Product ions for confirmation of C-CTX-1127**} \\ \hline
[M+Na]+ & u.m. & C-CTX-1127 [M+Na]+ \(m/z\) 1149.6 \\ \(m/z\) 1149.6 & & u.m. \\ \hline
[M+H]+ & u.m. & [M+H–nH2O]+ \(m/z\) 1109.6; \\ \(m/z\) 1127.6 & & 1091.6; 1073.6; 1055.6 \\ \hline \multicolumn{3}{c}{**Precursor and Product ions for confirmation of C-CTX-1-Me**} \\ \hline
[M+H–CH3–H2O]+ & u.m. & u.m. & [M+H–nH2O]+ \(m/z\) 1105.6; 1087.6; 1069.6 \\ \(m/z\) 1123.6 & u.m. & u.m. & specific fragments \(m/z\) 191.1; 108.9 \\ \hline \multicolumn{3}{c}{**Results for studied fish specimens**} \\ \hline Fish sampling (fishing spot) & 3 fish \({}^{a}\) (CI) & 1 fish \({}^{c}\) & 56 fish \({}^{d}\) \\
[fishing spot] & 1 fish \({}^{b}\) (SI) & (SI) & (SI) \\ \hline Cytotoxicity levels by & Positive & 1.4 ppb \({}^{*}\) & 0.006–0.75 ppb \({}^{**}\) \\ CBA-N2a (min–max) & C-CTX-1 & C-CTX-1 & C-CTX-1 \\ by LC-MS/MS & (4 fish) & (1 fish) & (20 fish) \\ \hline C-CTX congeners detected & C-CTX-1181 \({}^{*}\) & C-CTX-1157 \({}^{*}\), C-CTX-1127 \({}^{*}\), & C-CTX-1157 \\ by LC-MS/MS & & C-CTX-1181 \({}^{*}\) & C-CTX-1157 \\ \hline \hline \multicolumn{3}{c}{Abbreviations: u.m. = unmonitored ion. CBA-N2a = neuroblastoma cell-based assay. CI = Canary Islands (Spain). SI = Selvagens Islands. \({}^{*}\) Confirmation with a reference standard. \({}^{*}\) CTX-positive response (CBA-N2a). \({}^{**}\) Expressed in P-CTX-1 equivalents (FDA guidance: 0.01 \(\mu\)g/kg P-CTX-1 eq. = 0.01 ppb). \({}^{*}\) Epinphachs marginals (dusky group; \(n\)=1, implicated in CP) Serial spp. (amberjack, \(n=1\), implicated in CP) Serialism spp. (amberjack, \(n=1\), implicated in CP) Serialism spp. (amberjack, \(n=1\)), \({}^{b}\) Pagens Pagrans (red Porgy implicated in CP) [110]. \({}^{c}\) Serial fasciata (amberjack implicated in CP) [175]. \({}^{d}\) Kphalosus sextric (bermuda sea chub, \(n=5\)), Altarers scripts (scribibled leatherjacket, \(n=1\)), Sparisoma cetense (parroftish, \(n=14\)), Dipaldus cervins (zebra seabream, \(n=1\)), Bodinus cervins (barred hogfish, \(n=16\)), Ballises cepriss (grey ruggershi, \(n=4\)), Seras atitrala (blacktail comber, \(n=5\)), Sylvan viridensis (yellowmouth barraccuda, \(n=3\)), Serida lammerli (greater amberjack, \(n=1\)), Serida fasciata (lesser amberjack, \(n=2\)), Serida riviolana (longfin yellowful, \(n=2\)), Serida spp (amberjack, \(n=1\)) [176]. \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Summary of analytical approaches and results of C-CTX-1 confirmation and C-CTX identification using MRM and an ESI-triple quadrupole (6495 Agilent) on fish specimens from the North-East Atlantic Ocean (Macaronesia) [110,175,176]. Precursor ions are in bold. produced the characteristic ions associated with CTXs, including [M+H]\({}^{+}\), [M+NH4]\({}^{+}\), [M+Na]\({}^{+}\), [M+K]\({}^{+}\), and [M+H-nH2O]\({}^{+}\)[175]. C-CTX-1157 was also reported in a zebra seabram (_Diplodus cervinus_) from the Selvagens Islands that showed composite toxicity using CBA-N2a, although C-CTX-1 was not detected using LC-MS/MS [176].