## 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
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**References** & [109] & [110] & [175] & [176] & [112] \\ \hline \multicolumn{6}{c}{**LC conditions**} \\ \hline LC column & \multicolumn{6}{c}{Agilent Poroshell 120-EC C18 (50 \(\times\) 3 mm, 2.7 um)} \\ Mobile phases A and B & \multicolumn{6}{c}{A: 5 mM ammonium formate and 0.1\% formic acid in H\({}_{2}\)O; B: Methanol} \\ \hline \multicolumn{6}{c}{[M+Na]\({}^{+}\)\(m/z\) for precursor and product ions in MRM for **CTXs type 1**} \\ \hline
1133.6 (P-CTX-1) & + * & + * & + * & + * \\ \hline
1117.6 (P-CTX-2/-3) & + * & + * & + * & + * \\ \hline
1083.6 (GT-4B/CTX-4A) & + * & + * & + * & + * \\ \hline
1101.6 (M-secoC-TX-4B/-4A) & u.m. & + * & u.m. & u.m. & u.m. \\ \hline \multicolumn{6}{c}{[M+Na]\({}^{+}\)\(m/z\) for precursor and product ions in MRM for **CTXs type 2**} \\ \hline
1045.6 (CTX-3C/-3B) & + * & + * & + * & + * \\ \hline
1061.6 (51-hydroxyCTX3C) & + * & + * & + * & u.m. \\ \hline
1063.6 (2-hydroxy-CTX3C) & u.m. & + * & + * & u.m. & u.m. \\ \hline
1079.6 (2,3-dihydroxyCTX-3C) & + * & + * & + * & + * & u.m. \\ \hline
1063.6 (M-secoC-CTX3C) & u.m. & + * & u.m. & u.m. & u.m. \\ \hline
1077.6 (M-secoC CTXC-methylacetal) & u.m. & + * & u.m. & u.m. & u.m. \\ \hline \multicolumn{6}{c}{[M+Na]\({}^{+}\)\(m/z\) for precursor and product ions in MRM for **C-CTXs type 3**} \\ \hline
1163.7 (C-CTX-1) & + * & + * & + * & + * \\ \hline
1177.6 (C-CTX-1-Me) & u.m. & + & u.m. & + * & + * \\ \hline \multicolumn{6}{c}{**Results for different fish**} \\ \hline Fish sampling & 11 fish * (SI) & 3 fish \({}^{\text{c}}\) (CI) & 1 fish \({}^{\text{c}}\) & 56 fish \({}^{\text{f}}\) & 109 fish * (CI) \\ (fishing spot) & 9 fish \({}^{\text{b}}\) (M) & 1 fish \({}^{\text{d}}\) (SI) & (SI) & (SI) & (fhesh, liver) \\ \hline \multicolumn{6}{c}{Cytotoxicity levels} \\ by CBA-N2a (min-max) & not used & Positive CTX-like & 1.4 ppb * & 0.006–0.75 ppb ** & \multicolumn{1}{c}{<LOQ–1.365 ppb **} \\ by CBA-N2a (min-max) & \multicolumn{6}{c}{} \\ \hline Fish C-CTX-1 levels by \multirow{2}{*}{LC-MS/MS (min-max)} & C-CTX-1 * & C-CTX-1 * & C-CTX-1 * & \multicolumn{1}{c}{<LOQ–1.365 ppb **} \\ (8 fish, SI) & (4 fish) & (1 fish) & (20 fish) & (30 fish) & (20 fish) \\ \cline{2-6}  & \textless{LOQ–0.25 ppb *} & 0.12–0.76 ppb * & 0.84 ppb * & \multicolumn{1}{c}{<LOQ–0.48 ppb *} & \multicolumn{1}{c}{<LOQ–0.270 ppb *} \\ \hline \multicolumn{6}{c}{+ = monitored ion. u. m. = unmonitored ion. CBA-N2a = neuroblastoma cell-based assay. CI = Canary Islands (Spain). M = Madeira (Portugal). SI = Selvagens Islands (Portugal). * Expressed in C-CTX-1 equivalents (FDA guidance: 0.1 ug/kg C-CTX-1 eq. = 0.1 ppb); * expressed in P-CTX-1 equivalents (FDA guidance: 0.01 ug/kg F-CTX-1 eq. = 0.01 ppb); * Confirmation with a reference standard. \({}^{\text{a}}\)_Epinephelus marginatus_ (dusky group, \(n=1\)). _Balvagens scripts_ (barred hogfish, \(n=3\)). _Balvagens scripts_ (grey rigerigerfish, \(n=2\)), _Mysterroperas fisca_ (ligand grover, \(n=1\)), _Serrrans attracion_ (blacktail comber, \(n=1\)), _Skyrobens scatteri_ (berumda sea chub, \(n=2\)), _Spiravan vitrilensis_ (barracuda, \(n=1\)) [109]. \({}^{\text{b}}\)_Serial jacio_ _nom (longfish volurail, \(n=1\)), _Maltria nigra_ (blue marlin, \(n=1\)), _Dentex gibbons_ (pink dentree, \(n=3\)), _Seridiad tumorial_ (greater arberberalex, \(n=2\)), _Spiravan zsap_ (smooth hammerhead, \(n=1\)), _Ianus oxymitches_ (short mako, \(n=1\)) [109]. \({}^{\text{c}}\)_Epinephelus marginatus_ (dusky grouper, \(n=1\)), implicated in CP), _Seriola_ spp. (amberjack, \(n=1\), implicated in CP); _Lutjanus oxymapterus_ (cubera snapper, \(n=1\)) [110]. \({}^{\text{d}}\)_Pagnus_ _Pagnus_ (red popy, implicated in CP) [110]. \({}^{\text{e}}\)_Serida fascita_ (amberjack implicated in CP) [175]. \({}^{\text{f}}\)_Kaphuss sectra_ (berumda sea chub, \(n=5\)), _Altternus scripts_ (scribelle leaflet; \(n=1\)). _Spiravan cetes_ (parridrids, \(n=14\)). _Dipidus corvinicus_ (rebra seabrebarn, \(n=1\)), _Balvagens scorb_ (barred hogfish, \(n=16\)), _Balvics corvinicus_ (grey rigerfish, \(n=4\)), _Serrans attracion_ (blacktail comber, \(n=5\)), _Sphranrata virrilensis_ (yellowumothur barracuda, \(n=3\)), _Seriola dumerili_ (greater arberalex, \(n=1\)), _Seriola riscita_ (lesser amberjack, \(n=2\)), _Seriola rivisoma_ (longfin yellowtail, \(n=2\)), _Seriola spp._ (amberjack, \(n=1\)) [176]. \({}^{\text{g}}\)_Seriola_ spp. (amberjack, \(n=60\)), _Epinephelus marginatus_ (dusy gowerp, \(n=27\)), _Murana helena_ (black moray eel, \(n=11\)), _Dipidus vulgaris_ (common two-banded seabream, \(n=11\)) [112].  
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