## 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} \hline
**References** & [108] & [39] & [174] & [37] \\ \hline \multicolumn{5}{c}{**LC conditions**} \\ LC column & Phenomenex Luna & Phenomenex Luna & Phenomenex Kinetex & Phenomenex Kinetex C8 \\  & CS(2) (150 \(\times\) 2.0 mm) & C18 (100 \(\times\) 2 mm, 3 \(\mu\)m) & CS (75 \(\times\) 2.1 mm, 2.6 \(\mu\)m) & (75 \(\times\) 2.1 mm, 2.6 \(\mu\)m) \\ Mobile phase A & H\({}_{2}\)O & H\({}_{2}\)O & H\({}_{2}\)O & H\({}_{2}\)O \\ Mobile phase B & 100\% acetonitrile & 95\% acetonitrile & 95\% acetonitrile & 95\% acetonitrile & 95\% acetonitrile \\ Modifier (A and B) & 0.1\% formic acid & 5 mM ammonium acetate & 5 mM ammonium formate & 0.1\% formic acid \\ \hline \multicolumn{5}{c}{**MRM transitions for C-CTX-1 detection**} \\ \hline
[M+H–H\({}_{2}\)O]\({}^{+}\)\(m/z\) & & & 1123.6 \\ precursor ion & & & & \\ \hline
[M+H–H\({}_{2}\)O]\({}^{+}\)\(m/z\) & & & & \\ product ions & 1105.6; 1087.6; 1069.6 & 1105.7; 1087.7; 1069.8 & 1087.6; 1069.7 & 1105.6; 1087.6; 1069.6 \\ \hline \multicolumn{5}{c}{**Results for different fish**} \\ \hline Fish sampling & 1 barracuda \({}^{a}\) (fishing spot) & 153 lionfish (U) & 60 lionfish \({}^{b}\) & 77 fish \({}^{c}\) (Vl) \\  & (UO) & (VI) & 55 (SB), 5 (SM), 30 (G) & (VI) \\ \hline Cytotoxicity levels & 1.6 ppb \({}^{*}\) (cooked) & \textless{0.1 ppb \({}^{*}\) (43 fish)} & \textless{0.01 ppb \({}^{**}\) (5 fish, SB)} & \textgreater{}0.005 ppb \({}^{*}\) (29 fish) \\ by CBA-N2a & 2.1 ppb \({}^{*}\) (raw) & \textgreater{}0.1 ppb \({}^{*}\) (19 fish) & 0.01–0.1 ppb \({}^{**}\) (14 fish, SB)} & \textgreater{}0.1 ppb \({}^{*}\) (3 fish) \\ \hline C-CTX-1 confirmation & C-CTX-1 \({}^{*}\) & C-CTX-1 \({}^{*}\) & C-CTX-1 \({}^{*}\) & C-CTX-1 \({}^{*}\) (8 fish, SB) & (13 fish) \\ by LC-MS/MS & (1 fish) & (19 fish) & (8 fish, SB) & (13 fish) \\ \hline C-CTX congeners & C-CTX-1159, -1157 and -1143 & C-CTX-2 \({}^{*}\) & - & - \\ \hline \multicolumn{5}{c}{CBA-N2a = neuroblastoma cell-based assay: G = Guadeloupe (Lesser Antilles). SM = Saint Martin (Lesser Antilles). SB = Saint Barthélemy (Lesser Antilles). UO = unknown origin. VI = U. S. Virgin Islands (Lesser Antilles). * Expressed in C-CTX-1 equivalents (FDA guidance: 0.1 \(\mu\)g/kg C-CTX-1 eq. = 0.1 ppb). ** Expressed in P-CTX-1 equivalents (FDA guidance: 0.01 \(\mu\)g/kg P-CTX-1 eq. = 0.01 ppb). \({}^{*}\) Confirmation with a C-CTX-1 reference standard. \({}^{a}\) _Sphraema_ sp. (barracuda implicated in CP). \({}^{b}\) _Pterois roilatus. \({}^{c}\) Balistes retalta_ (queen triggerfish, \(n\) = 20). _Epinelphus gutututus_ (red hind, \(n\) = 20), _Ocyurus chrysurus_ (yellowtail snapper, \(n\) = 17), _Haemalon plumirai_ (white grant, \(n\) = 20) [37].} \\ \hline \end{tabular}
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Table 4: Summary of analytical approaches and results of C-CTX-1 confirmation by MRM using an ESI-quadrupole-linear ion trap instrument (QTRAP 4000, Applied Biosystems) in four studies on fish specimens from the Atlantic Ocean (Lesser Antilles) [37; 39; 108; 174]. The same approach also confirmed that invasive lionfish species (_Pterois volitans_) caught in the waters of the U. S. Virgin Islands [39] and Saint Barthelemy (French Antilles) [174] accumulated C-CTX-1. A 12% prevalence rate of ciguatoxic lionfish from the U. S. Virgin Islands, exceeding the FDA guidance level of 0.1 ppb C-CTX-1 eq., was established using CBA-N2a, and fish size did not correlate to toxicity level [39]. Selected reaction monitoring using LC-MS/MS confirmed the presence of C-CTX-1 in CBA-N2a-positive samples compared to reference standards [39]. In lionfish from Guadeloupe or Saint-Martin, no CTX-like activity was found, and C-CTX-1 was structurally confirmed using HPLC-MS/MS with a C-CTX-1 reference standard in the eight ciguatoxic samples from Saint-Barthelemy with composite cytotoxicities ranging from 0.157-0.33 ppb P-CTX-1 eq. [174]. Among the 77 samples of fish species from the U. S. Virgin Islands tested by [PERSON] et al. [37], 39 had cytotoxic activity determined using CBA-N2a.